Journal of the Brazilian Society for Virology Editors Editorial Board

Transcrição

VIRUS Reviews and Research
Journal of the Brazilian Society for Virology
Volume 17 (2), October 2012, Supplement 1
Annals of XXIII Brazilian Congress of Virology & VII Mercosur Meeting of Virology
September, 30 - October, 03, 2012, Rafain Hotel, Foz do Iguaçu, Paraná, Brazil
Editors
Romain Rolland Golgher, Editor
Fernando Rosado Spilki, Adjunct Editor
Editorial Board for this Supplement
Alexandre da Costa Linhares (PA, BR)
Aramis Augusto Pinto (SP, BR)
Carlos Frederico Menck (SP, BR)
Carlos M. Nozawa (PR, BR)
César Martins Chagas(RJ, BR)
Christian C. Niel (RJ, BR)
Clarice W. Arns (SP, BR)
Dean D. Erdman (USA)
Edson E. da Silva (RJ, BR)
Elliot Watanabe Kitajima (SP, BR)
Erna Geessien Kroon (MG, BR)
Hélio José Montassier (SP, BR)
José Albersio A. Lima (CE, BR)
José Antônio Jerez (SP, BR)
José Marcus S. Teixeira (DF, BR)
José Paulo G. Leite (RJ, BR)
John Woodall (RJ, BR)
Juan Arbiza (Uruguay)
Klaus E. Stewien (SP, BR)
Larry J. Anderson (USA)
Luisa Lina Villa (SP, BR)
Luiza Theresina M. de Souza (SP, BR)
Luiz Tadeu M. Figueiredo (SP, BR)
Maria Lúcia Rácz (SP, BR)
Marilda M. Siqueira (RJ, BR)
Moacyr Alcoforado Rebello (RJ, BR)
Nissin Moussatché (USA)
Olen M. Kew (USA)
Paolo Zanotto (SP, BR)
Paul Rota (USA)
Ricardo Ishak (PA, BR)
Roger I. Glass (USA)
Vera Gouvêa (RJ, BR)
Address
Rua Guaratinga 180/201 - 30315/430 Belo Horizonte - MG, Brasil.
Phone +55-31-3223-6239. Fax +55-31-3224-6239.
E-mail [email protected]
www.virusreviewsandresearch.com
XXIII Brazilian Congress of Virology & VII Mercosur Meeting of Virology
October 2012– Volume 17 (2) – Supplement 1 / Foz do Iguaçu, Paraná, Brazil
BRAZILIAN SOCIETY FOR VIROLOGY DIRECTORATE BOARD
(2011-2012)
Officers
President: Clarice Weis Arns, UNICAMP
Vice-President: Eurico de Arruda Neto, USP
First Secretary: Paula Rahal, UNESP
Second Secretary: Paulo Eduardo Brandão, USP
First Treasurer: Maria Angela Orsi, LANAGRO
Second Treasurer: Viviane Fongaro Botosso, INSTITUTO BUTANTAN
Executive Secretary: Fabrício Souza Campos, SBV
Councillors
Edson Elias da Silva, FIOCRUZ (2011-2012)
Maria Luisa Barbosa, INSTITUTO ADOLFO LUTZ (2011-2012)
Luiz Tadeu Figueiredo, USP (2011-2012)
Area Representatives
Basic Virology (BV)
Luciana Jesus Costa, UFRJ
Davis Fernandes Ferreira, UFRJ
Environmental Virology (EV)
Célia Regina Monte Barardi, UFSC
Fernando Rosado Spilki, Universidade FEEVALE
Human Virology (HV)
Maurício Lacerda Nogueira, FAMERP
Regina Maria Bringel Martins, UFG
Immunobiological Virology (IV)
Livia Melo Villar, FIOCRUZ
Silvia Maria Baeta Cavalcanti, UFF
Plant and Invertebrate Virology (PIV)
Alice Kazuko Inoue Nagata, EMBRAPA
Bergmann Morais Ribeiro, UNB
Veterinary Virology (VV)
Janice dos Reis Ciacci Zanella, EMBRAPA
Luizinho Caron, EMBRAPA
October 2012 Volume 17 (2) – Supplement 1 / Foz do Iguaçu, Paraná, Brazil
Organizing Committee
Amauri Alcindo Alfieri, UEL - Presidente do XXIII CBV
Alice K. I. Nagata, EMBRAPA
Bergmann Morais Ribeiro, UNB
Célia R. M. Barardi, UFSC
Clarice Weis Arns, UNICAMP – Presidente da SBV
Clarissa Damaso, UFRJ
Cláudia Maria Oliveira Simões, UFSC
Davis Fernandes Ferreira, UFRJ
Eduardo Furtado Flores, UFSM
Eurico de Arruda Neto, USP
Fernando Spilki, Universidade FEEVALE
Francisco Murilo Zerbini, UFV
Janice dos Reis Ciacci Zanella, EMBRAPA
João Pessoa Araújo Júnior, UNESP
José Alberto Caram de Souza Dias, IAC
José Paulo Leite, FIOCRUZ
Livia Melo Villar, FIOCRUZ
Luiz Tadeu Figueiredo, USP
Maria Angela Orsi, LANAGRO
Maurício Lacerda Nogueira, FAMERP
Paula Rahal, UNESP
Paulo Eduardo Brandão, USP
Regina Maria Bringel Martins, UFG
Viviane Fongaro Botosso, INSTITUTO BUTANTAN
Zélia Inês Portela Lobato, UFMG
Board of Examiners - Hélio Gelli Pereira Award
Invited Speakers
Paula Rahal, UNESP
Francisco Murilo Zerbini Junior, UFV
Alexandre da Costa Linhares, Dr., INSTITUTO
EVANDRO CHAGAS, PA, BRAZIL
Alice Kazuko Inoue Nagata, Dr., EMBRAPA
HORTALIÇAS, DF, BRAZIL
Ana Carolina Jardim, Dr., UNESP, SP, BRAZIL
Ana Cláudia Franco, Dr., UFRGS, RS, BRAZIL
Andréia Henzel, Dr., UFSM, RS, BRAZIL
Aparecida Yulie Yamamoto, Dr., FMRP-USP, SP,
BRAZIL
Ariel J. Pereda, Dr., INTA, ARGENTINA
Athos Silva de Oliveira, Dr., UNB, DF, BRAZIL
Caroline Rogotto Borges, Dr., UFSC, SC, BRAZIL
Celso Granato, Dr., UNIFESP, SP, BRAZIL
Célia Regina Monte Barardi, Dr., UFSC
Clarissa Damaso, Dr., UFRJ, RJ, BRAZIL
Colin Jeffries, Dr., SASA - SCIENCE & ADVICE FOR
SCOTTISH AGRICULTURE, UNITED KINGDOM
Cristina Carlan da Silva, Dr., FUNDAÇÃO
UNIVERSIDADE FEDERAL DO ABC, SP, BRAZIL
Curtis A. Suttle, PhD., UNIVERSITY OF BRITISH
COLUMBIA, VANCOUVER, CANADA
Daniel Perez, Dr., THE UNIVERSITY OF
MARYLAND, USA
Danielle B. Oliveira, Dr., ICB/ USP, SP, BRAZIL
Davis Fernandes Ferreira, Dr., UFRJ, RJ, BRAZIL
Dumith Chequer Bou-Habib, Dr., FIOCRUZ, RJ,
BRAZIL
Edison Luiz Durigon, Dr., USP, SP, BRAZIL
Elisabeth Lampe, Dr., FIOCRUZ, RJ, BRAZIL
Elizabeth P. B. Fontes, Dr, UFV, MG, BRAZIL
Elsa B. Damonte, Dr., UNIVERSIDAD DE BUENOS
AIRES, ARGENTINA
Fernando Lucas Melo, Dr., UNB, DF, BRAZIL
Francisco Murilo Zerbini Júnior, Dr., UFV, MG,
BRAZIL
Hiroyuki Katayama, PhD., UNIVERSITY OF
TOKYO, JAPAN
Isabel Guedes, Dr., UNIFES, SP, BRAZIL
Janice Reis Ciacci Zanella, Dr., EMBRAPA SUÍNOS
E AVES, SC, BRAZIL
Jeffries Coling, Dr., SASA - SCIENCE & ADVICE
FOR SCOTTISH AGRICULTURE, UNITED
KINGDOM
João Pessoa Araújo Junior, Dr., UNESP, SP, BRAZIL
João Renato Rebello Pinho, Dr., HOSPITAL
ISRAELITA ALBERT EINSTEIN, SP, BRAZIL
José A. Caram Souza Dias, Dr., IAC, SP, BRAZIL
José Carlos Couto Fernandez, Dr., IOC, RJ, BRAZIL
José Paulo Gagliardi Leite, Dr., FIOCRUZ, RJ,
BRAZIL
Juliana Echevarria, Dr., UFRJ, RJ, BRAZIL
Juliana Freitas-Astua, Dr., EMBRAPA MANDIOCA
E FRUTICULTURA CITRUS RESEARCH &
TECHNOLOGY, BA, BRAZIL
Juliano Bordigon, Dr., CARLOS CHAGAS
INSTITUTE, PA, BRAZIL
Jurema Schons, Dr., UPF, RS, BRAZIL
Karla Kirkegaard, PhD., STANFORD UNIVERSITY,
USA
Laura Sichero, Dr., ICESP, SP, BRAZIL
Luciana Helena Antoniassi, Dr., UNICAMP, SP,
BRAZIL
Luciana Jesus da Costa, Dr., UFRJ, RJ, BRAZIL
Luciana Kohn, Dr., UNICAMP, SP, BRAZIL
Luciano Tomazelli, Dr., ICB/USP, SP, BRAZIL
Lucio Gama, Dr., JONH HOPKINS UNIVERSITY,
USA
Maitê Vaslin de Freitas Silva, Dr., UFRJ, RJ,
BRAZIL
Marcelo Lopèz-Lastra, Dr., PONTIFICIA
UNIVERSIDAD CATÓLICA DE CHILE, CHILE
Maria Angela Orsi, Dr., LANAGRO, SP, BRAZIL
Maria Cristina Carlan da Silva, Dr., UFABC, SP,
BRAZIL
Maria Inês Zanolli Sato, Dr., ENVIRONMENT
COMPANY OF SÃO PAULO STATE, SP, BRAZIL
Marilyn J. Roossinck, Ph.D., THE PENNSYLVANIA
STATE UNIVERSITY, USA
Marina Gallo Calderón, Dr., CONSEJO NACIONAL
DE
INVESTIGACIONES
CIENTIFICAS
Y
TECNICAS, ARGENTINA
Martin Schutten, Dr., ERASMUS UNIVERSITY,
NETHERLANDS
Marylynn V. Yates, PhD., UNIVERSITY OF
CALIFORNIA, USA
Matías Victoria Montero, Dr., FIOCRUZ, RJ,
BRAZIL
Maurício Lacerda Nogueira, Dr., FAMERP, SP,
BRAZIL
Nancy Bellei, Dr., UNIFESP, SP, BRAZIL
Nicolas Gaidet, Dr., CIRAD, FRANCE
Nikolaos Vasilakis, Ph.D., UNIVERSITY OF
TEXAS MEDICAL BRANCH, USA
Paula Rahal, Dr., UNESP, SP, BRAZIL
Paula Radaelli, Dr., SYNGENTA, CE, BRAZIL
Paulo Michel Roehe, Dr., UFRGS, RS, BRAZIL
Paulo Sérgio Torres Brioso, Dr., UFRRJ, RJ,
BRAZIL
Peter Mertens, Dr., INSTITUTE FOR ANIMAL
HEALTH, PIRBRIGHT, UNITED KINGDOM
Poliane Alfenas Zerbini, Dr., UFV, MG, BRAZIL
Regina Barbosa Schröeder, Dr., HOSPITAL DOM
VICENTE SCHERER, RS, BRAZIL
Regina Maria Bringel Martins, Dr., UFG, GO,
BRAZIL
Ricardo Andrez Machado de Ávila, Dr., UFMG,
MG, BRAZIL
Sandra Vieira, Dr., FACULDADE DE MEDICINA
DA USP, SP, BRAZIL
Sarah da Silva Barreto, Dr., UNB, DF, BRAZIL
Simone Ribeiro, Dr., EMBRAPA CENARGEN, DF,
BRAZIL
Tatiana Michelon, Dr., UFCSPA, DF, BRAZIL
Thales Passos de Andrade, Dr., UNIVERSIDADE
ESTADUAL DO MARANHÃO, MA, BRAZIL
Timo Vesikari, PhD., UNIVERSITY OF TAMPERE,
MEDICAL SCHOOL, FINLAND
Victor Hugo Aquino Quintana, Dr., FCFRP-USP,
SP, BRAZIL
Vito Martella, Dr., UNIVERSITY OF BARI,
VALENZANO, ITALY
Viviane
Fongaro
Botosso,
INSTITUTO
BUTANTAN, SP, BRAZIL
Wyller Alencar de Mello, Dr., INSTITUTO
EVANDRO CHAGAS, PA, BRAZIL
Zelia Ines Portela Lobato, Dr., UFMG, MG, BRAZIL
Financial Support
CAPES
Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior
CNPq
Conselho Nacional de Desenvolvimento Cientifico
e Tecnológico
FUNCAMP
Fundação de Desenvolvimento da Unicamp
FUNDAÇÃO ARAUCÁRIA
Apoio ao Desenvolvimento Científico e Tecnológico
do Paraná
Exhibitors
ALLCROM
BIOMETRIX
BIOSAFE
LOBOV
NOVA ANALÍTICA
QIAGEN
SARSTEDT
SIGMA-ALDRICH
VECO
Organizer
Office Marketing Eventos
General Information
Secretary Schedule
September, 30th - 9am - 8pm
October, 1st - 7am - 8:30pm
October, 2nd - 7am - 2pm
October, 3rd - 7am - 17pm
Identification Card
The identification card will be required to attend
all activities of the meeting, including lunch area.
Media Desk (for lecturers only)
The media desk will be open as scheduled for the
secretary of the meeting.
Data - files with presentations - must be delivered
at the media desk at least 2 hours before the
scheduled time for the presentation. Please note
that personal computers will not be allowed in
lectures. Presentations will be copied and made
available to members of SBV after the meeting at
the institutional homepage unless unauthorised
by the speakers.
VIP Room
A VIP room will be available for lecturers, invited
persons and SBV staff.
Certificates
The certificates of presentation/participation
will be available at the secretary of the event on
the last day of the meeting. Identification cards
will be required.
Travel Agency
The official agency Centraltours Iguassu Service
will have an exclusive desk at the venue, offering
some tours:
Visit the Argentine and Brazilian Falls, Itaipu
Dam, Paraguay and Argentina in Shopping,
Duty Free Shop among other attractions. Every
night regular departures to the famous Latin
American Folkloric Show at Churrascaria Rafain,
watching a musical and dance while enjoying
a tasty barbecue with nobles meat. This is the
dish offered by Rafain Falls Steakhouse Show in
Foz do Iguaçu, a mixture that goes beyond the
cultural diversity of the three countries’ borders.
Special value including transportation + Dinner
+ Show : R$ 90.00 per person
Special Program for the day 02/10/2012 - From
2:00 pm
• Walk to the Falls Brazilian side (does not
include entrance fee for the Iguaçu National
Park)
Value R$ 20.00 per person
Value of ticket per person:
Brazilian = R$ 24.60
Mercosur = R$ 32.85
Other Nationalities = R$ 41.10
• Shopping: Tour to Shopping del Este at
Paraguay side Value R$ 20.00 per person
• Night Special: ballad (disco) in ONO MUSIC
HALL Begin: 11 p.m
DJ IVAN Scarparo (Sertanejo / Pop / Rock
/ Electronic / Latin / Funk)
Promotion:
• On purchase of 06 tickets wins a reserved
table.
• Purchase of 10 tickets wins a cabin.
** With availability and previous consultant**.
Scientific Program
Sunday, Sep. 30
Room 3
Pre-congress Seminar on Veterinary Virology in Brazil
Pre-congress
10:00 am – 12:30 pm Chairman: Paulo Michel Roehe, Dr., Fepagro Saúde Animal - IPVDF & UFRGS
Opening Remarks: Prof. Maria Angélica Miglino (CAPES)
Participants: Representatives of Brazilian academic and scientific institutions
Pre-congress
Pre-congress Workshop: Respiratory virus - “Human Challengers”
Chairwoman: Viviane Fongaro Botosso, Dr., Instituto Butantan
2:00 pm
• Martin Schutten, Dr., Erasmus University - “Influenza Antiviral Drug Resistance”
2:30 pm
• Wyller Alencar de Mello, Dr., Instituto Evandro Chagas - “Circulation of Seasonal and
Pandemic Influenza Viruses in the Amazon Region”
3:00 pm
• Danielle B. Oliveira, Dr., ICB/USP - “Molecular Diagnosis of Respiratory Viruses in
Hospitalized Children”
3:20 pm
• Sandra Vieira, Dr., Faculdade de Medicina da USP - “Impact of Laboratory Diagnosis of
Respiratory Viruses in the Pediatric Clinic”
3:40 pm
Coffee Break
Pre-congress
Pre-congress Workshop: Respiratory virus - “Animal Challengers”
Chairman: Edison Luiz Durigon, Dr., ICB/USP
4:00 pm
• Nicolas Gaidet, Dr., CIRAD FRANCE - “Ecological Drivers of Avian Influenza Virus
Infection in Wildfowl”
4:30 pm
• Luciano Tomazelli, Dr., ICB/USP - “Newcastle Disease in Wild Birds in Brazil: From the
Amazon to Antarctic”
4:50 pm
• Maria Angela Orsi, Dr., LANAGRO/Campinas, SP - “Newcastle Disease in commercial
aviculture”
5:10 pm
• Luciana Helena Antoniassi, Dr., UNICAMP - “Metapneumovirus and other Avian
Respiratory Viruses”
5:30 pm
7:00 pm - 9:00 pm
Discussion and Conclusions
Room 2
Opening Ceremony: Conference 1 - “Virus from pathogens to mutualists”
•
Marilyn J. Roossinck, Ph.D., The Pennsylvania State University
9:00 pm - 11:00 pm Cocktail Reception and Visit to the Exhibitions
Room 1
Mini-course 1 – “Innate immune response against virus: virus-host interplay”
•
•
Ana Cláudia Franco, Dr., UFRGS
Maria Cristina Carlan da Silva, Dr., UFABC
Monday, Oct. 01
Room 3
Mini-course 2 – “From the field to the genome: Biology and genetic structure of
7:30 am – 8:30 pm
Mini Courses
begomovirus populations”
• Alice Kazuko Inoue Nagata, Dr., Embrapa
• Francisco Murilo Zerbini Júnior, Dr., UFV
Room 4
Mini-course 3 – “Mapping of viral epitopes using proteomic techniques, molecular
biology and bioinformatics”
• Carlos Eduardo Fernandes dos Santos, Dr., UFMG
• Ricardo Andrez Machado de Ávila, Dr., UFMG
Room 5
Mini-course 4 – “Introduction to the general aspects of RNA virus entry, mechanisms of
replication and exit”
• Davis Fernandes Ferreira, Dr., UFRJ
• Luciana Jesus da Costa, Dr., UFRJ
Room 3
Round Table 1 and Selected Abstracts: Human virology - “Rotavirus vaccines:
current prospects and challenges”
Chairman: José Paulo Gagliardi Leite, Dr., IOC - “Rotavirus Specie A genotypes diversity”
• Alexandre da Costa Linhares, Dr., Instituto Evandro Chagas - “The Latin American
Experience”
HV860 - IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF
GASTROENTERIC VIRUSES AMONG CHILDREN TREATED IN HOSPITAL
IN GOIÂNIA-GO, AFTER THE INTRODUCTION OF ROTARIX
Almeida, T.N.V., Castro, I.A., Cunha, M.P., Souza, M.D., Cardoso, D.D.P., Fiaccadori, F.S.
Room 5
Round Table 2 and Selected Abstracts: Invertebrate virology - “Virus of
invertebrates and plants”
Chairman: Bergmann Ribeiro, Dr., UNB - “Genome of a baculovirus isolated from Perigonia lusca
(Lepidoptera: Sphingidae)”
• Fernando Lucas Melo, Dr., UNB - “Passion fruit`s caterpillar new baculovirus genome”
• Athos Silva de Oliveira, Dr., UNB - “Bean necrotic mosaic virus: a new and distinct
Brazilian tospovirus”
PIV1188 - TWO-STEP CLONING PROCEDURE FOR THE CONSTRUCTION
OF INFECTIOUS CDNA CLONES OF PEPPER MILD MOTTLE VIRUS
Monday, Oct. 01
Junqueira, B.R.T., Nicolini, C., Lucinda, N., Nagata,T.
PIV1408 - SEQUENCING OF A NEW VIRUS RELATED WITH THE COTTON
BLUE DISEASE IN BRAZIL
Round Table
Oral presentation
8:30 am - 10:30 am
Fausto, A.K.S, Vaslin, M.F.S.
Room 2
Round Table 3 and Selected Abstracts: Veterinary virology - “Animal vaccines”
Chairman: João Pessoa Araújo Junior, Dr., UNESP
• Vito Martella, Dr., Department of Veterinary Public Health, University of Bari,
Valenzano, Italy - “Emerging/novel viral pathogens of dogs, with special emphasis on
caliciviruses and astroviruses”
• Marina Gallo Calderón, Dr., Animal Virology Center, Buenos Aires - “Evolution of CPV in
Argentina. Clinical and epidemiological impact”
• Andréia Henzel, Dr., Setor de Virologia/UFSM - “Molecular analysis of feline calicivirus
isolates from Brazil compared to the vaccine strains”
VV790 - IDENTIFICATION AND TYPING OF PANTROPIC CANINE CORONAVIRUS (CCoV) STRAINS
Pinto, L.D., Barros, I.N., Budaszewzki, R.F., Antunes, J.R., Granados, O.F.O., Brandão,
P.E., Canal, C.W.
VV1427 - POPULATION DYNAMIC OF PORCINE PARVOVIRUS INDICATES
DECREASE OF VARIABILITY
Streck, A.F., Homeier, T., Danielle Gava, Foester, T., Truyen, U.
Room 4
Round Table 4 and Selected Abstracts: Environmental virology - “Waterborne
viruses”
Chairwoman: Célia Regina Monte Barardi, Dr., Department of Microbiology, Immunology and
Parasitology, Federal University of Santa Catarina
• Marylynn V. Yates, Dr., Professor of Environmental Microbiology, Department of
Environmental Sciences, University of California, Riverside, CA, USA - “New methods
for the detection of environmentally transmitted viruses”
• Hiroyuki Katayama, Dr., Urban Environmental Engineering Course, Department of
Urban Engineering, the University of Tokyo, Japan - “Determination of Recovery of
enteric viruses from environmental water”
•
•
Maria Inês Zanoli Sato, Dr., Environmental Analysis Department Head, EL CETESB,
Environment Company of São Paulo State - “Latest drinking water guidelines in Brazil
are the viruses important?”
Matías Victoria Montero, Dr., Fiocruz - “Norovirus detection in vegetables”
EV1130 - GIANT VIRUS OF THE MIMIVIRIDAE FAMILY ISOLATED
FROM RIO NEGRO RIVER, IN THE BRAZILLIAN AMAZON RAINFOREST:
MOLECULAR AND BIOLOGICAL CHARACTERIZATION
Campos, R.K., Boratto, P.V.M., Albarnaz, J.D., Silva, L.C.F., Dornas, F.P., Ferreira, P.C.P.,
Kroon, E.G., Abrahao, J.S.
EV766 - RAPID DETECTION OF NOROVIRUS IN NATURALLY CONTAMINATED FOOD: FOODBORNE GASTROENTERITIS OUTBREAK ON A
CRUISE SHIP IN BRAZIL, 2010
Round Table
Oral presentation
8:30 am - 10:30 am
Morillo, S.G., Luchs, A., Cilli, A., Timenetsky, M.C.S.T.
Room 1
Round Table 5 and Selected Abstracts: Basic virology - “Control of transcription
and translation in viral infections”
Chairwoman: Luciana Jesus da Costa, Dr., UFRJ - “HIV-1 replication is strongly dependent on
CAP-dependent translation in infected cells: implications of the expression of 2A protease from
Picornavirus for HIV infection”
• Marcelo Lopèz-Lastra, Dr., Chile - “Translation initiation of the full-length HIV-1 mRNA.
Functional and structural analysis of the 5’ untranslated region”
• Laura Sichero, Dr., ICESP - “Regulation of HPVs 18 and 16 early promoters by cellular
transcription factors”
Monday, Oct. 01
BV750 - THE TRANSCRIPTION FACTOR C-JUN IS DIFFERENTIALLY
REGULATED BY MEK/ERK AND MKK/JNK UPON VACV OR CPXV
INFECTION TO ASSIST DISTINCT VIRAL DEMANDS
Torres, A.A., Cruz, A.F.P., Leite, F.G.G., Soares-Martins, J.P., Pereira, A.C., Ferreira, P.C.P.,
Kroon, E.G., Bonjardim, C.A.
BV920 - BIOLOGICAL DIFFERENCES AMONG HUMAN PAPILLOMAVIRUS
TYPE 16 MOLECULAR VARIANTS
Sichero, L., Sobrinho, J.S., Villa, L.L.
10:30 am - 11:00 am Coffee-break and Visit to the Exhibitions
Room 2
11:00 am - 12:30 pm Conference 2: Basic virology - “Suppressing diversity in RNA viruses”
• Karla Kirkegaard, PhD., Stanford University
12:30 pm - 2:00 pm
2:00 pm - 3:00 pm
Lunch-break and Visit to the Exhibitions
Room 2
Conference 3: Environmental virology - “The role and diversity of viruses on sea”
Chairwoman: Isabel Paixão, Dr., UFF
• Curtis A. Suttle, Dr., University of British Columbia, Vancouver, Canada
Room 5
Round Table 6: Plant virology - “Preventing Phytoviruses in Agriculture:
Quarentenary Actions as First Barrier”
Round Table
Oral presentation
3:00 pm - 5:00 pm
Chairman: José A. Caram Souza Dias, Dr., IAC - "The Sprout/Seed-Potato Technology: addressing
the risk of introducing and spreading quarentenary soil-tuber born virus via imported seedpotato stocks”
• Colin Jeffries, Dr., SASA -Science & Advice for Scottish Agriculture, UK - “Phytosanitary
regulation and potato quarantine”
• Paula Radaelli, Dr., Syngenta - “Syngenta of Aracati plant quarantine: main activities,
advances and challenges”
• Paulo Sérgio Torres Brioso, Dr., UFRJ - “The Quarantened Phytoviruses of Brazil: A
review of their geographic locations and the threats they pose”
Room 3
Round Table 7 and Selected Abstracts: Human virology: - “Clinical Virology”
Chairman: Maurício Lacerda Nogueira, Dr., Famerp
• Celso Granato, Dr., UNIFESP - “Human Herpesvirus”
• Regina Barbosa Schröeder, Dr., Hospital Dom Vicente Scherer - “Polyomavirus in
transplant patients”
• Nancy Bellei, , Dr., UNIFESP - “Respiratory Virus”
• Aparecida Yulie Yamamoto, Dr., FMRP-USP - “Cytomegalovirus”
HV1190 - DETECTION OF HUMAN PARAINFLUENZA VIRUSES TYPES
1, 2, 3 AND 4 BY REAL TIME RT-PCR IN HEMATOPOIETIC STEM CELL
TRANSPLANT PATIENTS
Parmezan, S.N., Camargo, C.N., Bellei, N.
Room 1
Round Table 8 and Selected Abstracts: Immunobiologicals - “New RNA virus
targets for new drugs”
Chairwoman: Paula Rahal, Dr., UNESP
• Victor Hugo Aquino Quintana, Dr., FCFRP-USP - “Snake venom as a source of antiflavivírus compounds”
• Isabel Maria Vicente Guedes de Carvalho Mello, Dr., UNIFESP - “RNA virus replication
mechanisms and targets for antiviral drugs of direct action”
• Ana Carolina Jardim, Dr., UNESP - “Brazilian compounds: exploring nature sources to
future approaches against Hepatitis C”
Monday, Oct. 01
IV897 - CHARACTERIZATION OF THE COMPLETE B-IMMUNOME OF THE
RABIES VIRUS
Martins, T.G., De-Simone, S.G.
Round Table
Oral presentation
3:00 pm - 5:00 pm
IV1343 - THE SYNERGISTIC EFFECT OF COMBINED IMMUNIZATION
WITH A DENGUE DNA VACCINE AND CHIMERIC YELLOW FEVER/
DENGUE VIRUS LEADING TO STRONG PROTECTION IN MICE
Azevedo, A.S., Gonçalves, A.J.S., Freire, M.S., Galler, R., Alves, A.M.B.
Room 4
Round Table 9 and Selected Abstracts: Human virology - “Antiviral”
Chairwoman: Luciana Konecny Kohn, Dr., UNICAMP - “Perspective of Antiviral drugs from
microorganims sources”
• Caroline Rigotto Borges, Dr., UFSC - “Bioactive compounds from the Brazilian coast
invertebrates”
• Elsa B. Damonte, Dr., Universidad de Buenos Aires - “Identification of tomato genes
associated with begomovirus resistance”
HV815 - A RAPID IN SITU ENZYME-LINKED IMMUNOSORBENT ASSAY
FOR DENGUE VIRUS ANTIVIRAL MARINE SEAWEED SCREENING
Koishi, A.C., Zanello, P.R., Bianco, E.M., Bordignon, J., Duarte dos Santos, C.N.
HV1208 - EVALUATION OF THE ANTIHERPETIC ACTIVITY OF AQUEOUS
EXTRACTS OF HUSK FIBER FROM FOUR DIFFERENT VARIETIES OF
Cocos nucifera
Tinga, A.C.C., Silva, D.O., Alviano, C.S., Romanos, M.T.V., Alviano, D.S.
Room 2
Round Table 10 and Selected Abstracts: Veterinary virology - “Emerging viruses”
Chairwoman: Zélia Ines Portela Lobato, Dr., UFMG - “Bovine Vaccinia”
• Ana Cláudia Franco, Dr., UFRGS - “Cyclovirus, Avian gyrovirus 2”
• Peter Mertens, Dr., Institute for Animal Health, Pirbright, UK - “Bluetongue virus”
• Thales Passos de Andrade, Dr., Universidade Estadual do Maranhao - “White spot
syndrome (WSSD) and other emerging diseases of economically important marine
shrimp in Brazil”
VV1048 - ARBOVIRUS CIRCULATION IN WILD BIRDS OF PORTO ACRE –
ACRE STATE
Monday, Oct. 01
Round Table
Oral presentation
3:00 pm - 5:00 pm
5:00 pm - 5:30 pm
5:30 pm - 7:00 pm
7:00 pm - 8:30 pm
Martins, L.C., Chagas, L.L., Chiang, J.O., Ferreira, M.S., Buna, B.S., Costa, L.R.O.,
Vasconcelos, P.F.C.
VV1248 - EVIDENCE OF ARENAVIRUS CIRCULATION IN MATO GROSSO
DO SUL STATE – BRAZIL
Fernandes, J., Oliveira, R.C., Guterres, A., Serra, F., Gomes, R., Favacho, A., Bonvicino,
C.R., D’Andrea, P.S., Lemos, E.R.S.
Coffee-break and Visit to the Exhibitions
Room 2
“Hélio Gelli Pereira” Award
Room 3
“Technology, technical solutions for the contamination control”
• Luciano Figueiredo, Eng., VENCO
Poster Sections 1 and Visit to the Exhibitions
Room 1
•
•
Room 3
7:30 am – 8:30 am
Mini Courses
Room 2
Room 5
Tuesday, Oct. 02
Room 2
Round Table 11 and Selected Abstracts: Veterinary virology - “Swine Flu and
Avian Flu”
Chairwoman: Janice Reis Ciacci Zanella, Dr., Embrapa Suínos e Aves, - “Swine Flu in Brazil”
• Ariel J. Pereda, Dr., INTA, Argentina - “Avian Flu in South America or Swine Flu in
Argentina”
• Daniel Perez, Dr., The University of Maryland, USA - “Flu virus”
VV1463 - CROSS-SECTIONAL STUDY FOR THE SEROLOGICAL PROFILE
TO INFLUENZA H1N1 VIRUSES IN SWINE HERDS IN BRAZIL
Round Table
Oral presentation
8:30 am - 10:30 am
Rajão, D.S., Reis, J.K.P., Oliveira, F.G., Del Puerto, H.L., Alves, F., Braz, G.F., Costa, A.T.R.,
Guedes, R.M.C., Lobato, Z.I.P., Leite, R.C.
VV1494 - IMMUNOLOGICAL EVALUATION OF AN INACTIVATE VACCINE
AGAINTS SWINE INFLUENZA VIRUS (SIV) FOR POTENIAL USE IN PIGS
FROM PRODUCING FARMS IN COLOMBIA
Jaime, J.
Room 5
Round Table 12 and Selected Abstracts: Plant virology - “Plant Viruses”
Chairwoman: Alice Kazuko Inoue Nagata, Dr., Embrapa
• Juliana Freitas-Astua, Dr., Citrus Research & Technology - “Plant virus transmission by
Brevipalpus mites”
• Sarah da Silva Barreto, Dr., UNB - “Weeds as a source of begomoviruses for tomatoes”
•
Jurema Schons, Dr., UPF - “Occurrence of virus causing yellow dwarfism in cereals and
their vectors: current situation in Brazil”
PIV1108 - SYNONYMOUS SITE VARIATION DUE TO RECOMBINATION
EXPLAINS HIGHER BEGOMOVIRUS VARIABILITY IN NON-CULTIVATED
HOSTS
Lima, A.T.M., Ramos-Sobrinho, R., Gonzalez-Aguilera, J., Rocha, C.S., Silva, S.J.C., Xavier,
C.A.D., Silva, F.N., Duffy, S., Zerbini, F.M.
Tuesday, Oct. 02
PIV1380 - CHARACTERIZATION OF MACROPTILIUM YELLOW SPOT
VIRUS INFECTING COMMON BEAN AND MACROPTILLIUM
Round Table
Oral presentation
8:30 am - 10:30 am
Almeida, K.C., Silva, T.A.L., Lacorte, C.C., Ribeiro, S.G.
Room 3
Round Table 13 and Selected Abstracts: Human virology - “Immunovirology”
Chairwoman: Luciana Barros de Arruda, Dr., UFRJ
• Juliana Echevarria, Dr., UFRJ - “Monocytes from HTLV-1 infected patients are unable to
fully maturate into dendritic cells”
• Lucio Gama, Dr., Jonh Hopkins University, USA - “Innate immunity during the acute
phase of SIV and HIV infections”
• Juliano Bordignon, Dr., Carlos Chagas Institute - “Immune response in human dendritic
cells by Dengue virus infection”
• Dumith Chequer Bou-Habib, Dr., Oswaldo Cruz Foundation - “Effects of VIP and PACAP
Neuropeptides on HIV-1 replication in human primary macrophages”
HV1370 - IMMUNIZATION WITH DENDRITIC CELLS TRANSFECTED
WITH LAMP/GAG PLASMID INDUCES A POTENT ACUTE AND MEMORY
RESPONSE SPECIFIC TO HIV GAG
Lucas, C.G.O., Matassoli, F.L., Peçanha, L.M.T., Arruda,L.B.
10:30 am - 11:00 am Coffee-break and Visit to the Exhibitions
Room 2
11:00 am - 12:30 pm Conference 4: Veterinary virology - “Flu virus”
Chairwoman: Janice Reis Ciacci Zanella, Dr., Embrapa
• Daniel Perez, Dr., The University of Maryland - “Swine Flu and Avian Flu”
12:30 pm - 2:00 pm Poster Section 2 and Visit to the Exhibitions
1:00 pm - 2:00 pm
2:00 pm - 7:00 pm
11:00 pm
Lunch Break and Visit to the Exhibitions
Free time
Cultural Programm: Ono Music Hall*
Room 1
Wednesday, Oct. 03
•
•
Room 3
7:30 am – 8:30 pm
Mini Courses
Room 2
Room 5
8:30 am - 9:30 am
9:30 am - 10:00 am
10:00 am - noon
Noon - 1:30 pm
1:30 pm - 2:30 pm
Room 2
Conference 5: Human virology - “Dengue evolution”
•
Nikolaos Vasilakis, Ph.D., University of Texas Medical Branch - “Dengue - quo tu et quo
vadis?”
Coffee-break and Visit to the Exhibitions
Room 2
General Assembly of SBV, election of new Board of SBV and “Hélio Gelli Pereira”
Award
Lunch Break and Visit to the Exhibitions
Room 2
Conference 6: Veterinary virology - “Schmallenberg virus”
•
Peter Mertens, Dr., Institute for Animal Health, Pirbright, UK - “Schmallenberg virus
a novel member of the Simbu serogroup of the Orthobunyaviruses: another emerging
Arbovirus in Europe”
Room 2
Round Table 14 and Selected Abstracts: Human virology -“Molecular evolution
and genetic diversity of HIV, HBV and HCV”
Wednesday, Oct. 03
Chairwoman: Regina Maria Bringel Martins, Dr., UFG
• José Carlos Couto Fernandez, Dr., IOC - “HIV”
• João Renato Rebello Pinho, Dr., Hospital Israelita Albert Einstein - “HBV”
• Elisabeth Lampe, Dr., Fiocruz - “HCV”
HV941 - DETECTION OF RESISTANCE-ASSOCIATED MUTATIONS IN HCV
GENES NS3 AND NS5B TO EXPERIMENTAL DRUGS IN HCV TREATMENT-NAIVE PATIENTS FROM SOUTHERN BRAZIL
Vidal, L.E.L., Germano, F.N., Martinez, A.M., Silveira, J.M., Govea, G.S., Rodrigues, B.,
Soares, M.A., Santos, A.F.
HV1096 - ORIGIN AND TIME-SCALE OF HIV-1 SUBTYPE C EPIDEMIC IN
BRAZIL
Round Table
Oral presentation
2:30 pm - 4:30 pm
Delatorre, E.O., Bello, G.
Room 5
Round Table 15 and Selected Abstracts: Plant virology - “Virus-plant interactions”
Chairwoman: Francisco Murilo Zerbini, Dr., UFV
• Elizabeth P. B. Fontes, Dr., UFV - “Geminivirus-host interactions: Modulation of the
immune receptor NIK activity for tolerance to begomoviruses.”
• Juliana Freitas-Astua, Dr., Embrapa Mandioca e Fruticultura and Centro de Citricultura
Sylvio Moreira/IAC - “The unusual interaction between Citrus leprosis virus C and its
hosts”
• Simone Ribeiro, Dr., Embrapa Cenargen - “Identification of tomato genes associated
with begomovirus resistance”
• Poliane Alfenas Zerbini, Dr., UFV - “Characterizing host susceptibility factors in potyvirus
infection"
PIV1485 - DEVELOPMENT OF A NEW FREE TOOL FOR MAPPING VIRUS
GENOME USING LARGE AMOUNT OF DATA GENERATED BY NEXT-GENERATION SEQUENCING
Andrade, R.R.S., Vaslin, M.F.S.
PIV1230 - DNAJ PROTEIN IS INDUCED IN TOMATO INFECTION BY
PEPPER YELLOW MOSAIC VIRUS AND FAVOR VIRAL ESTABLISHMENT
IN FIRST STAGES OF INFECTION
Xavier, A.S., Bruckner, F.P., Cascardo, R.S., Zerbini, F.M., Alfenas-Zerbini, P.
Wednesday, Oct. 03
Room 1
Round Table 16 and Selected Abstracts: Basic virology: - “Virus-cell relationship”
Round Table
Oral presentation
2:30 pm - 4:30 pm
Chairman: Davis Fernandes Ferreira, Dr., UFRJ
• Maurício Lacerda Nogueira, Dr., FAMERP - “Identifying new cellular partners of yellow
fever NS5 protein”
• Clarissa Damaso, Dr., UFRJ - “Host-cell interactions during Cotia virus infection”
• Maitê Vaslin de Freitas Silva, Dr., UFRJ - “Plant virus infection alters micro RNAs and
small RNAs associated to transposons profiles in cotton”
BV870 - FLUORESCENCE SPECTROSCOPY INTERACTION STUDY
BETWEEN HRSV ATTACHMENT PROTEIN (G) AND QUERCETIN
Gomes, D.E., Teixeira, T.S.P., Paiola, L.C.C.C., Araújo, G.C., Cornélio, M.L., Fossey, M.A.,
Souza, F.P.
BV1272 - IDENTIFICATION OF NEW HIV-1 NEF-TARGETED PROTEINS
De Castro, R.O., Silva, M.E., DaSilva, L.L.P.
4:30 pm
Closing section and announcement of the XIV CBV
* event independent of Congress
Helio Gelli Pereira Award
The evaluation of several papers for the Award “Helio Gelli Pereira” will take a place on October, 1st from 5:30
pm – 7:00 pm. Presenters will have 10 minutes for oral presentation, and the end of the presentation will be
added five minutes to evaluator’s questions.
Prêmio “Hélio Gelli Pereira”
October 01st (Monday) - 5:30pm - 7:00pm
PLASMA LIPIDOMIC EXPRESSION SIGNATURE DISTINGUISHES HEPATITIS C-RELATED HEPATOCELLULAR
CARCINOMA AND LIVER CIRRHOSIS
Ana Maria Passos, Edson Lo Turco, Maria Lúcia C.G. Ferraz, Carla A.L. de Matos, Ivonete S.S. e Silva, Edson R.
Parise, Eduardo J. Pilau, Fabio C. Gozzo, Celso F.H. Granato
miRNAS AS REFERENCE GENES FOR QUANTITATIVE GENE EXPRESSION STUDIES USING RT-qPCR DURING
VIRAL INFECTION IN COTTON
Anna K. S. Fausto; Tatiane F. Silva; Elisson Romanel; Maite V. F. Silva
FIRST IDENTIFICATION OF CULEX FLAVIVIRUS (FLAVIVIRIDAE) IN BRAZIL
Daiane C. Machado, Adriano Mondini, Vinicius S. Santana, Patrícia T. K. Yonamine, Francisco Chiaravalloti
Neto, Paolo M.A. Zanotto, Mauricio L. Nogueira
GENETIC CHARACTERIZATION OF A NOVEL BOVINE PAPILLOMAVIRUS MEMBER OF THE
DELTAPAPILLOMAVIRUS GENUS
Michele Lunardi, Amauri A. Alfieri, Rodrigo A. A. Otonel, Brígida K. Alcântarab, Wagner B. Rodrigues, Antonio
B. Miranda, Alice F. Alfieri
EFFECTS OF HIV-1 IN INFLUENZA PANDEMIC INFECTION
Milene Mesquita, Marilda M. Siqueira, Dumith Chequer Bou-Habib, Thiago Moreno L. Souza
THE eIF3L PROTEIN INTERACTS WITH FLAVIVIRUS NS5 AND MODULATES YELLOW FEVER VIRUS
REPLICATION
Ana T. S. Morais, Danilo V. B. Duarte, Roberta V. M. Bronzoni, Maria C. F. S. Madrid, Laura H. V. G. Gil, Amanda
G. Oliveira, Cleslei F. Zanelli, Sandro R. Valentini, Paula Rahal, Mauricio L. Nogueira
Pag.
17
17
18
18
19
20
Oral Presentation
The oral presentation will be in the same time of the respective round table.
Posters Presentation
Day and Schedule of poster evaluation:
The poster must be fixed from 08:00am to
09:00am in the day of exhibition according to
the area of virology.
The poster must be remove after the section.
October 01st (Monday) – Section 01:
7:00pm - 8:30pm
Basic Virology: BV
Veterinary Virology: VV
October 02nd (Tuesday) – Section 02:
12:30pm - 2:00pm
Environmental Virology : EV
Human Virology: HV
Immunobiological Virology: IV
Plant and Invertebrate Virology: PIV
Helio Gelli Pereira Award
XXIII National Congress of Virology &
VII Mercosur Meeting of Virology
October, 2012 – Foz do Iguaçu, Paraná, Brazil
PLASMA
LIPIDOMIC
EXPRESSION SIGNATURE DISTINGUISHES
HEPATITIS C-RELATED HEPATOCELLULAR CARCINOMA AND LIVER
CIRRHOSIS
Passos, A.M., Lo Turco, E.G., Maria Lúcia
C.G. Ferraz, Carla A.L. de Matos, Ivonete
S.S. e Silva, Edson R. Parise, Eduardo J.
Pilau, Fabio C. Gozzo, Celso F.H. Granato
1. Department
of
Medicine,
Department
of
Surgery,
Department of Gastroenterology,
Federal University of Sao Paulo,
Pedro de Toledo Street 781, Sao
Paulo, SP, 04039-032, Brazil.
2. Chemistry Institute, University of
Campinas, Campinas, SP, Josue de
Castro Street, 13083-970, Brazil.
Hepatitis C (HC) is a major cause of
hepatocellular carcinoma (HCC). Late
diagnosis of HCC represents the main
factor for the poor survival of patients.
The most widely used biomarker for
HCC, alpha-fetoprotein (AFP), has
poor sensitivity and specificity and
has recently been removed from the
American Association for the Study of
Liver Diseases (AASLD) guidelines for
HCC management. Thus, identification
of sensitive and specific biomarkers
for HCC diagnosis is an urgent need.
In the present study, plasma lipid
patterns of patients with HC-HCC and
HC-liver cirrhosis (LC) were assessed
by
performing
matrix-assisted
laser desorption/ionization mass
spectrometry (MALDI-MS). Plasma
samples of 25 patients with HC-HCC
and 15 patients with HC-LC were
evaluated. Lipids were extracted from
plasma using the Bligh-Dyer protocol.
The extracts were subjected to MALDIMS. Data matrix was exported for
univariate and multivariate analysis.
“Hélio Gelli Pereira” - Award
17
A total of 2205 ions were initially
identified and 7 m/z signals were
highlighted as the most important
lipids for the discrimination of patients
with HC-HCC. The specific lipidomic
expression signature generated allows
an overall predictive accuracy of 93% of
HC-HCC and HC-LC. All 7 peaks showed
more than 4-fold change in HC-HCC (P <
0.01). The 7-peak algorithm was able to
distinguish the 2 groups at a sensitivity
of 96% and a specificity of 87%. MALDIMS specific peaks signature accurately
distinguished patients with HC-HCC
from those with HC-LC. The results
indicate the potential of this technique
and the selected peaks to improve the
surveillance of HCC in patients with
HC-LC.
miRNAS AS REFERENCE GENES FOR
QUANTITATIVE GENE EXPRESSION
STUDIES USING RT-qPCR DURING
VIRAL INFECTION IN COTTON
Anna K. S. Fausto; Tatiane F. Silva; Elisson
Romanel; Maite V. F. Silva
1. Universidade
Federal
do
Rio de Janeiro, Instituto de
Microbiologia, Laboratório de
Virologia Molecular Vegetal
2. Universidade Federal do Rio de
Janeiro, Instituto de Biologia,
Laboratório de Evolução Teórica
e Aplicada
The technology of Real time PCR
(RT-qPCR) is in expanding use in
the investigation of gene expression.
The technique of second generation
sequencing has contributed to the
identification of new genes, including
microRNAs (miRNAs). However, to
obtain reliable results using RT-qPCR, it
is necessary to normalize the data with
genes whose showed stability using
October 2012 – Volume 17 (2) – Supplement 1 – “Hélio Gelli Pereira” - Award
18
distinct samples conditions. miRNAs
have a regulatory role in eukaryotic
mRNAs, acting on different biological
functions. In the present study, the
stability of miRNAs and mRNAs were
evaluated in Gossypium hirsutum in
different tissues, different cultivars
and biotic stress caused by infection of
“Cotton leaf roll dwarf virus” (CLRDV).
Expression stabilities of the six miRNAs
were compared with five reference
genes already described for cotton
were evaluated. Four algorithms,
geNorm, NormFinder, BestKeeper and
ΔCt were used to identify the stability
of these expressions to provide an
accurate selection of reference genes.
Ghr-miR390 and Ghr-miR172 were
most stable genes to biotic stress.
The reference genes indicated by
algorithms were validated through
four case studies. The non-coding GhrmiR159, GhrmiR164, GhrmiR2118,
GhrmiR2910, GhrmiR3476 and coding
genes GhrDCL1, GhrDCL2, GhrDCL3
and GhrDCL4 were used to validate
the genes indicated to biotic stress.
This is the first study analyzing the use
of miRNAs as constitutive in cotton
during viral infection.
FIRST IDENTIFICATION OF CULEX
FLAVIVIRUS
(FLAVIVIRIDAE)
IN
BRAZIL
Daiane C. Machado, Adriano Mondini,
Vinicius S. Santana, Patrícia T. K.
Yonamine, Francisco ChiaravallotiNeto, Paolo M.A. Zanotto, Mauricio L.
Nogueira
1. Instituto de Biociências, Letras
e Ciências Exatas (Universidade
Estadual Paulista - IBILCE-UNESP)
2. Faculdade de Medicina de São José
do Rio Preto (FAMERP), Faculdade
de
Ciências
Farmacêuticas
(Universidade Estadual Paulista
- UNESP) - Campus Araraquara,
Universidade de São Paulo (USP),
Faculdade de Saúde Pública - USP,
Instituto de Ciências Biomédicas
Culex flavivirus (CxFV) was first
isolated in 2007 from Culex pipiens
in Japan and then identified in several
other countries. Characterization
of the CxFV showed that all strains
are related to cell fusing agent virus
(CFAV). In this manuscript we report
the first identification of CxFV in South
America. We have collected Culex sp
mosquitoes using BG-Sentinel traps
3 and manual aspirators. They were
pooled according to genus, sex and
location. Viral RNA was extracted and
Multiplex-Nested-PCR were performed
to test the presence of Flavivirus.
Positive samples were isolated in C6/36
cells and sequenced for phylogenetic
analyses. 265 female Culex mosquitoes
pooled in 83 pools were tested with
specific Culex flavivirus (CxFV), Saint
Louis Encephalitis virus (SLEV) and
West Nile virus (WNV) primers. Our
sequence data indicated maximum
sequence similarity of 97% with CxFV.
In this study we report the circulation
of Culex flavivirus in an urban setting
where Saint Louis encephalitis virus had
previously caused an outbreak. In terms
of public health, this is an important
finding due to the assumption that the
previous exposition of mosquitoes to
CxFV might lessen the susceptibility of
these mosquitoes to other flaviviruses.
GENETIC CHARACTERIZATION OF
A NOVEL BOVINE PAPILLOMAVIRUS
MEMBER OF THE DELTAPAPILLOMAVIRUS GENUS
Michele Lunardi, Amauri A. Alfieri,
October 2012 Volume 17 (2) – Supplement 1 – “Hélio Gelli Pereira” - Award
Rodrigo A.A. Otonel, Brígida K.
Alcântara, Wagner B. Rodrigues, Antonio
B. Miranda, Alice F. Alfieri
Laboratory
of
Veterinary
Microbiology, Veterinary Teaching
Hospital, Universidade de Cuiaba,
Laboratory of Animal Virology,
Department of Veterinary Preventive
Medicine, Universidade Estadual de
Londrina, Laboratory for Functional
Genomics
and
Bioinformatics,
Department of Biochemistry and
Molecular Biology, Oswaldo Cruz
Institute, Fiocruz
The family Papillomaviridae is
composed of 29 genera. While more
than 100 human papillomavirus
types were characterized, only twelve
bovine papillomavirus types had been
described. Recently, an investigation
revealed notable diversity among
bovine papillomavirus detected in
Brazilian herds. That study identified
four putative new BPV types. This
report describes the complete genomic
sequence and taxonomic position of
one of these novel PV types, the bovine
papillomavirus type 13. In order to
amplify the total genome sequence of
the bovine papillomavirus 13, the PV
genome was submitted to multiplyprimed rolling circle amplification.
To confirm that the band was indeed
papillomavirus DNA, PCR with
degenerate primers for cutaneous
papillomavirus was performed. Based
on the sequences generated, two primer
sets for long template PCR were selected
in order to amplify the most part of
the genome. The complete nucleotide
sequences of the cloned products
were determined by primer-walking
sequencing. The complete genomic
sequence of the bovine papillomavirus
13 has 7961 bp, with a GC content
19
of 45.1%. Bovine papillomavirus 13
contains eight ORFs, coding for E1, E2,
E4, E5, E6, E7, L1, and L2 proteins. The
phylogenetic analysis showed that the
novel bovine papillomavirus is sorted
into Deltapapillomavirus genus which
held a group dominated by artiodactyl
ruminant papillomavirus. This group is
notable in that papillomavirus infection
largely results in the development
of
fibropapillomas,
pathogenic
mechanism considered to be unique
among papillomaviruses.
EFFECTS OF HIV-1 IN INFLUENZA
PANDEMIC INFECTION
Milene Mesquita, Marilda M. Siqueira,
Dumith Chequer Bou-Habib e Thiago
Moreno L. Souza
Instituto Oswaldo Cruz/Fiocruz,
Laboratório de Vírus Respiratórios
e do Sarampo (LVRS), Laboratório
de Pesquisas do Timo (LPT)
HIV-1 persistently replicates in
lymphoid tissues, causing reduction
of CD4+ cells. This leads to profound
immunosuppression and increased
susceptibility
to
opportunistic
infections, such as influenza A virus
(FLU A), among others. FLU A is a singlestranded, negative-sense, enveloped
RNA virus, of the orthomyxoviridae
family, possessing eight gene segments.
This genomic feature facilitates viral
shift that enable new variants to emerge,
like pandemic H1N1 (H1N1pdm09), in
2009. Pandemic H1N1 virus caused 10
times more deaths than seasonal FLU
A (H3N2) virus, especially in specific
groups like children, elderly, pregnant
women and immunocompromised
individuals. Contradictory to this,
clinical outcomes of HIV-1-infected
individuals with H1N1pdm09 were
not different from those observed for
20
immunocompetent individuals. Since
HIV-1 imposes a novel homeostatic
equilibrium to its host, the life cycle
of influenza within this environment
may be highly affected. Considering
that over 30 million people in the
world are living with HIV-1 and that
their exposure to FLU A, which is a
seasonal pathogen, is quite common
– we investigated the in vitro biology
of these viruses. In this study, we
used
human
monocyte-derived
macrophages (MDM) from healthy
donors, MDCK lineage and parental
HeLa cell lineages. Although influenza A
does not establish productive infection
in MDM (with the exception of H5N1
virus), the infectivity of H1N1pdm09
inoculum in MDM was constant for
more than 5 days. However, when we
co-infected this MDM with HIV-1 (10
ng/mL of p24 antigen - R5-isolate
BaL), we observed a 1-log decrease
in H1N1pdm09 infectivity (n=8;
p<0.05). The reduction in H1N1pdm09
infectivity was neither due to massive
entry nor due to the effects of reactive
nitrogen oxygen species from HIV-1infected MDMs. We next observed that
interferon-inducible transmembrane
(IFITM) proteins, recently presented as
influenza and HIV-1 restriction factors,
are up-regulated more than 30% by
HIV-1 and gp120, even in epithelial
cells that would be susceptible to
H1N1pdm09 infection. Our results
demonstrate that studies such as this
may not only increase knowledge on the
physiopathology of HIV-1/influenza
co-infection, but may also contribute to
the identification/validation of novel
antiviral targets.
but may also contribute to the
identification/validation of novel
antiviral targets.
THE eIF3L PROTEIN INTERACTS
WITH
FLAVIVIRUS
NS5
AND
MODULATES YELLOW FEVER VIRUS
REPLICATION
Ana T. S. Morais, Danilo V. B. Duarte,
Roberta V. M. Bronzoni, Maria C. F.
S. Madrid, Laura H. V. G. Gil, Amanda
G. Oliveira, Cleslei F. Zanelli, Sandro
R. Valentini, Paula Rahal, Mauricio L.
Nogueira
1. Laboratório
de
Pesquisas
em Virologia, Departamento
de Doenças Dermatológicas,
Infecciosas
e
Parasitárias,
Faculdade de Medicina de São
José do Rio Preto-FAMERP
2. Departamento
de
Biologia,
Universidade Estadual Paulista
‘‘Julio de Mesquita Filho’’, Campus
São José do Rio Preto – IBILCE/
UNESP
3. Departamento de Virologia e
Terapia Experimental, Centro
de Pesquisas Aggeu Magalhães
CPqAM / FIOCRUZ
4. Departamento
de
Ciências
Biológicas, Faculdade de Ciências
Farmacêuticas,
Universidade
Estadual Paulista, Araraquara
The yellow fever virus (YFV) belongs
to the Flavivirus genus and causes an
important disease. NS5 is a viral protein
that contains the methyltransferase
and RNA-dependent RNA polymerase
domains. To identify the interaction
of YFV NS5 with cellular proteins, we
performed a two-hybrid screen using
YF NS5 RdRp domain as bait and a
human cDNA library. The eIF3L protein
was identified to interact with the YFV
RdRp domain in that screen in that
screen and we show that the interaction
occurs in the conserved terminal
October 2012 Volume 17 (2) – Supplement 1 – “Hélio Gelli Pereira” - Award
21
portion of the Interaction Domain of
RNApol. Mutations introduced into
the ID terminal portion demonstrated
that 5 amino acids are critical for
the interaction. This interaction
was confirmed in vitro assays and
by in vivo coimmunoprecipitations.
The significance of eIF3L for
replication of YFV was investigated
using overexpression of eIF3L in
BHK21-RepYF17D LucNeoIres cells.
Overexpression of eIF3L decreased
yellow fever virus replication. These
results indicates that the interaction of
eIF3L with YF NS5 plays an important
role in viral replication.
Oral Presentation
BV750 - THE TRANSCRIPTION
FACTOR C-JUN IS DIFFERENTIALLY
REGULATED BY MEK/ERK AND
MKK/JNK UPON VACV OR CPXV
INFECTION TO ASSIST DISTINCT
VIRAL DEMANDS
Torres, A.A., Cruz, A.F.P., Leite, F.G.G.,
Soares-Martins, J.P., Pereira, A.C.,
Ferreira, P.C.P., Kroon, E.G., Bonjardim,
C.A.
1. Universidade Federal de Minas
Gerais; UFMG; Av. Antônio Carlos,
6627 - Pampulha 31270-901 BELO HORIZONTE - MG
2. Grupo de Transdução do Sinal;
GTS; Universidade Federal do
Piaui
3. Medical College of Wiston,
Milwaukee, USA;
The Orthopoxviruses Vaccinia (VACV)
and Cowpox (CPXV) belong to the
Poxviridae family, that encloses the
more complex cytoplasmatic large
DNA viruses that infect animals.
Since virus-cell interaction plays a
decisive role in viral biology, our group
has been studying the activation of
mitogen-activated protein kinases in
response to these viruses infection.
We previously showed that these
Orthopoxviruses activate the protein
kinases ERK and JNK. Since c-Jun, a
common substrate downstrean of
these pathways, is associated with
the control of key cellular events, we
wondered whether c-Jun could play a
relevant role in the virus biology. By
Western Blotting assay, we showed
that c-Jun is activated upon infection
with both VACV and CPXV during the
whole infective cycle. Using wildtype (WT) or JNK 1/2 knock-out (KO)
mouse embryonic fibroblasts (MEFs),
Oral Presentation
23
we demonstrated that c-Jun activation
upon VACV infection is temporally
regulated by JNK after 6 hours of
infection. Differentially, activation of
c-Jun upon CPXV infection relies only
on JNK pathway. Using cell lines stably
transfected with plasmid expressing
c-Jun
dominant-negative
mutant
(DNcJun), we demonstrated that this
transcriptional factor has an impact
on the viral biology on a post-viral
replicative step, once release of viral
enveloped forms and also the plaque
phenotype of both VACV and CPXV,
were affected in DN cells. Since it has
been shown that cytokine production
is regulated by JNK during the infection
with diverse viruses, we sought to
investigate the expression of the
inflammatory cytokine interleukin-6
(IL-6) upon VACV and CPXV infection,
by means of quantitative reverse
transcription-PCR and ELISA. Our
results showed a higher stimulation of
IL-6 expression upon CPXV infection
when compared to VACV, which was
even higher in the KO and DN cells.
Altogether, these data suggest that two
closely related viruses can differently
activate the same signaling pathway, in
order to assist distinct viral demands.
BV870 - FLUORESCENCE SPECTROSCOPY INTERACTION STUDY
BETWEEN HRSV ATTACHMENT
PROTEIN (G) AND QUERCETIN
Gomes, D.E., Teixeira, T.S.P., Paiola,
L.C.C.C., Araújo, G.C., Cornélio, M.L.,
Fossey, M.A., Souza, F.P.
Dep. Física - IBILCE - Universidade
Estadual Paulista; Unesp; R.
Cristóvão Colombo, 2265 - Jd.
Nazareth, São José do Rio Preto
Human Syncytial Respiratory Virus
(hRSV) is responsible for lower
October 2012– Volume 17 (2) – Supplement 1 – Oral Presentation
24
Oral Presentation
respiratory tract disease as bronchiolitis
and pneumonia in infants. It is believed
that the key to the inhibition of viral
action is its membrane glycoproteins,
including the Attachment Protein (G),
responsible for the viral adsorption on
the cell surface. There are evidences
that compounds such as flavonoids
can decrease the viral infection, and
G protein can be a good target for the
action of these compounds.The aim of
this project includes cloning, expression
and purification of endodomain (G1)
and ectodomain (G2) of attachment
protein to perform interaction
potencial verification between endo/
ectodomain and quercetin trough
fluorescence spectroscopy to propose
a inhibitor to the virus. The virus
cDNA was transcript using the Hight
Capacity cDNA Reverse Trancription
Kit (Applied Biosystems®) and the
PCR was performed with the taq
DNA Polimerase (Recombinant) –
Fermentas® following manufacture
recommendations. The cloning of the
fragments was developed with TOPO
XL PCR Cloning Kit (Invitrogen®).
Following, pCR-XL-TOPO-G1 and pCRXL-TOPO-G2 constructs were subcloned
in pET28a vector to expression of
these proteins in BL21 DE3 bacteria
and the purification was performed
with a nickel resin. Interaction
potencial between endo/ectodomain
and quercetin was verified trough
fluorescence spectroscopy. Results
showed the amplification of a 135pb
(G1) and 699bp (G2) genes and the the
cloning of the proteins was obtained
with success. Expression presented
a expression of G1 (~12kDa) and G2
(~30kDa). Fluorescence spectroscopy
results
shown
fluorescence
suppression after titration indicating
interaction between G domains and
quercetin. These results are the first
step to further drug design. Our next
step is to explore better this interaction
and, once it is well understood, it will
be possible to develop drugs with high
potency and efficacy.
BV920 - BIOLOGICAL DIFFERENCES
AMONG HUMAN PAPILLOMAVIRUS
TYPE 16 MOLECULAR VARIANTS
Sichero, L., Sobrinho, J.S., Villa, L.L.
Instituto do Câncer do Estado de
São Paulo; ICESP; Av Dr Arnaldo,
251Faculdade de Medicina da USP;
FMUSP; Av Dr Arnaldo, 255Instituto
do HPV;
We compared E6/E7 protein properties
of three different HPV-16 molecular
variants: AA, E-P and E-350G. Primary
human foreskin keratinocytes (PHFK)
were infected with HPV-16 E6 and
E7 and evaluated for proliferation
and ability to grow in soft agar. E-P
infected keratinocytes presented the
lowest efficiency in colony formation
and proliferation. AA and E-350G
infected keratinocytes attained higher
capacity for in vitro transformation.
We also observed similar degradation
of TP53 among HPV-16 variants
tested. Furthermore, we accessed
the expression profile in early (p5)
and late passage (p30) infected cells
of 84 genes commonly involved in
carcinogenesis.
Most
differences
observed could be attributed to HPV16 E6/E7 expression. Nevertheless, we
detected different expression of ITGA2
and CHEK2 in keratinocytes infected
with AA and AA/E-350G late passage
immortalized cells, respectively. Our
results indicate differences among
HPV-16 variants that could explain, at
least in part, differences in oncogenic
potential attributed to HPV-16.
October 2012 Volume 17 (2) – Supplement 1 – Oral Presentation
Financial support: FAPESP 07/58590-7
and 08/57889-1; CNPq 573799/20083; Ludwig Institute for Cancer Research
BV1272 - IDENTIFICATION OF NEW
HIV-1 NEF-TARGETED PROTEINS
de Castro, R.O., Silva, M.E., da Silva,
L.L.P.
Faculdade Medicina Ribeirao Preto;
FMRP-USP; Av. Bandeirantes 3900,
Biocel 40, Ribeirao Preto - SP
Infection of human immunodeficiency
virus, HIV-1, results in impaired
function of the immune system
that leads to clinical manifestations
of AIDS. Nef is an HIV-1 accessory
protein that interacts with elements
of intracellular trafficking machinery
modulating protein expression at the
plasma membrane. In this process,
the most characterized effect of Nef is
the downregulation of CD4 and MHC-I
expression at the cell surface. Herein,
a proteomics approach was used to
determinate new proteins targeted
by HIV-1 Nef. Therefore, proteins
from cell surface were biotinylated,
pulled-down and 2D-DIGE was used
to determinate spots containing
proteins which expression was
specifically reduced in CD4+ T cells
(A3.01 T cell line) expressing Nef.
Proteins contained in these spots
were identified by mass spectrometry.
These included C1QBP (complement
component 1 q subcomponent
binding protein), LOC339779 (an
uncharacterized protein, here named
LOC), MYL6 (myosin, light chain 6,
alkali, smooth muscle and non-muscle)
and Peroxiredoxin 1 (PRDX1). The
sequence of these proteins was cloned
in pEGFP-C2 expression vector and
transfected in PEAK cells. Western blot
analysis showed that LOC, MYL6 and
Oral Presentation
25
PRDX1 had their expression reduced
when co-expressed with Nef. This
decrease was 44% (+-7%) for LOC,
36% (+-3,5%) for MYL6 and 34% (+11%) for PRDX. There was no effect
in C1QBP expression when it was coexpressed with HIV-Nef. This could be
due to GFP fusion in the N-terminal of
C1QBP. Analysis of protein localization
showed that MYL6 and PRDX1 were
expressed in plasma membrane and
co-localized with Nef when they were
co-expressed in PEAK cells. Interesting,
LOC was localized to punctated
structures throughout the cytoplasm
and this pattern was partially lost in
the presence of Nef. Therefore, these
findings suggested that LOC, MYL6 and
PRDX1 are new targets for HIV-1 Nef.
Financial support: FAPESP, CNPq and
FAEPA
EV766 - RAPID DETECTION OF
NOROVIRUS IN NATURALLY CONTAMINATED FOOD: FOODBORNE
GASTROENTERITIS OUTBREAK ON
A CRUISE SHIP IN BRAZIL, 2010
Morillo, S.G., Luchs, A., Cilli, A.,
Timenetsky, M.C.S.T.
Instituto Adolfo Lutz; IAL; Av Dr
Arnaldo, 355 Centro de Virologia
Cerqueira Cesar 01246-902
Norovirus (NoV) is a prevalent
pathogen of foodborne diseases;
however its detection in foods other
than shellfish is often time-consuming
and unsuccessful. In 2010, an outbreak
of acute gastroenteritis occurred on
a cruise ship in Brazil, and NoV was
the etiologic agent suspected. The
objectives of this study were report that
a handy in-house methodology was
suitable for NoV detection in naturally
contaminated food; and perform the
molecular characterization of food
26
Oral Presentation
strains. Food samples (blue cheese,
Indian sauce, herbal butter, soup, and
white sauce) were analyzed by ELISA,
two methods of RNA extraction, TRIzol®
and QIAamp®, following conventional
RT-PCR. The qPCR was used in order
to confirm the NoV genogroups. GI and
GII NoV genogroups were identified
by conventional RT-PCR after RNA
extraction using the TRIzol® method.
Two GII NoV samples were successfully
sequenced, classified as GII.4; and
displayed a genetic relationship with
strains from Asia continent also isolated
in 2010. GII and GI NoV were identified
in distinct food matrices, suggesting
that was not a common source of
contamination. TRIzol® extraction
followed by conventional RT-PCR was
a suitable methodology in order to
identify NoV in naturally contaminated
food. Moreover, food samples could be
processed within 8 hours, indicating
the value of the method used for NoV
detection, and its potential to identify
foodborne gastroenteritis outbreaks in
food products other than shellfish. This
is the first description in Brazil of NoV
detection in naturally contaminated
food other than shellfish involved in
a foodborne outbreak. Sponsor:PPGPLSP-CCD-SES/SP; IAL
EV1130 - GIANT VIRUS OF THE MIMIVIRIDAE FAMILY ISOLATED FROM
RIO NEGRO RIVER, IN THE BRAZILLIAN AMAZON RAINFOREST:
MOLECULAR AND BIOLOGICAL CHARACTERIZATION
Campos, R.K., Boratto, P.V.M., Albarnaz,
J.D., Silva, L.C.F., Dornas, F.P., Ferreira,
P.C.P., Kroon, E.G., Abrahão, J.S.
Universidade Federal de Minas
Gerais; UFMG; Av. Antonio Carlos,
6627, Pampulha, BH, MG
The Mimiviridae family comprises
giant DNA viruses which are studied
as putative pneumonia agents in
humans. Recent metagenomic studies
have detected DNA of viruses of this
family in natural aquatic ecosystems.
Although the Amazon rainforest is
known by its huge biodiversity, viruses
of this biome are poorly studied.
Thus, this work aimed the search of
viruses of the Mimiviridae family as
well as other giant viruses in the Rio
Negro river, AM, Brasil. Samples were
collected at various points of Rio Negro
river, in a region near Manaus. Water
samples underwent an enrichment
process in rice medium and were
filtered afterwards (in 200nm filters)
to retain the viruses in the filter.
Samples were then inoculated in
amoeba (Acathamoeba castellanii)
cultures aiming viral isolation and
were concomitantly submitted to PCR
testing, for amplification of the viral
helicase gene, which is conserved
in the viruses of the Mimiviridae
family. The helicase gene was partially
sequenced and phylogenetic analyses
were performed with the program
MEGA 5.05. Additionally, biological
analyses and electronic microscopy
images were done. The results of the
PCR tests indicated the presence of
the viral helicase gene in some water
samples of Rio Negro and one virus
was isolated from a sample by using
amoeba cultures. The sequencing,
phylogenetic analyses and electronic
microscopy imaging indicated that a
giant virus of the Mimiviridae family
was isolated, with nearly 600nm of
diameter, representing the virus with
the biggest diameter isolated in Brazil
to date. The presence of viruses of the
family Mimiviridae in Rio Negro river
corroborates previous studies which
indicates the involvement of these
viruses in aquatic ecosystems. Apoio:
CNPq, FAPEMIG, CAPES, MAPA.
HV815 - A RAPID IN SITU ENZYME-LINKED IMMUNOSORBENT ASSAY
FOR DENGUE VIRUS ANTIVIRAL
MARINE SEAWEED SCREENING.
Koishi, A.C., Zanello, P.R., Bianco, E.M.,
Bordignon, J., Duarte dos Santos, C.N.
1. Instituto Carlos Chagas; ICC/
FIOCRUZ-PR; . Algacyr Munhoz
Mader street 3775, 81350-010,
Curitiba, PR, Brazil
2. Universidade Federal do Paraná;
UFPR; Polytechnic Center, 81530130, Curitiba, PR, Brazil
3. Universidade Federal de Santa
Catarina; UFSC; Trindade Campus,
88040-900, Florianópolis, SC,
Brazil
Dengue, a mosquito-borne viral
disease, is a significant public health
problem that transcends geographical
boundaries being endemic in more
than 100 countries within tropical and
subtropical regions of the world. Despite
the important social and clinical impact,
there is no vaccine or specific antiviral
therapy for prevention and treatment
of dengue virus infection. Considering
the above, drug discovery research for
dengue is of utmost importance. Here
we propose a target-free approach
for dengue drug discovery based
on a novel, rapid, and economic 96well format in situ enzyme-linked
immunosorbent assay which can be
adapted for high-throughput screening.
The in situ ELISA was standardized for
Huh 7.5 cell line and infection with all
four serotypes of DENV, being DENV-1,
-2 and -3 clinical isolates from Brazil
Oral Presentation
27
and DENV-4 a laboratory strain. The in
situ ELISA was compared to the fociforming assay and the results presented
a high correlation (average r2= 0.95),
furthermore statistical analysis showed
an average S/B of 7.2 and Z- factor of
0.62; demonstrating assay consistency
and reliability. The assay was used to
screen the antiviral activity of a panel
of fifteen seaweed extracts at the
maximum non-toxic dose previously
determined by the MTT and neutral red
cytotoxic assays. Eight algae extracts
showed a dengue infection inhibition
in the post- infection treatment when
compared to the controls, with some
variations depending on the virus
serotype. Among these, four extracts
were chosen for further evaluation.
The pre-infection treatment showed no
significant inhibition of virus infection;
on the other hand, treatment during the
infection was highly effective, indicating
that these extracts must interfere in
early steps of the virus infection cycle.
Financial support: Fiocruz, CNPq, and
Fundação Araucária.
HV860 - IDENTIFICATION AND
MOLECULAR
CHARACTERIZATION OF GASTROENTERIC VIRUSES
AMONG CHILDREN TREATED IN
HOSPITAL IN GOIÂNIA-GO, AFTER
THE INTRODUCTION OF ROTARIX.
Almeida, T.N.V., Castro, I.A., Cunha, M.P.,
Souza, M.D., Cardoso, D.D.P., Fiaccadori,
F.S.
Universidade Federal de Goiás;
UFG; IPTSP/UFG - Rua 235 - s/n St. Universitário -CEP:74605050Goiânia/Goiás/Brasil
Acute
gastroenteritis
(AGE)
is
considered an important cause of
morbid-mortality among children less
than five years of age, worldwide. Of
28
Oral Presentation
importance among the viral agents are
group A rotaviruses (RVA), caliciviruses
(CVs), human astroviruses (HAstV)
and human adenoviruses (HAdV).
Because of RVA impact in Public Health,
vaccination has been considered an
effective alternative for prevention
of infection by theses agents. In this
context, in 2006, Brazil has included the
RVA (Rotarix) vaccine in the National
Immunization Program. Given this new
scenario, it is important to evaluate the
occurrence of different gastroenteric
viruses, in order to observe possible
changes in their circulation pattern and
its genomic variants. Therefore, in this
study 65 stool samples were obtained
from children less than five years of age
presenting with acute gastroenteritis.
The children were treated in two
hospitals in Goiânia-GO from 2008 to
2009. Viral detection was performed by
enzyme linked immunosorbent assay
(RVA and HAdV), polyacrylamide gel
electrophoresis (RVA) and polymerase
chain reaction (CV and HAstV), and
positivity rates of: 16.9% (11 / 65),
16.9% (11/65), 3.1% (2/65) and
6.1% (4/65) were observed for RVA,
CV, HAstV and HAdV, respectively.
Molecular characterization of the
RVA-positive samples, identified ten
samples as genotype G2 (VP7 gene),
and four as genotype P[4] and one as
P[9] P[11] (VP4 gene). The two HastVpositive samples were characterized
as genotype 1 (HAstV-1), whereas only
one of the four HAdV-positive samples
could be characterized as serotype
41 (HAdV-41). Of the 11 CVs-positive
samples, five were identified as GII
NoVs and six as SaVs. The data confirms
the circulation of gastroenteric viruses
in Goiânia-GO after the introduction
of the RVA vaccine, and reinforces the
need of the adoption of more effective
measures, in order to prevent these
infections.
HV941 - DETECTION OF RESISTANCE-ASSOCIATED
MUTATIONS
IN HCV GENES NS3 AND NS5B TO
EXPERIMENTAL DRUGS IN HCV TREATMENT-NAIVE PATIENTS FROM
SOUTHERN BRAZIL
Vidal, L.E.L., Germano, F.N., Martinez,
A.M., Silveira, J.M., Govea, G.S.,
Rodrigues, B., Soares, M.A., Santos, A.F.
1. Dep. de Genética, Universidade
Federal do Rio de Janeiro; UFRJ;
Rua Professor Rodolpho Paulo
Rocco, s/n, Ilha do Fundão, Cep
21.941-617
2. Division of Genetics, Instituto
Nacional de Câncer; INCA; Rua
André Cavalcanti, 37 - 4o andar
Bairro de Fátima, CEP 20231-050
3. Faculdade
de
Medicina,
Universidade Federal de Rio
Grande; FURG; Rua Gen. Osório,
s/nº - 4º andar - Centro – Rio
Grande / RS - CEP: 96201.900
Combination of pegylated interferon
and ribavirin is currently used in the
treatment of hepatitis C virus (HCV)
infection, leading to a sustained
virological response (SVR) in more than
50% of patients with chronic infection.
However, most patients infected with
HCV genotype 1 do not achieve SVR.
In view of these limitations, intense
investigation of drugs targeting viral
enzymes resulted in the development
of 40 new compounds, known as direct
acting antivirals (DAA). To characterize
natural DAA resistance-associated
polymorphisms, we obtained plasma
samples from203 HCV treatment-naïve
patients from Rio Grande / Brazil. Viral
RNA was extracted, subject to RT-PCR
and the resulting cDNA was used to
amplify the NS3 (protease/helicase)
and NS5b (polymerase) regions.
PCR products were sequenced and
aligned. Mutation analysis was carried
out through inferred amino acid
translation and phylogenetic analyses
were performed using NeighborJoining for HCV genotype assignment.
We amplified viral sequences from 95,
NS3(65) and NS5b(59), treatmentnaïve patients, and we found a
prevalence of 39% to genotype 1a,
28% to 3a, 23% to 1b and 10% to
2b. In NS3, we detected mutation
V36L, which confer resistance to
telaprevir,(genotype 1a/b in 4,5%
patients (2/44) and 100% (18/18) in
patients genotype 3a), Q80K, resistance
mutation to development drug TMC435,
found in 1,7% patients (1/60) in 1a/b
genotype and mutation M175L, that
confers resistance to boceprevir, 81%
(35/43) to 1a/b and 100% (17/17) to
3a.In NS5b we found mutation M71V,
resistant to Benzothiadiazine(90%
(9/10) in 1a/b and 91% (10/11) in 3a
genotypes); V138I in 100% (25/25)
in 1a/b and 3a; I447F in 65% (19/29)
of 1a/1b, I482L in 100% (9/9) of 3a,
and V499 in 100% (7/7) of 3a. After
that, we found some polymorphisms
in resistance mutations site, which
could be favorable or not to change to
resistant mutation. Thus, these natural
polymorphisms could harm future
treatment those patients with DAA.
HV1096 - ORIGIN AND TIME-SCALE
OF HIV-1 SUBTYPE C EPIDEMIC IN
BRAZIL
Delatorre, E.O., Bello, G.
Instituto Oswaldo Cruz - Fundação
Oswaldo Cruz; IOC - FIOCRUZ; Av.
Oral Presentation
29
Brasil, 4365, Manguinhos, Rio de
Janeiro - RJ, Brasil - CEP: 21040-360
The HIV-1 subtype C has spread
efficiently in the southern states of
Brazil. Phylogeographic studies indicate
that the HIV-1 subtype C epidemic in
southern Brazil was initiated by the
introduction of a single founder virus
population probably originated from
east Africa. The exact east African
country of origin of Brazilian HIV-1
subtype C remains unclear. The precise
time-scale of such an event is also
uncertain. Two independent studies
estimated the onset date of Brazilian
subtype C epidemic at around the early
1980s, while another study suggests
that this Brazilian epidemic could be
much older, dating back to between
1960 and 1970. Here we revisited
the origin and time-scale of HIV-1
subtype C epidemic in South Brazil. In
this study a large number (n = 352) of
subtype C pol sequences of east African
origin were retrieved from public
databases to explore relationships
between this strains and a subset of 30
HIV-1 subtype C Brazilian sequences
isolated at Rio Grande do Sul, Santa
Catarina and Parana. Phylogeographic
and evolutionary analyses were
conducted using Maximum-likelihood
and Bayesian methods. Phylogenetic
analysis of subtype C sequences
revealed that most (>70%) strains
from east Africa segregated in a single
regional-specific monophyletic group,
called CEA. All Brazilian sequences
form a highly supported monophyletic
subclade within the CEA lineage.
Bayesian coalescent-based analysis
indicated that the CEA clade most
probably originated in Burundi
between early and late 1960s, and was
subsequently disseminated to Ethiopia,
Kenya, Tanzania, Uganda and Brazil
30
Oral Presentation
between early 1970s and early 1980s,
where it further spread establishing
new local epidemics. These results
demonstrate that the origin of the
Brazilian subtype C epidemic could
be mostly probably traced to Burundi
and further suggest that the founder
subtype C strain was introduced in
the southern region at around 1975.
Financial support: CAPES
HV1190 - DETECTION OF HUMAN
PARAINFLUENZA VIRUSES TYPES 1,
2, 3 AND 4 BY REAL TIME RT-PCR
IN HEMATOPOIETIC STEM CELL
TRANSPLANT PATIENTS
Parmezan, S.N., Camargo, C.N., Bellei,
N.
UNIVERSIDADE FEDERAL DE SÃO
PAULO, UNIFESP, R. Pedro de Toledo,
781. 15° andar - CEP 04039-032
E-mail: sheilaparmezan@hotmail.
com
Human
Parainfluenza
Virus
(HPIV) is a common cause of
viral infection in patients with
hematologic hematopoietic stem cell
transplantation (HSCT). HPIVs are
divided into 4 different types, although
most clinical infections are due to types
1, 2, and 3. The incidence of HPIV has
been reported between 2% to 7%. In
Brazil studies of HPIV in HSCT patients
are scarce and effective diagnosis as
well as recognition and understanding
of incidence and risk factors those
viruses. The aim of this study was
to identify the presence of HPIV in
hospitalized and non hospitalized
HSCT with respiratory symptoms in a
Sao Paulo tertiary hospital. Included
patients were those who had a clinical
picture of acute respiratory infection
or possibly asymptomatic patients who
have contact with patients presenting
with infection. These patients were
evaluated by an infectious disease
physician who came into contact with
the laboratory staff when there were
suspected cases. The present study
analyzed the diagnostic for HPIV
1, 2, 3 and 4 of Real Time Reverse
Transcription-PCR (RT-PCR) in 202
nasal washes from patients (mean of 45
year of age, variation of 5 to 80 years)
attended between March 2008 to
December 2009 in a hematology ward
or outpatient bone marrow transplant
of São Paulo hospital. The most frequent
symptoms were coryza, cough, fever
and sore throat. Among analyzed
samples 11% were positive for HPIV,
10% (N= 20) HPIV type 3 and 1% (N=2)
HPIV type 4. Among positive samples,
only 24% (N=5) presented fever at
sampling, 86% (N=18) presented
coryza, 76% (N= 16) presented cough
and 10% (N=2) sore throat. This
study retrospectively examined the
frequency of symptomatic infections
by HPIV, showing the need to improve
strategies for infection control and
patient care and evaluate the outcomes
of these high risk patients.
HV1208 - EVALUATION OF THE ANTIHERPETIC ACTIVITY OF AQUEOUS
EXTRACTS OF HUSK FIBER FROM
FOUR DIFFERENT VARIETIES OF
COCOS NUCIFERA
Tinga, A.C.C., Silva, D.O., Alviano, C.S.,
Romanos, M.T.V., Alviano, D.S.
Instituto de Microbiologia Paulo
de Góes - UFRJ (2); IMPG - UFRJ (2);
Bl. I, sala 064 ss - CCS-UFRJ-Ilha do
Fundão-Rio de Janeiro/CEP21.941590Instituto de Microbiologia Paulo
de Góes - UFRJ (1); IMPG - UFRJ (1);
Bl. I, sala 050 - CCS-UFRJ-Ilha do
Fundão-Rio de Janeiro/CEP21941-
902
Plants have been used for treating
various diseases since ancient times.
The diffusion of knowledge about
these plants is given from generation
to generation and is empirical. Despite
the wide use of plants in popular
medicine, little is known about the
quality, efficacy and toxicity of most
of them. In our continuing efforts to
search for new antiviral agents from
traditional medicinal plants we set
out to study the anti-herpes simplex
viruses (HSV) activity of different
varieties of coconuts. In spite of the
availability of an effective antiviral agent
(acyclovir) to treat these infections,
resistant strains, have already been
isolated. Studies have shown various
biological activities associated with
the coconut as antitumor, analgesic,
anti-inflammatory,
antibacterial,
antileishmania and antiviral. The
current study aims to investigate the
anti-HSV activity of aqueous extracts
of husk fiber from four varieties
of Cocus nucifera (ACO: “comum”;
ACR: “olho de cravo”; AGI: “gigante”
and AVJ: “anão”). The extracts were
prepared, lyophilized, resuspended
in distilled water and filtered through
sterilizing conditions. Cytotoxicity
was performed on Vero cells, as host
system. In the antiviral assays were
employed concentrations of 62.5 µg/
mL to ACO, 31.3 µg/mL to ACR and AVJ
and 15.6 µg/mL to AGI, although CC50
was greater than 500 µg/mL to ACO,
ACR and AGI, and 95.2 µg/mL to AVJ.
Antiviral activity was evaluated by viral
titer reduction. All extracts showed
excellent activity for the two viruses
studied. ACO and ACR presented 99.9%
of inhibition to HSV-1 and HSV-2. AGI
was able to inhibit 94.2% and 99.8%
Oral Presentation
31
of the HSV-1 and HSV-2 propagation,
respectively, and AVJ showed 98.2%
of inhibition to HSV-1 and 98.3% to
HSV-2. According to these results we
can conclude that different varieties of
Cocus nucifera are promising for the
development of drugs for the treatment
of infections caused by herpes simplex
virus. Financial Support: CAPES, CNPq
and FAPERJ.
HV1370 - IMMUNIZATION WITH
DENDRITIC CELLS TRANSFECTED
WITH LAMP/GAG PLASMID INDUCES
A POTENT ACUTE AND MEMORY
RESPONSE SPECIFIC TO HIV GAG
Lucas, C.G.O. , Matassoli, F.L. , Peçanha,
L.M.T., Arruda,L.B.
UNIVERSIDADE FEDERAL DO RIO
DE JANEIRO; UFRJ; Rio de janeiro,
Cidade Universitário, CCS, Bloco I,
Lab.I-SS 048
We have demonstrated that association
of HIV1-p55Gag with LAMP (lysosomal
associated membrane protein) in the
form of DNA plasmid (LAMP/gag)
greatly enhanced the immune response
in immunized mice, in comparison
to native gag. However, it has been
proposed that a DNA vaccine alone
may not be effective in humans due to
low transfection efficiency. Dendritic
cells (DC) loaded with specific antigens
have been studied as a promising
vaccine strategy against tumors and
infectious disease, including HIV. Here,
we evaluated the immune response
induced by DCs transfected with LAMP/
gag in a mouse model and compared
the efficiency of this strategy with the
immunization with naked DNA. Mice
were inoculated with LAMP/gag DNA
(Lg) or with DCs transfected with
LAMP/gag (Lg-DC) and the activation
of acute and memory immune
32
Oral Presentation
responses were evaluated at 15 days
and 3 months after the immunization.
The activation of gag-specific T cells,
obtained from systemic and mucosal
tissues, were investigated by analysis
of cytokine production and expression
of chemokine receptors by ELISA and
FACS. Induction of serum and mucosa
anti-HIV antibodies was measured
by ELISA. Immunization with Lg-DCs
induced the activation of Gag-specific
polyfunctional TCD4+ and TCD8+ cells
at levels even higher than the ones
induced by immunization with naked
DNA. Lg-DCs also induced higher levels
of serum anti-HIV antibodies. Only the
animals immunized with transfected
DCs showed specific IL-17+ T cells in
the spleen and presented TGF-beta,
IL-17 and Gag-specific IgA in the
intestinal lavage. At 3 months after LgDC immunization we could still detect
polyfunctional activated T cells in the
spleen and high levels of anti-HIV IgG
in the serum. Our data demonstrated
that dendritic cells transfected with
LAMP/gag vaccine are immunogenic,
triggering a systemic and mucosal
response mediated by TCD4, TCD8 and
B cells. The response was sustained
for at least 3 months, suggesting the
development of memory immune
response.
IV897 - CHARACTERIZATION OF THE
COMPLETE B-IMMUNOME OF THE
RABIES VIRUS
Martins, T.G., De-Simone, S.G.
Instituto Oswaldo Cruz / Fundação
Oswaldo Cruz; IOC / FIOCRUZ; Av.
Brasil, 4365 Manguinhos - Rio de
Janeiro - RJ - Brasil CEP: 21040-360
Rabies is a zoonotic viral disease of
the central nervous system spread
from animals to humans in either
of two transmission cycles: urban
rabies which is mainly transmitted
by dogs, or wildlife rabies primarily
spread by bats and other wild animals.
The rabies virus (RABV) belongs to
the order Mononegavirales, family
Rhabdoviridae, genera Lyssavirus.
The viral genome consists of a nonsegmented single-stranded, negativesense RNA translated by five proteins,
and encapsidated with nucleoprotein
(N), and this N-RNA complex, together
with the phosphoprotein (M1) and
RNA-dependent RNA polymerase (L),
forms the RNP core. The RNP core
associated with the matrix protein
(M2) is condensed in the typical bulletshaped, characteristic of rhabdovirus.
The envelope of the lipid bilayer is
anchored on surface spikes trimeric
glycoprotein (G) surrounded by the
M-RNP structure. Although sufficient
knowledge is known about the
symptoms and the existence of effective
protection, little information is known
about the protective immune response.
Likewise, there is a need for an exclusive
real time diagnostic test. In this study,
all the RABV proteins were mapped
using the existing protective horse sera
by the parallel Spot-synthesis peptide
technique. A library of 716 peptides
with a length of 14 amino acids and
nine overlapping residues covering all
the virus proteins were synthesized
and evaluated for IgG reactivity. Thirtyone major epitopes of different sizes
were identified. Bioinformatics tools
and molecular modeling techniques
were used to determine that all
identified epitopes are situated at
the surface of the molecules. Possible
cross-reactive epitopes were identified
by using bioinformatics tolls in protein
data banks. The outcomes obtained in
this study will permit the development
of a biochip-peptide method valuable
for large-scale point-of-care RABV
serodiagnostics. Financial Support:
MCT-CNPq, FAPERJ, CAPES, FIOCRUZ
(PDTIS/IOC)
IV1343 - THE SYNERGISTIC EFFECT
OF COMBINED IMMUNIZATION
WITH A DENGUE DNA VACCINE AND
CHIMERIC YELLOW FEVER/DENGUE
VIRUS LEADING TO STRONG PROTECTION IN MICE
Azevedo, A.S., Gonçalves, A.J.S., Freire,
M.S., Galler, R., Alves, A.M.B.
Instituto Oswaldo Cruz/Fiocruz;
IOC/Fiocruz;
Avenida
Brasil
4.365 - Manguinhos, Rio de
Janeiro, RJInstituto de Tecnologia
em
Imunobiológicos/Fiocruz;
BioManguinhos; Avenida Brasil
4.365 - Manguinhos, Rio de Janeiro,
RJ
The dengue envelope glycoprotein (E),
the major component of virion surface,
is the main target for the development
of a dengue vaccine with induction of
neutralizing antibodies. Previously,
we have constructed and tested a
DNA vaccine (pE1D2), encoding the
ectodomain of the DENV2 E protein
(domains I, II and III). In the present
work, we tested these DNA vaccine
isolated and in combination with a
chimeric yellow fever/dengue 2 virus
(YF17D-D2). The YF17D-D2 virus
was also previously constructed by
replacing the prM and E genes from
the 17D yellow fever vaccine virus
by those from DENV2. Balb/c mice
were inoculated with these two
vaccines by different prime/booster or
simultaneous immunization protocols
and most of them induced a synergistic
effect on the elicited immune response,
mainly in neutralizing antibody
Oral Presentation
33
production. Furthermore, combined
immunizations remarkably increased
protection against a lethal dose of
DENV2, when compared to each
vaccine administered alone. In fact,
mice immunized either with the prime/
boost regimen or with simultaneous
inoculations did not present any
clinical sign of virus infection. The
cellular immune response was also
investigated and results showed that
immunization with the DNA vaccine
induced a robust production of IFNγ by CD8+ T lymphocytes, while
inoculation with only the chimeric
virus elicited significant a lower
response. Moreover, the combination
of both vaccine strategies did not lead
to an increase of such response, thus
revealing that the DNA vaccine was
main component for the induction of
IFN-γ. Financial support: PDTISFIOCRUZ, FAPERJ, CNPq, PRONEX and
INCTV.
PIV1108 - SYNONYMOUS SITE
VARIATION DUE TO RECOMBINATION
EXPLAINS HIGHER BEGOMOVIRUS
VARIABILITY IN NON-CULTIVATED
HOSTS
Lima, A.T.M., Ramos-Sobrinho, R.,
Gonzalez-Aguilera, J., Rocha, C.S., Silva,
S.J.C., Xavier, C.A.D., Silva, F.N., Duffy,
S., Zerbini, F.M.,
1. Dep. de Fitopatologia/BIOAGRO,
Univ. Fed. de Viçosa; UFV; Av. P.H.
Rolfs, s/n, Viçosa, MG 36570-000,
Brazil
2. Rutgers, The State University
of New Jersey; Dep. of Ecology,
Evolution and Natural Resources,
New Brunswick, NJ 08901, USA
Begomoviruses (single-stranded DNA
plant viruses) are responsible for
34
Oral Presentation
serious agricultural threats. In Brazil,
a number of novel begomoviruses
associated with cultivated and noncultivated hosts have been characterized
and an increasing body of evidence
points to a higher genetic variability
in begomovirus populations infecting
non-cultivated hosts. Here we present
a study contrasting the molecular
variability of two begomovirus
populations infecting a cultivated and
a non-cultivated host and discuss the
relative contribution of evolutionary
processes
their
diversity.
We
intensively sampled crops and weeds
in similarly sized geographic areas
known to harbor either Macroptilum
yellow spot virus (MaYSV) or Tomato
severe rugose virus (ToSRV). MaYSV
is a recently isolated species that was
previously only reported in weeds,
whereas ToSRV is the most widespread
tomato-infecting begomovirus in
Brazil. The MaYSV population was
notably more variable than the ToSRV
population, largely due a number of
recombination events in the 5'- portion
of its Rep gene. Although less variable, a
stronger evidence of adaptive selection
was shown for the ToSRV population.
In contrast, no evidence of adaptive
selection was detected on MaYSV,
indicating that adaptive selection does
not contribute to the higher variability
of this population. By mapping the
recombination events onto maximum
likelihood trees based on Rep and CP
sequences, we were able to distinguish
the individual contributions associated
with the evolutionary processes of
mutation and recombination. Using this
novel partitioning method, we assessed
recombination as the source of 0 and
42% of the standing genetic variability
of ToSRV Rep and CP, respectively,
and 36 and 16% of the variability of
MaYSV Rep and CP, respectively. These
results constitute a step towards
disambiguating the effects of various
evolutionary processes on viral genetic
variation. Financial support: CNPq,
FAPEMIG
PIV1188 - TWO-STEP CLONING
PROCEDURE FOR THE CONSTRUCTION OF INFECTIOUS CDNA CLONES
OF PEPPER MILD MOTTLE VIRUS
Junqueira, B.R.T., Nicolini, C., Lucinda,
N., Nagata,T.
Universidade de Brasília; UnB;
Campus Darcy Ribeiro Asa Norte
CEP 70910900
Pepper mild mottle virus (PMMoV)
is a tobamovirus, which consists of
monopartite single-stranded RNA
genome in positive polarity. The RNA
genome is surrounded by coat proteins
forming rod-shaped rigid particles. The
genome is capped at 5’ terminus and
has a tRNA-like structure at 3’ terminus
and possesses four open reading frames
(ORFs). Aiming to study the virushost interactions and the functions
of viral proteins the development of
the infectious clone of the virus was
attempted. To obtain cDNA of full-length
PMMoV genome, RNA extraction and
RT-PCR was performed using reverse
primer with MluI site and the forward
primer with T7 RNA polymerase
minimum promoter sequence. The
cDNA of full length genome of ~6.3
kb was successfully amplified by
RT-PCR. This cDNA fragments were
cloned into pCR4 vector and E. coli
DH5α strain was transformed
with this construct. The selected clone
was sent for sequencing and successful
cloning of cDNA of whole genome
was confirmed. Two clones were
subcloned into pUC19 to eliminate
endogenous T7 promoter sequence of
pCR4 plasmid and full-length PMMoV
RNA was transcribed in vitro after
the digestion with MluI for run-off
transcription. Nicotiana benthamiana
plants were inoculated with 2.0 to
3.0ug of synthesized transcripts per
leaf and maintained in green house for
one month. The virus recovery by the
infectious transcripts was confirmed
by
symptom
observation,
Dot
immunobinding assay (Dot-ELISA) and
transmission electron microscopy. One
N. benthamiana plant showed severe
mottle and top distortion symptoms,
which were very similar to symptoms
caused by wild type virus three weeks
post-inoculation. The positive signals
in Dot-ELISA observed in plant with
recovered virus were strong as in plants
with wild type virus and huge amount
of virus particles were observed
by electron microscopy by leaf dip
preparations. This study showed an
effective and simple protocol based
on two-step cloning to construct an
infectious clone of tobamovirus.
PIV1230 - DNAJ PROTEIN IS INDUCED
IN TOMATO INFECTION BY PEPPER
YELLOW MOSAIC VIRUS AND FAVOR
VIRAL ESTABLISHMENT IN FIRST
STAGES OF INFECTION
Xavier, A.S., Bruckner, F.P., Cascardo,
R.S., Zerbini, F.M., Alfenas-Zerbini, P.
Universidade Federal de Viçosa;
UFV; Campus universitário, Viçosa,
MG
During co-evolution between virus
and host, a complex interaction has
been developed involving several
mechanisms of pathogen attack and
host defense. Host defense responses
cause up- and downward shifts in
gene expression. However, the effects
Oral Presentation
35
of viral infection in the host’s gene
expression profile are still poorly
understood. To understand the process
of tomato infection by the potyvirus
Pepper yellow mosaic virus (PepYMV),
a subtractive library identified several
genes as differentially expressed
during the early stages of viral
infection. Among the induced genes
was the one encoding a DnaJ (HSP40)
protein. Members of the DnaJ multigene
family contain a highly conserved
70-amino acid signature region, the J
domain, and assist as co-chaperones of
HSP70s in various cellular processes.
The involvement of HSP proteins in
the enhancement or inhibition of
pathogenesis in a wide range of viral
infections has been described. To
study the role of the DnaJ protein in
plant potyvirus infection, expression
levels of the DnaJ gene were analyzed
in PepYMV-infected plants at 72 hours
post-viral inoculation (hpvi) and at 14
days post-viral inoculation (dpvi). Total
RNA was extracted, treated with DNase
and used as a template for qRT-PCR.
Results of three biological replications
confirmed the induction of DnaJ at 72
hpvi and 14 dpvi. To assess whether
this gene is directly related to the viral
infection process, functional analysis
by virus-induced gene silencing (VIGS)
using a Tobacco rattle virus (TRV)based vector was performed. Silencing
in plants infiltrated with TRV-DnaJ was
very effective and these plants showed
a reduction in viral load at 72 hpvi, but
not at 14 dpvi. Mock-inoculated, DnaJsilenced plants developed symptoms
which were strikingly similar to
those observed in non-silenced
plants infected by PepYMV. Ours
results suggests that DnaJ induction
contributes to early stages of PapYMV
infection, and that the activity of this
36
Oral Presentation
protein in the viral infection could
contribute to symptom developement.
Financial support: CAPES, CNPq,
Fapemig and International Foundation
for Science (IFS)
PIV1380 - CHARACTERIZATION
OF MACROPTILIUM YELLOW SPOT
VIRUS INFECTING COMMON BEAN
AND MACROPTILLIUM
Almeida, K.C., Silva, T.A.L., Lacorte, C.C.,
Ribeiro, S.G.
1. Embrapa Recursos Genéticos e
Biotecnologia, Cenargen, Parque
Estação Biológica - PqEB - Av. W5
Norte (final) Brasília - DF
2. Universidade de Brasília, UnB,
Rua Unb Darci Ribeiro s/n Campus Universitário Brasília DF E-mail: karolx121@hotmail.
com
Common bean (Phaseolus vulgaris L.)
is a staple food mainly in Latin America
Brazil is the world's largest producer
of common bean. Until recently, the
only reported begomovirus infecting
common bean in Brazil was bean
golden mosaic virus (BGMV). On a
recent survey, however our group
isolated
another
begomovirus,
Macroptilium yellow spot virus-MYSV,
infecting common beans in different
regions of Pernambuco and Sergipe
states. This virus is highly divergent
and partial characterization of different
bean samples resulted in only about
70-72% identity with BGMV isolates.
Diverging bean (PV) and Macroptillium
lathyroides (ML) isolates were selected
to obtain the complete genome.
Rolling-circle amplification (RCA)
products were digested with Kpn I
and BamHI rendering ~2.6 kb bands
which were cloned into pBluescript.
DNA-A obtained from samples MLPE75 and PV-PE171 were sequenced
and analyzed revealing 88% identity
with each other. In BLASTn searches,
PV-PE171-DNA-A
had
above
98% identity with MYSV BR:Bat1
(JN419012.1), BR:Inp1 (JN419018.1)
and BR:Pir1 (JN419015.1), isolated
from M. lathyroides e Calopogonium
mucunoides ML-PE75-DNA-A had an
identity of 99% and 98% with MYSV
strains BR: Mac1 (JN419009.1) and
BR:Bas1:09 (JN419005.1), isolated
from M. lathyroides. The identity was
above 95% also with MYSV isolated
from Canavalia sp and Calopogonium
mucunoides. RCA product obtained
from samples ML-PE75 and PVPE171 was inoculated by particle
bombardment and proved to be
infectious to common bean "Olathe"
and snap bean "Trepador" causing
leaf deformation and rugosity. The fact
that other begomovirus is infecting
common bean in the field and BGMV
and MYSV share common hosts is of
great concern since MYSV may arise as
an important virus disease to common
beans. Furthermore, mixed infections
can favor recombination events and
hence the potential emergence of novel
recombinant viral species. Financial
support: Embrapa, Fap-DF and CNPq.
VV1408 - SEQUENCING OF A NEW
VIRUS RELATED WITH THE COTTON
BLUE DISEASE IN BRAZIL
Fausto, A.K.S., Vaslin, M.F.S.
Universidade Federal do Rio de
Janeiro, UFRJ, Depto. Virologia, I.
Microbiologia, UFRJ, Rio de Janeiro,
RJ, Brazil E-mail: anna.karol@gmail.
com
Cotton blue disease (CBD) is an
important pathology present in
America, Africa and Asia that affects
cotton crops causing significant
economic losses. The disease is
transmitted by Aphis gossypii and its
causal agent is a virus from the family
Luteoviridae, genus Polerovirus, named
Cotton leaf roll dwarf virus (CLRDV).
Typical CBD symptoms include
stunting due to internodal shortening,
leaf rolling, intense green foliage, and
yellowing veins. In previous works,
using CLRDV molecular diagnosis,
we were able to identify a new virus
species infecting plants showing CBD
atypical symptoms (typical disease
symptoms plus withered and reddish
basal leaves). Analyses of a partial
polymerase sequence of these virus
isolates showed that they presented
low identity with CLRDV and can be
considered as representatives of a new
species of the genus Polerovirus. In
other to better characterize this new
species, named Cotton red leaf virus
(CoRLV), we submitted the isolate
CoRLV-PL3 to a deep sequencing
by "Illumina". All small RNA of an
infected plant were sequenced and
“contigs” were constructed. Contigs
matching viral genome were aligned
with related virus and used to design
specific primers. RT-PCR were
performed to three virus independent
isolates, although only CLRDV-PL3
showed amplification. To confirm
virus partial sequences obtained by
“contigs”, the amplicon sequence was
sequenced by Sanger method. The
sequence analysis showed that the
region amplified belonged to RdRp4
(RNA dependent RNA polymerase 4)
motif of Polerovirus. The nucleotide
sequence comparison by Clustal W
showed the maximum identity of 73%
with CLRDV and 71% with Pepper
yellow leaf curl virus. However, the
Oral Presentation
37
aminoacid sequence revealed more
similarity with Beet western yellows
virus (72%). The similarity with
CLRDV was 69%. These results suggest
that this new specie probably result of
recombination events between CLRDV
and an unknown virus, and it’s different
from other isolates found in previous
work. In order to obtain the complete
genome sequence of this virus, more
primers are been designed. Financial
Support: FAPERJ, CAPES.
PIV1485 - DEVELOPMENT OF A NEW
FREE TOOL FOR MAPPING VIRUS
GENOME USING LARGE AMOUNT OF
DATA GENERATED BY NEXT-GENERATION SEQUENCING
Andrade, R.R.S., Vaslin, M.F.S.
Janeiro, UFRJ, Lab. de Virologia
Molecular Vegetal, IMPPG, UFRJ,
Rio de Janeiro, RJ, Brazil E-mail:
[email protected]
Next-generation high-throughput DNA
sequencing (NGS) technologies have
produced large amount of data and
the principal challenge is its analyze.
Presently there are some good tools
to assembly genomes using a pool
of small length sequences (reads).
The most important step in NGS
analysis is mapping of reads with a
reference genome (similar sequence
of close specie or gene related), but
these kind of bioinformatics analysis
require robust memory capacity
and the software available aren’t
accessible for a research that do not
domine bioinformatics language or
are expensive. We developed a free
software with a friendly-user interface
developed in Java language compatible
for all major systems. This new tool is
the SearchSmallRNA programe, that is
38
Oral Presentation
available for download in http://www.
microbiologia.ufrj.br/informativo/
tome-nota/340-download-doprograma-buscasmallrna and allows
the users mapping virus genomes
efficiently. The program convert files
FASTAq (the format produced by
NGHS), FASTA or single list of reads to
a file of reduced size. Users can make
their analysis in Low Memory Mode, a
method that split large file into “pieces”
and store de partial results, avoiding
deadlocks or freezing of computers
with limited resources of memory.
SearchSmallRNA or BuscaSmallRNA,
uses a parallelization method which
reduces time of analysis. We tested
the software with data obtained
by deep-sequencing by Illumina
from cotton plants infected with the
polerovirus Cotton Leaf Roll Dwarf
Virus (CLRDV) that causes the cotton
blue disease. The complete data set of
some small interfering RNA (siRNA)
sequencing of diseased plant were
used and SeachSmallRNA was able to
construct the CLRDV complete genome
successfully. Other viral genome
from animal and plant virus as HIV,
MNSV, CMV, TuMV, PVX e TYLCV were
also mapped by the program using
previously data of NGS sequences.
These analysis demonstrated that
SearchSmallRNA can fast and safely
map virus genomes and is a good and
accessible tool for analysis of NGS data.
Financial Support: CAPES, FAPERJ
VV790 - IDENTIFICATION AND
TYPING OF PANTROPIC CANINE CORONAVIRUS (CCOV) STRAINS
Pinto, L.D., Barros, I.N., Budaszewzki,
R.F., Antunes, J.R., Granados, O.F.O.,
Brandão, P.E., Canal, C.W.
1. Universidade Federal do Rio
Grande do Sul, UFRGS, Av. Bento
Gonçalves 9090 Porto Alegre
2. Faculdade de Med. Vet. e Zoot da
Universidade de São Paulo , USP,
Av. Prof. Dr. Orlando Marques
de Paiva, 87 São Paulo E-mail:
[email protected]
Canine coronavirus (CCoV) affects
young dogs and has worldwide
distribution. Traditional strains were
restricted to the gastrointestinal
tract, causing vomiting, diarrhea,
dehydration, loss of appetite and, in
some cases, death, especially when the
animals were coinfected with other
viruses. Currently, CCoV is classified into
two genotypes: CCoV type I (CCoV-I)
and CCoV type II (CCoV-II) and, recently,
CCoV-II, known to be pantropic, has
been categorized into two subtypes,
CCoV IIa and CCoV-IIb. Both subtypes
were detected in other organs besides
the gastrointestinal tract. CCoV- IIa
causes severe systemic lesions that
could be fatal to young dogs. The aim of
the present study was to identify and
typify CCoV in organs of dogs which
died after presenting symptoms of
hemorrhagic gastroenteritis. Twenty
organs were collected from five dogs
aged 1 to 6 months, from different
breeds and gender, with or without
prior vaccination history. RNA was
extracted by TRIzol TM LS and the
reverse transcription was carried out
using the enzyme Superscript TM III
Reverse Transcriptase (Invitrogen,
USA), following the manufacture’s
recommendations.
The
partial
amplification of genes M and S were
obtained by PCR using specific primers
for CCoV. CCoV-II was detected in several
organs of this dogs. Fecal samples were
used to identify other intestinal viruses,
such as canine parvovirus type 2 (CPV-
2), canine distemper virus (CDV),
canine adenovirus types 1 and 2 (CAV-1
and CAV-2) and canine rotavirus (CRV).
From the five dogs tested, two showed
coinfection with CCoVII and CPV-2c,
one was positive only for CPV-2c, one
was positive only for CCoV-II, and one
was negative for investigated viruses,
all samples CPV-2c were confirmed
by sequencing. Sequential samples
were 99% similar to a previously
described Brazilian CCoV-II strain and
97% similar to European strains. In
conclusion, the new pantropic CCoV-II
found in organs of dogs suggests that
this agent is present in Brazil.
VV1048 - ARBOVIRUS CIRCULATION
IN WILD BIRDS OF PORTO ACRE –
ACRE STATE
Martins, L.C., Chagas, L.L., Chiang, J.O.,
Ferreira, M.S., Buna, B.S., Costa, L.R.O.,
Vasconcelos, P.F.C.
Instituto
Evandro
Chagas,
IEC, BR 316, Km 7 s/n Bairro
Levilândia- Ananindeua-PA E-mail:
[email protected]
Introduction: The state of Acre has a
rich biodiversity with about 50% of
the known birds of Brazil. Objective:
To study the prevalence of antibodies
to arboviruses in plasmas of wild
birds captured in the municipality of
Porto Acre-AC. Material and Methods:
A total of 48 wild birds belonging to
19 different species were captured,
between 2010 and 2011. Blood
samples were obtained for attempts
of viral isolation into newborn
mice; the plasmas were screened by
Hemagglutination Inhibition (HI) test
for detection of antibodies to a panel of
19 arboviruses as follows: Alphaviruses
(eastern equine encephalitis virus,
western equine encephalitis virus,
Oral Presentation
39
Mayaro virus and Mucambo virus)
Orthobunyaviruses (Guaroa virus,
Maguari virus, Tacaiuma virus,
Caraparu virus, Icoaraci virus, Belem
virus, Utinga virus, Oropouche virus and
Catu virus) and Flaviviruses (yellow
fever virus, Ilheus virus, Saint Louis
encephalitis virus, Cacipacore virus,
Bussuquara virus, and Rocio virus).
Results: None arbovirus was isolated
from samples investigated. By serology,
HI antibodies were found for 10
plasma samples (20.84%) belonging to
species of the families Thamnophilidae
(90%) and Thraupidae (10%).
Monotypic responses were detected in
one sample (10%) to eastern equine
encephalitis virus, four samples (40%)
to Saint Louis encephalitis virus and
six samples (60%) to Oropouche
virus. Cross reaction was found in
one sample (10%) to members of
the genus Orthobunyavirus and four
samples (40%) to members of the
genus Flavivirus. In five samples (50%)
was obtained serological response to
viruses of two or more tested genera.
The species Thamnophilus doliatus
e Rhegmatorhina melanosticta were
found presenting HI antibodies to
arboviruses. Conclusion: Immunity
to important arboviruses were found
in plasmas of wild birds captured in
Porto Acre-AC; the results confirm
that wild birds act as vertebrate hosts
for different arboviruses including
those pathogenic to humans. Financial
Support: IEC/CNPQ
VV1248 - EVIDENCE OF ARENAVIRUS CIRCULATION IN MATO GROSSO
DO SUL STATE – BRAZIL
Fernandes, J., Oliveira, R.C., Guterres,
A., Serra, F., Gomes, R., Favacho, A.,
Bonvicino, C.R., D’Andrea, P.S., Lemos,
E.R.S.
40
Oral Presentation
1. Fundação Oswaldo Cruz Instituto Oswaldo Cruz, FIOCRUZ
- IOC, Avenida Brasil 4365,
Manguinhos, 21040-900, Rio de
Janeiro/RJ, Brazil
2. Instituto Nacional do Câncer,
INCA, E-mail: jorlanfernandes@
hotmail.com
Arenaviruses are members of the
family Arenaviridae that consists of
a unique genus (Arenavirus) that
currently comprises 23 viral species.
It is assumed that humans usually
become infected with arenaviruses
contact with infected rodents or
inhalation of virus in aerosolized
droplets of secretions or excretions
from infected rodents. South America
arenaviruses belong to the Tacaribe
sorocomplex and five members are
kwon to cause hemorrhagic fever
in Argentina, Brazil, Bolivia and
Venezuela. In Brazil, the first report
of a hemorrhagic fever associated to
Sabia virus, whose reservoir is still
unknown, was done in 1990. There
are three other arenavirus circulating
in Brazil: Amapari, Flexal and Cupixi
virus. In this study, the presence of
arenavirus were investigated among
small mammals from Dois Irmãos do
Buriti and Sidrolândia municipalities.
Those municipalities are situated in
Mato Grosso do Sul State where there is
no description of arenavirus-associated
hemorrhagic fever, although they are
near areas where circulation of those
viruses are well documented. Two
fieldworks were conducted for rodent
trapping, the rodents were captured
in Sherman and Tomahawk live traps,
and processed in a laboratory installed
in the field. Rodent spleen and liver
samples were submitted to RT-PCR
amplification of partial S segment
using oligonucleotide primers of the
S segment of Junin virus. Twenty of
the 76 collected animals were positive
by molecular testing. The positives
animals compromise six different
species, and only one of them has been
recognized as a host for arenavirus.
These initial results showed a strong
evidence of arenavirus circulation in
Mato Grosso do Sul State, as well as a
high prevalence of these viruses among
the rodents from this region. Further
analyses must be made to identify the
specie or species of arenavirus, here
described, and the role played by the
positive rodents as hosts of these
viruses. Financial support : FIOCRUZ
and CNPq
VV1427 - POPULATION DYNAMIC OF
PORCINE PARVOVIRUS INDICATES
DECREASE OF VARIABILITY
Streck, A.F., Homeier, T., Gava, D.,
Foester, T., Truyen, U.
University of Leipzig, , Leipzig,
Germany Embrapa Swine and Poultry
Research Center, CNPSA, Concórdia,
SC, Brazil E-mail: afstreck@hotmail.
com
The porcine parvovirus (PPV) is
considered to be a major cause of
reproductive failure in swine. In the
last 15 years, several reports described
new PPV biotypes containing amino
acid substitutions located in the
capsid surface. Analysis of the PPV
evolutionary dynamic revealed that the
virus displays a substation rate in the
structural protein gene close to those
usually finding in RNA viruses. That facts
raise concern about the commercial
vaccines, leading to the hypothesis
that the emerging biotypes are a result
of an escape mechanism from the
current vaccine used. To address these
questions, the population dynamic
of PPV isolates from swine herds was
analyzed using all PPV viral protein
gene complete and partial sequences
originated from swine and deposited
in GenBank. The population dynamic
history of the virus was calculated
using Bayesian Markov Chain Monte
Carlo method with a Bayesian skyline
coalescent model. Additionally, an in
vitro model was performed by twenty
consecutives passages of the Challenge
strain (a virulent field strain) and
NADL2 strain (a vaccine strain) in PK15
cell-line supplemented with polyclonal
antibodies raised against the vaccine
strain. The Bayesian analysis indicated
a decrease in the population diversity
over the years and in consequence the
presence of around eleven dominant
PPV strains. In agreement, the in vitro
study revealed that a lower number of
mutations appear for both viruses in
the presence of anti-PPV antibodies in
comparison with the control passages
without antibodies. As hypothesis, the
antibodies pressure may reduce the
neutral selection, which should play a
major role in the new mutations drift.
In swine, the PPV vaccines largely used
in the last 30 years probably reduced
the genetic diversity of the virus. In
this scenario, vaccine failures and the
non-vaccinated populations (e.g. wild
boars) may have an important impact
in the emergence of new biotypes.
Financial support: Capes and DAAD.
VV1463 - CROSS-SECTIONAL STUDY
FOR THE SEROLOGICAL PROFILE TO
INFLUENZA H1N1 VIRUSES IN SWINE
HERDS IN BRAZIL
Rajão, D.S., Reis, J.K.P., Oliveira, F.G., Del
Puerto, H.L., Alves, F., Braz, G.F., Costa,
A.T.R., Guedes, R.M.C., Lobato, Z.I.P.,
Oral Presentation
41
Leite, R.C.
1. Escola
de
Veterinária,
Gerais, UFMG, Av. Presidente
Antônio Carlos 6627, CP 567, Belo
Horizonte, MG, Brasil 31270-901
2. Instituto
de
Pesquisas
Veterinárias Especializadas Ltda,
IPEVE, R. Esmeralda 786, Belo
Horizonte, MG, Brasil 30411-191
E-mail: [email protected]
Influenza A virus (IAV) is one of
the main pathogens that causes
respiratory disease in pigs and
the humoral immune response is
important to prevent the infection and
to decrease clinical manifestation of
the disease. The circulation dynamics
of specific pathogens in a herd can
be accessed by the analysis of its
serological profile. Therefore, the
objective of this work was to study the
serological profile of influenza virus
in commercial swineherds in Brazil. A
cross-sectional study was performed
in seven Brazilian herds located in
Minas Gerais, São Paulo, and Paraná
states, sampled before (H1 to H3a) and
after the human influenza H1N1 2009
pandemic (H3b to H6). Serum samples
from 10 animals in each production
category (sows, lactating piglets,
nursery, grower, and finisher pigs)
were evaluated by hemagglutination
inhibition test (HI) against classical
swine (cH1N1) and pandemic (pH1N1)
H1N1 influenza viruses. All herds
sampled before the pandemic (H1 to
H3a) were seronegative. Serological
profile patterns were similar in all herds
evaluated after 2009 H1N1 pandemic
(H3b to H6), in which maternal derived
antibodies (MDA) seemed to decay in
nursery phase. Seropositive animals
were detected in all categories, with
42
Oral Presentation
191 (46.9%) animals seropositive for
cH1N1 and 196 (48.1%) for pH1N1.
Some differences were observed in
antibody titers between herds, and
H3b and H4 appeared to have low
viral circulation, but H5 seemed to be
going through an influenza outbreak.
H6 showed a slightly different pattern,
with an increase in mean antibody titer
of lactating piglets, probably due to IAV
infection even in the presence of MDA.
These results show the serological
profile of Brazilian swineherds infected
with influenza A virus and provide
information that allows to perform
vaccination and introduce the correct
control measures at the right moment
to prevent the spread of the disease.
VV1494 - IMMUNOLOGICAL EVALUATION OF AN INACTIVATE VACCINE
AGAINTS SWINE INFLUENZA VIRUS
(SIV) FOR POTENIAL USE IN PIGS
FROM PRODUCING FARMS IN
COLOMBIA
Jaime, J.
Universidad Nacional de Colombia, ,
Thirty 9 week old fattening pigs
were randomly divided into 3 groups
(group A n=10; group B n=10; control
n=10). Pigs were vaccinated IM
twice at week 1 and 3 of the study.
The group A was vaccinated with a
bivalent inactivated vaccine (A/SW/
COL0102/2009/pH1N1 and A/SW/
COL0401/2008/cH1N1)
employing
aluminium hydroxide as adjuvant
(Treatment A) and the group B with
a bivalent inactivated vaccine (same
viruses together) employing an
emulsion (Emulsigen®) as adjuvant
(Treatment B). Serum samples were
collected weekly for 9 times after the
first vaccination for HI assay. From
each sampling 5 serum samples were
randomly chosen from each group
for IL-4, IL-10 and INF-γ analysis by
ELISA at weeks 1,3,5,7,9 and whole
blood samples were collected at
weeks 1, 5 and 9 for flow cytometry
(FC) analysis. Despite that not finding
significant differences between both
inactivated biologicals, treatment B
generated higher antibodies’ titers
during the study compared with the
treatment A. Moreover, the pandemic
strain employed generated the highest
antibodies’ titers, compared with
the classical strain. IL-4 and IL-10
concentration not showed significant
difference
between
treatments,
but treatment B generated higher
concentrations of IL4 and IL-10
compared to treatment A. For INF-γ,
no significant differences between
treatments were found; vaccine
containing
aluminum
hydroxide
produced higher levels compared
with the emulsion. Employing FC, we
found in both treatments at week 5
posvaccination an activated cellular
response by populations of CD4 and
CD8, compared with week 1 and 9.
The CD69 molecule was measured to
determine the activation of CD4+CD8+
by both vaccines, which showed that
its expression was highest at week
5 of the study. Vaccines employed
were able to generate humoral and
cellular response, however, the best
adjuvant stimulated the two types of
immune response was the emulsion.
Both vaccines can eventually protect
animals against circulating strains
in Colombia. Financial support:
Universidad Nacional de Colombia,
Ministerio de Agricultura y Desarrollo
Rural de Colombia (MADR), Asociación
Colombiana de Porcicultores.
Posters Basic Virology - BV
XXIII Brazilian Congress of Virology &
44
Basic Virology: BV
BV722 - GENOMIC CHARACTERIZATION OF A NOVEL SEQUENCE OF
APEU VIRUS (ORTHOBUNYAVIRUS)
Pinto, C.A., Moura, C.S.S., Magalhães,
C.L.B., Assis, M.T.A., Bonjardim, C.A.,
Kroon, E.G., Peregrino, P.C.P.
Gerais, UFMG, Av. Antônio Carlos,
6627 - Pampulha - Belo Horizonte
- MG CEP 31270-901
2. Universidade
Federal
de
Ouro Preto, UFOP, E-mail:
[email protected]
Apeu virus (APEUV) is a group C
arbovirus, member of Bunyaviridae
family and Orthobunyavirus genus.
The orthobunyaviruses’ genome is
composed of three segments of singlestranded negative-sense RNA named S
(~1000 bp), M (~4500 bp) and L (~7000
bp). Group C viruses are potential
emergent human pathogens. However,
studies involving these viruses are rare
and their molecular characteristics are
poorly known, which complicates their
correct identification. To date, only
partial sequences for segments S (762
bp), M (1851 bp) and L (1254 bp) from
APEUV BeAn848 were characterized.
The aim of this work is to conclude
the sequencing of APEUV’s (BeAn848)
genome. To achieve this goal, viral RNA
was extracted from the supernatant
of Vero cells infected with Apeu and
submitted to RT-PCR using specific
primers, and the cDNA was amplified
by PCR. Then, DNA was purified and
cloned into pGEM vector, amplified in
DH5alfa E. coli, and the positive clones
were sequenced. The results showed
two novel sequences located in regions
1-2047pb and 3566-5555pb in viral
L segment (4036 nucleotides). The
amino acid sequence deduced from
the region 3669-3741 corresponds to
motifs D and E from region 3 of RNApolymerase, which is highly conserved
between members of Bunyaviridae
family. The deduced amino acid
sequence from the region 35665555pb showed identity with LACV
(48%), AKAV (48.2%), BUNV (48.5%),
TAHV (49.2%), OROV (51%) and CARV
(81.6%). These results indicate the
need of molecular characterization of
group C Orthobunyaviruses in order
to allow its correct classification and
identification.
BV728 - EVALUATION OF OXIDATIVE
STRESS IN MURINE MODEL OF
HEPATIC INFECTION BY CARAPARU
VIRUS (BUNYAVIRIDAE)
Almeida, L.T., Bernardes, C.S., Costa,
A.C.T., Camini, F.C., Silva, M., Souza,
M.O., Pedrosa, M.L., Lima, W.G., Pinto,
C.A., Ferreira, P.C.P., Magalhães, J.C.,
Magalhães, C.L.B.
1. Universidade
Federal
de
Ouro Preto, UFOP, Campus
Universitário Morro do Cruzeiro,
Bauxita, Ouro Preto, MG
Gerais, UFMG, Campus Pampulha,
Belo Horizonte. MG
3. Universidade Federal de São
João Del Rei, UFSJ, Campus Alto
Paraopeba, Ouro Branco, MG
The group C viruses (Bunyaviridae)
were first described in the Brazilian
Amazon region during the 1950s. A
total of 14 distinct group C viruses
have been isolated from humans,
mosquitoes, wild rodents, marsupials,
and bats. Caraparu virus (CARV) was
first isolated in 1956 from a sentinel
October 2012 Volume 17 (2) – Supplement 1 / Foz do Iguaçu, Paraná, Brazil - Posters - Basic Virology: BV
monkey and subsequently, it has been
isolated from the blood of a febrile
forest worker and from arthropods. In
countries of South America, group C
bunyaviruses are among the common
agents of human febrile illness,
and have caused multiple notable
outbreaks of human disease in recent
decades. Recent evidence suggests
that oxidative stress could play an
important role in the pathogenesis
of inflammatory viral diseases. In
experimental infected mice, CARV can
cause hepatitis. To investigate if CARV
infection induces hepatic oxidative
stress, BALB/c mice were infected
subcutaneous with 10*5 plaqueforming units and 3, 7 and 14 days pi
the animals were sacrificed and liver
obtained for dosages. Control animals
were inoculated subcutaneously with
MEM (Eagle’s minimum essential
medium) in the same conditions as
infected animals. Lipid peroxidation
and protein oxidation levels were
measured by the thiobarbituric acid
(TBARs) and protein carbonyls,
respectively. Total glutathione (GSH)
was measured to assess the antioxidant
status. The infected animals developed
liver inflammatory disease 3 and 7
days pi with a favorable recovery 14
days pi. The involvement of oxidative
stress was not observed, since no
differences were observed in TBARs
and protein carbonyl levels between
infected and controls mice. However,
GSH levels increased significantly in
infected animals, on different days
pi. Therefore, this study indicates
that an increase of antioxidant status
may be related to the response after
hepatic CARV infection, controlling
the levels of oxidative stress on this
tissue. Thus, this study contributes to
better understanding the pathogenic
Basic Virology: BV
45
mechanisms of bunyavirus infection.
BV729 - INFECTION BY CARAPARU
VIRUS (BUNYAVIRIDAE) MODULATES
ACTIVITY AND GENE EXPRESSION
OF ANTIOXIDANTS ENZYMES IN
MICE LIVER
Bernardes, C.S., Almeida, L.T., Costa,
A.C.T., Camini, F.C., Silva, M., RossoniJúnior, J.V., Pedrosa, M.L., Lima, W.G.,
Pinto, C.A., Ferreira, P.C.P., Magalhães,
J.C., Magalhães, C.L.B.
1. Universidade
Federal
de
Universitário Morro do Cruzeiro,
Bauxita, Ouro Preto, MG
Gerais, UFMG, Campus Pampulha,
Belo Horizonte. MG
João Del Rei, UFSJ, Campus Alto
Paraopeba, Ouro Branco, MG
E-mail:
carolina_bernardes@
msn.com
Oxidative stress has been recognized
to be an important contributor to viral
pathogenesis. The cell has extensive
machinery to ensure maintenance
of oxidative homeostasis. Important
components of this machinery are
the superoxide dismutase (SOD) and
catalase enzymes. Disruption of the host
antioxidant enzymes is associated with
many disease states. Caraparu virus
(CARV) is an arthropod borne virus that
belongs to the group C (Bunyaviridae),
isolated in 1956 on Brazil. In humans,
CARV causes a disease characterized
by flu-like symptoms, and in mice
CARV causes hepatitis. Despite the
public-health importance of the
bunyaviruses, little is known about the
pathogenic characteristics of the group
46
Basic Virology: BV
C circulating in Brazil. The purpose
of this study was to investigate the
effect of CARV infection on modulation
of the antioxidants enzymes in mice
liver. BALB/c mice were infected
subcutaneous with 10*5 PFU of CARV
and control animals were inoculated
with MEM. Animals were sacrificed 3,
7 and 14 days post inoculation (dpi)
and liver samples were collected for
determinate catalase and SOD activity,
gene expression of catalase, Cu/
ZnSOD (cytosolic form) and MnSOD
(mitochondrial form). The infected
animals developed liver inflammatory
disease 3 and 7 dpi with a favorable
recovery 14 dpi. The infected animals
showed an increased of catalase
activity at 3 dpi, while a decreased
was observed 7 dpi, returning to
normal levels 14 dpi. In relation to SOD
activity, the infected animals showed
a decreased of activity at 3 dpi, while
an increased was observed 14 dpi.
The real-time PCR analysis showed
a decreasing and increasing in the
catalase gene expression levels 3 and
14 dpi, respectively. The Cu/ZnSOD
gene expression levels decreasing 3
and 7 dpi, returning to normal levels
14 dpi. The MnSOD gene expression
levels increasing 14 dpi. Then, our
data indicates that CARV infection
modulates the antioxidants enzymes
on liver and that this event may be
involved on disease evolution.
BV739 - DEVELOPMENT OF A
REVERSE
TRANSCRIPTION-POLYMERASE CHAIN REACTION FOR
AMPLIFICATION OF THE DOMAIN III
OF THE ENVELOPE PROTEIN GENE
OF DENGUE VIRUS SEROTYPES 1
AND 2
Dias, E.H.V., Freitas, G.R.O., Oliveira,
T.F.M.S., Chávez, J.H., Yokosawa, J.
Universidade Federal de Uberlândia,
UFU, Av. Pará, 1720, Bloco 4C, andar
superior; C. Umuarama; Uberlândia,
MG E-mail: edigarhenriquevaz@
yahoo.com.br
The dengue is an arboviral disease
caused by dengue virus (DENV), which
is represented by four serotypes and
belongs to the family Flaviviridae
and genus Flavivirus. Transmission
of the virus occurs mainly by bite of
the mosquitoes Aedes aegypti and
albopictus. According to the World
Health Organization, dengue leads the
ranking of the most important human
arboviruses. This study aims the
implementation of an RT-PCR method
for amplification of the segment
corresponding to the domain III (DIII)
of the envelope protein gene of dengue
virus serotypes 1 and 2, followed by
sequence analysis of the amplified
products for characterization of the
viral strains found in Uberlândia,
MG and nucleotide sequences of
the envelope protein gene were
obtained from GenBank for DENV-1
and DENV-2, respectively. Alignment
of these sequences was carried out
and oligonucleotides developed for
RT-PCR and nested PCR and different
conditions of the RT-PCR were tested.
Optimal conditions for DENV-I DIII
amplification were achieved and
for DENV-2 DIII they still need to
be established. Financial Support:
FAPEMIG
BV741 - DETECTION OF DENGUE
VIRUS IN AEDES AEGYPTI AND
AEDES ALBOPICTUS IN DIVINÓPOLIS, MINAS GERAIS
Taranto, M.F.R., Santos, M. dos, Souza,
J.P., Andrade, A.C.S.P., Camargos, V.N.,
Alves, S.N., Santos, L.L.dos, Pereira,
L.H.S., Oliveira, C.D.L., Taranto, A.G.,
Magalhães, J.C., Kroon, E.G., Drumond,
B.P., Figueiredo, L.B., Ferreira, J.M.S.
João del Rei, UFSJ, Av. Sebastião
Gonçalves Coelho, 400 Chanadour
- Divinópolis-MG
Gerais , UFMG, Av. Antônio Carlos,
6627 Pampulha - Belo HorizonteMG
3. Universidade
Federal
de
Juiz de Fora , UFJF , Rua José
Lourenço Kelmer, s/n - Campus
Universitário São Pedro - Juiz de
Fora-MG
João del Rei , UFSJ, Rod.: MG 443,
KM 7 Ouro Branco - MG E-mail:
[email protected]
Infection with Dengue virus (DENV)
is considered as a major public health
problem in Brazil. The epidemiological
situation of dengue in Minas Gerais
State (MG) is considered highly
endemic, in 2010 were recorded a total
of 261.915 cases. Divinopolis, a city
of MG, have been reported over 5.000
cases of the disease. According to the
latest epidemiological bulletin of May
2012, the state has already notified
23.357 cases and Divinopolis is among
the 30th cities with the largest number
of cases in this state, totaling 131
notifications. An epidemiological study
was carried out in order to monitoring
of the locomotion and vector behavior
of dengue vector and DENV detection
in Divinopolis at public places from
April/2011 until May/2012. In this
context, traps for collection of Aedes
eggs containing wood vanes were
manufactured and placed in 44
Basic Virology: BV
47
places, which were distributed in six
areas of the city (North, Northeast,
Southeast, Southwest, West, and
Central). Subsequently, eggs of each
area were counted and the pallets
were submersed in water containing
fish meal in plastic glasses and covered
with tulle. On the sixth day of larvae
eclosion, stages L3 and L4, larvae were
identified as A. aegypti or A. albopictus
and stored at -80 °C. The RNA extraction
and viral RNA detection using primers
described by Lanciotti et al. (1992)
were performed with QIAGEN kit and
RT-PCR, respectively. A total of 11.976
larvae were stored in 399 pools. As
a result, 72% and 28% of larvaes
were identified as A. aegypti and as
A. albopictus, respectively. Currently,
viral RNA of 1.686 larvae was extracted
showing that DENV was detected in a
pool of 30 A. aegypti larvae from West
Regional in May/2011. These findings
provide support for intensifying
preventive and educational actions
where was detected a high incidence of
dengue vector. This work can contribute
to reduce the occurrence of dengue
cases and consequently, the rates of
morbidity and mortality in Divinopolis
city. FAPEMIG, CNPq, CAPES
BV756 - MEK1/2 INHIBITOR
(U0126) REDUCES YELLOW FEVER
VIRUS REPLICATION BOTH IN VITRO
AND IN VIVO
Torees, A.A., Oliveira, L.C., Albarnaz,
J.D., Cruz, A.F., Palhares, R.M., Cardozo,
P.L., Xavier, E.R.S., Pantuzzo, C.M.,
Peregrino, P.C., Kroon, E.G., Bonjardim,
C.A.
6627- Pampulha E-mail: licetorres@
gmail.com
48
Basic Virology: BV
The Yellow fever virus (YFV), prototype
species of Flavivirus genus, is a small
enveloped virus, which icosahedral
capsid contains a single positivestranded RNA genome of 11 kb. About
900 million people live in risk areas
for yellow fever in tropical regions of
South America and Africa. Currently,
there is no specific treatment against
the YFV and the available vaccine
is not completely safe, leading to
complications such as neurotropic and
viscerotropic diseases. Furthermore,
the vector mosquito is endemic in
Brazilian urban areas. The goal of this
work was characterize the MEK1/2
pharmacological inhibitor (U0126)
effect on the vaccine YFV strain (YFV17D, Fiocruz) replication, both in
vitro and in vivo. Our data showed
that U0126 inhibited the YFV-17D
replication in Vero cells, in a dosedependent way (~2log10 inhibition in
the 20µM non-cytotoxic concentration).
By electron microscopy (CM-UFMG),
we saw a significant increase in the
number of Endoplasmic Reticulum
(ER)-associated vesicles, where viral
replication machinery is located,
as well as typical virions of ~40nm
inside vesicles in YFV-17D-infected
cells (24 and 48 hours post-infection,
hpi). In the U0126-treated cells, these
structures were not observed (24hpi),
or ER-associated vesicles and virions
were rarely observed (48hpi). When
intracranially inoculated with YFV-17D,
BALB/c mice showed loss of weight
after the 6th day post-infection (dpi),
reaching paralysis or 25% loss of initial
weight in the 8th or 9th dpi, when they
were euthanized. We detected YFV,
by plaque assay, only in the brains of
infected mice.When mice were pretreated with U0126 (30µg/animal/
day), we found a >1log10 decrease in
the viral titers in comparison to vehicletreated infected mice. Taken together,
our results strongly suggest that
MEK1/2 plays a functional role during
YFV life cyle both in vivo and in vitro,
and it could be used as a therapeutic
target for antiviral treatment of postvaccination complications.
BV757 - DEVELOPING CELL CULTURE
MODELS TO STUDY THE INTERACTION OF DENGUE VIRUS WITH THE
INVERTEBRATE HOST
Ferreira, F.V., Vilela, A.P.P., Souza, E.A.,
Santos, J.R., Kroon, E.G., Marques, J.T.,
6627- Pampulha
2. Laboratório
de
VírusDepartamento de Microbiologia,
ICB-UFMG, Av. Antônio Carlos,
6627- Pampulha Laboratório de
RNAi3. Departamento de Bioquímica
e Imunologia, ICB-UFMG, Av.
Antônio Carlos, 6627- Pampulha
E-mail:
fvianaferreira@gmail.
com
The World Health Organization
estimates about half of the world’s
population live in areas at risk of
infection
with
arthropod-borne
viruses (arboviruses). More than
one hundred arboviruses infect
humans and cause diseases. Most of
these diseases, such as Dengue, lack
efficient therapeutic, prophylactic
and surveillance tools. Targeting
the invertebrate host is a potential
approach to prevent transmission
of arboviruses. Different pathways,
including the RNA Interference (RNAi),
Jak-STAT and NF-�B signaling, mediate
innate antiviral immunity in insects. An
in vitro cell-based system to study the
interaction between arboviruses and
the invertebrate host is an important
tool to dissect the role of these antiviral
pathways. Here, we developed and
characterized cell culture infection
models in Aedes aegypti Aag2 cells
and Drosophila S2 cells with Dengue
virus (DENV). We chose both Aedes
aegypti and Drosophila cells because
the former is the major vector in
nature and the latter a great animal
model to study invertebrate antiviral
immunity. Our results show successful
DENV replication in both cell types. By
quantitative PCR (qPCR), we detected
viral RNA starting at 24 hours post
infection and progressively increasing
until the last time post infection of 168
hours. We also detected virus plaque
forming units (PFU) in cell extracts and
supernatants starting at 48 hours post
infection. Hence, we have characterized
profile of DENV replication in two
different invertebrate cell culture
models that will allow us to study the
different immune pathways involved in
Dengue virus infection.
BV797 - STUDY OF THE INTERACTION BETWEEN THE HUMAN
PROTEIN P54NRB WITH NS5 OF
YELLOW FEVER AND KUNJIN VIRUS
Terzian, A.C.B., Madrid, M.C.F.S., Carmo,
A.C.V., Bronzoni, R.V.M., Khromykh,
A.A., Nogueira, M.L.
FACULDADE DE MEDICINA DE SÃO
JOSÉ DO RIO PRETO, FAMERP, Av.
Brigadeiro Faria Lima, 5416 - Vila
São Pedro - 15090-000, São José do
Rio Pr E-mail: anacarolinaterzian@
gmail.com
Yellow Fever Virus (YFV) causes
a hemorrhagic fever and it is the
Basic Virology: BV
49
prototype of genus Flavivirus. Kunjin
virus (WNKUN), the Australian
subtype of West Nile Virus (WNV), is
naturally attenuated and is used to
develop vaccine candidates against
more pathogenic WNV strains.
Flavivirus replication is a complex
mechanism that involves interaction
between viral RNA and cellular and
viral proteins. NS5 is the largest and
highly conserved viral protein and it is
critical for many functions, including
replication, RNA capping and virushost interactions. The p54nrb protein
is a nuclear transcription factor
associated with nuclear membrane and
exhibits multifunction characteristics
in nuclear processes in eukaryotic
cells. Interaction between NS5 and
p54nrb may influence localization
and transport of proteins and viral
RNA within the cell. The purpose of
this study was to characterize the
interaction between p54nrb and NS5.
Previously, we identified by Yeast
2-hybrid screening and confirmed by
plasmid linkage that p54nrb interacts
with NS5 of YFV. The interaction was
mapped using NS5 deletions and the
results suggested that interaction
domain is located in the C-terminal
region of YFV NS5. Although, Flavivirus
replication is cytoplasmic, IF assays
showed co-localization of p54nrb with
YFV NS4 in the perinuclar region and
with NS5 in the nucleus. In contrast,
WNKUN NS5 co-localized with p54nrb
in the perinuclear region and p54nrb
was identified by mass spectrometry
analysis as one of the proteins coprecipitated by monoclonal antibodies
to WNKUN NS5 protein. RT assay of viral
RNA showed the accumulation of YFV
particles in Hela cells overexpressing
p54nrb. However, replication of
WNKUN replicon and virus was not
50
Basic Virology: BV
affected by the overexpression of
p54nrb. The results suggest the role
for p54nrb in the YFV and WNKUN
replication; however, more detailed
studies are needed to characterize the
interactions between NS5 and p54nrb.
BV800 - COMPARISON OF THREE
ASSAYS TO DETERMINE DENGUE
VIRUS TITRATION IN CELL CULTURE
Barros, H.L.S., Freitas, G.R.O., Polloni,
L.C., Dias, E.H.V., Oliveira, T.F.M.S.,
Yokosawa, J., Chávez, J.H.
UFU, Uberlândia, Av. Pará, 1720,
Bloco
4C,Campus
Umuarama
Uberlândia MG. CEP 38400902
E-mail: [email protected].
br
Dengue virus (DENV) belongs to the
Flavivirus genus of the Flaviviradae
family. This genus includes the most
important arboviruses in public
health, due to their morbidity and
mortality. The dengue fever, caused
by DENV infection, is responsible for
worldwide morbidity and mortality,
causing about 50 to 100 millions of
cases annually. Thus, the goal of this
work is the comparison of three viral
titration techniques for DENV. MTT
assay is extensively used for viral
titration, although no data is available
in the literature for DENV titration.
Thus, the comparison of this assay with
two other established methods will
provide useful information. This study
standardized and compared Plaque
Formation Unit (PFU) and TCID50%
(Tissue Culture Infectious Dose 50%)
with the MTT method. All assays were
performed with LLC-MK2 cell type. As
results, MTT titration assays of DENV1, DENV-2 and DENV-3 could not be
calculated, because no expressive
differences of absorbance between the
viral dilutions and the cellular control
were observed. However, DENV-3
exhibited the most intense cytopathic
effect. PFU titration assay with DENV-1
and DENV-2 in LLC-MK2 cells showed
no lysis plaque formation. TCID50%
was performed with serotypes 1, 2 and
3 of DENV and no extensive cytopathic
effect was observed in the most
diluted concentrations. Dengue Titers
obtained by TCID50% were 5x10^4,05,
5x10^5,74, and 5x10^6,33 for DENV1, DENV-2 and DENV-3, respectively.
DENV-4 will be also titrated by the
same techniques. Considering the three
techniques employed in this study,
DENV titration by TCID50% was more
appropriate than the PFU and MTT
techniques in LLC-MK2 cells. Financial
support: CNPQ/UFU/FAPEMIG
BV801 - FLAVIVIRUS SURVEILLANCE IN LARVAE OF MOSQUITOES IN
UBERLÂNDIA, MG
Polloni, L.C., Rodrigues, J.L.A., Melo,
L.D., Souza, L.P.F., Barros, H.L.S., Freitas,
G.R.O., Dias, E.H.V., Arruda, J.H., Oliveira,
T.F.S., Yokosawa, J., Chávez, J.H.,
UFU, Uberlândia, Av. Pará, 1720,
Bloco
4C,Campus
Umuarama,
Uberlândia MG. CEP 38400-9 E-mail:
[email protected]
Flaviviruses,
including
Dengue,
West Nile, Saint Louis encephalitis
virus, are major emerging human
pathogens, affecting millions of
individuals worldwide. These viruses
are transmitted by the bite from an
infected arthropod, mainly mosquitoes.
Dengue virus is primarily transmitted
by Aedes mosquitoes, particularly A.
aegypti. Other Aedes species such as
A. albopictus, A. polynesiensis and A.
scutellaris can also transmit the disease.
Mosquitoes from the genus Culex are
related to Saint Louis encephalitis and
West Nile transmission. Viral presence
in larvae of mosquitoes strongly
suggests transovarial transmission,
as previously described. Uberlândia
is the main town in the Triângulo
Mineiro region, in the state of Minas
Gerais, Brazil. Dengue outbreaks in
humans are well documented in such
region, but few studies involving the
arthropod vector are performed in the
city. This is an ongoing study which
aims to perform virus surveillance
in mosquitoes and larvae using a
Flavivirus genus-specific RT-HemiNested-PCR assay. Larvae of mosquitoes
were collected in Uberlândia during
January, 2012. The action was part
of LIRA, a program which estimates
the index of Aedes larvaes in urban
areas. All larvae were morphologically
classified and separated in pools (n=
137) of A. aegypti (58,70%/ n= 80), A.
Albopictus (4,30%/ n= 6) and Culex sp
(37%/n= 51), considering the urban
area. Pools were stored at -70oC and
RNA was extracted with TRI® reagent
(Sigma). RT-Hemi-Nested-PCR assay
was performed with specific primers
for different flaviviruses, including
dengue, Saint Louis encephalitis and
Rocio virus. A total of 32 samples were
processed. One pool of A. albopictus
tested positive for DENV-1 and DENV3, further analisys are required to
confirm this result. Viral surveillance
in mosquitoes and larvae are
extremely important for epidemiology
and the control of future flaviviruses
outbreaks. Financial Support: CNPQ/
UFU/FAPEMIG
BV804 - CHARACTERIZATION OF A
NOVEL PROTEIN ANTIVIRAL FROM
Basic Virology: BV
51
LONOMIA OBLIQUA USING BIOINFORMATICS TOOLS AND ACTIVITY
ANALYSIS BY REAL TIME
Carmo, A.C.V., Mendonça, R.Z., Giovanni,
D.N.S., Yamasaki, L.H.T., Figueiredo,
C.A., Oliveira, M.I., Santos, F.C.P., Curti,
S.P., Rahal, P.
1. Instituto Butantan - Laboratório
de Parasitologia, IB, Av Vital
Brasil, 1500, Butantã - São PauloSP 05503-000
2. Laboratório
de
Estudos
Genômicos, IBILCE-UNESP, UNESP,
São José do Rio Preto Núcleo de
Doenças Respiratórias, Centro
de Virologia, Instit, IAL, Avenida
Doutor Arnaldo, 355 - Pacaembu
São Paulo, 01246-000
3. Núcleo de Doenças de Transmissão
Vetorial, Centro de Virolog, IAL,
Avenida Doutor Arnaldo, 355 Pacaembu São Paulo, 01246-000
The control of viruses is of great
interest to the public health
area. Several studies have been
conducted that show the presence of
pharmacologically active substances
in the hemolymph of insects. Recently
we have demonstrated the existence
of a potent antiviral in the hemolymph
of Lonomia obliqua caterpillar. This
protein was able to reduce at 106 times
the replication of herpes virus and in
10,000 fold the rubella virus. Assays
using RT-PCR to determine viral RNA
present in no treated and rAVLO treated
infected cells also showed a reduction
in the same scale. The analysis of this
protein by bioinformatics suggests
that this protein is globular, secreted
with a signal peptide which is cleaved
between amino acids 16 and 17. The
52
Basic Virology: BV
studies also allows us to infer that this
antiviral protein has the ability to bind
to MHC class I. It was found that there
are several protein binding sites on the
weak and strong bases with various
HLA. The bioinformatic analysis also
shows a strong presence of α-helices
in the N-terminal region and allowed
to classify the antiviral protein as α/β
type of structure, as we detected the
presence of more than 30% α-helix
and 20 % of β-sheet found separately
along the protein chain. In the BLAST
sequence analysis of cDNA antiviral
protein, no sequence similarity was
found in Genbank, suggesting that it is
from a novel protein family. It can be
inferred by an analysis of this region
that the possible antigenicity region
would be between the 70-110 amino
acids, showing high accessibility. This
high antigenic region on the surface,
can be a possible region to interaction
with other proteins. Financial support:
FAPESP (08/57263-5), CAPES.
BV806 - PROTEIN ASSOCIATION IN
POLYMERIC NANOPARTICLES AND
POTENTIAL USE IN IMMUNIZATION
Félix-Bastos, V.A., Dionísio, M.,
Yokosawa, J., Grenha, A.M.M.
1. Universidade
Federal
de
Uberlândia, UFU, Av. Pará 1720 Campus Umuarama CEP 38400902- Uberlândia - MG
2. Universidade do Algarve, Ualg,
Campus Gambelas, 8005-139,
Faro, Portugal E-mail: victor_
[email protected]
Polymeric nanoparticles are an
innovative way of delivering proteins
for
therapeutic
or
preventive
approaches.
Chitosan
(CS)/
carrageenan (CRG) nanoparticles, with
or without tripolyphosphate (TPP),
were previously proposed by our group
as a potential tool for the delivery of
proteins such as ovalbumin and insulin.
These nanoparticles are now proposed
for non-invasive immunization, taking
benefit from mucosal delivery routes,
like the nasal or pulmonary. CS/CRG/
TPP nanoparticles were prepared
by polyelectrolyte complexation/
ionic gelation. Bovine serum albumin
(BSA, 67 kDa, pI = 4.7) was used as
model antigen. Its association to the
nanoparticles was tested in theoretical
loadings of 15 and 30% (m/v)
respective to the amount of CS in the
nanoparticle formulation. BSA was
directly mixed with CS solution prior
to nanoparticle formation. Afterwards,
a CRG-TPP solution was added to
the CS-BSA solution, under magnetic
stirring, so that ionic interaction
takes place. Nanoparticle suspension
was centrifuged and BSA association
efficiency measured indirectly in
the supernatant (the amount of free
protein that did not form nanoparticles,
was determined). The results showed
an association efficiency of 74 ± 9%
and 49 ± 22% for loadings of 15 and
30% of BSA, respectively. As BSA is
capable of triggering an immunological
response, future work will focus on
the evaluation of the potential of BSAloaded CS/CRG nanoparticles in this
regard. Additionally, a chimeric protein
of hepatitis C virus (HCV) is proposed
to be associated to the nanoparticles
and its behavior tested. Financial
support: Fundação para a Ciência e
Tecnologia, Portugal (projeto PTDC/
SAU-FCF/100291/2008 and PEst-OE/
EQB/LA0023/2011); Fundação de
Amparo à Pesquisa do Estado de Minas
Gerais – FAPEMIG; *Félix-Bastos has
been awarded with a scholarship from
Programa de Mobilidade Internacional,
Diretoria de relações internacionais e
interinstitucionais – UFU.
BV807 - IN VITRO CITOTOXIC AND
ANTI-DENGUE VIRUS ACTIVITY OF
BIGNONIACEAE AND POLYGONACEAE PLANT SPECIES
Rodrigues, R.A.L., Marinho, P.E.S.,
Gomes-Ruiz, A.C., Brandão, G.C.,
Evangelista, K.S., Ferreira, P.C.P.,
Bonjardim, C.A., Oliveira, A.B., Kroon,
E.G.
1. Instituto de Ciências Biológicas,
ICB/UFMG, Avenida Antônio
Carlos, 6627, Pampulha, Belo
Horizonte, Minas Gerais
2. Viriontech do Brasil Industria
Insumos e Serviços em Biotec,
VIRIONTECH, Rua Gustavo da
Silveira, 2100, Santa Inês, Belo
Horizonte, Minas Gerais
3. Faculdade de Farmácia, FAFAR/
UFMG, Avenida Antônio Carlos,
6627, Pampulha, Belo Horizonte,
Minas Gerais E-mail: ralr_0703@
yahoo.com.br
Dengue is considered the most
important arboviral disease of
humans and it is estimated that 100
million dengue cases occur every year
around the world. However, the main
strategies of dengue prevention and
control have focus on public health
measures against virus vector, once
there is no therapeutic agent or specific
vaccine against Dengue virus (DENV).
In the last decade, the research for
new medicines has been improved,
especially after the introduction of large
scale in vitro biological models, which
allowed a consistent statistic analysis
of the results. Previous studies have
Basic Virology: BV
53
shown that some botanical families
have species with relevant antiviral
activity, between them, Bignoniaceae
and Polygonaceae. In the present study,
plants extracts from those families were
evaluated against DENV-2. The extracts
were fractionated using Sephadex
LH 20® column and the fractions
were combined according to their
TLC profiles, resulting in an amount
of 22 fractions, between them, 15 of
Bignoniaceae and 7 of Polygonaceae.
The in vitro citotoxicity and antiviral
activity were evaluated by MTT
colorimetrical method. The results
were expressed by the 50% citotoxic
concentration o (CC50), 50% effective
concentration (EC50) and values of
selective index (SI = CC50/EC50). Seven
out of the 22 tested fractions have
antiviral activity with EC50 values in
the range of 4,18 + 0,7 ug/ml to 32,12
+ 24,2 ug/ml and SI between 4,13 and
61,56. From the active fractions, five
belongs to Anemopaegna sp. and two
belongs to Polygonum spp. The results
obtained in this work demonstrate that
chemical constituents of those fractions
are promissory as anti-dengue agents.
Chemical and molecular studies will
be realized in future with the objective
to determinate the active substances
present in those fractions and the
action mechanism. Financial support:
Viriontech, CNPq, FINEP
BV817 - INHIBITORY EFFECT OF A
FLAVONOID FROM KALANCHOE DAIGREMONTIANA AGAINST HERPES
SIMPLEX VIRUSES, IN VITRO
Saraiva, G.F., Gomes-Ürményi, F.G.,
Cavalcanti, J.F., Costa, S.S., Romanos,
M.T.V.
Universidade
Federal
do
Rio de Janeiro, UFRJ, E-mail:
54
Basic Virology: BV
[email protected]
Dengue fever is the most prevalent
arboviral disease worldwide. Despite
this threat for human health, no specific
chemotherapy or safe vaccination
for dengue virus (DENV) infection is
currently available. Therefore, there
is a requirement for effective antiviral
agents and therapeutic strategies for
DENV infection. Since the first report
about the role of heparan sulfate (HS)
in the initial interaction for DENV
attachment to vertebrate cells, diverse
HS-like glycosaminoglycans were
evaluated as antiviral agents against
DENV. Seaweeds represent a natural
source rich in sulfated polysaccharides,
compounds mimicking HS. In this study,
the anti-DENV-1 activity of six DLgalactans from Cryptonemia seminervis
marine alga (crude polysaccharide
fraction [P6], polysaccharides purified
by treatment with KCl 2M [P7], partial
depolymerization [16], subfractions
from P16, fractionated by anionexchange
chromatography
[P15,
P17 and P18]) were evaluated. The
experiments were made in C6/36 cell
culture, and all six sulfated galactans
showed CC50 values higher than 200
µg/mL (maximum concentration
tested). Our results show that antiviral
activity varied according molecular
weight (MW). P6 (332 KDa) and P7
(362 KDa) showed 99.9% of inhibition
and after partial reductive hydrolysis
there was a decrease in this activity.
P15 (231 KDa) presented inhibition of
65%, no activity was observed to P16
(51 KDa) and P17 (60 KDa) and 85%
of inhibition was observed to P18 (63
KDa). Despite the low MW, P18 had
the highest degree of sulfation of all
galactans evaluated. Studies are being
conducted to determine the doseresponse curve and mechanisms of
action. Preliminary results showed that
P6 presented an ED50 value of 1.43 µg/
mL and P7 of 0.79 µg/mL and selective
index values superior to 139.8 and 253,
respectively. The results show that
sulfated galactan from C. seminervis
can be seen as a new pharmacological
compound to dengue virus infection
treatment.
BV819 - SLEEPING WITH THE
ENEMY: VACCINIA VIRUS ISOLATION
FROM A HOUSEHOLD ENVIRONMENT DURING A BOVINE VACCINIA
OUTBREAK IN BRAZIL
Assis, F.L., Borges, I.A., Mesquita, V.S.,
Ferreira, P.C.P., Trindade, G.S., Kroon,
E.G., Abrahão, J.S.
Gerais,
UFMG,
Av.
Antônio
Carlos, 6627, Pampulha Belo
Horizonte - MG, 31270-901 E-mail:
[email protected]
The Vaccinia virus (VACV) is the
causative agent of zoonotic outbreaks
that affect cattle and buffaloes in
Brazil and Asia, respectively, leading to
economic losses. Since 1999, several
VACV have been isolated during
outbreaks in Brazil and have been
molecular and biologically featured.
Phylogenetic approaches can cluster
the Brazilian VACV (VACV-BR) into
two distinct groups. This molecular
dichotomy is correlated to in vivo and
in vitro phenotypes. VACV infections
in humans are frequently related to
occupational contact with sick animals
during the milking, and had never
been related with fomites neither
household environments. In August
2011 a bovine vaccinia (VB) outbreak
was reported on Carangola county,
Minas Gerais state, Brazil. During this
outbreak several farms were affected,
involving humans and dairy cattle. In
the studied property, clinical samples
were collected from affected humans,
three sick cows, besides the indoor
environments (IE). The samples were
submitted to molecular diagnosis and
virus isolation. Samples from the two
affected patient, sick animals and an IE
sample from affected patient’s pillow
were positive for both vgf and ATIbased PCR. Further, we were able to
isolate virus from a human sample and
from a patient’s pillow, both previously
positive in molecular diagnosis. We
sequenced fragments of the A56R and
A26L genes from the isolated virus.
The phylogenetic study shows that
this isolate clusters with other VACVBR, of group 1. This isolate was named
Carangola virus (CARV). This was the
first time that a VACV-BR was isolated
from a household environment during a
VB outbreak. VACV infections have been
frequently associated to occupational
activities and manly after direct
contact with sick animals or between
humans. The isolation of a VACV from
a household environment raises new
questions about non-occupational risk
factors related with BV transmission
chain. Financial Support: FAPEMIG,
CNPq, CAPES.
BV827 - SEQUENCING AND ANALYSIS
OF VIRULENCE RELATED GENES
BRAZILIAN VACCINIA VIRUSES
Carvalho, C.M., Assis, F.L., Rezende, I.M.,
Ferreira, J.M.S., Kroon, E.G., Trindade,
G.S., Santos, J.A., Drumond, B.P.
1. Universidade Federal de Juiz de
Fora, UFJF,
Gerais, UFMG,
Basic Virology: BV
55
João del Rei, UFSJ, E-mail:
camilamarques.bio@hotmail.
com
Vaccinia virus (VACV) has been
recognized as the agent of an emerging
zoonotic disease in the last decades,
in Brazil. It is known that at least
two different VACV populations are
circulating in Brazil (VACV-BR1 and
-BR2), presenting different genetic
and biological properties. VACV-BR1
contains strains Araçatuba, Cantagalo,
Pelotas2, GuaraniP2 and Serro and
VACV-BR2 groups VBH, BAV, GuaraniP1
(GP1) and Pelotas1 (P1V). In order to
better characterize and understand
the phylogenetic relationship of
P1V, GP1 and Serro (isolated from
horse, cow and human, respectively)
some virulence related genes were
sequenced and analyzed, as follows:
A53R (tumor necrosis factor receptor),
B16R (IL1 beta receptor), C3L
(complement binding protein) and
K3L (protein kinase RNA-dependent
inhibitor). Sequences were aligned and
analyzed using tools implemented in
the software MEGA 4.0. Phylogenetic
analyses demonstrated that P1V
clustered within VACV, Horsepox virus
and Rabbitpox virus and that Serro
presented a closer relationship with
Horsepox virus what is in agreement
with previous studies. K3L sequence
of P1V was identical to sequences of
GP1, BAV, and VBH (group VACV-BR2).
On the other hand, in A53R, B16R,
C3L nucleotide and deduced proteins
sequences, nucleotide and amino acid
differences were observed, differing
P1V from other Brazilian VACV. No
sequences of P1V or GP1 presented
premature stop codons, in contrast to
B16R sequence of Serro. This study
reinforces the genetic variability
observed in Brazilian VACV what
56
Basic Virology: BV
could be reflected in their biological
properties. Indeed, VACV belonging
to group VACV-BR2 have already been
demonstrated to be more virulent in
murine model (as P1V and GP1) than
viruses from VACV-BR1 group (in
which Serro is included). The genetic
polymorphism regarding the gene
B16R could be also responsible for
those biological differences observed
between those virus populations what
should be further tested. Financial
Support:FAPEMIG,
CNPq,
CAPES,
PROPESQ/UFJF
BV842 - ANTIVIRAL ACTIVITY OF
ARISTOLOCHIACEAE
EXTRACTS
AGAINST ROTAVIRUS
Diniz, J.S., Silva Jr., W.F., Cecilio, S.G.,
Magalhães, C.L.B., Ferreira, J.M.S., De
Magalhães, J.C.
1. Universidade Federal de São João
del Rei, UFSJ, Campus Centro
Oeste Doa Lindu, Divinópolis,
Minas Gerais
del Rei, UFSJ, Rodovia MG 443,
Km 7, Campus Alto Paraopeba,
Ouro Branco, Minas Gerais
3. Universidade Federal de Ouro
Preto, UFOP, Campus Morro do
Cruzeiro, Bauxita, Ouro Preto,
Minas Gerais E-mail: [email protected]
Rotavirus (RV), one of the main causative
agents of gastroenteritis, is responsible
for 2 million hospitalizations and over
600,000 deaths of children under
5 years annually. Currently, two RV
vaccines and some effective antiviral
drugs against RV are available. However,
recent studies have shown mutations
in antigenic regions, highlighting the
importance of alternative treatments
against rotavirus. Our study aimed
to investigate the antiviral activity of
Aristolochia cymbifera extracts against
RV and investigation of anti-rotavirus
elements. Cytotoxicity and antiviral
activity of hydro-alcoholic (HIE),
dichloromethane (DIE) and hexane
(HE) Aristolochia cymbifera extracts
were evaluated by assaying the
number of cells that remained viable
after being exposed to the virus and
extracts (antiviral testing) or only the
extracts (Cytotoxicity assay). For both
tests, were used MA104 cells, grown
in DMEM medium containing 6% fetal
bovine serum (FBS) at 37ºC, under an
atmosphere of 5% CO2. After 48h of
infection, in presence of the extracts,
metabolic cell viability was evaluated
by adding 20μL/well of MTT and
reading in a ELISA spectrophotometer
at 492nm. All extracts proved to be
toxic to cells with a CC50 of 19.3 mg/
mL (HIE), 23.3 g/mL (DIE) and 36.7
mg/mL (HE). Presence of metabolically
active cells and higher absorbance
than the wells with virus control
(100% death cell) indicated that the
rotavirus caused slight reduction in
cytopathic effect at 20 and 10μg/mL
(DIE), 33μg/mL (HE) and 20μg/mL
(HIE). Our results indicate that extracts
of A. cymbifera (or its components)
are toxic to mammalian cells, while
also showing promising antirrotavirus
activity.
BV849 - ALPHAVIRUS GENOME
DELIVERY OCCURS AT THE PLASMA
MEMBRANE AND IS A TEMPERATURE DEPENDENDENT PROCESS
Vancini, R., Ferreira, D., Hernandez, R.,
Brown, D.
1. North Carolina State University,
NCSU, 345 Polk Hall, Raleigh, NC
- USA
Janeiro, UFRJ, Ilha do Fundão - Rio
de Janeiro- RJ E-mail: rgvancin@
ncsu.edu
Entry mechanisms of enveloped viruses
(viruses with a surrounding outer
lipid bilayer membrane) are usually
classified as being either endocytotic or
fusogenic. Different mechanisms have
been proposed for Alphavirus entry
and genome delivery. Controversial
observations led to a general belief
that alphaviruses can infect cells either
by protein-assisted fusion with the
plasma membrane in a pH independent
manner or by endocytosis and fusion
with the endocytic vacuole in a low
pH environment. Here, the mechanism
of Sindbis virus penetration has been
revisited using direct observation of
the process by electron microscopy
under a set of different temperatures.
In temperatures non-permissive for
endocytosis or any vesicular transport,
events occur which allow the entry of
the virus genome into the cells, allowing
the temperature dependence of the
process to be observed. Consequently,
the delivery of the viral RNA does not
require low pH mediated endocytosis
or low pH fusion, supporting the
growing body of evidences that
propose an alternative mechanism
for alphavirus entry. Arboviruses
are agents of significant human and
animal disease, therefore strategies
to control infections are needed and
include development of compounds
which will block critical steps in the
early infection events. In this study, an
accurate view of the entry process is
presented, providing details that have
been previously overlooked.
Basic Virology: BV
57
BV852 - FLAVIVIRUS INFECTION
FROM MOSQUITOES IN VITRO
REVEALS CELL ENTRY AT THE
PLASMA MEMBRANE
Vancini, R., Kramer, L., Ribeiro, M.,
Hernandez, R., Brown, D.
1. North Carolina State University,
NCSU, 345 Polk Hall, Raleigh, NC
- USA
2. Wadsworth Center - State
University of New York at Albany,
SUNY , Albany, New York, NY - USA
Dengue and West Nile viruses are
enveloped RNA viruses that belong to
genus Flavivirus and are considered
the important mosquito-borne viral
pathogenic agents worldwide. Despite
their clinical importance, there are
neither vaccines nor antiviral drugs
to prevent or treat dengue and west
nile infections. A potential target for
intervention strategies is the virus cell
entry mechanism. This mechanism,
however, remains controversial and
poorly described. Previous studies
about flavivirus entry process had
focused on the effects of biochemical
and molecular inhibitors on viral entry
leading to controversial conclusions
regarding
the
participation
of
cytoskeleton components, clathrinmediated endocytosis (CME) and
cholesterol for infection. It has been
recently proposed that there is an
alternate pathway for cell penetration
employed by Sindbis virus that does
not involve exposure to low pH,
endocytosis or the fusion of virus
membrane with the host cell membrane.
This mechanism was also proposed
for the interaction of flaviviruses
with host cells and suggests that an
alternate pathway may exist for both
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Basic Virology: BV
virus families. In the present study
we analyzed the early events in the
infection process by means of electron
microscopy and immuno-gold labeling
of viral particles during cell entry, and
took advantage of a new approach for
infecting cells with viruses obtained
directly from mosquitoes. Our
preliminary results show that, Dengue
and West Nile viruses did not present
direct fusion with plasma membrane
of host cells and also did not enter by
endocytosis. These data suggest that
medically important flaviviruses infect
cells by a mechanism that involves
direct penetration of the host cell
plasma membrane.
BV864 - ANALYSIS OF THE
ANTIVIRAL ACTIVITY OF UBIQUITIN
LIGASE RNF125 AGAINST HIV-1
Pezzuto, P., Aguiar, R.S., Abreu, C.M.,
Britto, A.M.A., Tanuri, A., Giannini,
A.L.M.
Universidade Federal do Rio
de Janeiro, UFRJ, Av. Brigadeiro
Trompowski S/N - CCS Ilha do
Fundão - CEP:21941-590 E-mail:
[email protected]
RNF125 is an ubiquitin ligase, localized
on cell membranes and expressed
in immune cells. This protein was
identified in a functional screen for
T-cell regulators that shows RNF125
negatively regulates T-cell activation.
This protein contains a RING domain,
three zinc-finger-like domains, a UIM
domain and a myristoilation site.
RNF125 targets the double strand RNA
sensor RIG-I to degradation, which
is important in the establishment of
innate immunity. RIG-I potentially
recognizes HIV-1 RNA, contributing to
the production of IFN type 1 to control
virus infection. Recently, RNF125 was
identified as a new cellular factor able
to inhibit HIV-1 replication, by as yet
undefined mechanisms. The present
study seeks to assess the effects of
the protein RNF125 against HIV-1,
elucidating the molecular mechanisms
involved in this process. For that, Jurkat
cells transiently expressing RNF125
or mutant form of this protein were
infected with HIV-1 virus harboring
the luciferase infectivity reporter
gene (NL4.3-Luc). HIV-1 replication
levels were quantified by luciferase
activity. RNF125 inhibited 40% of the
virus replication. In addition, the virus
production by cells expressing RNF125
wasn´t affected but these particles
were less infective. In the presence
of RNF125 was a significant increase
of Gag-Pol precursor production
and RNF125 compromises the p66/
p51 heterodimer ratio in reverse
transcriptase enzyme. These effects
were reverted by RNF125 mutant
that block catalytic domain (RNF125
C37,40A). Our results suggest that
the cellular factor RNF125 blocks
HIV replication and potentially could
become a novel therapeutic target
against HIV-1. CAPES, CNPq e FAPERJ
BV873 - DIRECT ACTION OF PLA2-CB
AND CROTOXIN OF CROTALLUS
DURISSUS TERRIFICUS VENOM ON
DENGUE VIRUS TYPE 2
Muller, V.D.M., Cintra, A.C.O., Figueiredo,
L.T.M., Sampaio, S.V., Aquino, V.H.,
1. Faculdade
de
Ciências
Farmacêuticas de Ribeirão Preto,
FCFRP USP-RP, Av. do Café, s/nº. Campus Universitário - Ribeirão
Preto - SP - 14040-903
2. Faculdade de Medicina de
Ribeirão Preto, FMRP USP-RP,
Av. Bandeirantes, 3900 - Monte
Alegre - CEP: 14049-900 E-mail:
[email protected]
The re-emergence of dengue virus
as a significant human pathogen has
lead to an increasing need for effective
antivirals drugs. Natural products
of plant or animal origin have an
extensive chemical diversity and are
an inexhaustible source of compounds
with promising biological activities.
The venoms of animals are a source
of several chemicals with biological
and pharmacological activity. In
a preview study, we showed that
PLA2-CB and crotoxin, isolated from
Crotallus durissus terrificus snake
venom have a potent anti-dengue
and anti-yellow fever virus activity,
which occurs in the early stage of viral
replication. In the present study, we
evaluated the direct action of PLA2CB and Crotoxin on the viral particle.
DENV-2 (strain NGC, 1.17x103 PFU)
was incubated with PLA2-CB (8ng/
uL), crotoxin (8ng/uL) or PBS for 1h at
room temperature. The mixture were
treated with 8µL of RNAse A (20µg/
mL) or PBS for 1h at 37°C. The viral
RNA was extracted and degradation
was evaluated by a real-time RT-PCR.
The viral RNA was degraded when
DENV-2 was treated with PLA2-CB
or crotoxin and RNAse-A, showing
that these toxins have direct action
on viral particle. In other experiment,
different concentration (8, 4 or 0,0004
ng/uL) of toxins were incubated with
1.17x103 PFU of DENV-2 for 1 h at
room temperature. Subsequently,
these mixtures were diluted hundred
times and used to infect the cells.
The toxins concentration used in cell
monolayer (0.08, 0.04 or 0.000004 ng/
uL) was used to pre-treatment the cells
for 1h with posterior cell infection.
Basic Virology: BV
59
The viral title was determined by realtime RT-PCR. PLA2-CB and crotoxin
at 8 or 4 ng/uL inhibit 100% the viral
replication; while no inhibition on viral
replication was observed in the cells
pre-treated with these toxins (0.08,
0.04 or 0.000004 ng/uL). These finds
reinforce our previous results, and
suggest that PLA2-CB and crotoxin
have a direct action on viral particle.
Financial support: FAPESP.
BV874 - SCREENING OF A NOVEL
ANTIVIRAL INHIBITOR OF FLAVIVIRUS
Pacca, C.C., Marques, R.E., Teixeira,
M.M., Leite, A.C.L., Espindola, J.W.P.,
Oliveira-Filho, G.B.O., Nogueira, M.L.
1. FACULDADE DE MEDICINA DE SAO
JOSE DO RIO PRETO, FAMERP, Av.
São Pedro - 15090-000
2. UNIVERSIDADE FEDERAL DE
MINAS GERAIS, UFMG, Av. Antônio
Carlos, 6627, Pampulha Belo
Horizonte - MG, 31270-901
PERNAMBUCO, UFPE, Av. Prof.
Moraes Rego, 1235 - Cidade
Universitária, Recife - PE - CEP:
50670-901 E-mail: carolpacca@
gmail.com
The members of the Flaviviridae family,
including Yellow Fever Virus (YFV) and
Saint Louis Encephalitis Virus (SLEV),
are important human pathogens with
increasing impact around the globe.
Moreover, in recent years the Flavivirus
genus has gained further attention due
to re-emergence of diseases caused by
its members, such as dengue. Infections
by Flavivirus cause serious morbidity
and mortality worldwide, but effective
60
Basic Virology: BV
antiviral
chemotherapeutics
for
humans are not available. Therefore, it
is critical to develop new therapeutics,
preferentially targeting viral proteins.
In order to identify compounds with
antiviral activity against YFV and SLEV,
we tested five compounds for the ability
to inhibit viral replication in vitro. The
compounds, that possess different
structural features identified as ftalylthiazole and fenoxytiosemicarbazone
derivatives were tried in view to identify
a potential lead candidate. To evaluate
the inhibitory effect of the compounds,
we inoculated Vero E6 cells with YFV
and SLEV, separately, and treated the
cells with each compound for seven
days. We observed a marked inhibition
of viral replication at concentrations
that presented minimal toxicity to the
cells. Further testing demonstrated that
two compounds (a ftalyl-thiazole and a
fenoxytiosemicarbazone derivatives)
were more effective, reducing viral
replication in 60% and 75% for YFV
and SLEV, respectively. These data were
confirmed by immunofluorescence and
flow cytometry. The effectiveness of the
selected compounds in inhibiting viral
replication will be confirmed by in vivo
experiments.
BV882 - EXPRESSION AND PURIFICATION OF A DENV-3 NS1
RECOMBINANT POLYPEPTIDE FOR
POTENTIAL USE IN THE EARLY
DIAGNOSIS OF DENGUE INFECTIONS
de Sousa, C.S., Lima, M.R.Q., Baptista,
M.L., da Silva, M., Nogueira, R.M.R., dos
Santos, F.B.
Instituto Oswaldo Cruz, IOC, Av.
Brasil 4365 Manguinhos E-mail:
[email protected]
The dengue virus (DENV) is associated
with explosive epidemics and has
become a major public health
worldwide. Dengue diagnosis can be
performed using several approaches,
however sensitive and specific assays
to diagnose in the early stage of fever
are desirable. Previous studies have
shown the role of NS1 antigen capture
tests for the early dengue diagnosis,
however a significant lower sensitivity
was observed in DENV-3 cases. The use
of recombinant antigen may eliminate
the problems associated with the
standardization of DENV antigen
prepared in mouse brain or cell culture.
The cost of most kits for diagnosis is
prohibitive for many dengue-endemic
countries and the in-house production
of recombinant polypeptides could
provide a safe and valuable resource
for serodiagnosis. Here we aimed
to produce a DENV-3 recombinant
NS1antigen potentially useful for
the early diagnosis of dengue. To
express the DENV-3 NS1 recombinant
polypeptide, the DNA fragment
amplified was cloned into TA vector
pCR 2.1. The insert was subcloned
into expression vector pQE32 in
frame with the vector’s hexahistidine
tag. The resulting plasmid was
transformed into E. coli M15 (pRep4)
and for expression, a single colony was
grown and induced by the addition of
isopropyl-�-D-thiogalactoside (IPTG).
The purification of hexahistidinetagged polypeptide was performed
by nickel affinity chromatography
under denaturing conditions and the
specificity of the protein was addressed
by immunoblot analysis using antihistidine antibodies. We identified
and produced in E. coli a candidate
diagnostic polypeptide corresponding
to the C-terminal portion (179 amino
acids, 20.3kDa) of the DENV-3 NS1
protein (pD2-7). The polypeptide was
successfully purified under denaturing
conditions, however the low expression
did not yield enough for the purification
under native conditions. Its reactivity
needs to be accessed against human
DENV-positive sera. Financial support:
FAPERJ, CNPq, CAPES, PDTIS and
FIOCRUZ
BV896 - EVALUATION OF ANTIVIRAL
ACTIVITY OF YEAST AND FILAMENTOUS ENDOPHYTIC FUNGI
EXTRACTS OF VARIOUS BRAZILIAN
ECOSYSTEMS AGAINST DENGUE
VIRUS (DENV-2)
Silva, K.L.S., Marinho, P.E.S., SilvaFernades, A.T., Rodrigues, R.A.L., GomeRuiz, A.C., Johann, S., Vieira, M.L.A.,
Rosa, L.H., Rosa, C.A., Kroon, E.G.
1. Laboratório
de
Vírus,
Gerais, ICB/UFMG, Av. Antônio
Carlos, 6627, Campus Pampulha,
Belo Horizonte, Minas Gerais
2. Laboratório de Ecologia e
Biotecnologia de Leveduras,
UFMG, ICB/UFMG, Av. Antônio
Carlos, 6627, Campus Pampulha,
Belo Horizonte, Minas Gerais
E-mail:
ludmilakaren@gmail.
com
Dengue is a significant public health
threat, with 50 to 100 million cases
per year and approximately 3.5 billion
of people at infection risk. Over the
past 30 years infection rates have
dramatically increased, in part due to
urbanization. Dengue virus (DENV)
infections, transmitted by Aedes
mosquitoes, can be caused by any of
the four DENV serotypes (DENV1-4).
There is no vaccine or effective antiviral
treatment available for dengue virus,
and mosquitoes control measures have
Basic Virology: BV
61
largely failed to curb dengue incidence
in most parts of the world. Therefore,
novel approaches for protection are
required, and it is very important to
develop antiviral drugs against this
virus. The aim of this study was to
discover new antiviral compounds
from extracts of yeast and filamentous
endophytic fungi of different Brazilian
ecosystems. Citotoxicity and antiviral
activity assays were conducted in
LLCMK-2 cell lines using MTT assay.
The results were expressed by the
50% citotoxic concentration (CC50),
50% effective concentration (EC50)
and values of selective index (SI =
CC50/EC50). Altogether 128 extracts
were tested and 25 showed antiviral
activity against DENV-2. Six extracts
showed a SI less than 4, one extracts
showed a SI between 4 and 10, and one
extracts showed a SI superior to 10.
Concluding, about 6% of the extracts
tested showed an antiviral activity for
DENV-2, as showed in CC50 and CE50.
These results justify further studies
to clarify the mechanisms of action
of these extracts. Financial Support:
CNPq, CAPES, FAPEMIG
BV902 - PRODUCTION OF A RECOMBINANT PROTEIN FROM LONOMIA
OBLIQUA
CATERPILLAR
WITH
ANTIVIRAL ACTIVITY IN BACULOVIRUS/SF-9 CELLS SYSTEM
Mazzoni, M.K.F., Mazur, T.R., Carmo,
A.C.V., Giovanni, D.N.S., Araujo, R.L.,
Martins, L.M., Veiga, A.B.G., Moraes,
P.R.H., Mendonça, R.Z.
1. Laboratório de ParasitologiaInstituto Butantan, IB, Avenida
Vital Brasil,1500 - Butantã- São
Paulo -SP - CEP:05503-000
2. Universidade Federal de Ciências
62
Basic Virology: BV
da Saúde de Porto Alegre, UFRGS,
Porto Alegre/RS E-mail: mkfm@
email.it
The viral infection control is of great
interest in Public Health. Several
studies have shown the presence of
active compounds in the hemolymph
of arthropods, some of which are of
interest for the development of new
pharmacological drugs. Recently we
have demonstrated the existence
of a potent antiviral in hemolymph
of Lonomia obliqua caterpillar.
This purified protein reduced virus
production (TCID50 ml–1) more
than 157 fold (from 3.3±1.25x107
to 2.1±1.5x105) to measles virus,
61 fold to polio virus (2.8±1,08x109
to 4.58±1.42x107) and 64 fold to
H1N1 influenza virus. This study
aims to build recombinants bacmids
containing sequences encoding this
antiviral protein in baculovirus/SF-9
cell system and and test the antiviral
activity of recombinant protein. To
synthesize cDNA, RNA of L. obliqua
was extracted and used in polymerase
chain
reactions
using
reverse
transcriptase polymerase (RT-PCR)
with specific primers for the antiviral
protein, based on the sequence of the
cDNA libraries of L. obliqua tegument
and spicules. Restriction sites were
inserted in the cDNA for connection
to the donor plasmid pFastBac1TM
(Invitrogen).
The
recombinant
plasmid was selected in Escherichia
coli DH5α and subsequently used
in the transformation of DH10Bac
E. coli, to obtain the recombinant
bacmids. This bacmid, containing the
sequence of a protein with antiviral
activity was used for expression of
this protein in baculovirus/SF-9
cells system. In order to investigate
the antiviral effects on picornavirus
(EMC encephalomiocardite), whole
hemolymph and recombinant protein
(1% v/v) were added to the L929
cells cultivated on 96-well plates,
1 hour before infection with 100
TCID50 of virus. Samples of the cell
cultures were collected daily and
analyzed to determine the percentage
of cells with cytopathic effect (CPE).
The recombinant protein was able to
block the replication of 100 TCID50
of picornavirus (EMC), showing that
the recombinant antiviral protein
remains fully active. Financial support:
FAPESP (08/57263-5), CAPES, CNPq.
Imunobiológicos
BV903 - RECOMBINANT ANTIVIRAL
PROTEIN OF LONOMIA OBLIQUA
PRODUCED IN A BACTERIAL EXPRESSION SYSTEM
Giovanni, D.N.S., Carmo, A.C.V.,
Carvalho, N.D., Mazzoni, M.K.F., Mazur,
T.R., Cantinha, R.S., Martins, L.M.,
Barros-Battesti, D.M., Mendonça, R.Z.
1. Laboratório de Parasitologia Instituto Butantan , LP-IB, Av
Vital Brasil, 1500, Butantã - São
Paulo-SP 05503-000
2. Laboratório Especial de Coleções
Zoológicas, I. Butantan, LECZ-IB,
Av Vital Brasil, 1500, Butantã São Paulo-SP 05503-000 E-mail:
[email protected]
Infections viral are of great interest in
Public Health, mainly those caused by
influenza. Studies with hemolymph of
arthropods have shown the presence
of active principles of interest for the
development of new pharmacological
drugs as antiviral. In 2009 we have
demonstrated the existence of a potent
antiviral in hemolymph of L. obliqua.
This purified protein reduced virus
production (TCID50 ml–1) by more
than 157 fold (from 3.3±1.25x107
to 2.1±1.5x105) to measles virus,
61 fold to polio virus (2.8±1,08x109
to 4.58±1.42x107) and 64 fold to
H1N1 influenza virus. Recently we
constructed a recombinant bacmid for
expression of an antiviral protein in
baculovirus/insect cell system. This
expressed protein showed good activity
antiviral. The addition in infected
cultive reduced in herpes virus titers
106 in comparation to those in the
control culture. Like the experiments
carried out with the rubella and
EMC virus, the use also caused a
105 decrease both. However, this
expression system is very expensive
and laborious. Therefore we cloned and
expressed this protein with antiviral
activity in bacterial system. To antiviral
sequence was made a PCR with specific
primers for the antiviral protein, based
on the sequence previously cloned in
our laboratory. Restriction sites were
inserted in the primer for connection to
the plasmid pET28a. First reaction was
performed with the restriction enzyme
BamHI, followed by digestion with the
enzyme HindIII. The PCR product and
vector pET28a was digested with the
same enzymes. After binding insert
in the vector, the construction was be
selected in E. coli TOP10. The antiviral
protein was induced with 0.3mM IPTG
at 0.4 DO at 37 ° C for 4 hours. After
expression sonic bacteria were then the
supernatant used for tests of activity.
Protein was detected in western blot
using anti-His, and the tests with EMC
virus showed a protection of 27 times
compared to control. Financial support:
FAPESP, CAPES.
BV906- INHIBITION OF HEPATITIS C
VIRUS USING siRNAS TARGETED TO
Basic Virology: BV
63
THE VIRAL GENOME AND CELLULAR
PROTEINS HSPS
Braga, A.C.S., Carneiro, B.M., Batista,
M.N., Rahal, P.
UNESP São José do Rio Preto, IBILCEUNESP, Cristovão Colombo, 2265
Hepatitis C is a consequence of
infection by hepatitis C virus (HCV) and
it is estimated that approximately 170
million people are chronically infected
worldwide. Recent studies have been
demonstrated interactions between
viral and host proteins during the HCV
replication cycle and these interactions
might be used for development of new
therapies against hepatitis C. The
families of HSPs (heat shock proteins)
consist of cellular proteins that show
interaction with HCV proteins and
the inhibition of these proteins could
reduce viral replication. In this study
we inhibited, using siRNA, the cellular
proteins Hsp90 and Hsp27 alone or in
combination with the inhibition of viral
regions 5’UTR, NS3 and NS5A. We used
a stable Huh-7 cell culture expressing
subgenomic HCV replicon SGR-JFH1 to
inhibitions tests by siRNA molecules.
After 72 hours of siRNAs transfections,
cells were lysed and submitted to
protein expression and viral RNA
levels analysis. All siRNA molecules
directed to viral genome showed
efficient inhibition of viral replication
and the best response was obtained by
siRNA directed to 5’UTR region (94.2%
inhibition). For the inhibition of cellular
proteins the Hsp90 (4 logs reduction)
showed the greatest reduction of viral
replication (43.4% inhibition) and
using of this molecule together with the
siRNA molecule directed to the 5’UTR
showed an inhibition of 91% of viral
replication. Despite the inhibition of
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Basic Virology: BV
5’UTR region has been more effective
in reducing viral replication than the
inhibition of cellular protein Hsp90,
the joint inhibition achieved good rates
of viral inhibition and this approach
could become an important strategy
in the suppression of HCV as the use
of two molecules together can reduce
the viral escape. Financial support:
FAPESP, CAPES
BV909 - HYSTOPATHOLOGICAL AND
ULTRASTRUCTURAL ANALYSIS OF
CARDIAC TISSUE OF BALB/C MICE
INFECTED BY DENGUE VIRUSES
Rasinhas, A.C., da Silva, M.A.N., Jácome,
F.C., Carvalho, G.C., Barth, O.M., BarretoVieira, D.F.
Fundação do Instituto Oswaldo
Cruz, FIOCRUZ, Av. Brasil, 4365 Manguinhos, Rio de Janeiro E-mail:
[email protected]
The dengue virus (DENV) is an arbovirus
of the Flaviviridae family, being
responsible for the tropical disease
known as the Dengue Fever (DF).
While cardiac manifestations of the
disease are uncommon, they are often
associated with Dengue Hemorrhagic
Fever (DHF), a significantly more
severe form of the disease. Due to the
viral infection of cardiomyocytes and
endothelial cells, these manifestations
can lead to cases of atrioventricular
conduction disorder, supraventricular
arrhythmia
and
myocarditis.
Upon microscope observation, the
presence of lymphocytic infiltrate
and myocytolysis of cardiac cells is
considered a conclusive diagnosis
of myocarditis. Even so, cases of
myocarditis caused by DENV have
proven to be mostly benign throughout
the course of the disease, without long
term complications. Several studies
have already shown the susceptibility of
mice to DENV infections, showing that
the virus can be detected in the spleen,
lung, liver and brain. In this study,
cardiac tissue of adult BALB/c mice
infected and reinfected experimentally
with a non-neuroadapted DENV was
analyzed. For the primary infection
the animal was inoculated with
DENV-1 or -2 by intravenous route
and euthanized 72h post-infection
(p.i.). For analysis of the secondary
infection the mice were infected with
the DENV-2 followed by a reinfection
with DENV-1 and euthanized 72h p.i.
Tissue fragments were processed
following the standard techniques of
photonic and transmission electron
microscopy. Heart tissue samples have
shown cardiac cells with a slight loss
of cytoplasm, areas with inflammatory
infiltrate, disorganization of cardiac
fibers and inflammatory cells.
Alterations observed in present studies
were similar to the ones described
in DHF human cases of the disease,
which show mice represent indeed
an adequate model for the study of
heart infection by the DENV. Financial
support: IOC, Faperj, CNPq
BV910 - STUDY OF STRUCTURE AND
FUNCTION OF HUMAN RESPIRATORY SYNCYTIAL VIRUS SH PROTEIN
Araújo, G.C., Oliveira, R.J., Teixeira,
T.S.P., Gomes, D.E., Leite, V.B.P., Fossey,
M.A., Souza, F.P.
Estadual
Paulista,
UNESP,
R.
Nazareth - São José do Rio Preto
Human Respiratory Syncytial Virus
(hRSV) is leading cause of respiratory
infection in infants and children
worldwide. Its genome encodes 11
proteins including the surface proteins
F, G and SH. The function of protein SH is
not very well understood. Experiments
reported that expressed in bacteria
there is increased permeability of
compounds with low molecular
weight, suggesting the formations of
ion channels. Studies on SH regarding
structure and function are fundamental
for a better understanding of its
mechanism. The aim of this study is to
propose tertiary and quaternary threedimensional model of protein SH. The
model of SH protein was generated by
I-TASSER server and their functional
and structural features analyzed by
PredictProtein and PsiPred servers.
These models were analyzed by
Molecular
Dynamics
simulation
for refinement with analysis of the
hydrophobicity of the protein central
region, studies of behavior in lipid
membrane and possible formation of
oligomers. Prediction of SH protein
model resulted in a linear model with
one alpha-helix between amino acids
20-42. Analysis by PsiPred indicated
that this region is a transmembrane
region. Molecular dynamics simulation
showed that in solution, the protein
changes its linear conformation for a
globular conformation confirming the
hydrophobicity of the central domain.
Based on these analyzes it is expected
to propose the protein structure
and understand its function in viral
infectivity.
BV914 - DENGUE SURVEILLANCE: ASSESSMENT OF A SENTINEL SYSTEM
TO DETECT DENGUE SEROTYPES IN
MOSQUITOES
Fávaro, E.A., Parra, M.C.P., Ozanic, K.,
Dibo, M.R., Mondini, A., Colombo, T.E.,
Machado, D.C., Chiaravalloti, F.N., Eiras,
A.E., Nogueira, M.L.
Basic Virology: BV
65
1. Faculdade de Medicina de São
José do Rio Preto-SP, FAMERP, Av.
São Pedro CEP: 15090-000
2. Universidade de São Paulo, USP,
Av. Professor Almeida Prado,
1280 - Butantã São Paulo, CEP:
05508-900
Gerais, UFMG, Av. Antonio Carlos
- Pampulha- Belo Horizonte-MG CEP: 31270-901
4. Universidade Estadual Paulista,
UNESP, Rod. Araraquara-Jaú KM 1
Bairro Machados. CEP 15090-901
E-mail:
eliane_favaro@yahoo.
com.br
Introduction: Dengue is one of the most
important viral diseases worldwide. It
can be caused by four different serotypes
(DENV 1-4) that are transmitted by the
bite of Aedes mosquitoes. To decrease
dengue cases, health authorities
must follow adequate entomological
and epidemiological surveillance
methods and vector control activities
are usually targeted. The use of adult
traps has currently been applied
as an alternative form of vector
surveillance. Material and Methods:
We assessed the presence of dengue
infected mosquitoes captured by adult
traps, in association with dengue
human cases, as potential sentinel
events. MosquititoTM traps and BGSentinelTM traps were installed in
different houses of São José do Rio Preto,
a medium-sized city of São Paulo State,
Brazil. After mosquito identification,
they were pooled according to gender,
genus/species and site of collection for
posterior viral extraction and RT-PCR
assays for DENV detection. The data
were spatially analyzed using ArcGIS
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Basic Virology: BV
software. Results: One hundred thirtyfive mosquitoes were collected. Eightyfive specimens belonged to Aedes
genus and fifty to Culex genus. Fiftynine pools were analyzed and four
pools were positive for DENV-1, one
pool was positive for DENV-2 and two
pools were positive for DENV-4. This is
just the initial phase of our study and
these preliminary data reflects what
was found within human samples.
BV917 - INTERACTIONS BETWEEN
YELLOW FEVER VIRUS NS4B AND
HOST PROTEINS BY PROTEOMICS
ANALYSIS
Vidotto, A., Morais, A.T.S., Pacca, C.C.,
Mohana-Borges, M., Gil, L.H.V.G.,
Nogueira, M.L.
José do Rio Preto, FAMERP,
Laboratório de Pesquisa em
Virologia - Brigadeiro Faria Lima,
5416 - São Pedro
Janeiro, UFRJ,
3. Laboratório
de
Genômica
Estrutural, Instituto de Biofísica
Carlos Chagas Filho Centro de
Pesquisas Aggeu Magalhães,
CPqAM-FIOCRUZ, Departamento
de
Virologia
e
Terapia
Experimental - CPqAM-FIOCRUZ
E-mail:
alessandravidotto@
yahoo.com.br
Yellow fever is caused by Yellow Fever
Virus (YFV), the prototype of the
Flavivirus genus. The virus replication
is highly dependent on host factors.
YFV NS4b is a non-structural protein
that is reported to viral replication
and immune evasion, but its role and
cellular partners are not yet known.
Proteomics has been applied to study of
the interaction between virus and the
host cell proteins. We have expressed
GST fusion YFV NS4b protein in E. coli
and the synthesis of the protein was
confirmed by Western blot analysis
using specifics antibodies. NS4b-GST
protein was purified by Glutathione
SepharoseTM 4B affinity column. GST
Pull down studies were performed
using GST and GST-NS4b as a bait
and cellular extracts to search virushost protein interactions using onedimensional electrophoresis coupled
to mass spectrometry (MS). Through
this assay, various proteins that interact
with NS4b were identified. These
proteins were identified by MAS and
classified by their cellular roles, such
as: entry into host cell, initiation of viral
infection, regulation of viral genome
replication, viral transcription, cell-cell
signaling, interferon signaling pathway,
signal transduction, defense response,
platelet activation, RNA processing,
translation, protein maturation, postGolgi vesicle-mediated transport, cell
proliferation, cell cycle checkpoint, DNA
repair, actin cytoskeleton organization,
apoptosis, protein ubiquitination,
proteolysis, fatty acid transport and
glycolysis. We also evaluated the role of
these interactions in viral replication
by protein overexpression in the
BHK LucNeo Replicon YF17D cells.
In addition, we are developing coimmunoprecipitation and functional
studies, also testing the action of
a drug, in order to elucidate the
mechanisms of virus replication and
infection. The findings of this wok will
provide important information for
understanding Flavivirus infection and
generate potential targets for antiviral
drugs, improving the virus diseases.
BV919 - PRELIMINARY EVALUATION OF ANTIHERPES ACTIVITY OF
CAFFEIC ACID DERIVATIVES
Rigotto, C., Marostica, L.L., Martínez,
M.D., Reginatto, F.H., Duran, F., Simões,
C.M.O.
Universidade Federal de Santa
Catarina, UFSC, Campus Universitário
da Trindade, Florianópolis, SC,
Brasil. Caixa postal 476 E-mail:
[email protected]
Among the several biological responses
elicited by caffeic acid derivatives, the
inhibition of human immunodeficiency
virus type 1 (HIV-1) appeared recently
as the most promising pharmacological
activity. Herpes Simplex Virus (HSV)
are responsible for infections of oral,
ocular and genital regions, and efforts
have been made to find new drugs for its
treatment, mainly due to its resistance
to the most common available
treatment (e.g. acyclovir valacyclovir,
fancyclovir and pencyclovir). Synthetic
analogues derived from natural
products are an interesting source of
bioactive molecules with promising
pharmacological activities, including
antiviral activity, which justifies the
research in this area. In this study,
we evaluated the antiherpes activity
of twenty four synthetic caffeic
acid derivatives. Cytotoxicity was
evaluated on Vero cells by using
MTT assay, and antiviral activity was
tested against HSV-1 (KOS strain)
by viral plaque number reduction
assay. Results were expressed as
50% cytotoxic concentrations (CC50)
and 50% viral replication inhibitory
concentrations (IC50), respectively, in
order to calculate the selectivity index
(SI=CC50/IC50) of each tested sample.
Among the tested compounds, seven
inhibited HSV-1 replication showing SI
Basic Virology: BV
67
values between 2.1 and 12.76. Further
studies are required to elucidate the
mechanism of antiherpes action of
the most active derivatives as well as
to perform chemical modifications in
order to enhance their antiviral activity.
Financial Support: CAPES, CNPq.
BV929 - ANTIVIRAL ACTIVITY
OF EXTRACTS OF PLOCAMIUM
BRASILIENSE AND PLOCAMIUM
CARTILAGINEUM AT IN VITRO
REPLICATION OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 1
Barcelos, I.O., Fonseca, R.R., Meneses,
L.C., Pereira, P.S., Ribeiro, C.P., Giongo,
V., Teixeira, V.L., Castello-Branco, L.R.R.,
Cirne-Santos, C.C., Paixão, I.C.N.P.
1. Universidade
Federal
Fluminense, UFF-RJ, Rua Outeiro
de São João Baptista, S/Nº Niterói, Rio de Janeiro
2. INSTITUTO OSWALDO CRUZ, IOC,
AV. BRASIL - RIO DE JANEIRO/RJ
E-mail:
ingridbarcelos@gmail.
com
Human immunodeficiency virus type
1 (HIV-1) etiologic agent of Acquired
Immunodeficiency Syndrome (AIDS)
has been one of main targets from
different parts of the world studies
due to its emergence as an important
infectious agent. HIV has slow
progression in the organism and can
stay in latent state for years. System
immune cells are the target of the virus,
and turn human organism susceptible
to the acquisition of opportunistic
infections, leading to development of
AIDS. Emergence of new viral strains
resistant to antiretroviral therapy has
been one of main issues for maintaining
infected individuals survival. The
aim of this study is to search new
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Basic Virology: BV
compounds with anti-HIV-1 activity,
seeking treatment new strategies and
alternatives to combat AIDS. In this
study we used raw extracts of red
algae Plocamium cartilagineum and
Plocamium brasiliense, and MT-2 cells
were maintained in culture and used
for cytotoxicity and antiviral assays.
Viruses isolate HIV-1 IIIB were used
for antiviral assays. Results showed
that the extracts were considered
non-cytotoxic to cells. All extracts
inhibited the viral replication in a
dose-dependent manner, but extracts
EBPBHiD and EBPCHiD inhibited the
viral replication between 61 to 99%.
Extract EBPBHiD did not present
virucidal activity and extract EBPCHiD
was able to inactivate about 30% of
the viral particles at 37° C. A potent
inhibitory activity from EBPBHiD and
EBPCHiD on pretreated cells during 2
hours, 1 day and 5 days was observed.
The formation of syncytia was also
inhibited by extracts EBPBHiD and
EBPCHiD in a dose-dependent way with
inhibitory activity varying between 31
and 96%. Thus, we conclude that the
extracts EBPBHiD and EBPCHiD are
promising to continue studies in vitro
and in vivo of their mechanism of action
and future development of drugs with
antiviral action.
BV943 - PRELIMINARY EVALUATION
OF ANTIHERPES ACTIVITY OF A
BUTANOLIC FRACTION AND THREE
ISOLATED C-GLYCOSYLFLAVONOIDS
FROM WILBRANDIA EBRACTEATA
Quiroz, C.G., Rigotto, C., Gazola, A.C.,
Schenkel, E.P., Simões, C.M.O.
Universidade
Federal
de
Santa Catarina, UFSC, Campus
Trindade, Laboratório de Química
Farmacêutica Universidade Federal
de Santa Catarina, UFSC, Campus
Trindade, Laboratório de Virologia
Aplicada E-mail: carlosguillermo.
[email protected]
Natural products are an inexhaustible
source
of
bioactive
molecules
with
promising
pharmacological
activities, including antiviral activity,
which justifies researches in this
area. Wilbrandia ebracteata Cogn
(Cucurbitaceae), usually known as
‘‘taiuiá’’, is a medicinal plant used in
South American folk medicine against
skin affections, as purgative and emetic,
and to treat inflammatory conditions.
Herpes Simplex Viruses (HSV) are
responsible for many infections of
oral, ocular and genital regions, and
efforts have been made to find new
drugs for their treatment, mainly due
to their resistance to the most common
available treatment (e.g. acyclovir). The
aim of this study was to evaluate the antiHSV-1 activity (KOS and 29R strains)
of a n-butanolic fraction enriched in
C-glycosylflavonoids obtained from the
roots of “taiuiá”, and three compounds
isolated from this fraction: spinosin,
swertisin and isovitexin. Cytotoxicity
on Vero cells and anti-HSV-1 activity
were assessed by MTT and viral
plaque number reduction assays,
respectively. Results were expressed as
50% cytotoxic concentrations (CC50)
and 50% viral replication inhibitory
concentrations (IC50), respectively, in
order to calculate the selectivity index
(SI=CC50/IC50) of each tested sample.
According to the obtained results,
the n-butanolic fraction showed
antiherpes activity only against the
acyclovir resistant strain (29R) with
a SI of 4.48 (CC50=125.00 µg/mL
and IC50=27.89 µg/mL). The isolated
flavonoids
evaluated
separately
were not active against both strains.
Additional experiments are currently
under development to evaluate the
synergism among these flavonoids, as
well as to define the mode of action in
order to determine the step of the viral
multiplication cycle where impairment
could occur. Financial support: CAPES
and CNPq (PEC-PG fellowship of the
first author).
BV944 - EFFECTS OF THE CORTICOIDS DEXAMETHASONE AND
PREDNISOLONE ON INFLUENZA A IN
VITRO REPLICATION
Sacramento, C.Q., Nascimento, D.O.,
Martorelli, A., Fintelman-Rodrigues, N.,
Bozza, F.A., Souza, T.M.L.
Fundação
Oswaldo
Cruz,
FIOCRUZ,
Rua
Leopoldo
Bulhões,1480,Manguinhos. Pavilhão
HPP,sala B109b.Rio de Janeiro.
E-mail: carolina.sacramento@ioc.
com.br
Influenza is the main cause of acute
respiratory infection. Although it
is generally self-limited, influenza
infection may become more severe
in some populations, such as elder
patients, imunossupressed individuals,
pregnant women and children.
The infection caused by the 2009
pandemic influenza A/H1N1 virus
had a more aggressive course in these
populations, leading them to develop
pneumonia, acute lung injury/acute
respiratory distress syndrome (ALI/
ARDS), multiple organ failure and
exacerbation of underlying diseases.
Endogenous glucocorticoids play an
important anti-inflammatory role.
Systemic corticosteroid therapy has
been used as an adjuvant treatment of
pneumonia associated and ALI/ARDS
in hospitalized patients with severe
influenza infection. However, corticoids
Basic Virology: BV
69
effects in the outcome of these
patients, in viral load and shedding are
controversial. The aim of this study is
to analyze the effects of two corticoids
in clinical use, dexamethasone (dex)
and prednisolone (pred) on influenza
A in vitro replication. MDCKs were
infected with influenza A/H3N2/
England/72 (H3N2) at four different
multiplicity of infection (MOI; ranging
from 0,03 to 0,00003) and treated
with different concentrations of dex
or pred (50; 10; 5; 1; 0,5; 0,1; 0,05
ug/ml). Viral titers were measured
during 3 days by neuraminidase (NA)
activity, and cytopathic effect (CPE)
was observed for comparison. Both
corticoids tested were able to decrease
influenza A NA activity. In the first 24
hours post-infection (hpi), CPE was
observed in the highest MOI (0,03)
tested and NA activity was reduced.
After 48hpi, there was an inhibitory
effect in NA activity in the inferior
MOIs. NA was also reduced in the lower
MOI (0,00003) 72hpi and there was no
difference between the others MOIs
and the controls. Our data suggest that
dex and pred can inhibits influenza A
replication on a time-dependent and
MOI-independent manner. Further
experiments will be done to better
understand the mechanism of action of
the two corticoids.
BV949 - THE ANTI-POLIOVIRUS
ACTIVITY OF CAESALPINIA FERREA
SULFATED POLYSACCHARIDE AND
QUERCETIN
Lopes, N., Faccin-Galhardi, L.C., Espada,
S.F., Ricardo, N.M.P.S., Linhares, R.E.C.,
Nozawa, C.
1. Universidade
Estadual
de
Londrina, UEL, Rod. Celso Garcia
Cid, Pr 445 Km 380, Cx. Postal
70
Basic Virology: BV
6001, CEP 86051-980
2. Universidade Federal do Ceará,
UFC, Av. da Universidade, 2853,
Benfica, Fortaleza, CE, CEP 60020181 E-mail: nay_lopes@hotmail.
com
Substances of natural origin have
aroused great interest, being exploited
in the search for new drugs capable of
controlling viral infections. Caesalpinia
ferrea is a plant found throughout the
tropical region of Brazil, popularly
known as pau-ferro or jucá. Quercetin
is the main representative of the
flavonoids of the flavonols subclass and
several medicinal properties, including
antiviral, are related. Poliovirus (PV)
is an enterovirus, member of the
Picornaviridae family, the causal agent
of paralytic poliomyelitis due to the
invasion of the central nervous system.
Despite extensive efforts to eradicate
the wild virus, the disease remains
endemic in some African and Asian
countries. The aim of this study is to
investigate the antiviral activity of a
sulfated polysaccharide of C. ferrea
(SPLCf) and quercetin to PV in HEp2 cells culture. The 50% cytotoxic
concentration (CC50) of SPLCf and
quercetin were >3000 µg/ml and
>1000 µg/ml, respectively, determined
by MTT assay. The antiviral action was
assayed by plaque reduction assay, by
the use of the following protocols, the
time-of-addition assay (-2h, -1h, 0, +1h,
+2h), the inhibition of virus adsorption
and the virucidal effect. For the most
significant results, we performed the
immunofluorescence assay (IFA). We
also performed RT-PCR for monitoring
the activity of the compounds in
the synthesis viral nucleic acid. The
compounds showed a great activity
at time 0h with IC50/selective index
of 1.73 µg/ml/>1734 for SPLCf, and
8.37 µg/ml/>119.5 for quercetin. IFA
demonstrated 100% and almost 90%
of viral inhibition at the concentration
of 10 µg/ml for SPLCf and 12.5 µg/ml
for quercetin, respectively. The RT-PCR
showed a significant inhibition of RNA
synthesis at the same concentrations
of the compounds. The results suggest
the interference of the SPLCf and
quercetin in several stages of the viral
replication, therefore, representing
potential inhibitors of PV. Financial
support:
CAPES/CNPq/Fundação
Araucária/UEL
BV958 - MORPHOLOGICAL STUDIES
OF MICE LUNG TISSUE INFECTED
WITH DENGUE VIRUS SEROTYPES 1,
2 AND 3
Jácome, F.C., Rasinhas, A.C., da Silva,
M.A.N., Carvalho, G.C., Barth, O.M.,
Barreto-Vieira, D.F.
Fundação Oswaldo Cruz, Fiocruz,
Av. Brasil 4365, Manguinhos E-mail:
[email protected]
Dengue virus (DENV), an arbovirus
belonging to the Flaviviridae family,
Flavivirus genus, has 4 serotypes:
1(DENV-1), -2, -3 and -4. One challenge
for the studies of dengue pathogenesis
is the lack of an animal model that is
not immunossupressed nor inoculated
intracranially. This work shows
alterations in lung of immunocompetent
mice inoculated intravenously with
non-neuroadapted
virus.
Adult
BALB/c mice were inoculated with
DENV-1, -2 and -3 with doses of
10000TCID50/0,1mL. The virus titers
(DENV-1: 107.5TCID50/0.1mL, DENV2: 106.66TCID50/0.1mL, DENV-3:
107.23TCID/0.1mL) were calculated
by the Reed & Muench method. For
primary infection studies (DENV-1
or -3), the mice were euthanized 72h
after infection. For reinfection studies
they were inoculated with DENV-2
(at 2 months of age), 2 or 4 months
later reinoculated with DENV-1 and
euthanized 72h after reinfection. For
histological studies, the tissue was
fixated in millonig, embedded in parafin
and stained with hematoxylin and
eosin. For ultrastructural studies, the
mice were perfused and lung tissue was
processed with standard techniques
for observation in transmission
electron microscope (TEM). For virus
isolation, supernatant of macerated
lung and sera samples of infected mice
were inoculated in C6/36 cell line. The
cells were processed for observation in
TEM as mentioned above. Interalveolar
septa swelling, presence of erythrocytes
inside alveolar spaces, inflammatory
infiltrate into the peribronchiolar space,
mononuclear and polymorphonuclear
cells and platelets inside capillaries,
vascular congestion, hemorrhagic foci
and cellular debris presence in the
bronchiolar lumen were observed in
the lung tissue. DENV particles were
observed in C6/36 cells inoculated
with the supernatant of lung macerates
and with the animal sera. These studies
show that BALB/c mice are permissive
for DENV and that morphological
alterations observed in lung tissue are
similar to those seen in human cases.
Financial Support: Faperj, IOC, CNPq
BV960 - EXPERIMENTAL MURINE
MODEL FOR THE PATHOGENESIS
STUDY OF DENGUE VIRUSES
Barreto-Vieira, D.F., Jácome, F.C.,
Rasinhas, A.C., da Silva, M.A.N., Barth,
O.M.
Av. Brasil 4365, Manguinhos E-mail:
Basic Virology: BV
71
[email protected]
A great difficulty to study dengue
virus (DENV) infection in humans
and for a virus vaccine developing
is the absence of a suitable animal
model which presents a disease
with similar aspects of the Dengue
haemorrhagic fever and Dengue shock
syndrome. In the majority of models
the animals are immunocompromised
and/or inoculated by routes like the
intracerebral, with neuroadapted
DENV. Tissues of adult BALB/c mice
infected
with
non-neuroadapted
DENV-1 and DENV-2 serotypes from
patient sera were analyzed. The tissue
fragments were processed following
the standard techniques of fotonic
and transmission electron microscopy.
In primary infection with DENV-1
and DENV-2 morphogical alterations
were observed inside hepatic, lung,
kidney and cerebellum tissues. DENV1 particles and specific DENV antigen
was observed in C6/36 cells inoculated
with the supernatant of spleen and
lung macerates and with the animal
sera. Ultrastructural studies of alveolar
macrophages of animals infected with
DENV-2 showed DENV-like particles
inside the rough endoplasmic reticulum
and Golgi complex, suggesting viral
replication. DENV particles were
ultrastructurally
identified,
and
immunolocalized inside C6/36 cells,
inoculated with the supernatant (liver,
lung kidney and cerebellum) of tissue
macerates. The corporal temperature
in the majority of mice increased
after the second day post-infection.
Elevated enzyme levels of alanine
aminotransferase
and
aspartate
aminotrasferase were observed. In
secondary infections morphological
alterations were observed in liver,
lung and heart. The tissue injuries
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Basic Virology: BV
were more severe than those seen
in animals with signs of primary
infection. DENV-1 particles, specific
DENV-1 antigen and DENV-1 RNA were
present in C6/36 cells inoculated with
the animal sera. These studies confirm
the susceptibility of BALB/c mice to
infection and reinfection by DENV1 and DENV-2 and those they can be
used as a model for testing of drugs
and vaccine candidates against DENV.
Financial Support: Faperj, IOC, CNPq
BV963 - BIOLOGICAL AND IMMUNOLOGICAL CHARACTERIZATION OF
THE BRAZILIAN SMALLPOX VACCINE
Medaglia, M.L.G., Lucas, C.O., Arruda,
L.B., Damaso, C.
1. Instituto de Biofísica Carlos
Chagas Filho, IBCCF, Rua Carlos
Chagas s/n Ilha do Fundão - Rio
de Janeiro
2. Instituto de Microbiologia Paulo
de Góes, IMPPG, Rua Carlos
Chagas s/n Ilha do Fundão - Rio
de Janeiro E-mail: mlmedaglia@
gmail.com
Smallpox is caused by variola virus
(Orthopoxvirus, Poxviridae), and was
eradicated in the late 1970s due to a
worldwide immunization campaign
using live vaccinia virus (VACV). Several
countries maintain stocks of smallpox
vaccine in view of the general concern
of inadvertent use of variola virus as
a biological weapon. However, given
the adverse events observed following
vaccination, new generation vaccines
have been designed and attempts have
been made to isolate attenuated clones
from effective vaccine strains used in
the past. In Brazil, IOC was the VACV
strain used by Instituto Oswaldo Cruz
to manufacture the smallpox vaccine.
Herein we plaque purified two clones
of VACV-IOC, named B141 and B388
and proceeded with their biological
and immunological characterization.
Both clones generated similar viral
yields and protein profile in BSC40 cells. In human Hep2 cells, B388
had a 6-fold lower virus production.
Inoculation of Balb/c mice with 10^6
PFU of both clones via tail scarification
produced milder lesions compared to
the virulent strain WR. Mice inoculated
intranasally with 10^5 PFU of B388
and doses as high as 10^7 PFU of B141
presented 100% survival rates. Virus
titration of spleen and liver of those
mice revealed no viral spread up to 7
days post infection (p.i.). In contrast,
all mice died when inoculated with
10^5 PFU of VACV-WR and the virus
was already detected in spleen and
liver after 3 days p.i. Immunization of
mice with both B388 and B141 clones
induced the activation of specific T
and B cells, detected by ex vivo IFN-g
production and secretion of neutralizing
antibodies in the sera, in which the
clone B388 promoted a more potent
response. Lethal challenge 4 weeks
post-immunization showed 100%
and 50% survival rates for B141 and
B388 immunized mice, respectively.
The genome sequences of both clones
are currently being determined. These
data will contribute to further research
on safer vaccines development using
the brazilian smallpox vaccine.
BV965 - NITRIC OXIDE PRODUCTION AND APOPTOSIS IN HUMAN
HEPATIC CELLS INFECTED WITH
DENGUE VIRUS
Costa, L.S., El-Bacha, T., Campos, S.H.,
Conceição, T.M., Da Poain, A.T.
Janeiro, UFRJ, Av. Carlos Chagas
Filho S/N CCS Bloco E/18 E-mail:
[email protected]
Dengue is an infectious disease that
affects millions of people over the world.
It is caused by dengue virus (DENV)
and transmitted by Aedes aegypti
mosquitoes. DENV can produce a
subclinical infection, a mild self-limiting
disease, dengue fever (DF) or the lifethreatening dengue hemorrhagic fever
(DHF) that can lead to shock and death.
Experimental evidences suggest that
the liver is an important site of virus
replication and serious damage of
this organ has been found in severe
cases of DENV infection. It has been
shown that the human hepatocyte
cell line, HepG2 produces cytokines,
lipid mediators, and nitric oxide (NO)
upon infection, which may be involved
in disease manifestations. The aim of
the present work was to characterize
NO production in HepG2 cells during
the course of DENV infection. Cells
were grown in appropriated medium
and infected with DENV, M.O.I 1.
DENV replication in HepG2 cells
was evaluated by plaque assay and
flow cytometry. NO production was
investigated by intracellular NO
quantification and inducible Nitric
Oxide Synthase (iNOS) expression
and apoptosis was measured using
a double staining method with The
Vybrant Apoptosis Assay Kit#2. Our
results showed that infected cells
present an increase in NO production.
Additionally, we demonstrated for
the first time that DENV promoted an
increase in iNOS mRNA expression
24 hours post-infection, followed by
increase in the synthesis of this enzyme
30 hours post infection. This increase
in iNOS accounted for a significant 35%
increase in NO production 48 hours
Basic Virology: BV
73
post-infection. Apoptosis was detected
after 48h post infection, suggesting NO
involvement in cell death. Given the
fact that NO modulates mitochondria
bioenergetics,
mitochondrial
dysfunction might be involved in cell
death observed at this time point of
infection. The molecular mechanisms
underlying these events are unknown
and are under investigation. Financial
Support: CNPq, FAPERJ
BV973
ULTRASTRUCTURAL
STUDIES OF C6/36 CELLS INFECTED
WITH DENGUE VIRUSES
da Silva, M.A.N., Gomes, G.M., Jácome,
F.C., Barth, O.M., Vieira, D.F.B.
Fundação Oswaldo Cruz, Fiocruz, Av
Brasil, Manguinhos, Rio de Janeiro,
Brasil E-mail: marquinhosans@
hotmail.com
This study purpose to analyze
ultrastructural aspects of dengue virus
(DENV) serotypes 1 (DENV-1), -2 and
-3 that circulated in state of Rio de
Janeiro between years of 2000 and
2011. The DENV strains were isolated
from patient sera and propagated in
Aedes albopictus mosquito cell line
(C6/36 cells). The titers of the viruses
(DENV-1: 107.5 TCID 50/0.1mL, DENV2: 106.66 TCID 50/0.1mL and DENV3: 107.23/0.1mL) were calculated by
the method of Reed & Muench (1938).
100L of strain of the viruses were
inoculated in C6/36 cells that were
grown in L-15 medium supplemented
with 1% non-essencial aminoacids,
10% tryptose phosphate broth, and
10% fetal bovine serum. The tubes were
kept at 28ºC and observed daily for viral
cytopathic effects for six days. C6/36
normal cell monolayers were used as
negative control. After observation
period the monolayers were fixed
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Basic Virology: BV
in 1% glutaraldehyde in cacodylate
buffer (0.2 M, pH 7.2), post-fixed
with 1% buffered osmium tetroxide,
dehydrated in acetone, embedded in
epoxy resin and polymerized at 60ºC
during three days. The blocks were
cut to ultra-thin sections of 50-70
nm thickness using a diamond knife
adapted to an ultramicrotome. The
sections were picked up onto copper
grids and stained with uranyl acetate
and lead citrate and observed in a
transmission electron microscopy
(TEM). C6/36 cells were infected with
all three serotypes. Cytopathic effects
were observed between fourth and
fifth days in all monolayer. 25%, 50%
and 30% of cytopathic effect was
observed in five days in monolayer
inoculated with DENV-1, DENV-2 and
DENV-3, respectively. Ultrastructural
studies showed typical DENV particles
and nucleocapsids that occur in
great number in the endoplasmatic
reticulum; virus particles inside
cytoplasmatic vesicles and syntitium
were observed too. Cell monolayers of
negative control were always free from
DENV-like particles. Morphological
differences between DENV-1, -2 and -3
particles not were observed.
BV980 - ANALISYS OF VIRULENCE
FACTORS INVOLVED IN SEVERAL
ASPECTS OF VIRUS-CELL INTERACTION DURING POXVIRUS INFECTION
Schnellrath, L.C., Attias, M., Damaso, C.
Instituto de Biofísica Carlos Chagas
Filho, IBCCF, Av. Carlos Chagas Filho,
373, Bloco G - Cidade Universitária RJ E-mail: [email protected]
Poxviruses encode a wide variety
of virulence factors, many of which
are linked to the antiviral pathway
triggered by interferons (IFNs). The
signaling pathway triggered by IFN
leads to an increased expression of
the double stranded RNA-dependent
protein kinase (PKR), inhibiting
protein synthesis in infected cells.
Correlation between induction of
autophagy and activation of PKR has
been reported. The absence of genes
that encode inhibitors of IFN-related
pathways causes host restriction and
loss of pathogenicity. Therefore, our
goal is to study the involvement of
these proteins in several aspects of
virus-cell interaction during infection.
Our results show that when the action
of IFN-related pathways was not
counteracted by poxviruses, infection
led to phosphorylation of both PKR and
eukaryotic initiation factor 2 (at alpha
subunit), as measured by Western
Blot, subsequently inhibiting protein
synthesis as evaluated by metabolic
labeling with radioactive methionine.
Consequently, we observed a nonproductive infection in cell culture
with near 3-log inhibiton of virus yield.
It was also possible to detect evident
autophagy, since a punctate pattern
of LC3 after 8 hours post-infection,
as well as co-localization of LC3 and
LAMP and autophagosome formation
in the cytoplasm were observed. By
immunofluorescence assays, we were
able to confirm the existence of this
process during infection using the
inhibitor of autophagy 3MA, which
blocked the number of cells with
punctate pattern of LC3 in nearly 95%.
However, inhibition of autophagy by
3MA was not able to recover the virus
replication. Thus autophagy was not
responsible for the non-productive
infection. Nevertheless, the activation
of PKR is not sufficient or not involved
in the induction of autophagy during
infection in some cell lines. We are
currently investigating the intricate
relationships of several signaling
pathways that could lead to the
induction of autophagy during poxvirus
infection.
BV981 - MOLECULAR MODEL OF
INTERACTION BETWEEN HRSV G
PROTEIN AND FLAVONOIDS
Araújo, G.C., Teixeira, T.S.P., Gomes,
D.E., Fossey, M.A., Souza, F.P.
Estadual
Paulista,
UNESP,
R.
(hRSV) is one of the main agents of
acute respiratory infections (ARI).
One of the key targets of adaptive host
immune response is the hRSV G protein,
which is responsible for attachment
to host cell. There are evidences that
compounds such as flavonoids can
decrease viral infection in vitro. The
present study aimed to check, through
computational tools, the possible sites
of interaction between G protein and
flavonoids. Three-dimensional model
of G protein and the flavonoids were
determined by Rosetta and FROG2,
respectively. Q-SiteFinder program was
used for of interaction sites prediction.
Docking studies were made by GLIDE
program. The model obtained for
G protein is mostly composed by
random coils and alpha helixes, typical
of a transmembrane protein. The
flavonoids docked in a polar region
of the protein and the main forces of
these interactions are hydrophobic,
electrostatic and hydrogen interaction,
being hydrophobic and hydrogen
interaction the main forces of
non glycosilated and glycosilated
flavonoids,
rescpectively.
The
Basic Virology: BV
75
interaction energy ranged from -9,74
to -4.71 kcal/mol being the flavonoid
Kaempferol-3-O-α-L-Arabinopiranosil(2�1)-α-L-Apiofuranoside-7-O-α-LRhamnopyranoside with the lowest
interaction energy. Knowledge of G
protein structure is of great importance
to elucidate the mechanism of viral
infectivity, and the results obtained in
this work allow us, in a later step, to
direct further experimental studies
to discover effective inhibitors for
attachment protein.
BV988 - HERPES SIMPLEX TYPE 1
ACTIVATES GLYCOLYSIS THROUGH
ENGAGEMENT OF THE ENZYME
6 - P H O S P H O F RU C TO - 1 - K I NAS E
(PFK-1)
Abrantes, J.L., Alves, C.M., Costa, J.,
Almeida, F., Sola-Penna, M., Fontes,
C.F.L., Souza, T.M.L.
1. Programa de Pós-Graduação em
Química Biológica, Instituto d,
UFRJ, AV. CESAR PERNETTA, S/N,
CCS, BLOCO H, SALA 26
2. Laboratório
de
Vírus
Respiratórios e do Sarampo, ,
IOC, AV. BRASIL, 4365, HPP, SALA
B109B
Viruses such as HIV, HCV, Mayaro
and HCMV affect cellular metabolic
pathways,
including
glycolysis.
Although some studies have suggested
that the inhibition of glycolysis affects
HSV-1 replication and that HSV-1infected eyes have increased lactate
production, the mechanisms by which
HSV-1 induces glycolysis have never
been investigated in detail. In this
study, we observed an increase in
glucose uptake, lactate efflux and ATP
content in HSV-1-infected cells. HSV-1
triggered a MOI-dependent increase in
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Basic Virology: BV
the activity of phosphofructokinase-1
(PFK-1), a key rate-limiting enzyme
of the glycolytic pathway. After HSV-1
infection, we observed increased PFK1 expression, which increased PFK-1
total activity, and the phosphorylation
of this enzyme at serine residues. HSV1-induced glycolysis was associated
with increased ATP content, and
these events were critical for viral
replication. In summary, our results
suggest that HSV-1 triggers glycolysis
through a different mechanism than
other herpesviruses, such as HCMV.
Thus, this study contributes to a better
understanding of HSV-1 pathogenesis
and provides insights into novel targets
for antiviral therapy.
BV990 - CAFFEINE INHIBTS HCV
SUBGENOMIC REPLICON REPLICATION IN VITRO
Batista, M.N., Carneiro, B.M., Braga,
A.C.S., Silva, R.C.M.A., Nogueira, M.L.,
Rahal, P.
1. UNESP - São José do Rio Preto
, IBILCE/UNESP, Rua Cristóvão
Colombo, 2265
2. FAMERP - Faculdade de Medicina
de S.J. Rio Preto, FAMERP , Av.
Brigadeiro Faria Lima, 5416
E-mail:
batista_m.n@hotmail.
com
Hepatitis C is a liver infection caused by
hepatitis C virus (HCV), which infects
hepatocytes. Usually the infection
does not generate an adequate host
immune response causing, in most
cases, a chronic condition. Hepatitis
C has been considered the major
worldwide cause of cirrhosis and
hepatocellular carcinoma. PEG-INF in
association with ribavirin is the best
current treatment although it shows
a low sustained virological response
for some genotypes along with severe
side-effects and high cost, therefore
new treatments are being sought. This
study aimed to identify the effects of
pure caffeine on the HCV replication in
vitro. Hepatocellular carcinoma, Huh7 cells were cultured and transfected
with subgenomic replicon of HCV
genotype 2a (pSGR-JFH-1). Initially the
cells expressing the SGR-JFH1 replicon
were transferred to 96 wells plates
and after 24hours different caffeine
concentrations were added: 0,0001mM;
0,001mM; 0,1mM; 1mM and 10mM.
Cells were incubated for 24h, 48h and
72h followed by MTT cytotoxicity assay.
The same procedure was adopted using
PEG-IFN-α at concentrations: 0,1ng/
mL; 1ng/mL; 10ng/mL; 100ng/mL;
1000ng/mL. Viral RNA expression was
evaluated by qPCR with Taqman probe
to 5’UTR region of HCV. We observed in
samples treated with caffeine a dosedependent effect at viral replication,
inhibiting the viral replication around
80% on its major viable concentration.
Furthermore the combination of
caffeine and IFN showed an increase
in the inhibitory effect when compared
to IFN treatment alone. These data
was confirmed by protein expression
analysis using NS3 primary antibody.
This work demonstrated the capacity of
pure caffeine to inhibit HCV replication
in vitro and its potential as a new
antiviral therapy against HCV alone or
in association with conventional drug
treatment. Financial Support: FAPESP
BV991 - EFFECTS OF HIV-1 IN
INFLUENZA PANDEMIC INFECTION
Mesquita, M.M.A., Siqueira, M.M., BouHabib, D.C., Souza, T.M.L.
Fiocruz, IOC-Fiocruz, Rua Leopoldo
Bulhões 1480, Manguinhos E-mail:
[email protected]
HIV-1 persistently replicates in
lymphoid tissues, causing reduction
of CD4+ cells. This leads to profound
immunosuppression and increased
susceptibility
to
opportunistic
infections, such as influenza A virus
(FLU A), among others. FLU A is a singlestranded, negative-sense, enveloped
RNA virus, of the orthomyxoviridae
family, possessing eight gene segments.
This genomic feature facilitates viral
shift that enable new variants to emerge,
like pandemic H1N1 (H1N1pdm09), in
2009. Pandemic H1N1 virus caused 10
times more deaths than seasonal FLU
A (H3N2) virus, especially in specific
groups like children, elderly, pregnant
women and immunocompromised
individuals. Contradictory to this,
clinical outcomes of HIV-1-infected
individuals with H1N1pdm09 were
not different from those observed for
immunocompetent individuals. Since
HIV-1 imposes a novel homeostatic
equilibrium to its host, the life cycle
of influenza within this environment
may be highly affected. Considering
that over 30 million people in the
world are living with HIV-1 and that
their exposure to FLU A, which is a
seasonal pathogen, is quite common
– we investigated the in vitro biology
of these viruses. In this study, we
used
human
monocyte-derived
macrophages (MDM) from healthy
donors, MDCK lineage and parental
HeLa cell lineages. Although influenza A
does not establish productive infection
in MDM (with the exception of H5N1
virus), the infectivity of H1N1pdm09
inoculum in MDM was constant for
more than 5 days. However, when we
co-infected this MDM with HIV-1 (10
ng/mL of p24 antigen - R5-isolate
Basic Virology: BV
77
BaL), we observed a 1-log decrease
in H1N1pdm09 infectivity (n=8;
p<0.05). The reduction in H1N1pdm09
infectivity was neither due to massive
entry nor due to the effects of reactive
nitrogen oxygen species from HIV-1infected MDMs. We next observed that
interferon-inducible transmembrane
(IFITM) proteins, recently presented as
influenza and HIV-1 restriction factors,
are up-regulated more than 30% by
HIV-1 and gp120, even in epithelial
cells that would be susceptible to
H1N1pdm09 infection. Our results
demonstrate that studies such as this
may not only increase knowledge on the
physiopathology of HIV-1/influenza
co-infection, but may also contribute to
the identification/validation of novel
antiviral targets. Financial support:
Faperj, CNPq e POM-IOC
BV993 - ENHANCEMENT OF HEPATIC
AND SERUM LIPID ACCUMULATION
DURING DENGUE VIRUS INFECTION
IN A MOUSE MODEL OF THE SEVERE
DISEASE
Siqueira, L.O.F., Dias, M.S., AssunçãoMiranda, I., Da Poian, A.T.
Janeiro, UFRJ, Cidade Universitária,
Rio de Janeiro - RJ E-mail: lorena_
[email protected]
Dengue is one of the most widespread
arboviroses, whose etiologic agent
is a flavivirus, the dengue virus
(DENV). There are evidences that liver
damage is characteristic of severe
manifestation of the disease associated
with abnormal lipid metabolism of
infected patients. The aim of this study
was to investigate changes in lipid
metabolism of serum and liver tissue
in vivo in an animal model of severe
dengue. We quantified both hepatic
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Basic Virology: BV
and serum lipids and lipoproteins as
well as the serum level of the hepatic
transaminases through colorimetric
assays and evaluated by real-time PCR
the gene expression associated with
(a) lipid biosynthesis, the enzyme fatty
acid synthase (FAS); and (b) transport
of lipids, the MTTP protein. Mice were
infected intraperitoneally with 100
PFU of a mouse-adapted strain of
DENV (n=5) or apyrogen PBS (n=5).
The animals were sacrificed by cervical
displacement 6 days after infection.
Blood samples were collected through
the ocular plexus for determination of
viral titer, platelet count, hematocrit
and serum analyses. Liver samples
were collected for assessment of
viral replication by quantification
of infectious particles, analysis by
qPCR and lipid quantification. The
infected animals showed severe
thrombocytopenia and several signs
of liver damage, such as hepatomegaly
with the livers showing curved edges
and whitish steatotic appearance and
an increase of serum levels of the
transaminases. The viremia was 104
PFU/mL. Viral replication in the liver
reached 105 PFU/mL. We observed a
significant increase in both serum and
hepatic cholesterol and triglyceride
in infected group, indicating an
accumulation of lipids. FAS expression
increased ~ 2.5 fold and the expression
of MTTP was reduced, suggesting an
increase of lipid production in liver. The
results showed strong changes in lipid
metabolism linked with viral infection
and liver damage, and the study of
these changes will be important for the
understanding the pathology and viral
behavior.
BV996 - EVALUATION OF ANTIVIRAL
ACTIVITY FROM MARINE SPONGES
AGAINST HERPES SIMPLEX VIRUS
TYPE 1
Bianchi, B.R., Kohn, L.K., Santos,
M.M.A.B., Passarini, Michel, R.Z.,
Bonugli-Santos, R.C., Sette, L.D.,
Berlinck, R.G.S., Porto, P.S., Caserta,
L.C., Arns, C.W.
1. Universidade
Estadual
de
Campinas/FCM, FCM/UNICAMP,
Universidade
Estadual
de
Campinas/IB,
IB/UNICAMP,
Cidade Universitária Zeferino Vaz Rua Monteiro Lobato, 255 bl
E
IB/UNESP,
Universidade
de
São Paulo, USP/São Carlos,
Universidade
Estadual
de
Campinas/CPQBA,
CPQBA/
UNICAMP,
E-mail:
bianca.
[email protected]
Herpes simplex virus type 1 (HSV-1)
belongs to the Herpesviridae family
and is the etiologic agent of herpes
labial in humans. It is easily transmitted
and cause latent infections. Marine
organisms represent a barely tapped
source of bioactive natural products,
but only few of them display antiviral
activity. The objective of this study was
to evaluate antiviral activity of active
extracts from marine Sponge against
Herpes simplex virus type 1 (HSV1). Antiviral activity was studied to
determine the stage of the replicative
cycle of the virus is acting: (1) Cells were
first treated with the sample for 1 hour
and after that the virus was inoculated
(phase adsorption or penetration); (2)
the viruses were inoculated into the
cells for 1 hour and after that sample
was added the (replication phase) and
(3) finally the virus was incubated with
the sample and then this was added
on the cells (virus inactivation). The
determined MNTC for compound was
50 µg/mL and have potential antiviral
activity against HSV-1, with 90% of
inhibition in the phase of adsorption
viral for Monanchora arbuscula,
99% in the replication phase for
Didemnun ligulum and 90% in the
virus inactivation for Hemimycale
sp. and 83% for Gennaria disticha.
Further studies are underway in order
to establish the exact antiviral modeof-action and in order to identify the
compound responsible for this activity.
BV998 - INDUCTION OF THE IFN
ALFA, INF BETA, INF LAMBDA1 AND
INF LAMBDA2 GENE TRANSCRIPTION UPON INFECTION BY TAHYNA
VIRUS IN A549 CELLS
Apolinário, T.M., Almeida, G.M.F.,
Oliveira, D.B., Bonjardim, C.A., Kroon,
E.G., Ferreira, P.C.P.
Gerais, UFMG, Av. Antônio Carlos
6627 Pampulha, CEP 31270-901,
Belo Horizonte, Minas Gerais E-mail:
[email protected]
Tahyna vírus (TAHV) is a member
of the Bunyaviridae family, genus
Orthobunyavirus, belonging to the
California Encephalitis subgroup. It is
and arbovirus that infects humans and
is endemic from Europe, Asia and Africa.
TAHV has an enzootic life cycle with
numerous different mosquito vectors
and small mammals as amplifying
hosts. The symptoms during human
infections include fever, headaches,
conjunctivitis, jaundice, sore throat
and, in rare cases, encephalitis. The
virus genome is composed of three
negative single stranded RNAs named
after their relative sizes: S (small), M
(medium) and L (large). Interferons
Basic Virology: BV
79
(IFN) are important cytokines
usually secreted by infected cells and
that generate an antiviral state in
surrounding cells. This work aims to
evaluate the transcription of IFNalfa,
INFbeta, INFlambda1 and INFlambda2
genes in cells infected by TAHV. To do
that, A549 cells were grown in 6 well
plates and infected with either TAHV
or the Vesicular Stomatitis virus (VSV).
Cells were harvested and the total RNA
was extracted and used as template
in RT-PCR. The generated cDNAs
were then used in real-time PCRs in
order to quantify the transcripts using
specific primers. We observed that
the expression of the IFNalfa, INFbeta,
INFlambda1 and INFlambda2 cell
genes were very low when compared
to expression levels seen in cells
infected with VSV as a positive control,
as it is known that the late is a potent
inducer of IFN expression in infected
cells. The expression of IFN genes in
cells infected with TAHV was similar to
that seen in uninfected cells. Financial
support: CNPq, CAPES and FAPEMIG
BV1005 - MORPHOLOGICAL AND
ANTIGENIC
CHARACTERIZATION
OF BEAN781455 VIRUS: A POSSIBLE
NEW ARBOVIRUS ISOLATED FROM
BIRD
(RAMPHOCELUS
CARBO)
CAPTURED IN PORTO ACRE, ACRE,
BRAZIL
Queiroz, A.L.N., Medeiros. D.A.B.A.,
Diniz, J.A.P., Vasconcelos, P.F.C.
Instituto Evandro Chagas, IEC,
Rodovia BR-316 km 7 s/n - Levilândia
- 67030-000 - Ananindeua / Pará /
Brasil E-mail: alicezootec@yahoo.
com.br
The Brazilian Amazon has natural
ecosystems with ideal conditions
for the occurrence and maintenance
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Basic Virology: BV
of Arboviruses. About 200 different
arboviruses have been isolated in
this region, 100 new to science and
34 associated with human infection,
which becomes relevant the taxonomic
characterization of these viruses.
The study aimed to analyze the
viral isolate BEAN781455 obtained
from the bird Ramphocelus carbo
captured in Porto Acre / AC, in order
to describe the morphological and
antigenic properties of it. The viral
isolate BEAN781455 killed suckling
mice, and caused cytopathic effect
(CPE) in cultures of VERO cells. Viral
titration was performed by counting
the number of lysis plaques produced
on to monolayers of VERO cells;
viral replication was confirmed by
indirect immunofluorescence assay
(IFA). Antigenic characterization was
based on a complement fixation test
(CF) performed against antigens /
serum of arboviruses isolated in the
Brazilian Amazon. In order to verify the
presence of hemagglutinin, sucroseacetone antigens were prepared and
hemagglutination
inhibition
test
perfomed. The particle morphology
was analyzed by transmission electron
microscopy (TEM), analyzing of
infected VERO cells. The viral strain
BEAN781455 was isolated in newborn
mice which died on 4th day postinfection (pi). In VERO cells, CPE was
observed three days pi. The title of the
viral stock was 3.7 log 10 pfu / 0.2 mL
with the peak of the title reached two
days pi. By CF, the viral antigen reacted
only with its respective homologous
antiserum. Viral hemagglutinin was
not detected. Electron micrographs of
infected VERO cells revealed bulletshaped particles and the particles
were frequently seen budding in
clusters from the plasma membrane.
The results suggest that the viral
sample BEAN781455 is an ungrouped
virus and a member of the family
Rhabdoviridade. Molecular biology
studies have been conducted to classify
sample in a genus and viral species.
BV1024 - ANTIVIRAL ACTIVITY
OF ASTERACEAE FAMILY PLANT
EXTRACTS AGAINST DENGUE VIRUS
2 (DENV-2)
Marinho, P.E.S., Rodrigues, R.A., Silva,
L.K.S., Cursino, A.E., Brandão, G.C.,
Braga, A.O., Kroon, E.G.
Universidade EStadual de MInas
gerais, UFMG, - Avenida Antonio
Carlos 6627 – CEP: 31.270-901 – BH/
MG. E-mail: [email protected]
The absence of specific medication for
dengue is one of the main problems
related to the disease that currently
has limited treatment to reduce the
symptoms of infection. Many species of
plants belonging to different families
have been used for the treatment
of diseases or related symptoms to
possible viral infections. Dengue is one
of the major public health problems in
the world. In Brazil, more than 750.000
cases of dengue were registered in
2011 (MS, Brasil). The development
of a herbal medicine against Dengue
virus, may enable reduction of the
incidence and mortality in affected
regions. In this work, the antiviral
activity of ethanolic plant extracts of the
Asteraceae family were tested against
Dengue virus 2 (DENV-2). In a first
step, the 50% cytotoxic concentration
(CC50) was determined and then the
50% effective concentration of the
viral effect (EC50) for each extract
was calculated from concentrationeffect-curves after linear regression
analysis. Both tests were made in cells
LLCMK2 and the cellular viability
was evaluated by MTT technique. The
therapeutic index or selective index
(IS) is defined as CC50 over EC50. Until
now, 51 samples have been tested and
40 of them showed no activity, 10 were
moderately active with IS between 4
and 10, and 1 showed antiviral activity:
Bidens sp. (IS: 11,35), against DENV-2.
This extract will be tested for virucidal
activity against DENV-2 and other
serotypes of the virus in an attempt to
understand the mechanisms of action
of these extracts, which are good
candidates for fractionation to identify
active principles. Financial Support:
INCT Dengue, Pronex-Dengue, CNPq,
FAPEMIG, CAPES
BV1032 - EXPRESSION OF THE NS1
AND NS3 PROTEIN OF DENGUE VIRUS
TYPE 3 IN PROKARYOTIC CELLS
Oliveira, A.S., Amarilla, A.A., Figueiredo,
L.T.M., Aquino, V.H.
Universidade de São Paulo, USP, Av.
do Café, s/nº. Campus Universitário
- Ribeirão Preto - SP - 14040-903
Dengue viruses (DENV 1-4) represent
a major emerging arthropod-born
pathogen, which cause disease of great
public health importance in tropical and
subtropical countries. DENV belongs
to genus Flavivirus, family Flaviviridae
and is transmitted by mosquitoes of the
genus Aedes. The RNA genome encodes
a large polyprotein that give rise to
three structural (prM, C and E) and
seven nonstructural (NS1-5) proteins.
NS1 exists in two forms, soluble and on
the cell surface, participates in the viral
replication and, possibly, is involved in
the pathogenesis. NS3 is a citoplasmatic
protein that associates with cell
membrane and has several enzymatic
Basic Virology: BV
81
activities related to viral polyprotrein
processing and viral replication. The
aim of this work was to express the
NS1 and NS3 protein of DENV-3 in
prokaryotic cells. NS1 and NS3 genes
of D3BR/RP1/2003 strain, isolated in
Ribeirão Preto-SP, was amplified by RTPCR. The amplicons were inserted in
the cloning vector TOPO XL (Invitrogen,
USA) using the T4 DNA ligase enzyme
(Invitrogen, USA) and the resulting
plasmid was used to transform E. coli
TOP 10 strains. Colonies containing
the desired plasmids were selected by
restriction enzymes digestion (Bam
HI and Hind III) and DNA sequencing
of the corresponding plasmids. The
selected plasmid was digested again
with the two restriction enzymes and
the obtained insert was ligated into the
expression vector pQE-30 (QIAGEN,
USA), previously digested with the
same restriction enzymes. E. coli
BL21(DE3) strain was transformed
with the ligation product. The presence
of the insert was confirmed with double
digestion (Bam HI and Hind III) and
DNA sequencing. Colonies containing
this plasmid were subjected to protein
expression induction with IPTG
leading to the production of NS1 and
NS3 recombinant proteins, which were
confirmed by imunoblotting. These
proteins could be used as tools for
diagnostic methods or basic research.
BV1034 - ENZIMATIC ACTIVITY
ANALYSIS OF NS3 MUTANT HELICASE
OBTAINED FROM A GENOTYPE 3A
RELAPSE PATIENT
Provazzi, P., Mukherje, S., Alicia, M.,
Hanson, Rahal, P., Frick, D.N.
1. São Paulo State University UNESP, UNESP, Rua Cristovão
Colombo, 2265- São José do Rio
82
Basic Virology: BV
Preto, São Paulo
2. University of Wisconsin, ,
Milwaukee,
Milwaukee,
WI,
53217
The Hepatitis C virus (HCV) infects
170 million people worldwide and
is responsible for acute and chronic
hepatitis, cirrhosis and hepatocelular
carcinoma. Nevertheless, affordable
and highly effective treatment
options are not yet available. The HCV
Genotype 3a is common in Brazil, and
in clinical evaluations it is associated
with a mild illness manifestation
and a better response to the antiviral
therapy. Because the processing of the
viral polyprotein is essential for HCV
replication, the NS3 protein has been
considered to be a primary target for
the development of anti-HCV drugs. In
previous work we identified the amino
acid substitution W501R on RNA
binding site of NS3 helicase genotype
3a of a relapse patient. The objective of
the present work was to evaluate the
effect of a of W501R substitution on a
genotype 3a NS3 helicase. The helicase
NS3 variant sequence was cloned,
expressed in E. coli cells to assess their
level of expression, and purified from
soluble fraction. The methodologies
of MBHA, FP-SSB-DNA binding and
ATP assay were used to evaluate the
helicase activity in RNA and DNA
unwinding, DNA-helicase binding
and ATPase, respectively. The MBHA
procedure showed that the activity of
DNA and RNA unwinding was faster
in NS3 helicase wild type genotype 3a
and genotype 1b enzymes than in the
NS3 helicase W501R mutant protein.
By the FP-DNA binding analysis was
observed a weaker DNA–NS3 helicase
W501R mutant binding than the DNA
– NS3 helicase wild type binding. The
evaluation of ATP hydrolysis activity
revealed a decrease in the velocity of
NS3 helicase W501R mutant ATPase
activity when compared to the NS3
helicase wild type and Con-1 enzyme.
We believe that evaluation of the NS3
helicase protein activity can provide
key information about the NS3 and
consequently the replication viral
and the Hepatitis C establishment.
Additional studies with replicon cells
and possible helicase inhibitors will
provide more conclusive results.
Financial support: FAPESP, CAPES,
CNPq, NIH grant RO1 AI088001 and
the UWM Research Foundation.
BV1066 - ANTIVIRAL PROPERTIES
OF DRIMYS BRASILIENSIS AGAINST
EQUINE HERPEVIRUS
Parreira, R.M., Simoni, I.C., Fávero, O.,
Fernandes, M.J.B.
Instituto
Biológico,
IB,
Av.
Conselheiro
Rodrigues
Alves,
1252 Universidade Presbiteriana
Mackenzie
Equine herpesvirus type 1 (EHV-1)
is an important ubiquitous enzootic
equine pathogen. The demand for
more effective and affordable antiviral
drugs is one of strategies to control the
viral infections. Drimys brasiliensis
is a plant from the Atlantic Forest,
Winteraceae family and among
the various therapeutic properties
includes the barks infusion to treat
ulcer, cancer, malaria, general pains
and respiratory problems. Previous
antiviral studies with various barks
and leaves extracts of D. brasiliensis
demonstrated an in vitro inhibition on
EHV-1, at non cytotoxic concentrations
of 500μg/mL. This study aimed to
continue the investigation with one
of these extracts, the crude aqueous
extract from leaves (DBAq) in order to
study its mode of action on this virus.
For this, the extract was evaluated
on initial phase of viral replication
and on direct and extracellular effect
(virucidal action). For virucidal assay,
equal parts of DBAq at 500 μg/mL and
virus (log10 dilutions) were mixed
and incubated for 1h at 37ºC; then
these mixtures were inoculated into
cells. For assay penetration, the cells
were firstly inoculated with virus and
maintained for 30 min at 4ºC; after this
period, the DBAq extract was added
and the cells were incubated at 37ºC.
Controls without extract and/or virus
were made in both assays. These assays
were made on Vero cells using the viral
titer reduction assay and expressed in
inhibition percentage (IP). The results
demonstrated that DBAq inhibited
both the viral penetration and directly
inactivated the virus by 99%. This plant
extract also exhibited antiviral activity
for other herpesviruses (bovine and
suid type 1) in other studies. Taken
together, these data indicate that D.
brasiliensis is promising as antiviral
source.
BV1070 - DITERPENES ISOLATED
FROM MARINE BROWN ALGAE AND
OXOQUINOLINE DERIVATIVES WITH
HIV MICROBICIDE POTENCIAL:
STUDIES OF CYTOTOXICITY AND
ANTIVIRAL ACTIVITY USING HUMAN
CERVICAL EXPLANTS AND CELL
LINES.
Stephens, P., Paixão, I., Lyra, P.O.,
Branco, C., Amorim, L., Batalha, P.,
Santos, F., Souza, M.C., Launeville, V.,
Osako, K., Cunha, A.C.
Fundação Oswaldo Cruz, FIOCRUZ,
Av. Brasil 4365 - Pav. Lônidas
Deane - RJ - Manguinhos Fundação
Basic Virology: BV
83
Oswaldo Cruz/Instituto Oswaldo
Cruz, FIOCRUZ/IOC, AV. Brasil 4365
- Manguinhos - RJ- cep. 21045900
According to UNAIDS 2009, there
were more than 33 million people
living with HIV worldwide. Brazil has
a population of approximately 192
million inhabitants and (from 1980
to 2010) more than 470.000 AIDS
cases were diagnosed, with more than
34.000 new cases per year. Currently,
there is no effective HIV/AIDS vaccine
or cure, although the introductions
of antiretroviral drugs significantly
improved the prognosis of infected
individuals with access to treatment.
However, the emergence of drugresistant viral strains is increasing.
Therefore numerous studies have
been developed, such as preventive
strategies in order to find some
low-toxicity and low-cost anti-HIV
substances. The literature has been
shown that studies using the ex vivo
model (human cervical explant) are
suitable for histopathological analysis
as well as for drug testing. The aim of
this study is to evaluate the cytotoxicity
and anti-viral activity of diterpenes
isolated from marine brown algae and
oxoquinoline derivatives in cervical
explants (epithelial and stromal
tissues) and PM-1 cells. We used
human cervical explants obtained
from fertile age women from the
Hospital Federal de Bonsucesso (HFB),
Rio de Janeiro, Brazil. Cytotoxicity
assays were performed by the MTT
3-(4,5-dimetiltiazol-2-il)2,5-difenil
brometo de tetrazolium assay and
measurement of ELISA p24 antigen
in
supernatants
from
explant
cultures and cell lines treated with
marine diterpenes and oxoquinoline
derivatives and both infected by
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Basic Virology: BV
HIV-1. The diterpenes isolated from
marine brown algae and oxoquinoline
derivatives studied by our group have
important effects on HIV replication,
as we have observed more than 90%
viral inhibition. Cytotoxicity levels
lower than 30% were observed in both
classes of substances. Further preclinical studies are needed to better
evaluate these substances as potential
candidates for microbicides or sistemic
drugs. Financial Suport: FIOCRUZ,
Brazilian Ministry of Health, UNESCO,
CNPq, FAPERJ, CAPES, FOPESQ-UFFPROPPI.
BV1115 - MOLECULAR AND EPIDEMIOLOGIC ANALYSIS OF INFLUENZA
A/H1N1 PANDEMIC IN CANCER
PATIENTS
Andrade, V.M.M., Dias, M.M.A.,
Resende, P., Siqueira, M.M., Souza,
T.L.M.
Instituto Oswaldo Cruz, IOC/
FIOCRUZ, Rua Leopoldo Bulhões
1480,
Manguinhos
E-mail:
[email protected]
Among the respiratory viruses,
Influenza is identified as the main
cause of global public health impact
and has a large genetic diversity, due to
processes of antigenic drifts and shifts.
This can be illustrated by the emergence
of influenza virus A/H1N1 in 2009.
Recent data on the clinical course of
H1N1pdm infection stimulated further
investigation on this virus infection on
specific groups at higher risk, such as
cancer patients. They have an atypical
manifestation of influenza infection
and are more likely to have severe
outcomes. In addition, influenza may
be shaded for long periods of time in
these patients. In a pandemic context,
these patients might become “human
reservoirs” of virus, which may have
direct implications on its spread.
Nevertheless, the information on the
evolution of H1N1pdm infection in
patients with cancer is still poor or
absent in Brazil. Through sequence
analysis of H1N1pdm from respiratory
secretions, we observed that two
patients with acute lymphoblastic
leukemia (ALL) had periods of
persistent virus H1N1pdm of 45 and
58 days. These are the longest period
of excretion of the new virus has been
reported so far. We evaluated the
presence of cumulative mutations in
these samples of one patient during
one month after the onset of symptoms
and found continuous virus evolution.
We found that there was no significant
variation in the HA gene in relation to
other strains circulating in different
regions of the world in the same
period. We analyzed samples of coinfected individuals with H1N1pdm/
HIV-1, fragments encoding the protein
haemagglutinin, neuraminidase and
matrix. Regarding the HA gene, the
strains isolated from patients have a
significant genetic diversity compared
to other strains of H1N1pdm described
in various parts of the world. Financial
support: CNPq, FAPERJ
BV1124 - ASPECTS OF THE IMMUNE
RESPONSE OF THE CENTRAL
NERVOUS SYSTEM OF MICE IN EXPERIMENTAL INFECTION BY THE
JURUAÇÁ; VIRUS IN VIVO
Ferreira, N.C., Diniz, J.A.P.
1. Universidade Federal do Pará,
UFPA, Rua Augusto Corrêa, nº 01,
bairro Guamá, Belém-PA
2. Instituto Evandro Chagas, IEC,
Avenida Almirante Barroso,
nº492, bairro Marco, Belém-PA
E-mail:
natalie.chaves@gmail.
com
The central nervous system (CNS) was
long regarded as an immune-privileged
site. It is now clear that inflammatory
reactions may occur in the CNS, as in
neurodegenerative, demyelinating and
caused by microorganisms diseases.
Few studies have been conducted
on the neuropathology of viruses
isolated in the Amazon region, as is
the case of the Juruaçá virus, a possible
member of the family Picornaviridae.
The occurrence of reactive gliosis,
as well as the absence of cytopathic
effect (CPE) in primary cultures of
CNS cells, suggested that the Juruaçá
virus infection newborn mice have
caused an immune and inflammatory
disease. The aim of this study was to
examine the immune response of the
CNS in mice infected with the Juruaçá
virus, from the expression of cytokines
and nitric oxide (NO). For detection of
cytokines and of NO were performed
enzyme immunoassays (ELISA) and
tests for quantification of nitrite using
the Griess reagent, respectively, and
for the detection of viruses in primary
cultures of CNS cells was performed
for indirect immunofluorescence (IFI).
It was observed that newborn mice
showed a significant increase in the
production of IL-12 and IFN-γ and
expression of NO after infection by the
virus under study. The other cytokines
tested showed a different expression
for each new experiment. The increase
in the expression of pro-inflammatory
cytokines IL-12 and IFN-γ and in
the production of NO induced by the
Juruaçá virus, points to an intense
inflammatory response that may have
caused the death of the infected mice.
Financial support Instituto Evandro
Basic Virology: BV
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Chagas (IEC), Universidade Federal do
Pará (UFPA) and Conselho Nacional
de Desenvolvimento Científico e
Tecnológico (CNPq)
BV1131 - PRELIMINARY ANTIHERPES SCREENING OF PREPARATIONS
OBTAINED FROM VACCINIUM SPP.
AND SAMBUCUS SPP. FRUITS
Schneider, N.F.Z., Petrova, V., Kennelly,
E.J., Simões, C.M.O.
Catarina, UFSC, Florianópolis, SC,
Brazil
2. Lehman College, , New York, USA
E-mail: nairaschneider@yahoo.
com.br
Herpes Simplex Viruses (HSV) are
responsible for many infectious
diseases during the lifetime of the host.
Prolonged therapy with the available
antiherpes drugs had induced drugresistance, hence, the development of
new antiherpes agents are still needed.
Berries, such as blueberry, elderberry
and cranberry, are well known as
“super fruits”, which are a rich source
of anthocyanins and have shown to be
beneficial to health for their potential
antioxidant, antiviral and antitumoral.
Hence, this study investigated the
cytotoxicity and the antiherpes activity
of crude extract samples of some berries
species (Vaccinium corymbosum,
V.angustifolium,
V.myrtillus,
V.macrocarpon, Sambucus nigra and
S.ebulus), which were cultivated in
Bulgaria and USA. The cytotoxicity
and the potential antiherpes activity of
these samples were assessed on Vero
cells by MTT and viral plaque reduction
assays, respectively. The tested viruses
were HSV types 1 (HSV-1, KOS and
29R strains - sensitive and resistant
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to acyclovir, respectively). Results
were expressed as 50% cytotoxic
concentrations (CC50) and 50% viral
replication inhibitory concentrations
(IC50), respectively, in order to calculate
the selectivity index (SI=CC50/IC50) of
each sample. The results obtained from
this screening showed that the extracts
of V.corymbosum, V.macrocarpon and
V.mirtillus showed SI = 2 (0.84/0.4 mg/
mL), 4 (1.09/0.27 mg/mL) and 12.5
(1.5/0.12 mg/mL), respectively, for
KOS strain. These samples also showed
activity for 29R strain with SI =1.6
(0.84/0.53 mg/mL), 4.2 (1.09/0.26
mg/mL) and 7.5 (1.5/0.2 mg/mL),
respectively. S.ebulus and S.nigra
showed activity only against 29R
strain, with a SI of 8 for both extracts
(2.06/0.26 mg/mL and 1.76/0.22
mg/mL). Additional experiments
are currently under development to
better chemically characterize these
extracts and to detect the compounds
responsible for the activity, as well as
to elucidate the mechanism of antiviral
activity. Financial support: CNPq/
CAPES, Brazil.
BV1137 - FULL-LENGTH GENOME
DETERMINATION OF BIMITI VIRUS
USING
THE
PYROSEQUENCING
METHOD
da Silva, D.E.A., Inada, D.T., Vianez
Junior, J.L.S.G., Cardoso, J.F., Lima, C.P.S.,
Sousa, E.C.J., Nunes, K.N.B., Nunes,
M.R.T., Vasconcelos, P.F.C.
Instituto Evandro Chagas, IEC, Rod.
BR-316, KM-7, S/N, LEVILÂNDIA,
67030-000-ANANINDEUA-PARÁBRASIL E-mail: daisysilva@iec.
pa.gov.br
The Bimiti virus (BIMV) was firstly
isolated from Culex spissipes in
Trinidad & Tobago in 1955 and
was classified as a member of the
Orthobunyavirus genus belonging to
the Guamá serogroup. In the Brazilian
Amazon, BIMV (strain BeAn100519)
was only isolated in 1966 from the
blood of a Proechimys guyannensis
rodent, and later from sentinel mice
and Culex mosquitoes. The purpose of
this study was to sequencing the fulllength genome of BIMV (BeAn100519)
by pyrosequencing method. The
viral genome assembling obtained
by the de novo assembly method was
implemented in Newbler v2.6 software.
For removal of contaminants, the
BLASTn program was used together
with shell script. The BLASTn program
was also used to search for a genomic
reference and to serve as a model for
the implementation of the reference
mapping assembly by the Geneious
v.4.8.5 software for the generation of
scaffold genomic segments of the virus.
The de novo assembly method showed
satisfactory results. It has used 47% of
the reads from the sequencing, for the
construction of a total of 1616 contigs
with N50 equal to 425 nt and an average
size of around 1025 nt. This length
was sufficient to complete the three
genomic segments of the virus after the
assembly by reference. This procedure
counted with the concatenation of
these contigs together with the reads
initially used for the generation of
scaffold. After submitting the generated
scaffolds in the BLASTn program, the
genome showed similarity to genomes
of three distinct viruses, chosen with
the best statistical values, all belonging
to the family Bunyaviridae (L segment:
Zungarococha virus [e-value:]/ M
segment: Apeu virus [e-value]/ S
segment: Cachoeira Porteira virus
[e-value]). This study showed that the
pipeline used was efficient in order
to cover the complete genome of the
BIMV, highlighting its homology with
members of the Bunyaviridae family
which confirm the serological studies
carried out to group the virus.
BV1138 - EVALUATION OF SUBFRACTIONS OF ETHYL ACETATE EXTRACT
OF STRYPHNODENDRON ADSTRINGENS IN THE REPLICATION OF
HERPESVIRUS AND POLIOVIRUS
Espada, S.F., Lopes, N., Galhardi, L.C.F.,
Santos, J.P., de Mello, J.C.P., Linhares,
R.E.C., Nozawa, C.
1. Universidade
Estadual
de
Londrina, UEL, Rodovia Celso
Garcia Cid, Pr 445 Km
2. Universidade
Estadual
de
Maringá , UEM, Av. Colombo,
5.790 E-mail: samanthafespada@
hotmail.com
Stryphnodendron adstringens (Mart.),
popularly known as barbatimão,
is used in empirical medicine
as
adstringent,
antidiarrheal,
antimicrobial, hypoglycemic, cicatrizer,
analgesic, anti-inflammatory and for
gastric ulcer treatment. The extracts
obtained from the stem bark are rich
in tannins. Antiviral activity of crude
extract and aqueous and ethyl acetate
fractions of S. adstringens against
bovine herpesvirus type 1 (BoHV1) and poliovirus type 1 (PV-1) has
been reported in HEp-2 cells. Twelve
subfractions (F3.1 - F3.12) obtained
by submitting ethyl acetate fraction to
column chromatography were evaluate
for potentiation of antiviral activity for
PV-1, BoHV-1 and herpes simplex virus
type 1 (HSV-1). The 50% cytotoxic
concentrations (CC50), determined
by MTT assay, for F3.1 – F3.12 were
277.5; 705; 120; 300; 125; 55; <125;
Basic Virology: BV
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248.7; 178; 7; >1000 and >1000 µg/
ml, respectively. The antiviral activity,
determined by plaque reduction assay,
was performed by the addition of the
subfractions, at the concentrations
of 1.5 and 100µg/ml, at the time zero
of infection (simultaneously to the
infection), and the 50% inhibitory
concentration (IC50) were, respectively,
0.13 and 10.2 µg/ml for PV-1; 2 and
20.2µg/ml for BoHV-1 and 0.19 and
16.5 µg/ml for HSV-1. The subfractions
F3.1 and F3.2 showed no antiviral
activity for HSV-1 up to 100μg/ml. The
selectivity index (SI) (CC50/CI50) of
the subfractions varied from 923 to 5.2
for PV-1, from >307.6 to 3.8 for BoHV1 and >5263.1 to >10 for HSV-1. The
promising results obtained by testing
subfractions of ethyl acetate extract
straighten the antiviral property of S.
adstringen, moreover, an insight into
specific compound(s) that account for
the antiviral activity and the replication
steps that are inhibited.
BV1141 - IDENTIFICATION OF HOST
CELL FACTORS CONTROLLING INTRACELLULAR TARGETING OF HIV-1
ENVELOPE GLYCOPROTEINS
Estela, A.P., Aguiar, R.S., da Silva, L.L.P.,
Janeiro, IB/UFRJ, R. Brigadeiro
Trompowsky s/n, 21943-970,
Rio de Janeiro, RJ, Brazil
2. Universidade de São Paulo,
FMRP/USP, Av dos Bandeirantes,
3900, 14049-900, Ribeirão Preto,
SP, Brazil E-mail: estelapereira@
usp.br
HIV-1 infection process begins with
fusion between the virus envelope
and the host cell plasma membrane,
which allows delivery of the capsid
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Basic Virology: BV
containing the viral genomic RNA into
the cytoplasm. This crucial membrane
fusion event is catalyzed by the action
of envelope (ENV) glycoproteins. The
Env protein precursor, known as gp160,
is synthesized in the endoplasmic
reticulum and processed by cellular
proteases during trafficking through
the secretory pathway. Processing
of gp160 generates a soluble surface
and a transmembrane glycoprotein,
respectively gp120 and gp41. gp41
anchors the gp41/gp120 complex to
membranes and is believed to contain
sorting information in its cytosolic tail
(CT). Proper incorporation of gp41/
gp120 complexes into virus particles
is an essential step of the HIV-1
replication cycle, but the mechanism
of
Env
glycoprotein
targeting
to viron assembly sites remains
incompletely understood. To identify
host cell machinery implicated in the
intracellular trafficking of gp41, we
used a yeast two hybrid based system
to test interaction between the gp41CT
and various clathrin adaptor proteins.
We detected a robust interaction of
gp41 CT with the µ1A subunit of the
AP-1 complex. AP-1 is localized at
TGN and endosomal membranes and
mediates protein transport between
these compartments. Interestingly,
gp41 CT did not interact with the
isoform µ1B, thought to be specifically
involved in polarized sorting, or the
equivalent subunits of AP-2, AP-3 or
AP-4 complexes (µ2, µ3 and µ4). Our
results also show that interaction with
µ1A requires a motif in gp41CT highly
conserved among different groups
of HIV and SIV, which follows the
consensus YXXØ (where Ø is a bulky
hydrophobic amino acid). The function
of this potential sorting determinant
on gp41CT and its interaction with
µ1A in ENV incorporation and viral
infectivity is currently being tested in
our lab. Financial support: CNPQ, Próreitoria de Pós-graduação USP
BV1147 - AZADIRACHTA INDICA
SULFATED
POLYSACCHARIDES
INHIBIT THE INFECTION OF HERPES
SIMPLEX VIRUS-1 IN HEP-2 CELLS
Faccin-Galhardi, L.C., Lopes, N., Espada,
S.F., Yamamoto, K.A., Santos, J., Ray, B.,
Linhares, R.E.C., Nozawa, C.
1. Universidade
Estadual
de
Londrina, UEL, Rodovia Celso
Garcia Cid, PR 445, Km 380,
campus Universitario
2. University of Burdwan, , The
University of Burdwan, Rajbati,
Bardhaman - 713 104, West
Bengal, INDIA E-mail: lgalhardi@
sercomtel.com.br
Azadirachta indica A. Juss, popularly
known as neem, has been extensively
used in Ayurvedic medicine by Indian
population for over 2000 years. It is used
traditionally for the healing of various
diseases. Natural products and their
derivatives provide an excellent source
for new antiviral drugs. The present
study aims at evaluating the activity
of two polysaccharides (P1 and P2)
isolated from the leaves of Azadirachta
indica and their sulfated derivatives
(P1S and P2S) against herpes simplex
virus-1 (HSV-1). The cytotoxicity of
the compounds was analyzed by MTT
and the antiviral effect was determined
by plaque reduction assay in different
protocols. The polysaccharides did
not show any cytotoxic effects on
HEp-2 cells at the highest tested
concentration (200 µg/ml). P1 did not
inhibit HSV-1 infection in all treatments
and P2 showed low percentage of viral
inhibition (63.5%) only for 200 µg/ml,
with inhibitory concentrations (IC50)
of 177 µg/ml and selectivity index (SI)
of 8.1. However, their sulfated forms
exhibited significant antiviral effect
with IC50 of 28.5 µg/ml and SI of
56.1 for P1S and 80.5 µg/ml and 19.9
for P2S, respectively. The compounds
demonstrated better inhibitory effect
when added concomitantly at the time
of the infection and demonstrated
a dose-dependent curve inhibition.
Lower effect was observed when the
compounds were added before and
after viral infection and in the virucidal
and adsorption inhibition tests. P1S
and P2S inhibited about 90% of
protein synthesis as demonstrated by
immunofluorescence assay, at higher
concentration, for time 0 hour. Viral
DNA synthesis was also inhibited by
sulfated polysaccharides however
in all tested concentrations. We
suggested that the sulfated forms of
A. indica polysaccharides act against
HSV-1 by inhibiting the initial stage of
viral replication. This study provides a
scientific basis for the past and present
empirical and ethnopharmacologic
use of this plant. Financial support:
CNPq, CAPES, Fundação Araucária and
PROPPG/UEL.
BV1151 - CORRELATION OF DENGUE
CASES AS TO RAINFALL, SEASONALITY, BUILDING INFESTATION RATE BY
AEDES AEGYPTI IN THE COUNTY OF
JATAÍ, STATE OF GOIÁS
Policarpo, O.F., Costa, V.G., Moreli, M.C.
Universidade Federal de Goiás,
UFG, Rodovia BR 364, Km 192
Parque Industrial 3.800 E-mail:
[email protected]
Introduction: Dengue belongs to the
genus Flavivirus, family Flaviviridae,
Basic Virology: BV
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being transmitted to humans by
mosquito Aedes aegypti. In worldwide
around 2.5 billion people live in risk
areas and each year occur about
21,000 deaths. Objective: this study
aimed to link dengue cases to its spatial
distribution in the city, to analyze
the correlation between the building
infestation rate (BIR), rainfall data,
and viral seasonality in the city of Jataí,
State of Goiás. Material and methods:
Data from cases dengue 2007 and 2010,
were provided by the Health Medical
Center of the city of Jataí and the BIR
as an indicator of risk of transmission
was provided by the Epidemiological
Department. The city was divided into
regions by software Google Earth. The
Student´s t-test was used to perform
the statistical analysis (p=0,05).
Results: Between 2008-2010, 1.191
dengue cases were confirmed, of which
83.6% took place in 2010, and most
cases were indentified in the Northeast
region of the city. The annual rainfall
was 2240 mm/2008, 2230 mm/2010
and 2050 mm/2009. As for the BIR,
all were <1% (2008>2009>2010).
There was no association between
dengue cases and periods of dry and
rainy seasons (p=0.29). Discussion:
There was correlation between
rainfall and BIR, only in 2008, while
the disagreement of the other years
could be due to extensive areas of
land without buildings, resulting in
error in the calculation of the BIR, or
on the way and quality inspection of
the property. Moreover, studies report
that rain excess can derail the focus of
the mosquito breeding sites and thus
reduce viral transmission. Additionally,
did not observed seasonal viral,
reflecting in the vector circulation
throughout the year. Conclusion: The
rates used in this study are useful as
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indicators in the dengue transmission,
but multiple climatic factors must
be considered. Furthermore, the city
presents a great vector circulation,
resulting in the constant increase of
dengue cases, even in lower rainfall
periods. Thus, further research must
be conducted to discover the serotypes
of dengue in this region. Financial
support: FUNAPE
BV1152
INDUCTION
OF
AUTOPHAGY DURING COTIA VIRUS
INFECTION
Afonso, P.P., Attias, M., Damaso, C.
Instituto de Biofísica Carlos Chagas
Filho, UFRJ, CCS - bloco G Rio de
Janeiro RJ E-mail: ppafonso@biof.
ufrj.br
Cotia virus SPAn 232 (COTV) is
a poxvirus isolated in 1961 from
sentinel mice in São Paulo, Brazil. Our
group has recently reported that COTV
probably represents a new poxvirus
genus based on the anlysis of COTV
genome. Combined next-generation
sequencing technologies were used to
determine the full-lenght genome of
near 185 kb. COTV has novel genes and
an interesting panel of ORFs involved
in immunomodulatory functions.
We are currently investigating novel
aspects of virus-host cell interactions.
Analysis of COTV-infected cells by
transmission electron microscopy
revealed the presence of myelinic
figures in the cytoplasm, which
resemble autophagosomes, as well
linear membranes associated with
viral particles. Some of them enclosed
virus particles and others had virus
particles disposed along the external
face. They were not visualized in noninfected cells and accumulated during
late stages of the virus cycle with
distinct multiplicities of infection,
suggesting a possible role in virus
morphogenesis. Immunofluorescence
assays revealed that infected cells
presented a punctuate pattern of LC3
distribution triggered late in infection.
LC3-positive vesicles colocalized with
a lysosome marker, LAMP-2, indicating
that autophagy progressed to stages
of autophagosome maturation. The
number of infected cells showing an
LC3 punctate pattern varied from 85
to 91%. These levels were similar
to the positive controls (starvation
and rapamycin treated cells), which
showed 62% and 90%, respectively.
Treatment of infected cells with an
autophagy inhibitor 3-MA reduced
the number of infected cells showing
a LC3 punctate pattern by 66% and
decreased virus intracellular titres by
33%. Western blot analysis showed the
conversion of LC3-I to LC3-II during
infection, which was intensified in the
presence of an inhibitor of autophagic
flux chloroquine, suggesting that COTV
infection induces autophagic flux.
Support: CNPq, Faperj, INPeTAm
BV1156 - EFFECT OF NATURAL AND
SYNTHETIC ANTIVIRAL SUBSTANCES
ON VACCINIA VIRUS REPLICATION
Medaglia, M.L.G., Fernandes, M.M.,
Rezende, B.C., Alves, F.O., Vegi, P.F.,
Santos, M.S., Bernardino, A.R., Noseda,
M.D., Duarte, M.E., Mourão, P.A.,
Damaso, C.
1. Instituto de Biofísica Carlos
Chagas Filho, IBCCF-UFRJ, CCS
bloco G Rio de Janeiro, RJ
2. Instituto
de
Química
Universidade
Federal
Fluminense, IQ-UFF, Depto Física
e Química - UNIFEI, DFQ-UNIFEI,
Depto Bioquímica e Biologia
Molecular-UFPr, DBBM-UFPR,
3. Hospital
Universitário
Clementino Fraga Filho-UFRJ,
HUCCF-UFRJ, Av Rodolpho Rocco
- Cidade Universitária, Rio de
Janeiro E-mail: mlmedaglia@
biof.ufrj.br
Vaccinia virus (VACV) is the prototypic
member of the genus Orthopoxvirus
(Poxviridae). Some VACV strains are
used as smallpox vaccine in several
countries and adverse effects following
vaccination are frequently described.
Nevertheless, there is no antiviral
therapy available to treat these cases.
In addition, there are frequent reports
of Cantagalo virus infection in dairy
cattle and milkers in Brazil. Cantagalo
virus (CTGV) is a strain of vaccinia
originally isolated from pustular lesions
in cows in 1999. Here we present
the results of the antiviral effect of
distinct compounds on the replication
of different strains of VACV. Sulfated
galactan (SG) and Lambda-2T (LT)
are a highly anionic polysaccharides
extracted from marine algae. Noncytotoxic concentrations of SG were
incubated with CTGV and VACV-WR
during the adsorption period in BSC40 cells. We observed an inhibition
of viral plaques by nearly 80% at 2.5
ug/ml for CTGV and only 67% at 5
ug/ml for VACV-WR at 48 hours postinfection. LT, on the other hand, was
particularly efficient in inhibiting
VACV-IOC entry by nearly 90% at 1
ug/ml. Similar levels of inhibition of
CTGV entry by SG were detected by
immunofluorescence assays. We also
used recombinant CTGV expressing
B-galactosidase under control of a viral
promoter. We detected an inhibition of
80% in B-gal activity when 15 ug/ml
Basic Virology: BV
91
SG was added during CTGV adsorption
to cells. Comparing to GAGs, SG had
a greater effect on the inhibition of
virus adsorption since heparin and
heparan sulfate inhibited virus plaque
formation by 80% at 20 ug/ml. We
have also started an investigation on
the effect of pyrazoles and derivatives
on the replication of VACV-WR. Of
6 pyrazoles tested so far, only the
5’-amino-pyrazole MPQ-7 at 100 uM
presented antiviral effect reducing the
size of virus plaques after 24 hours
of infection. This work suggests that
polysaccharides isolated from marine
algae as well as pyrazole derivatives
present promising results as anti-VACV
agents. Financial support: CNPq, Capes,
Faperj, INPeTAm, PIBIC-UFRJ.
BV1165 - EVALUATION OF THE
EFFECT OF LEPTODACTYLUS LABYRINTHICUS SKIN SECRETION
MOLECULES ON THE RABIES VIRUS
PENETRATION IN MAMMALIAN
CELLS
Cunha Neto, R.S., Vigerelli, H., Sciani,
J.M., Jared, C., Antoniazzi, M.M., Chaves,
L.B., Silva, A.C.R., Pimenta, D.C.
Instituto Butantan, IBU, Av. Vital
Brasil, 1500, 05503-900 Instituto
Pasteur, IP, Av. Paulista, 393, 01311000 E-mail: renecunhaneto@yahoo.
com.br
Rabies is a zoonosis characterized as
a lethal progressive acute encephalitis
caused by Rabies virus (RV). Toxinology
is a field in which different organisms,
such as amphibians, are studied,
regarding their venoms, poisons and
secretions as sources of biologically
active substances, from which
components with therapeutic action
can be obtained. The aim of this study
was to evaluate whether molecules
92
Basic Virology: BV
obtained from the skin secretion of
Lepdodactylus labyrinthicus could
interfere on the RV penetration in
mammalian cells. The skin secretion
was mechanically collected from L.
labyrinthicus specimens that were
submerged in deionized water and
manually compressed. This skin
secretion solution was lyophilized,
ressuspended
in
appropriated
buffers and submitted to biochemical
characterization. The presence of
proteins in this secretion was evaluated
by electrophoresis (SDS-PAGE). The
skin secretion solution was filtered
in 10 kDa cut-off membrane and was
fractionated by Reversed Phase High
Performance Liquid Chromatography
(RP-HPLC), using a C18 monolithic
column. Cytotoxic tests with the
fractions were performed using BHK21 cell line. The evaluation of the
possible neutralizing effect of fractions
on RV, PV strain, penetration in BHK21 was assessed through tests based
on Rapid Fluorescent Focus Inhibition
Test (RFFIT) and the fractions able to
reduce cell infection were characterized
by mass spectrometry. The SDSPAGE analyses revealed that proteins
are present in this skin secretion;
therefore; the RP-HPLC processed
skin secretion solution was previously
filtered through 10 kDa membrane.
Fourteen fractions were collected.
None of them was cytotoxic and four
were able to decrease the RV infection.
Mass spectrometry analyses revealed
that the fractions contain molecules
ranging from 100 to 800 Da. The active
fractions are currently undergoing
purification and thorough biochemical
characterization. The virological tests
will then be repeated with the purified
molecules.
BV1166 - EFFECTS OF INDOLE
ALKALOIDS ISOLATED FROM THE
SKIN SECRETION OF RHINELLA JIMI
ON RABIES VIRUS INFECTION IN
BHK-21 CELL LINE
Vigerelli, H., Sciani, J.M., Jared, C.,
Antoniazzi, M.M., Caporale, G.M.M.,
Silva, A.C.R., Pimenta, D.C.
Instituto Butantan, IBu, Av. Vital
Brasil, 1500, 05503-900 Instituto
Pasteur, IP, Av. Paulista, 393, 01311000 E-mail: hugovigerelli@gmail.
com
Rabies is an acute infectious disease
caused by a virus that affects the
central nervous system, which
mechanism of infection is associated
to the cell penetration via the nicotinic
acetylcholine receptor. Alkaloids, in
spite of their neurotransmitter role,
can also be employed as therapeutic
agents. These molecules can be isolated
from several plants, but also from a
large number of amphibians. The aim
of this study was to assay alkaloids
extracted from the skin of amphibians
as possible interfering agents in the
process of infection of the rabies virus in
mammalian cells. Amphibian (Rhinella
jimi) skin secretions were collected
through
mechanical
stimulation.
A liquid-liquid partition (H2OCH¬2¬Cl2) was performed and the two
resulting solutions were purified by
RP-HPLC, in a C18 column. Structural
characterization was performed by
mass spectrometry. Cytotoxic tests
of the isolated compounds were
performed over BHK-21 cells. Briefly,
96-well microtiter plates containing
the cells were incubated for 24h in the
media containing different dilutions
of the purified molecules. For the
virologic test, fixed strain PV (Pasteur
Virus) was used on fluorescence
inhibition test and fluorescent foci
inhibition test, with both simultaneous
and time course treatment of the
cells with the virus and the fractions.
Sixteen fractions were obtained by RPHPLC. The cytotoxic tests revealed that
9 fractions were toxic to BHK-21cells.
Fraction 14 was able to reduce rabies
virus infection in both tests, apparently
showing competition effects. Mass
spectrometric analyses showed that this
fraction contains two indole alkaloids,
N`,N`-dimethyl 5-hydroxytryptamione
(bufotenine) and N`,N`,N`-trimethyl
5-hydroxytryptamine (5-HTQ), which
are currently undergoing purification.
The two individual components will be
retested for biological activity in order
to evaluate which retains the biological
effect so that more in depth assays can
be performed.
BV1186 - QUERCETINS FROM
BAUHINIA LONGIFOLIA INHIBIT
MAYARO VIRUS
Yamamoto, K.A., Meneses, M.D.F.,
Salles, T.S., Santos, A.E., Kuster, R.M.,
Soares, M.R., Ferreira, D.F.
Filho, 373, CCS, Bloco I - Cidade
Universitária E-mail: kristie.bio@
gmail.com
According to World Health Organization,
viral infections represent an important
share of world’s population mortality.
Every year, new viruses continue to
emerge and such demand for new
substances has stimulated the search for
antiviral agents. In recent years, natural
compounds have been associated with
low toxicity and high selectivity against
various human and animal viruses.
Mayaro virus is an arbovirus (genus
Alphavirus, family Togaviridae) that
Basic Virology: BV
93
presents clinical significance both for
epidemic outbreaks and also because it
can cause symptoms that overlap with
the clinical diagnosis of dengue fever.
In this work, partitions of crude extract
of Bauhinia longifolia (Leguminosae
Family) into ethyl acetate (AcOEt) and
butanol (BuOH), as well as four isolated
quercetins (AES1-4) were tested
against MAYV in Vero cell culture. The
compounds citotoxicity was evaluated
by neutral red incorporation method
and the 50% toxicity for Vero cells were
795 (AES1), 941 (AES2), 411 (AES3),
265 (AES4), 3116 (AcOEt) and 418 µg/
mL (BuOH), with a selectivity index
of 94 (AES2), 4 (AES4), 623 (AcOEt)
and 208 (BuOH). Antiviral activity
was evaluated by virus yield inhibition
assay and titrated by plaque assay. Cells
treated with different concentrations
of AES2, AcOEt and BuOH 1 hour postinfection inhibit viral yield in a dosedependent manner more than 90% at
25 µg/mL, except for AES4. AES3 did
not show significant antiviral effect.
The 1 hour pre-treatment assay with
AES2, AcOEt and BuOH at 100 µg/mL
was able to protect the cells of viral
infection up to 98.7%; 66.9% and
85.7%, respectively. Finally, a strong
direct effect on viral particle was seen
for AES2, AcOEt and BuOH at 100
µg/mL, showing no infectious virion
yield. These compounds seem to affect
all tested steps of viral replication.
At the moment, further tests are
being analyzed in order to elucidate
the mechanism of action of these
substances. Financial Support: CAPES,
FAPERJ, INBEB
BV1198 - IN VITRO EFFECTS OF A
TRIAZOLIC COMPOUND AGAINST
OSELTAMIVIR RESISTANT STRAINS
OF INFLUENZA
94
Basic Virology: BV
Fintelman-Rodrigues, N., Alves, C.M.,
Ferreira, V.F., Souza, T.M.L.
1. Instituto Oswaldo Cruz, FIOCRUZ,
Manguinhos, Rio de Janeiro
2. Instituto de Química, UFF,
Niterói, RJ Laboratório de vírus
respiratórios e do sarampo, LVRS,
UFF, , E-mail: nataliafintelman@
gmail.com
Resistance to the neuraminidase
inhibitors (NAIs oseltamivir, zanamivir
and peramivir) most commonly arises
in residues around the enzyme active
site. These may only affect binding
of a subset of the inhibitors, eg.
H274Y (H275Y) affects the binding
of oseltamivir and peramivir, but not
zanamivir. Only known mutations
can be detected by sequence analysis.
However, phenotypic analysis in the
enzyme inhibition assay can detect
altered binding of the inhibitors
due to mutations in unknown sites.
Hence both phenotypic and genotypic
surveillance needs to be carried out to
ensure all potential resistant variants
are identified. While oseltamivir
and zanamivir are licensed globally,
oseltamivir is the primary drug of
choice for the treatment of influenza.
However, there are concerns about
resistance arising to oseltamivir,
especially in light of the global
spread of the oseltamivir resistant
seasonal influenza virus in 2008. As
the NA is a validated target, efforts
to develop alternative NA inhibitors
by screening natural products and
synthetic compounds are pivotal.
Triazolic compounds have a broad
range of biological activity, including
anti-platelet, anti-psychotic and anti
TB activity. Screening for triazolic
compounds against NA activity of wild
type and oseltamivir resistant influenza
A and B strains, we found compound
4, a polycyclic hydroxylated triazole,
with no halogens, as the most potent
molecule. In yield reduction assays the
EC50 was 0.38 M, compared to 0.03 M
for oseltamivir. The toxicity for MDCKs
cells, the CC50 was >2000 M, giving a
selectivity index of 5000 for compound
4 and 66,000 for oseltamivir. In the NASTAR enzyme inhibition assay the IC50
was around 10 nM against influenza
A, B, and H275Y and E119V mutant
NAs. Passaging virus in the presence of
compound 4 led to emergence of a virus
with mildly reduced sensitivity after 4
passages. It had a G249P mutation, and
retained sensitivity to oseltamivir. The
G249 is 20-30 Å from the active site.
Altogether, our results suggest that
the chemical structure of compound 4
is a promising one for development of
novel anti-influenza inhibitors.
BV1206 - HIV-1 NEF REGULATES
PR ACTIVITY AND ITS ABSENCE
REDUCES MATURE VIRAL PARTICLES
PRODUCTION AND INTEGRASE INCORPORATION
Mendonça, L.M., Poeys, S.C., Abreu,
C.M., Tanuri, A., Costa, L.J.
Departamento de Virologia, IMPPG
- UFRJ, Av Carlos Chagas, 373
Departamento de Genética, IB UFRJ, Av Carlos Chagas, 373 E-mail:
[email protected]
Nef is a viral accessory protein
expressed throughout the replicative
cycle of primate lentiviruses as HIVs
and SIVs. The major phenotype
associated with Nef expression is the
increase on virus infectivity, however,
the mechanism by which Nef achieves
this function is not fully elucidated.
Although many studies have described
the role of Nef on the early stages of
HIV-1 replicative cycle, its contribution
on the late stages is less explored. Our
group has previously described the
interaction of Nef with the p6*-PR
region of the GagPol precursor. Since
both p6* and PR are involved with
protein processing, we explored the
role of Nef on maturation, viral protease
activity and assembly. In order to check
if Nef has an effect on PR function we
performed an in vitro kinetic analysis
of PR activity in the presence of a GST.
Nef chimera, or in its absence. We also
verified the level of protease activity
in the context of the virus particle,
comparing the proteolytic processing
of Gag and GagPol in HIV-1 WT or its
nef-deleted counterpart and verified
the levels of mature particle production
and enzyme incorporation by Western
Blot. In our assays, Nef inhibited the PR
activity in vitro in a dose-dependent
manner.
Moreover,
nef-deleted
viruses showed enhanced Gag and
GagPol processing. This accelerated
processing has dire consequences to
the virus, as loss of enzymatic content,
as Integrase, on the budding virions,
as well as diminished production of
mature particles observed by sucrose
gradients. All these results converge to
a model in which Nef acts as a regulator
of PR activity. The lack of Nef would
therefore cause the PR to become over
activated, leading to a faster processing
of viral proteins culminating in
abnormalities in enzyme content and
less mature particles production. This
model also explains why viruses that
do not express Nef can be up to 40
times less infective than the wild type
counterparts in different cell lines.
Financial Support: Capes, CNPq and
FUJB.
BV1222 - IMPROVEMENT OF ARARA-
Basic Virology: BV
95
QUARA HANTAVIRUS ISOLATION BY
SHELL VIAL CULTURE PROCEDURE
Figueiredo,
G.G.,
Sabino-Santos,
Jr.G., Carvalho, A.C.D., Volpin, M.R.L.,
Figueiredo, L.T.M.
Centro de Pesquisa em VirologiaFMRP- USP, CPV- FMRP- USP,
Av. Bandeirantes, 3900 E-mail:
[email protected]
Hantaviruses belong to the family
Bunyaviridae,
genus
Hantavirus,
and comprehend viruses distributed
worldwide. They are enveloped viruses
with three single-stranded segments
of negative-sense RNA genome: the
small (S), medium (M) and large (L)
segments. The S segment encodes a
nucleocapside protein, the M segment
two glycoproteins (Gn and Gc), and
the L segment a RNA polymerase.
American hantaviruses are causative of
a severe Hantavirus Cardiopulmonary
Syndrome
(HCPS).
Hantaviruses
are carried by rodent hosts and
transmitted to humans in infectious
aerosols of animal excreta. Hantavirus
isolation is a laborious process and in
our experience, using the conventional
method, only allows Araraquara
hantavirus (ARAV) detection in
Vero-E6 cell culture supernatants after
the fourth passage of the virus. In the
present study, we have used the shell
vial culture procedure to improve
ARAV isolation. Initially, in the BSL3 laboratory, a lung from a Necromys
lasiurus rodent infected by ARAV was
macerated. It was used to infect Vero
E6 cell monolayers in T25 flasks with
Earle essential medium. The flasks
were submitted to centrifugation,
2,000 rpm, 2 hours, at 25 ºC. After this
procedure, Earle essential medium
with 5% fetal bovine serum was added
to the flasks that were incubated at
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Basic Virology: BV
37ºC. After 7 days, it was observed
that the majority of the cells died and
the ARAV genome was detected in
the cell supernatant by RT-PCR. This
supernatant was passed to new cell
cultures that were incubated at 37ºC,
for 14 days. Thus, following this new
virus isolation procedure, hantavirus
genome was detected by RT-PCR since
the first passage and ARAV antigens
were observed in infected cells at
passages two and three by indirect
immunofluorescent test. Our results
show that the centrifugation, as part
of the shell vial procedure, enhances
approximation and adsorption of the
hantavirus into the cells improving
virus isolation. Financial support:
FAPESP
BV1234 - ADAPTATION EVALUATION
OF A COMMERCIAL IMMUNOASSAY TEST TO ANTI-HCV MARKER IN
DRIED BLOOD SPOT (DBS) SAMPLES
Marques, B.L.C., Lewis-Ximenez, L.L.,
Lampe, E., Villar, L.M.
Av. Brasil, 4365 - Manguinhos, Rio
de Janeiro E-mail: miss.marques@
gmail.com
Several studies have demonstrated the
importance of DBS samples as a good
alternative specimen once it represents
a non-invasive blood collection with
no need of phlebotomist. Moreover,
samples can be storage and sent to
other laboratories without freezing.
The present study aims to compare
two commercial enzyme immunoassay
(EIA) for anti-HCV detection among
DBS samples. Ten individuals were
selected and gave paired sera and
DBS samples, where 5 were anti- HCV
reactive and 5 were anti-HCV negative
among sera samples by commercial
EIA 1 (HCV Ab, Radim). DBS samples
were assayed by EIA 1 increasing
sample volume ten-fold compared to
serum. Using commercial EIA 2 (Murex
Anti-HCV kit, Abbot), manufacturer’s
instructions and sample volume (five
and nine-fold increase compared to
sera) were evaluated. Using EIA1,
100% of concordance for anti-HCV
detection was observed among sera
and DBS. Using EIA2 among DBS
samples according manufacturer’s
protocol gave 80% of concordance
compared to sera results, where two
false negative results were obtained
(mean OD value=1.13). When sample
volume was increased 5 and 9- fold
compared to sera samples, 100% of
concordant results among sera and
DBS samples were observed. Mean OD
values for anti-HCV positive samples
were 3.63 and 3.98 using five and nine
fold increase, whereas mean OD values
for negative samples were 0.070 and
0.226 using five and nine fold increase.
It is concluded that both EIAs can be
used for anti-HCV detection among
DBS samples, but sample volume has
to be increased. Financial Support:
FAPERJ, CNPQ.
BV1238 - ANALYSIS OF CLINICAL
STRAINS OF HUMAN RESPIRATORY
SYNCYTIAL VIRUS AND SUSCEPTIBILITY TO NEUTRALIZATION BY
PALIVIZUMAB
Vilela, M.R.S., Criado, M.F., MouraNeto, J.P., Arruda, E.A.
Av. Bandeirantes, 3900
2. Universidade
Federal
do
Amazonas, UFAM, v. General
Rodrigo Octávio Jordão Ramos,
3000 E-mail: mayaravilela@
hotmail.com
The Human Respiratory Syncytial Virus
(HRSV) is the most common cause of
acute respiratory infection (ARI) in
early age causing annual epidemics
of bronchiolitis and pneumonia
worldwide. According to World Health
Organization (WHO), RSV infection is
responsible to 64 million cases with
approximately 160,000 deaths per year.
Premature infants, newborns, children
and adults with cardiopulmonary
or immunodeficiency disorders are
more susceptible to develop severe
RSV disease. The best anti-RSV
intervention currently available is the
passive prophylaxis using monoclonal
antibody (mAb), such as Palivizumab
(PZ). PZ is a humanized mAb that bind
HRSV F protein between residues 258
to 275. Despite the higher efficiency
of PZ recent studies have been shown
that specific point mutations in HRSV
F protein can cause neutralization
resistance and selection of escape
mutants. This study aims to investigate
the amino acid changes in the F protein
of Brazilian clinical strains of HRSV
and correlate it with susceptibility
to neutralization by PZ “in vitro”. We
analyzed ninety one HRSV positive
samples from patients attempted in
the Clinical Hospital of University São
Paulo, School of Medicine of Ribeirão
Preto (HCFMRP/USP) during the
years 2010 to 2011. We observed by
real time PCR (qPCR) using Taqman
methodology that HRSV subgroup B
was prevalent compared to subgroup A
(89% versus 11%) in 2010, alternating
its proportion in 2011 (69% subgroup
A versus 31% subgroup B). The HRSV
F gene from selected samples was
amplified by conventional PCR and when
required by nested PCR followed by
amplicon purification and sequencing
Basic Virology: BV
97
reaction. Nucleotide sequences were
analyzed using DNASTAR software.
The standardization of neutralization
assay was performed in HEp-2 cells
monolayer using 1000 pfu of HRSV
VR-26 (Long strain) from ATCC in the
presence of different concentrations
of PZ (5 to 100 µg/mL) during four
days post-infection (pi). HRSV without
PZ were used as negative control. Our
results shown that 15 to 100 µg/mL
of PZ inhibit cytopathic effect of HRSV
during 4 days pi and control without PZ
shown large syncytium. Neutralization
assay using selected HRSV clinical
samples are undergoing. This study
is extremely important due to high
annual incidence of HRSV infections
and high spending on public health.
BV1250 - STANDARIZATION OF A
RAPID ONE-STEP RT-PCR ASSAY FOR
DETECTION OF BRAZILIAN ARENAVIRUS
Pádua, M., Machado, A.M., Souza, W.M.
, Machado, A.R.S.M., Figueiredo, L.T.M.
Centro de Pesquisa em Virologia Fac Med Rib Preto - USP, CPV - FMRP/
USP, Av. Bandeirantes - 3900 - Monte
Alegre - Ribeirão Preto - 14040040
E-mail: michellydepadua@hotmail.
com
Arenaviridae Arenavirus are cause of
hemorrhagic fevers with a high case
fatality rate. In South America, the most
important Arenaviruses are: Junin in
Argentina, Guanarito in Venezuela,
Machupo in Bolivia and Sabiá in
Brazil. These viruses are transmitted
to humans by exposure to aerosols of
rodent excreta, or by direct contact
with infectious material from these
animals. In Brazil, little is known about
Arenaviruses. Recently, our research
group has developed an ELISA for
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detection of antibodies to Arenavirus.
In the present study, we show a
new one-step reverse transcriptionpolymerase chain reaction (RT-PCR)
for Arenavirus. For the test, degenerate
primers were designed to anneal in
highly conserved regions of the gene
of nucleocapsid protein of American
Arenavírus (Amapari, Sabiá, Junin and
Flexal). The method was developed
using Amapari virus (AMAV) as
the positive control. The virus was
cultivated in Vero E6 cells, and RNA
extracts of AMAV were submitted to
the RT-PCR, showing amplicons of 324
nucleotides. This amplicon was cloned
in vector TOPO 2.1 (Invitrogen) and
it was quantified, diluted and used
in the RT-PCR at different dilutions
to establish a standard curve that
allows quantify tested material. It was
determined that the limit detection of
the test was 10 copies/μl of viral RNA.
Other RNA viruses, such as Coxsackie
B5, Influenza A, Human Respiratory
Syncytial
virus
A,
Hantavirus
Rio
Mamore,
Parainfluenza-2,
Metapneumovirus, Oropouche, and
Rhinovirus-39 were tested by the
one step RT-PCR in order to verify
the specificity of the test, and showed
negative results. This one-step RT-PCR
is reproducible, specific and sensitive,
being an useful tool for diagnosis and
research with Arenavirus. Financial
Support: FAPESP.
BV1254 - STUDY OF THE MECHANISM
OF ACTION OF MONOTERPENES AND
AMINOMETHILNAPHTHOQUINONES
IN THE IN VITRO REPLICATION OF
HERPES SIMPLEX VIRUS TYPE 1
Ribeiro, C.P., Meneses, L., Vargas, M.,
Barros, C., Martins, V.G., Giongo, V.A.,
Amorin, L.M.F., dos Santos, T.F.Q., da
Silveira, F.C.A., Silva, G., Neves, A.,
Macedo, N.P.V., Ribeiro, M.S., Pinto, A.,
Paixão, I.C.P.
Universidade Federal Fluminense,
UFF, RUA OUTEIRO DE SÃO JOÃO
BATISTA S/N, CENTRO-NITERÓIRJCEP-24020-140
E-mail:
[email protected]
Virus infection by herpesvirus
simplex type-1 may cause different
diseases, such as cutaneous, genital
infections and encephalitis. Acyclovir
is the reference compound used in
antiviral therapy. With the emergence
of drug-resistant strains of HSV
against the current antiviral drugs,
new antiviral agents, especially those
with different modes of action, are
urgently needed. In this work, we
studied the mechanism of action of
perillyl alcohol (POH) and acid (PA),
and the aminomethilnaphthoquinones
derivatives in the in vitro replicatiton of
herpes simplex type 1. Vero cells were
grown on 96, 24 or 6 well plaques in
DMEM medium. The HSV-1 strain used
was KOS, which is sensitive to acyclovir.
Our results demonstrate that the
substances tested in this work showed
higher anti-HSV-1 activity than ACV
in the concentration range that were
not cytotoxic to Vero cells. However,
the substances did not show virucidal
activity, so they do not inactivate the
viral particule. According to the value of
Selective index (S.I.) the most promisor
substance between the monoterpenes
was PA (S.I. - 9568,42), and among
the
aminomethilnaphthoquinones
derivatives was the substance R423
(S.I. - 1525,29). We observed that the
synthetic substances can’t inhibit the
activity of the Na+K+ ATPase pump.
The substances which have chlorine
radical, R423 and R424, may be acting
in the pretreatment phase, and that
R424 can also act in the γ phase of
the viral multiplication cycle. The
substance R425 may be affecting
penetration of the virus into the host
cell. The in silico analysis of ADMET
parameters revealed that substances
that showed lower theoretical risk of
mutagenicity, tumorigenicity, irritant
and reproductive effects were R423
and R424. The substance R424 showed
the highest values of drugscore and
druglikeness. Our data show that all
these substances have a promising
profile for further in vitro and in vivo
assays toward development of new
strategies in anti-HSV-1 therapy.
BV1256 - EXPRESSION OF ALIX-V
IMPAIRS CD4 DOWNREGULATION
BY HIV-1 NEF
Amorim, N.A., Silva, E.M.L., da Silva,
L.L.P.
Ribeirão Preto - USP, FMRP - USP,
Av. Bandeirantes, 3900 - Monte
Alegre - CEP: 14049-900 Ribeirão
Preto - SP
2. Faculdade
de
Ciências
Farmacêuticas de Ribeirão Preto
- USP, FCFRP - USP, Av. do Café, s/
nº. - Campus Universitário - CEP:
14040-903 Ribeirão Preto - SP
Nef is an HIV accessory protein
that promotes viral replication and
progression to full-blown AIDS. A
prominent function of Nef is the
downregulation of specific cell surface
proteins related to immune response,
such as CD4 and MHC-I. It is believed that
Nef redirects its targets to degradation
in lysosomes by interfering with
intracellular membrane trafficking,
but the mechanisms are not fully
Basic Virology: BV
99
understood. Our previous research
indicated that Nef induces recruitment
of CD4 to multivesicular bodies (MVBs)
through a pathway that requires
functional ESCRT machinery and is
independent of cargo ubiquitination. It
has been shown that Nef interacts with
Alix/AIP1, an ESCRT associated protein
that assists with cargo recruitment
and intralumenal vesicle formation in
MVBs. Here we investigated the role
of Alix as a “connector” in the process
which Nef induces the degradation
of CD4. For this, we overexpressed
a truncated version of Alix (Alix
V-domain) that contain the potential
Nef binding motif, but lacks the
motifs required for interaction with
ESCRTs. We reason that Alix V-domain
would act as a dominant negative by
preventing interaction of Nef with
endogenous Alix molecules. We used a
combination of flow citometry, western
blot and pulse-chase analyses to test
this. Interestingly, we observed the Alix
V-domain impairs the degradation, but
not the internalization of CD4 by Nef.
Moreover, we show that this effect of
Alix VD is not due to a general block
in lysosomal targeting, as indicated
by the lack of effect in EGF uptake
and degradation assays. In summary,
our data indicates that induced
endocytosis and lysosomal targeting
are two separate functions of Nef in
CD4 downregulation and that the
latter requires interaction with Alix.
Financial Support: FAPESP
BV1265 - HIV NEF PROTEIN ALTER
PHENOTHYPING ASSAYS
Poeys, S.C., Mendonça, L.M., da Costa,
L.J.
UNIVERSIDADE FEDERAL DO RIO DE
JANEIRO, UFRJ, CCS - IMPG - Cidade
100
Basic Virology: BV
Universitária, Ilha do Fundão, RJ
- CEP: 21941-590 E-mail: sandro_
[email protected]
Nef is an accessory protein of HIV
and several roles have been ascribed
for this protein; including the down
regulation of CD4 and MHC-I from the
cell surface. All functions attributed
to Nef are achieved by interaction
with different cellular proteins. Nef
also interacts with viral proteins such
as GagPol and Protease. Most of the
phenothyping assays for HIV-1 are
based on viral strains deleted on the Nef
gene. Since Nef interacts with Protease
its absence could not represent the
real EC values obtained in this assays
when assaying protease or maturation
inhibitors. The objective of this work
is to demonstrate that Nef influences
phenothyping assays. For this, 293T
cell were transfected with two
different infectious clone (NL-4.3 and
NL-4.3ΔNef) and treated with different
concentrations of the protease inhibitor
lopinavir; the produced viruses in each
condition were titrated using TZM-bl
indicator cell line (β-Gal as reporter)
and the IC50 was calculated using the
GraphPad Prism. The IC50 of the wild
type viral isolate was 2,8nM while for
the nef-deleted virus was 4,8nM. This
difference was statistically significant
(p=0,043). We found that virus deleted
in Nef have a lower number of infectious
particles produced, however, this virus
is lower sensitive to the protease
inhibitor was demonstrated by the
higher IC50. Phenothyping assays
using an lymphocytic cell line (MOLT)
will be made to confirm these results.
BV1268 - THE INTERACTION
BETWEEN THE VIRAL NEF AND THE
CELLULAR ALIX/AIP-1 PROTEINS
HAVE A ROLE FOR THE INCREASE IN
HIV-1 INFECTIVITY
da Silva, G.P.D., Mendonça, L.M., Costa,
L.J.
Janeiro, UFRJ, Cidade Universitária,
Rio de Janeiro - RJ E-mail:
[email protected]
Nef is an accessory protein expressed
early during the replication cycle of the
primate lentiviruses HIV and SIV. This
protein plays essential roles in viral
infectivity and disease progression
to AIDS. It is established that Nef
has different cellular interaction
partners to perform various functions
attributed to this protein, however
the function related to the increase in
virion infectivity and viral replication
in
primary
lymphocytes
and
macrophages has not been established.
Nef can mediate down-regulation
of CD4 cell surface expression and
prevent apoptosis of HIV-1-infected
T cells. It has been reported that the
multi-modular cellular protein Alix/
AIP1 has a central and required role
in targeting the ESCRT machinery to
the midbody for membrane abscission
in cytokinesis, and is essential for
the budding of certain enveloped
viruses such as HIV-1. We have been
investigating the role of the interaction
between Nef and the cellular protein
Alix/AIP1. Nef interaction with Alix/
AIP1 have been previously mapped
to the amino acid residues YPLTF
present at the positions 135-139 on
the C-terminal of the Nef protein from
the HIV NL4-3 isolate. The objective
of this study was to elucidate whether
this interaction directly influences the
increase in viral infectivity. For this,
assays were carried out in Alix/AIP1 siRNA knockout cultures of Hella
cells. The knockout was performed
using transfection with plasmid pBAsi
Alix/AIP1 # 2 and pBAsi Alix/AIP1
expressing microRNA against Alix/AIP1. Alix/AIP-1 expression was monitored
by western blotting of cell lysates. In
parallel, experiments were performed
in Hek293T cells and we could
observed that this cell linage does not
express Alix/AIP-1 during the first four
passages in culture. Virus produced in
these cells during the first passages
were dependent on Nef for the increase
in viral infectivity. Interestingly,
levels of Alix/AIP-1 increased at each
passage and this correlated with a less
dependence on Nef for the production
of fully infectious HIV-1 viral progeny.
These results are being confirmed in
Alix/AIP-1 knockouts of HeLa cells.
Our results demonstrate that Alix/
AIP-1 plays a role for the increase in
HIV-1 infectivity by Nef and that the
expression of this cellular protein has
a temporal regulation in Hek 293T a
cell line highly use in studies of several
aspects of the replication of HIV-1.
BV1271 - BRADYKININ ENHANCES
RNA VIRUS INFECTION IN HUMAN
BRAIN MICROVASCULAR ENDOTHELIAL CELLS
PAPA, M.P., RUST, N.M., Scovino, A.M.,
Da Silva, Carneiro, M.M., Silva, C.,
Eduardo, C. Peçanha, L.M.T., Scharfstein,
J., Arruda, L.B.
Filho, 373, CCS - Bloco I, Lab ISS
- 048
2. Fundação
Oswaldo
Cruz,
FIOCRUZ, Av. Professor Moraes
Rego, s/n - Campus da UFPE Recife
3. Fundação
Oswaldo
Cruz,
Basic Virology: BV
101
FIOCRUZ, Av. Augusto Lima, 1715
- Barro Preto Belo Horizonte MG E-mail: michelle_premazzi@
hotmail.com
Introduction: Bradykinin (BK) is
a vasoactive peptide generated
in
pathophysiological
conditions
including inflammation associated to
infection. Activation of BK receptors
(BK1R and BK2R) in endothelial
cells (EC) modulates cell survival and
production of inflammatory mediators
and may be involved in the control of
virus replication. Dengue virus (DENV)
and Sindbis virus (SINV) belong to
Flaviviridae and Togaviridae families,
respectively, and both present ssRNA
as genome. SINV infection causes rash
and arthritis, characterized by swelling,
and leukocyte infiltrate into the joint
due to inflammatory alterations of
the underlying endothelium. DENV
infection causes symptoms ranging
from classical fever to dengue
hemorrhagic fever, associated to
inflammation and altered vascular
permeability caused by changes in
endothelial cells. Although clinically
different, infection by DENV and SINV
involves the release of inflammatory
mediators and alterations in the
vascular endothelium. We investigated
if these infections would influence the
response of ECs to BK and the effect of
BK on the viral replication. Methods:
Human brain microvascular endothelial
cells (HBMECs) were infected with
SINV or DENV and the expression of
BKR mRNA and protein were analyzed
by qRT-PCR and flow cytometry,
respectively. Virus replication was
evaluated by plaque assay and qRTPCR. Cell death was analyzed by
flow cytometry. Results: Infection of
HBMECs by SINV and DENV induced
an increased expression of BK1R and
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Basic Virology: BV
BK2R. BK added to cultures increased
viral replication in a BK2R and PI3K
dependent manner. Apoptosis was
detected in HBMECs infected with both
viruses and addition of BK delayed
this process. Conclusions: Infection of
HBMECs with SINV or DENV increases
the responsivity to BK. The signaling
mediated by the peptide enhances viral
replication, possibly, by prolonging the
survival of infected cells through the
induction of anti-apoptotic pathways.
Financial support: CNPq, FAPERJ.
BV1282 - STUDY OF THE EFFECTS OF
PROTEIN TRANSLOCATION HSLU7
CELL AFTER INFECTION OF YELLOW
FEVER VIRUS
Gavioli, A.F., Ribeiro, M.R., Terzian,
A.C.B., Nogueira, M.L.
Universidade Estadual Paulista,
UNESP/IBILCE,
Faculdade
de
Medicina São José do Rio Preto,
FAMERP, E-mail: arieligavioli@
ig.com.br
Yellow fever virus (YFV) causes
disease with significant morbidity and
mortality in tropical regions. Viruses
such as YFV use several strategies
for recruitment and alteration of
the biochemical cellular process.
Replication of YFV and the interactions
between viral and cellular proteins are
widely unknown. The cellular splicing
mechanisms is essential to diversify
the gene expression and increase
it’s proteomic potential. The cellular
protein hSlu7 plays an important
role in the second catalytic reaction
step of the alternative splicing. Some
physiological conditions of cellular
stress can alter the concentrations
of the protein causing nuclearcytoplasmic translocation and affects
cellular splicing. We initially detected
an interaction between NS5 protein of
YFV and hSlu7 in a yeast two-hybrid
screening. We also further characterize
this interaction biochemically. Now
we show that in cells infected with
YFV hSLU7 shuttles from nucleus
to cytoplasm, as occurs in cells
submitted to stress. The translocation
of this protein between nucleus and
cytoplasm may represent a viral
mechanism of regulation of the cellular
gene expression. We will now further
analyze the interaction between human
protein hSlu7 and the viral protein NS5,
as well as the effects of this interaction
on the cellular splicing mechanisms
after YFV infection. Financial Support:
CAPES, CNPq
BV1289 - QUANTIFICATION AND
DETECTION OF EPSTEIN-BARR DNA
IN ADENOIDS, PALATINE TONSILS,
NASOPHARYNGEAL
SECRETIONS
AND PERIPHERAL BLOOD OF
PATIENTS WITH CHRONIC ADENOTONSILLAR DISEASES BY REAL-TIME
PCR
Pestana, N.F.
Faculdade de medicina de Ribeirao
Preto , fmrp - usp, Av. Bandeirantes
3900 - Monte Alegre
The
Epstein-Barr
virus
(EBV)
is a DNA virus belonging to the
Gamaherpesviridae family that cause
infection in 95% of humans. This
virus is the main agent of infectious
mononucleosis.
EBV
infects
B
lymphocytes, where it can cause a
lytic infection, with production of new
viruses, or establish latency, keeping
its genome in episomal state with
expression of few genes. Adenotonsillar
chronic diseases have a large impact
on public health, however the genesis
of these diseases is poorly understood.
How EBV can persist in palatine tonsils
and adenoids, the aim of this study was
to evaluate the impact of the presence of
EBV in high and small titles in palatine
tonsils, adenoids, nasal secretions
and peripheral blood with the degree
of inflammation of these tissues in
patients with chronic adenotonsillar
diseases. For this, samples were
collected from 121 patients with
chronic
adenotonsillar
diseases
without respiratory symptoms who
underwent adenotonsillectomy in the
HCRP. The detection and quantification
of EBV was performed by qPCR, after
validation by amplification of β-actin
gene. The frequency of detection of
EBV was 0,71%, and 81 patients had
detectable virus in the palatine tonsils,
while 68 had EBV in adenoids, 28 in
nasal secretions and 24 in peripheral
blood. The median of the viral load was
2,6x105 genome copies / g of tissue
in the palatine tonsils and 4,0x105
copies / g in the adenoids. Although
approximately 50% of patients had
high viral load of EBV in adenotonsillar
tissue (> 10x105 copies / g), there was
no relationship between high viral
load in these tissues with the degree
of inflammation of the palatine tonsils
and adenoids, which refutes EBV as
a cause of adenotonsillar chronic
hypertrophy. However, the presence of
high viral loads of EBV in the palatine
tonsils and adenoids was significantly
associated with co-detection of this
virus in nasal secretions and peripheral
blood, illustrating the importance of
these organs in viral spread. Indeed,
we can conclude that the palatine tonsil
and adenoid of patients with chronic
adenotonsillar disease are reservoirs
of EBV, which often allow the viral
replication and viral shedding.
Basic Virology: BV
103
BV1291 - PORPHYRINS TREATMENT
REDUCE DENGUE VIRUS REPLICATION IN HUMAN HEPATOCYTES AND
MACROPHAGES
Figueiredo, C.M., Neris, R.L., CruzOliveira, C., Penna, L.P., Da Poian, A.T.,
Assunção-Miranda, I.
de
Janeiro,
UFRJ,
Avenida
Brigadeiro Trompovisky,CCS,Bloco
D,Laboratório de Imunidade e
Inflamação E-mail: camilam.f@
hotmail.com.br
Dengue virus (DenV) is an arbovirus
of Flaviviridae family. High incidence
of dengue desease put in evidence
the necessity to develop strategies for
infection prevention and treatment.
Characterization of molecules with
anti-dengue activity presents an
alternative to control the disease.
Previously, our group demonstrated
that pre-incubation of DenV with
heme and others metalloporphyrins,
like
cobalt-protoporphyrin
IX
(CoPPIX) and tin-protoporphyrin IX
(SnPPIX), results in DenV particle
inactivation. The aim of this work was
to investigate porphyrins’ ability to
control DenV replication in infected
human hepatocytes (HepG2 cells) and
peripheral blood macrophages. HepG2
cells were maintained in culture at 37°C
in a 5% CO2 atmosphere and infected
with DenV at a very low multiplicity of
infection (MOI) of 0,2. After 6 hours of
infection the cells were treated for 40
hours with heme 75 µM, CoPPIX 25 µM
or SnPPIX 25 µM. Viability of HepG2
cells and the viral replication was
assessed 72 hours post-infection (hpi)
by MTT assay and quantification by
plaque assay in baby hamster kidney
cells (BHK), respectively. Treatment
with CoPPIX and heme provided a
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Basic Virology: BV
complete protection of infected HepG2
cells when compared to the levels of
cell viability of the control not infected.
Treatment with heme and CoPPIX also
promoted a decrease in the number of
infectious particles in the supernatant
by two orders of magnitude. Protection
effect was not evident in SnPPIX posttreatment. Porphyrins concentration
tested did not alter significantly the
viability of uninfected HepG2 cells.
Human macrophages were infected
with a MOI of 2 and treated with
heme, CoPPIX and SnPPIX for 48hs,
immediately after infection. Secretion
of TNF and RANTES in cell culture
supernatants were measured by ELISA.
The treatment provided a reduction of
approximately 50% of TNF and RANTES
in the supernatant when compared to
untreated and infected macrophages.
Based on these results we conclude
that porphyrins are molecules able to
protect the infected cells and decrease
the inflammatory response induced
by infection. Furthermore, these
compounds are capable to reduce DenV
replication, suggesting a therapeutic
potential for this class of molecules.
Financial Support: CNPq; FAPERJ
BV1307 - TRANSFECTION-NEUTRALIZATION WITH ANTIBODIES:
AN ANTIVIRAL RABIES THERAPY
MODEL
Castilho, J.G., Batista, H.B.C.R.,
Rodrigues, A.C., Carnieli-Junior, P.,
Oliveira, R.N., Silva, C.R., Caporale,
G.M.M., Carrieri, M.L., Kotait, I.,
Brandão, P.E.
1. Instituto Pasteur, IP, Av. Paulista,
393, Cerqueira Cesar
2. Faculdade
de
Medicina
Veterinária e Zootecnia da USP,
FMVZ/USP, Av. Prof. Dr. Orlando
M. Paiva, 87, Cidade Universitária,
São Paulo E-mail: juliana.
[email protected]
Rabies, which is a lethal human disease,
continues to represent a serious
public health problem, particularly in
developing countries where canine
rabies is endemic, and is responsible
for 55,000 human deaths every year.
In light of this, the present study
sought to investigate the efficiency
of transfection with antibodies
as a possible antiviral therapy for
rabies. We used two anti-rabies virus
polyclonal antibodies produced in
equines: the first was from unpurified
hyperimmune serum (total antibody)
and the second was a commercial
antibody used in post-exposure antirabies prophylaxis in humans which
contains only the Fab’ fragment of
purified specific immunoglobulins.
To investigate the effectiveness of
transfection-neutralization
with
these antibodies, N2A cells were
infected with different infectious
doses (0.1; 1.0; 10 and 100 TCID50)
of the rabies virus (antigenic variant
2) in 96-well plates and incubated
for 24 hours. The infected cells were
then transfected with the different
antibodies using a cationic reagentlipofectamine 2000 (treatment group);
for the negative transfection control,
only minimum essential medium was
used (control group). After incubation
for 11 hours, the plates were fixed
with 80% acetone and analyzed by
direct immunofluorescence using a
fluorescein isothiocyanate-conjugated
antinucleocapsid rabies antibody. The
ability of the antibodies to neutralize
the rabies virus was determined by
counting the number of fluorescent
foci for each of the infectious doses
and comparing the results with the
control group to give the percentage
inhibition. Fluorescent focus inhibition
was 90.3%, 89.2%, 100% and 100%
for the total antibody and 65.7%,
94%, 100% and 100% for the Fab’
antibody in cells infected with 100,
10, 1.0 and 0.1 TCID50, respectively.
In the presence of a high viral load
(100 TCID50), inhibition with total
antibody (90.3%) was greater than
with purified antibody (65.7%). When
cells were infected with lower viral
loads, fluorescent focus inhibition for
both antibodies was similar. These
findings indicate that transfection
with antibodies can be used as a tool
in antiviral therapy and as a means
of potentially treating rabies. This
mechanism could also serve as a model
for other viral infections. Financial
Support: FAPESP
BV1317 - ANTIVIRAL ACTIVITY OF
PORPHYRINS ON ENVELOPED VIRUS
REPLICATION
Neris, R.L.S., Cruz-Oliveira, C., Penna,
L.P., Figueiredo, C.M., Da Poian, A.T.,
Bozza, M.T., Assunção-Miranda, I.
Instituto de Microbiologia Paulo de
Góes, IMPPG, UFRJ, Rio de Janeiro
Instituto de Bioquímica Médica,
IBqM, UFRJ, Rio de Janeiro E-mail:
[email protected]
Viral envelope is involved in binding
and fusion to host cell. These
interactions are essential in promoting
an efficient enveloped virus entrance
and replication in target cells. Each
year million people widespread the
world are infected by enveloped
viruses. Among these viruses we can
include Dengue Virus (DenV), Yellow
Fever (YFV), Sindbis (SinV), Mayaro
(MayV) and Vesicular Stomatitis
Basic Virology: BV
105
Virus (VSV). Antiviral compounds
that targets viral envelope membrane
could inhibit a wide range of enveloped
viruses. Porphyrins are amphipathic
organic compounds with a tetrapirrolic
ring that can carry metallic ions on
its center. Our previous group data
shows that porphyrins like Heme,
Tin-Protoporphyrin IX (SnPPIX) and
Cobalt-Protoporphyrin IX (CoPPIX)
have antiviral activity against DenV and
YFV. The aim of this work was to test the
antiviral activity of porphyrins on other
enveloped virus like SinV, MayV and
VSV. Viral stocks were pre-treated with
different concentrations (50, 100, 200,
300 and 500 µM) of Heme, CoPPIX and
SnPPIX for one hour at 37ºC in the dark.
After that, viral titer was obtained by
plaque assay in BHK cells. Cell viability
of treated and non-treated virus
infected BHK cells was determined by
MTT assay. Cytopatic effect on BHK
cells was observed through optical
microscopy and the presence of viral
proteins on BHK cells was assessed by
fluorescence microcopy. Pre-treatment
with CoPPIX 200 and 300 µM was
able to reduce MayV, SinV and VSV
titer at least in 4 orders of magnitude.
CoPPIX 500 µM treatment promoted
a reduction to undetectable levels of
SinV, MayV and VSV particles. Antiviral
activity of SnPPIX was only observed
in MayV infection. SnPPIX 200 μM
promotes a reduction on the number of
MayV infectious particles at 5 orders of
magnitude. Different from that of DenV
and YFV, pre-treatment with heme was
not efficient to reduce SinV, MayV and
VSV infectivity. CoPPIX protect cells
from cytopatic effect induced by SinV
and MayV infection, while SnPPIX was
able to protect cells only from MayV
infection in BHK cells. This protection
was not evident to heme pre-treatment.
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Basic Virology: BV
Viral proteins weren’t detected by
immunofluorescence microscopy in
cells infected with CoPPIX 500 µM pretreated SinV. Given these evidences, we
conclude that SnPPIX and CoPPIX are
capable to inactivate others enveloped
viruses besides DenV and YFV. Other
studies must be made to understand
the porphyrins mechanism of action.
Financial Support: Faperj and CNPq
BV1350 - IMMUNE RESPONSE TO
APEU VIRUS (ORTHOBUNYAVIRUS)
INFECTION IN MURINE MODEL.
Chaves, A.T., Bozzi, A., Costa, F.C., Santos,
J.R., Oliveira, J.G., Corrêa-Oliveira, R.,
Ferreira, P.C.P.
6627 - Bloco F4-127 - Pampulha
Cx. Postal 486
2. Centro de Pesquisas René
Rachou/Fundação
Oswaldo
Cruz , CPqRR/FIOCRUZ, Avenida
Augusto de Lima 1715 _ Barro
Preto_ BH- MG CEP: 30190-002
E-mail:
chaves.anathereza@
gmail.com
Arthropod-borne viruses have emerged
as a major human health concern.
Viruses transmitted by mosquitoes
are the cause of the most serious and
widespread human diseases worldwide
and are ubiquitous in areas where
humans live. The Apeu virus (APEUV)
belongs to group C arboviruses and
is a member of Bunyaviridae family
and Orthobunyavirus genus. Although
APEUV has already been isolated for
more than five decades, the role of the
in vivo or in vitro immune response in
the pathogenesis of APEUV remains
unknown. The aim of this study was to
investigate the infection kinetics of as
well as the immune response profile
triggered by APEUV infected Swiss
mice. Animals were experimentally
infected with APEUV at different times
(12, 24, 48, 72 and 120 hours). The
spleen cells proliferative response
was evaluated in vitro using the
MTT
[3-(4,5-dimethylthiazolyl-2)2,5-diphenyltetrazolium
bromide]
colorimetric assay. Our data have
shown that at 48 hours post-infection
with APEUV proliferative response
was increased when compared to the
other periods evaluated. A significant
decrease was observed in proliferative
response 72 hours post-APEUV
infection. Cytometric Bead Array
(CBA) immunoassay kit was used for
semi-quantitative analysis of several
cytokines (IL-2, IL-4, IL-6, IFN-gamma,
TNF, IL-17A and IL-10) in the serum
of infected mice. We observed an
increase (p <0.05) in the secretion of
pro-inflammatory cytokines (IL-6, IFNgamma, TNF) in serum of animals 72
hours post-APEUV infection. With the
MTT method, it is suggested that the
peak of cell proliferation was reached
in 48 hours of culture, after in vitro
stimulus. APEUV infection causes a
pro-inflammatory profile in 72 hours
post-infection. We suggest the period
of 72 hours for further analyses of
immunological profile upon infection
with the virus. Financial support:
CNPq, CPqRR/FIOCRUZ
BV1366 - EFFECT OF THE TOSPOVIRUS NSS PROTEIN ON THE
TRANSCRIPTIONAL ACTIVITY OF
THE DIFFERENT ANTICARSIA GEMMATALIS NUCLEOPOLYEDROVIRUS
BACULOVIRUS
PROMOTERS
IN
INSECT CELL LINES
Vieira, A.M., Morgado, F., Nagata, T.,
Ribeiro, B.M., Resende, R.O., Oliveira,
V.C.
Universidade de Brasília, UnB,
Campus Universitário Darcy Ribeiro
RNA silencing-inhibiting proteins have
been identified in several plant viruses.
Among the best-studied examples are
the NSs (non-structural) of the Tomato
spotted wilt virus – TSWV. Here,
wes evaluated the influence of the
protein NSs on the activity of several
baculovirus promoters in Trichoplusia
ni (BTI-Tn-5B1-4) and Anticarsia
gemmatalis (UFL-AG-286) insect cell
lines. Recombinant baculoviruses
derived from Anticarsia gemmatalis
nucleopolyedrovirus were constructed
to place the luciferase gene under the
control of promoters active in the early,
ie-1 (vAgie1) and gp64 (vAggp64);
early/late, vp39 (vAgvp39,); or very
late, ppolh (vAgppolh,) stages of virus
replication. As well, a recombinant
baculovirus carrying the TSWV-NSs
gene under the control of the viral
polyhedrin (polh) gene promoter was
previously constructed. The cells were
separately infected at a MOI of 10 per
cell with vAgie1, vAggp64, vAgvp39
or vAgppolh or co-infected (MOI of 5
for each virus) with vAcNSs. After 12,
24, 48 and 72 h post-infection (p.i.),
the cells were collected and lysed. The
luciferase activity was measured by
Luciferase Assay Substrate (Promega)
in a Spectramax M5 (Molecular
Devices). A enhanced of 2,4 (vAcNSs +
vAgie1) and 2,23 (vAcNSs + vAgvp39)
times in the luciferase activity was
obtained in BTI-Tn-5B1-4 cells when
vAcNSs was co-infected compared
to vAgie1 and vAgvp39 alone, at 48
h pi. At 72 h p.i. the co-infection with
vAcNSs + vAgppolh showed significant
increase of 14,65 times. This increase
Basic Virology: BV
107
in the luciferase activity by vAcNSs
was not observed in UFL-Ag cell line.
In conclusion, the silencing suppressor
TSWV-NSs protein enhanced the
expression of the luciferase protein
under the control of the ie1, vp39 and
ppolh AgMNVP promoters.
BV1387 - INHIBITION OF THE UBIQUITIN-PROTEASOME
SYSTEM
DECREASES HIV-1 RELEASE BUT
INCREASES THE INFECTIVITY OF A
NEF-DEFICIENT VIRUS
Cunha, M.S., Costa, L.J.
Filho, 373, CCS Bloco I, subsolo -sala
048 E-mail: marcela.scw@gmail.
com
Nef is an accessory protein encoded
only by primate lentiviruses (HIVs
and SIVs) and is associated with
pathogenicity and the increase in viral
infectivity by a still not characterized
mechanism. Since Nef is expressed
early during the viral replication cycle,
studies on Nef focused mainly on its role
during the initial stages. Our group has
demonstrated that Nef binds to GagPol
and thus can act during the late stages
of the replication cycle of HIV-1 and
SIVs. A previous work demonstrated
that budding of HIV-1 is dependent
on ubiquitin-proteasome system. To
further investigate the role of Nef in
this step of viral cycle, we compared
the release and infectivity of HIV-1
WT and HIV-1 ΔNef in the presence
or absence of a proteasome inhibitor
(MG132). For this purpose, HEK 293T
cells were transfected with HIV-1 WT
(wild type) or Nef-deficient HIV-1
(ΔNef) vectors. After 24h, the medium
was changed and 5µM of MG132 was
added. Supernatants and lysates were
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Basic Virology: BV
collected after 5 hours and analyzed by
Western blot (WB). To estimate viral
infectivity, supernatants were used
to infect TZM-bl indicator cells. The
release of HIV-1 WT was reduced by
2.2-fold upon treatment with MG132,
while for HIV-1 ΔNef the reduction was
greater, achieving 3.8-fold. The viral
infectivity for both viruses was equally
reduced by a 2.8–fold when treated
with MG132. Therefore, for HIV-1
ΔNef, despite the fewer amount of
viruses being released, the infectivity
is proportionally increased, while for
HIV-1 WT the infectivity is reduced
and cannot be explained only by the
reduction of viral release. Our results
indicate that both WT and nef-deleted
viruses are dependent on ubiquitinproteasome system for efficient viral
release as previously described for
HIV-1 WT. However, these viruses
behave differently regarding levels of
release and infectivity, indicating that
Nef acts during this stage of the viral
replication cycle.
BV1391 - HIV PROMOTES BIP
INCREASED EXPRESSION BY UPR
ACTIVATION IN CELLS FROM HIV-POSITIVE PATIENTS UNDER DIFFERENT
ANTIRETROVIRAL THERAPIES
Borsa, M., Dahmer, M., Tonello, Y., Pinto,
A.R.
Universidade
Federal
de
Santa Catarina, UFSC, Campus
Universitário Reitor João David
Ferreira Lima, Florianópolis, SC,
Brazil E-mail: nanaborsa@gmail.
com
The Unfolded Protein Response (UPR)
is a mechanism initiated whenever
protein folding in the endoplasmic
reticulum (ER) is compromised. The
UPR pathway has 3 sensors of ER
stress, PERK, IRE1, and ATF6, which
bring cell back to homeostasis through
transcriptional
and
translational
controls that promotes an increased
expression of chaperones genes as
BiP. Viral infection can influence
protein folding and activate UPR signal
pathways, but the HIV infection impact
on UPR activation remains unclear. The
aim of this study was to analyze the HIV
infection impact on the BiP expression
in cells from HIV-positive individuals
under different antiretroviral therapy.
B lymphocytes, T CD4+ lymphocytes
and monocytes were obtained from
10 healthy individuals and 15 HIVpositive patients clustered in 3
groups: (1) treatment-naive, (2) under
antiretroviral therapy with or (3)
without protease inhibitors. Protein
lysates from these cells were submitted
to Western Blot analysis through
anti-BiP MAb. The bands obtained
after development were quantified
(ImageJ) and data was submitted
to ANOVA statistical analysis. Cells
from HIV negative volunteers showed
similar BiP expression between
cellular subpopulations. In HIV
positive individuals there was a higher
expression of BiP in B lymphocytes
and T CD4+ lymphocytes. Analysis of
bands intensity quantification showed
significantly higher expression of BiP
in B lymphocytes from HIV positive
volunteers in comparison with cells
from non-infected individuals. These
non-equal phenotypes suggest that
HIV infection induces ER stress and
consequent increased expression of
BiP as an effort to refold unfolded
proteins. In B lymphocytes BiP
increased expression may be related
to neighboring cell UPR activation
through infected cells signaling, which
is amplified by the secretory properties
of B lymphocytes. This activation does
not seem to be related to viral loads
presented by the HIV-positive patients
or their therapy outlines. Financial
support: CAPES and CNPq.
BV1433 - PHYLOGENETIC ANALYSIS
OF PANDEMIC H1N1 INFLUENZA A
VIRUS CIRCULATING IN THE SOUTH
AMERICAN REGION
Goñi, N., Moratorio, G., Coppola, L.,
Ramas, V., Comas, V., Soñora, M.,
Chiparelli, H., Cristina, J.
Facultad de Ciencias - Universidad
de la Republica, UDELAR, Igua 4225
- 11400 Montevideo Ministerio
de Salud Publica, MSP, Av. Navarro
3051, 11200 Montevideo
The first influenza pandemic of this
century was declared in April of 2009,
with the emergence of a novel H1N1
Influenza A virus strain (H1N1pdm).
Understanding the evolution of
H1N1pdm populations within the
South American region is essential
for studying global diversification,
emergence, resistance and vaccine
efficacy. In order to gain insight into these
matters, we have performed a Bayesian
coalescent Markov Chain Monte Carlo
analysis of hemagglutinin (HA) and
neuraminidase (NA) gene sequences of
all available and comparable HA and NA
sequences obtained from H1N1pdm
IAV circulating in the South American
region. High evolutionary rates and
fast population growths characterize
the population dynamics of H1N1pdm
strains in this region of the world. A
significant contribution of first codon
position to the mean evolutionary
rate was found for both genes studied,
revealing a high contribution of nonsynonymous substitutions to the
mean substitution rate. In the 178
Basic Virology: BV
109
days period covered by these studies,
substitutions in all HA epitope regions
can be observed. HA substitutions
D239G/N and Q310H have been
observed only in Brazilian patients.
While substitution D239G/N is not
particularly associated to a specific
genetic lineage, all strains bearing
substitution Q310H were assigned to
clade 6, suggesting a founder effect.
None of the substitutions found in the NA
proteins of H1N1pdm strains isolated
in South America appears sufficiently
close to affect the drug binding pocket
for the three NA inhibitor antivirals
tested. A more detailed analysis of NA
proteins revealed epitope differences
among 2010 vaccine and H1N1pdm
IAV strains circulating in the South
American region.
BV1435 - DEVELOPMENT OF QUICK
STANDARTIZATION
PROCEDURE
OF PURIFICATION CONDITIONS
FOR ORDER CAUDOVIRALES BACTERIOPHAGE BY ION EXCHANGE
CHROMATOGRAPHY
Dias, R.S., Souza, F.O., Silva, L.C.F.,
Duarte, V.S., Assao, V.S., Fonseca,
L.A.B.V., Oliveira, L.L., Silva, C.C., Silva,
E.A.M., de Paula, S.O.
Universidade Federal de Viçosa,
UFV, Av P. H. Rolfs s/n Viçosa, Minas
Gerais, Brasil E-mail: rosousa318@
yahoo.com.br
Bacteriophages are the most abundant
life forms on earth and have influence
on the microbial world. The activity
of the phage is specific, attacking the
target bacterial cells, without affecting
the normal microbial flora of the host.
We developed a method to standardize
phage purification conditions, which is
capable of providing pure viral particles,
in a faster and less laborious way, using
110
Basic Virology: BV
ion exchange chromatography in a FPLC
system. Tests were conducted in 15 mL
flasks, in different pH values to obtain
the isoelectric point of proteins of the
viral capsid, allowing to determine the
pH at which the net charge of the virus
particle was negative. DEAE-Sephadex
resin was equilibrated with buffers at pH
values to be analyzed (4.5 to 9.5). Viral
suspensions were mixed with buffers
and kept under stirring for at least 30
minutes. The presence of genome of
the bacteriophage in the supernatant
indicated the lack of binding of the
virus to the resin. To elute, low pH
buffer and high concentrations of NaCl
were tested and aliquots were analyzed
for the presence of viruses by agarose
gel electrophoresis. After determining
the
conditions
of
purification,
chromatographic assays were done
connected to a FPLC apparatus. Peaks
were collected and presence and
purity of the viruses were confirmed
by electrophoresis and scanning
spectrophotometer. The bacteriophage
was present in more than one aliquot
(aliquots 21, 22, 23 and 24). However,
aliquot 24 showed higher protein
concentration and purity. Electron
microscopy showed that it is a caudate
virus, therefore belonging to the order
Caudovirales, family Podoviridae. We
can conclude that this is an interesting
technique to purify unknown phage
by FPLC systems, primarily due to the
speed and low cost. Financial support:
FAPEMIG, CAPES
BV1442 - GENETIC DIVERSITY OF
HIV-1 IN A SINGLE ROUND REPLICATION WITH PBMC&ACUTE;S FROM
DIFFERENT DONORS
Soane, M.M., Alkmim, W.T., Santos,
C.M., Peixoto, J.M., Zukurov, J.P.L.,
Simão, L.N., Bueno, J., Janini, L.M.R.
Universidade Federal de São Paulo,
UNIFESP, Rua Pedro de Toledo, 781,
16ºAndar, Vl Clementino, São PauloSP E-mail: michelsoane@hotmail.
com
The HIV-1 has an important genetic
diversity, fact explained by the inherent
infidelity of the reverse transcriptase
enzyme that the virus uses to transcribe
its RNA genome into DNA so that it
may be integrated into the human
genetic material and propagated
along with it. The lack of proofreading
mechanisms, high turnover of virions,
and propensity for recombination also
contributes to the extensive variability
of HIV. These parameters provide the
virus quasispecies with an impressive
capacity to adapt to immunologic or
pharmacologic pressures. The aim of
this study was to assess the genetic
variability of HIV-1 during early
stages of infection. PBMCs from three
healthy donors were infected with
pseudosubtype virus in cell culture.
After a single replication cycle proviral
clones were obtained by Single Genome
Amplification. A 1200pb fragment
obteained from pol gene was sequenced
from all clones. Observed mutations
were not uniformly distributed among
clones occurring more frequently
between the nucleotides 300 to 350,
450 to 500pb and 1000pb to 1050 in
clones generated using PBMCs from
patient 1 with the entropy of 0.3 bit
and between the nucleotides 200
to 400pb, 500 to 650 pb and 800 to
900pb with 0.2, 0.3 and 0.3 entropy,
respectively from patient 2. Finally, the
nucleotides 1 to 50, 350 to 400pb, 450
to 600 and 900 to 950pb (0.35-0.70
entropy) in clones generated using
PBMCs from patient 3. The distribution
of sites where mutations occur is
different between different clones
and PBMCs patients. Our experiments
demonstrated a rapid accumulation of
mutations in pseudotypes suggesting
that HIV-1 may undergo a diversity
expansion soon after transmission as
part of a strategy to regain the genetic
diversity lost during the transmission
bottleneck. Furthermore, the selective
pressure exerted by host on HIV plays
a major role in variability during early
stages of infection. This may contribute
to the successful development of HIV
vaccines, drugs and to monitoring of
HIV infected individuals. Financial
support: FAPESP
BV1464 - DOUBLE-NEGATIVE T
CELLS DURING VACCINIA AND APEU
VIRUS INFECTION IN VITRO
Villani, F.N.A., Silva, T.E., Kroon, E.G.,
Oliveira, J.G., Ferreira, P.C.P., CorrêaOliveira, R.
1. Centro de Pesquisas René Rachou
- FIOCRUZ-MG, CPqRR/FIOCRUZ,
Av Augusto de Lima, 1715 Barro
Preto Belo Horizonte-MG CEP
30190002
2. Instituto de Ciências BiológicasUFMG, ICB/UFMG, Av Antônio
Carlos, 6627 Pampulha Belo
Horizonte-MG CEP 31270901
E-mail: fernanda.villani@cpqrr.
fiocruz.br
In the last 30 years we have seen a
marked appearance of emerging viral
diseases, with approximately 100 new
viruses identified, some being related to
major epidemics in humans. The Apeu
virus (APEUV), a Orthobunyavirus
of group C, has been described in
sentinel animals in Amazon forest
which is a potential emerging virus.
Also, the reemergence of pathogenic
viruses with high dissemination rate
Basic Virology: BV
111
as the Poxvirus is a reality. Zoonotic
human infections caused by Vaccinia
virus (VACV), a Orthopoxvirus, in
Brazil reached worldwide recognition.
Recently, it has been shown that CD4CD8- T cell population (DN T cells)
play an important role in the antiviral
immune response. The aim of this
work was to evaluate the activation
profile of DN T cells expressing alphabeta or gamma-delta T-cell receptors
(TCR) by APEUV and VACV in healthy
humans. For that, peripheral blood
mononuclear cells (PBMC) were
isolated from four donors and exposed
to UV-inactivated APEUV and VACV
(WR isolate). The expression of
surface markers (CD4, CD8, alpha-beta
TCR and gamma-delta TCR) and the
intracellular cytokines (IL-1beta, IL-6,
IL-10, IL-17A, IFN-alpha, IFN-beta, IFNgamma and TNF-alpha) were assessed
by flow cytometry. We observed a
higher frequency of alpha-beta DN T
cells expressing IL-10, IFN-alpha and
TNF-alpha in VACV stimulated cells
as compared to non-stimulated cells.
IFN-gamma expression was higher in
VACV-stimulated gamma-delta DN T
cells compared to non-stimulated cells.
We did not observe differences in the
expression of these markers in cells
stimulated with APEUV. Further studies
will be conducted to investigate the role
of these subpopulations in the immune
response against APEUV. Our results
show that DN T cells from healthy
humans are highly activated in vitro by
VACV and suggests an important role
of these cells in the course of human
infection with this virus. Financial
support:
CAPES/CNPq/FAPEMIG/
CPqRR/Plataforma Citometria PDTIS/
Fiocruz Area: Human Virology
BV1482 - EVALUATION OF GFP EX-
112
Basic Virology: BV
PRESSION USING SEMILIKI FOREST
VIRUS EXPRESSION SYSTEM
Puglia, A.L.P., Pereira, C.A., Rezende,
A.G., Novaski, I.A., Jorge, S.A.C., Astray,
R.M.
Instituto Butantan, IBu, Avenida
Vital Brasil 1500
Introduction: Semliki Forest Virus
(SFV) has been studied for recombinant
protein production in infected
mammalian cells, for therapeutics or as
a vaccine vector. Expressing the green
fluorescent protein (GFP) reporter,
SFV vectors allow the quantification of
gene delivery Objective: To evaluate by
fluorescence microscopy the kinetics
of expression of GFP in BHK-21 cells
infected with SFV-GFP. Methodology:
The recombinant SFV were produced
in vitro by transfection of BHK-21 with
expression and structural SFV RNAs.
After 24 h SFV-GFP were harvested from
supernatant and quantified by qRTPCR. BHK-21 cells cultivated in α-mem
medium with 10% SFB were infected
with SFV-GFP when reached 80% of
confluence. Cells were incubated at
37ºC in a chamber containing 5% CO2
coupled to the fluorescence microscope
(IX81 da Olympus®). During 24 hours,
images were acquired in bright field
and fluorescence DIC microscopy.
Results/Discussion: The first GFPpositive cells were detected four hours
post-infection. During the incubation
period it was evidenced an increasing
number of fluorescent cells until 24
hours post-infection. The infected cells
showed fluorescence in different times
post infection. This is probably due to
different amounts of virus that infected
each cell, since SFV-GFP is unable to
replicate. In general, the percentage
of GFP positive cells in the population
was low, but the infected cells showed
high fluorescence, which may indicate
the presence of a heterogeneous cell
population, with cells presenting
distinct permissibility of infection.
BV1498 - GENETIC DIVERSITY GENERATION IN HIV-1 IS RELATED TO
VIRAL TROPISM
Nascimento-Brito, S., Zukurov, J.P.L.,
Antoneli, F., Bosco, F., Maricato, J.T.,
Oliveira, G., Volpini, A., Coimbra, R.S.,
Araujo, F.M.G., Torrieri, R., Leite, L.R.,
Janini, L.M.R.
Paulo, UNIFESP, r Pedro de Toledo
669 6 andar fundos CEP04039032
São Paulo SP
2. Centro
de
Excelência
em
Bioinformática, CEBio-Fiocruz,
r Araguari 741, 3 andar
CEP30190110 Belo Horizonte,
MG E-mail: [email protected]
HIV has been for the last decades a
major pathogen of human beings. As
etiological agent of AIDS, it has attracted
significant interest of researchers. HIV1 genetic variability is a hallmark of
this virus, and is implicated with many
aspects of the epidemics. Another
important feature of this virus is
differences in tropism during infection.
Most of infected individuals present at
early stages of infection the so called
R5 strain of HIV-1, characterized by the
usage of CCR5 as co-receptor for viral
entry into cells. Late during infection is
described the occurrence of X4 strains,
characterized by the usage of CXCR4
as co-receptor. Usually the raising of
X4 virus correlates with the worsening
health condition of the subject. In
viral particles, these differences in
tropism are represented by differences
in gp120, a component of the spikes
Basic Virology: BV
113
protruding from virus surface. It has
been demonstrated that leucocytes
infected with X4 or R5 HIV-1 show
different transcription pattern of
cellular genes. Here we show, by using
deep sequencing, a different profile of
genetic variability in HIV-1 harboring
spikes from X4 and R5 strains after a
single round of viral replication. These
differences are particularly present
in env coding region of viral genome.
Our results suggest that R5 viruses
generate more genetic variability in
progeny compared to X4 viruses, and
it correlates with the fact of R5 viruses
are more often found at the beginning
of infection. By generating more
genetic variation, R5 HIV-1 would be
most likely capable to counteract the
bottleneck effect of person to person
transmission, and with this adapt to
new host organism.
Posters Environmental Virology - EV
EV685 - VIRUCIDAL ACTIVITY OF
CHEMICAL
BIOCIDES
AGAINST
MIMIVIRUS,
A
PUTATIVE
PNEUMONIA AGENT
Campos, R.K., Andrade, K.R., Dornas,
F.P., Ferreira, P.C., Bonjardim, C., Kroon,
E.G., Abrahão, J.S.
Gerais, UFMG, Av. Antonio Carlos
6627, Pampulha Belo Horizonte/
MG CEP 31270-901 Cx Postal 486
Acanthamoeba polyphaga mimivirus
(APMV), the largest known virus,
has been studied as a putative
pneumonia agent, especially in
hospital environments. Despite the
repercussions of the discovery of APMV,
there has been no study related to the
control of APMV and the susceptibility
of this virus to disinfectants. This work
investigated the virucidal activity
against mimivirus of chemical biocides
commonly used in clinical practice for
the disinfection of hospital equipment
and rooms. APMV was dried on
sterilized steel coupons, exposed to
different concentrations of alcohols
(ethanol,1-propanol and 2-propanol)
and
commercial
disinfectants
(active chlorine, glutaraldehyde and
benzalkonium chloride) and titrated
in amoebas using 22 the TCID50
value. The stability of APMV on an
inanimate surface was also tested in
the presence and absence of organic
matter for 30 days. APMV showed a
high level of resistance to chemical
biocides, especially alcohols. Only
active chlorine and glutaraldehyde
were able to decrease the APMV titers
to undetectable levels. Dried APMV
showed long-lasting stability on an
inanimate surface (30 days), even in
the absence of organic matter. The
Environmental Virology: EV
115
data presented herein may help health
and laboratory workers plan the
best strategy to control this putative
pneumonia agent from surfaces and
devices.
EV707 - CHARACTERIZATION OF
THE FIRST BRAZILIAN VIROPHAGE
ISOLATED FROM RIO NEGRO WATER
SAMPLES, AMAZON
Boratto, P.V.M., Campos, R.K., Ferreira,
P.C.P., Kroon, E.G., Abrahão, J.S.
Laboratório de Virus- Depto. de
Microbiologia - ICB- UFMG, UFMG,
Av. Antônio Carlos, 6627 - Pampulha
- C.P. 486 - CEP 31270-901 - BH,MG
Virophages are agents that use
replication machinery of other virus
to produce its own progeny, reducing
the infectivity of these “hosts”. They
are usually associated with infections
caused by giant viruses (130- 750nm).
The first described virophage, the
Sputnik virus, was discovered in 2008,
in a water-cooling tower located in
Paris, in association with a virus of the
Mimiviridae family. Further studies
described the characterization of
other phycodnaviruses virophages
in antartic lakes. The purpose of this
study was to prospect virophages in Rio
Negro, AM, Brazil. Water samples were
collected and enriched in water-rice
medium and, subsequently, submitted
to successive filtrations. Molecular
and biological assays were performed
with suspect samples, including realtime PCR, transmission electron
microscopy, infectivity reduction tests
and one-step-growth-curve assays
using mimivirus and Acanthamoeba
castelannii cell culture. The PCR results
indicated the amplification of a gene
fragment that encode for the proteins
October 2012 Volume 17 (2) – Supplement 1 / Foz do Iguaçu, Paraná, Brazil - Posters - Environmental Virology: EV
116
of the virophage capsid. Icosahedral
structures (40nm) were observed by
the electron, associated with mimivirus
factories. Infectivity reduction tests and
one-step-growth-curve assays showed
a decrease of 99.9% of mimivirus
cytopathic effects and replication,
which is consistent with virophages
biology. Our data indicate the isolation
of the first Brazilian virophage,
the fifth in the world. Although the
Amazonian biome has one of the richest
biodiversity in the planet, the diversity
of viruses is poorly studied, as such as
their ecological relationships. Based
on our data and others studies, we can
speculate that the virophages could
participate on the biological control of
giant viruses in the Amazonian biome.
EV721 - SURVEILLANCE OF ENTEROVIRUS, POLIOVIRUS AND HUMAN
ADENOVIRUS IN SAMPLES FROM
SPRING WATER SOURCES IN FLORIANÓPOLIS/SC
Nascimento, M.A., Fongaro, G., Barardi,
C.R.M.
Catarina, UFSC, Campus Trindade,
MIP, UFSC CEP: 88040-900 E-mail:
[email protected]
The microbiological quality of
environmental waters is still evaluated
by means of fecal indicator bacteria.
Human adenoviruses (HAdV) are
viral agents of multiple pathogenesis
and they have been found in surveys
of polluted waters. Poliovirus (PV)
belongs to enteroviruses group and
must be monitored on environment,
once the wild virus has not been
eradicated and individuals immunized
with attenuated oral vaccine shed
vaccine-derived poliovirus in their
feces, which can suffer genetic drifts
and regain neurovirulence. The
present study aimed to evaluate the
incidence of HAdV, enteroviruses and
PV in spring water sources from six
sites of Florianópolis/SC, with a total
of 60 samples analysed. Four sites were
located in different points of Lagoa do
Peri, which is one of the main drinking
water supplier in Florianopolis; one
site was a surface water source used
for drinking without treatment by a
small school and the last one a surface
water source treated by a community
association before consumption. The
viruses were detected by RT-PCR
(enteroviruses) and qPCR (HAdV).
Positive samples of enteroviruses by
RT-PCR were tested for viral infectivity
and distinction of enteroviruses
from polioviruses by cell culture
using passages on RD and L20b cells
respectively. Positive HAdV samples
by qPCR were submitted to DNAse
treatment, to check viral integrity. The
results showed that HAdV was present
in 95% of the analysed samples with an
average of 2.93x107 gc/L with 74% of
integrity by DNAse and enteroviruses
was present in 40% of the samples.
All of the enteroviruses showed to be
infectious and non-polio, since they
showed cytophatic effects (ECP) on
RD cells and not on L20b cells. The
high incidence of HAdV and infectious
enteroviruses indicates contamination
of these water sources with human
effluents, and portrays the lack of health
care, justifying the urgent need to add
viral parameters in the water quality
surveillance. Financial support:CNPq
Universal 470808/2009
EV770 - SPATIAL AND TEMPORAL
ANALYSES OF BACTERIOPHAGES IN
TWO TROPICAL MARINE ENVIRONMENTS OF RIO DE JANEIRO STATE,
BRAZIL
Barbosa, J.E.F., Pedrosa, L.G.M., Pereira,
P.S., Salomon, N.S., Vogel, V.A.R., Paula,
B.F., Ferreira, D.F., Crapez, M.A., Silva,
R., Damaso, C., Amorim, L.F.F., Giongo,
V.A., Paixão, I.C.P.
1. UNIVERSIDADE FEDERAL DO RIO
DE JANEIRO, UFRJ, Departamento
de
Virologia,
Instituto
de
Microbiologia Prof. Paulo de
Góes,
2. UFRJ UNIVERSIDADE FEDERAL
DO RIO DE JANEIRO, UFRJ,
Programa de Biologia Molecular,
Instituto de Biofísica Carlos
Chagas Filho,UFRJ
3. UNIVERSIDADE
FEDERAL
FLUMINENSE, UFF, Departamento
de Biologia Celular e Molecular,
Instituto de Biologia, UFF Niterói
br
Indubitably, Earth’s ocean represents
the world’s largest biosphere of
marine phages. However, in a spite
of the abundance little is yet known
about this distribution and diversity
in tropical aquatic ecosystems. Here,
we evaluated for the first time marine
phages and their relationship with
host and environments variables in
two coastal regions of Rio de Janeiro,
Brazil. In this research was analyzed
the presence of phages in surface
seawater samples (27) collected from
the eutrophic Guanabara Bay (GB) and
the upwelling region of Arraial do Cabo
(AC), during winter, spring and summer
of 2011. A detailed study of physical
chemical and nutrients parameters
were done (T=18.7-28.1°C, S=30-35,
pH=7.86-8.73, Conductivity=47.1-52.9
ms, DO=5.36-12.5 mgL-1, Chl-a =2.32-
117
22.58μg.L-1, NH4+=1.69-28.98μM.L-1,
NO2-=0.14-4.15 μM.L-1, NO3-=0.128.15μM.L-1 and PO43-=0.06-3.29
μM.L-1). A strong positive correlation
(PC) were observed for bacteria (BAC)
and Chl-a (r= 0.84) in AC stations,
showing the dominance of primary
productivity in this region. Otherwise,
in GB stations were verified a weak PC
for BAC and Chl-a (r=0.36) and a high PC
for BAC and PO43 (r=0.70) indicating
a possible “Bottom-Up” control. It
was observed in summer samples the
highest bacterial abundances for AC
(13.4 ±0.7 x107cells ml-1) and for GB
(11.8 ±0.5x107cells ml-1). These data
were strongly correlated with bacterial
biomass production, as measured
by
3H-thymidine
incorporation
for
AC
(18.7±0.2x10-5gCl-1h-1)
and GB (23.4±0.4x10-5gCl-1h-1).
The 20L of seawater samples were
filtered based on virus concentration
through the method of adsorption
and elution in polarized membranes
allowing molecular analyses. In this
study, PCR primers CPS1/CPS2 were
successful in yielding PCR products
of approximately 165 bp from virus
communities concentrates from both
sites studied. Besides, marine phages
were also examined by TEM and until
now we observed only Podoviridae
family. This is the first report of marine
phage in seawater samples collected
from coastal regions of Rio de Janeiro
and a better characterization of tropical
phages diversity is still needed.
EV878 - RELATIONSHIP BETWEEN
MICROBIOLOGICAL AND PHYSICO-CHEMICAL PARAMETERS AND
OCCURRENCE
OF
ROTAVIRUS
GENOMES IN DRAINAGE BASIN OF
JUIZ DE FORA, MG.
Assis, A.S.F., Cruz, L.T., Pinto, M.A.O.,
118
Bessa, M.E., Drumond, B.P., Otenio,
M.H., Vieira, C.B., Miagostovich, M.P.,
Rosa e Silva, M.L.
1. Universidade
Federal
de
Juiz de Fora, UFJF, R. José
Lourenço Kelmer,s/n, Campus
Universitário-S.Pedro - Juiz de
Fora, MG
2. Embrapa Gado de Leite - Juiz de
Fora, MG, Embrapa - JF/MG, Rua
Eugênio do Nascimento, 610 Dom Bosco - Juiz de Fora, MG
3. Instituto Oswaldo Cruz , IOC/
FIOCRUZ-RJ, Av. Brasil, 4365 Manguinhos - Rio de Janeiro, RJ.
E-mail: andressasilvino@yahoo.
com.br
Several enteric viruses are present
in aquatic environments due to
contamination by sewage effluents,
even in the absence of fecal coliforms,
which are the microbial indicators
of water quality assessment. These
viruses are frequently associated
to waterborne viral gastroenteritis,
including group A rotaviruses (GARV).
Infection by GARV is a significant
public health problem, especially in
developing countries. The present
study aimed to investigate the presence
of GARV in surface waters of the Bacia
Hidrografica do Corrego de Sao Pedro
(BHCSP), in the city of Juiz de Fora,
Minas Gerais state, correlating with
microbiological and physico-chemical
parameters for water quality. From July
2011 to May 2012, 2L of surface water
were collected at 8 sites along the basin,
in six campaigns, totalizing 48 samples.
Putative present viral particles were
concentrated by adsorption-elution
in negatively charged membrane,
followed by centrifugation. The viral
RNA, extracted by the silica method,
was submitted to RT-PCR for detection
of the virus genome. Fecal coliforms
were quantified and physico-chemical
parameters (conductivity, chlorine, pH,
salinity, temperature and turbidity)
were determined in each site in all
campaigns. The presence of genetic
material of GARV was detected in
25,0% (12/48) of the studied samples.
Bacteriological
analyses
showed
that 53,7% (27/48) of analyzed
water samples exceeded the values
established by the CONAMA N357/05
for class 1 and 2 waters. GARV were
detected in 19.0% (4/21) of the water
samples considered into the values
acceptable of the microbiological
quality. Statistical analyzes only showed
significant correlation between GARV
detection and turbidity (p=0,000). The
data of this study confirm that the lack of
coliforms does not necessarily exclude
other pathogens, such GARV and point
to the need the establishment of viral
parameters to assess water quality.
"Financial support": CNPq, CAPES,
FAPEMIG, EMBRAPA and PropesqUFJF.
EV925 - EVALUATION AND IDENTIFICATION
OF
VIRUS
LIKE
PARTICULES (VLP) IN THE SIDERASTREA STELLATA CORAL REEF IN
BRAZILIAN STATE PARK COASTAL
SEAS CONSERVATION UNIT (PECS)/
RJ
Pereira, P.S., Barbosa, J.E.F., Pinto, A.,
Teixeira, V.L., Giongo, V., Paixão, I.C.N.P.
Universidade Federal Fluminense,
UFF-RJ, Rua Outeiro de São João
Baptista, S/Nº - Niterói, Rio de
Janeiro
E-mail:
pris.santana@
hotmail.com
The reef systems include a reservoir of
high genomic diversity, and therefore
constitute an ecosystem with high
biodiversity and productivity, and
can even be compared to rainforests.
In recent decades, coral reefs have
suffered an unprecedented decline due
to human intervention compromising
the reef systems health. It is therefore
necessary to establish the mechanisms
responsible for the loss of their quality
of life systems. The main objective of this
study was to observe micro-organisms
presence in coral Siderastrea stellata
with the scope of understanding the
microbial loop functioning and assess
marine viruses participation in coral
diseases. The samples were taken in a
site within the waters of a Brazilian state
park coastal seas conservation unit.
The participation of marine viruses
as regulators of bacterial metabolism
and marine life diversity is the basis of
our research on interactions between
diseases of coral reefs and microbial
loop. The evaluation of the presence
of virus in coral was performed
through epifluorescence microscopy
and polymerase chain reaction (PCR)
techniques with promising results for
G20 primer that is a conserved fragment
of the virus capsid, incorporation of
H³-thymidine , virus quantification
and physical-chemical analyses of
water column. Results showed an
amplification of DNA from marine
virus
demonstrating
association
of virus to coral. Likewise there
was incorporation of H³-thymidine
supporting the results obtained in
quantification and PCR. There was a
satisfactory result of bacterial culture
which was assessed by epifluorescence
microscopy that allowed to calculate
bacterial total biovolume. Analyses
of transmission electron microscopy
(MET) was performed and genome
analyses will be realized by sequencing
119
in near future.
EV940 - USE OF FLOW CYTOMETRY
AND PLAQUE ASSAY TO DETECT INFECTIOUS HUMAN ADENOVIRUSES
IN WATER MATRICES
Moresco, V., Damazo, N.A., Barardi,
C.R.M.
Catarina,
UFSC,
Laboratório
de
Virologia
Aplicada,
MIP,
CCB, Florianópolis, SC E-mail:
[email protected]
Human adenoviruses (HAdV) are one
of the most prevalent and persistent
enteric viruses in the aquatic
environment and are responsible for
several waterborne outbreaks. The
current methods used to detect HAdV
in water samples are usually based
on molecular techniques, although
these methods do not predict viral
infectivity. An alternative to evaluate
the presence of infectious viruses is
to infect permissive cells in vitro. In
order to determine if different water
matrices can interfere on in vitro HAdV
replication, the aim of this study was to
evaluate the detection limit of HAdV2
in two water matrices: fresh water
and treated drinking water using flow
cytometry (FACS) and plaque assay
(PFU). For both assays, A549 cells were
infected with known amounts of HAdV2,
using serial tenfold dilutions either
in PBS or in the water matrices. FACS
and PFU were performed as described
by Barardi et al., 1998 and Cromeans
et al., 2008 respectively, with some
modifications. The results obtained in
the FACS assay showed no significant
differences between the HAdV2 diluted
in PBS or in the water matrices, with a
percentage of fluorescent cells ranging
from 32 to 1.48% according to virus
120
dilution, corresponding respectively
to 1.30 E+06 and 1.13 E+05 infected
cells/mL. PFU evaluation showed
a similar detection limit of HAdV2
diluted in PBS and in treated drinking
water, with viral titres of 1.4 E+03 and
1.7 E+03 PFU/mL respectively. On the
other hand, the detection of HAdV2
in fresh water decreased 2 logs (9.0
E+01). This lower value is probably
due to water components which can
inhibit cell infection. The FACS method
provided faster (72h) and more
sensitive analysis when compared to
plaque assay (7 days), allowing the
immunodetection in the early stages
of virus replication, while PFU requires
cell to cell spread. These two methods
will be further applied to evaluate the
presence and infectivity of HAdV2 in
water matrices. Financial support:
CNPq Universal 471755/2011-7;
CAPES
EV962 - DETECTION AND QUANTIFICATION OF HEPATITE A VIRUS (HAV)
AT THE MAIN SOURCE OF WATER
SUPPLYING IN BELEM, BRAZIL AND
ITS RELATION WITH THE CONCENTRATION OF COLIFORMS
Santos, D.A.S., Garza, D.R.G., Fernandes,
J.S., Teixeira, D.M., Spada, P.P., Gurjão,
T.C.M., Vale, E.R., Corrêa, V.C., Sousa,
N.R., de Paula, V.S., Mascarenhas, J.D.P.,
Gabbay, Y.B., de Sá Morais, L.L.C.
Laboratório de Microbiologia
Ambiental-Rodovia BR 316 SN,
Ananindeua-Pa
2. Instituto Oswaldo Cruz-Fiocruz,
IOC/FIOCRUZ, Av. Brasil, 4365
Manguinhos-Rio de Janeiro-RJ
The survey for waterborne pathogens
is currently an important mission of the
health surveillance teams, especially
with regard to water intended for
human consumption. Brazil lacks data
concerning the occurrence of enteric
viruses in public water supplies,
especially about HAV which is highly
endemic in the North of the country.
For this study, monthly samples were
collected in three points of the Utinga
water reserve. The chromogenic
substrate method was used for
colimetric assays. Viral particles were
concentrated and eluted in filtering
membranes, RNA was extracted and
reverse transcribed. Viral cDNA was
detected by nested PCR and quantified
by real-time PCR. In the collection
point located at the Bolonha Lake,
colimetric assays revealed values that
varied from 1.97x102 (MPN/100 mL)
to 7.7x103 (MPN/100 mL). HAV was
detected in 4 of 12 samples by NestedPCR, but only two of these were
positively quantified by real-time PCR
(average of 521,702.95 copies/µL).
At the point located at the Água Preta
channel, thermotolerant coliforms
varied from 2.01x102 (MPN/100 mL)
to 2.91x103 (MPN/100 mL). HAV was
found in 6 of 12 samples, of which only
one was successfully quantified by
real-time PCR, yielding an average of
312,983.39 copies/µl. No samples from
the point located at ETA Bolonha were
positive for coliforms, nevertheless,
HAV was detected in 2 samples
through nested-PCR, yielding copy
numbers of 285,278.89 and 44,256.35,
respectivly. Nested PCR proved to be
more sensible to detect viral RNA.
High concentrations of thermotolerant
colifoms in the surface waters of the
Utinga supply highlighted the need to
protect this important water source
from local environmental impacts,
such as the release of sewage in natura.
The quality of water from the Bolonha
output is seriously compromised in
virological terms, highlighting the
absence of an obligatory relation
between the presence of coliforms and
viruses in water.
EV975 - IS IT NECESSARY TO CONCENTRATE VIRAL PARTICLES FROM
SOIL MATRICES FOR MOLECULAR
DETECTION OF ENTERIC VIRUSES?
Staggemeier, R., Bortoluzzi, M., Heck,
L.M.S., Bianchi, E., Da Luz, R.B., Fabre,
R.B., Soliman, M.C., Fontana, T.,
Pacheco, A.M., Kluge, M., Fleck, J.D.,
Vecchia, A.D., Heinzelmann, L., Spilki,
F.R., Almeida, S.E.M.
Universidade Feevale, Feevale, ERS239, 2755, Novo Hamburgo, RS, CEP
93352-000 E-mail: rstaggemeier@
gmail.com
The protocols available for recovery
and detection of viral particles from
soil and sediment samples are highly
variable regarding in sensitivity, costs
and complexity. The most of virus
concentration methods are based on
acid precipitation, organic flocculation,
and polyethylene glycol precipitation
(PEG-6000). The development of
methodologies that allow for proper
detection of viral pathogens in the
environment is of great importance
in studies of environmental virology.
The present study developed a
methodology for virus concentration in
sediments able to extract the virus from
particle material. This study compared
the standard PEG concentration
method with no concentration.
The PEG method was used as often
reported on literature and for the nonconcentration technique 1 g of the
solid (sediment) was added to 1 ml of
121
Eagle's minimum essential medium
(E-MEM, Nutricell; pH7,4). The solution
was homogenized by vortexing for
1 minute and then centrifuged at
14,000 rpm for 10 minutes. The
supernatant was used for viral DNA /
RNA extraction. The identification of
viral genomes present in the samples
was performed by conventional PCR
for adenovirus (AdV), rotavirus (RV),
enteroviruses (EV). We analyzed 20
sediment samples from farms in both
methodologies. Through the technique
of PEG, only 3 samples were positive
for AdV. Using the non-concentration
technique 7 samples were positive for
AdV and 6 for RV. In both techniques
there was no identification of positive
samples for EV. The protocol with
no previous concentration showed
greater sensitivity, effectiveness and
shorter execution time. Furthermore,
it requires low cost of reagents
and equipment commonly found in
laboratories, making it an attractive
alternative than virus concentration
soil and sediment samples. Financial
support: CNPq, CAPES, FAPERGS,
Feevale.
EV977 - VIRAL DETECTION IN
WATER AND SEDIMENTS SAMPLES
FROM RURAL AREAS OF ROLANTE
AND RIOZINHO, RS, BRAZIL
Staggemeier, R., Bortoluzzi, M., Bianchi,
E., Da Luz, R.B., Fabre, R.B., Soliman,
M.C., Fontana, T., Pacheco, A.M., Kluge,
M., Fleck, J.D., Vecchia, A.D., Rodrigues,
M.T., Baldasso, N.A., Spilki, F.R., Almeida,
S.E.M.
1. Universidade Feevale, Feevale,
ERS-239, 2755, Novo Hamburgo,
RS, CEP 93352-000
2. Empresa
de
Assistência
122
Técnica e Extensão Rural do
RS, ASCAR-EMATER/RS, E-mail:
[email protected]
The Rio dos Sinos watershed is located
in the northeastern of Rio Grande Sul.
The municipalities of Rolante and
Riozinho are part of the watershed
and located right above the Guarani
Aquifer. Enteric viruses in the soil have
the ability to migrate through it by
the successive adsorption-desorption
phenomena, thus, providing risk of
contamination of groundwater by the
ease of penetration of viral particles in
the soil. Among these viruses are the
adenovirus (AdV), enterovirus (EV)
and the rotavirus genogroup A (GARV),
which causes diseases in humans and
animals. This study aimed to evaluate
the presence of AdV, EV and GARV in
water and sediment samples from rural
properties of Riozinho and Rolante, Rio
Grande do Sul, Brazil. Twenty sediment
(100 g) samples and 55 water (500
mL) samples from springs, artesian
wells, dams and streams were collected
and submitted to extraction of DNA/
RNA, followed by, when necessary,
the synthesis of cDNA by reverse
transcription. The viral detection
for EV and GARV was performed by
polymerase chain reaction (PCR), and
detection for AdV by Real Time PCR
(qPCR). Of the 55 water samples, 87,3%
(48/55) were positive for AdV, 25,5%
(14/55) for GARV and 1,8% (1/55)
for EV. From sediment samples, 80%
(16/20) were positive for AdV and 30%
(6/20) for GARV. The results suggest
intense contamination of groundwater,
surface water and soil in the region,
furthermore, the presence of these
pathogens in soil may contribute to
an increased risk of contamination of
groundwater. Financial support: CNPq,
CAPES, FAPERGS, Feevale.
EV984 - DETERMINATION OF THE
RECOVERY EFFICIENCY OF HUMAN
AND MURINE NOROVIRUS ON
BERRIES
Melgaço, F.G., Victoria, M., Corrêa, A.A.,
Miagostovich, M.P.
1. Oswaldo Cruz Institute-Oswaldo
Cruz Foundation, IOC-Fiocruz, Av.
Brazil, 4365, Manguinhos, Rio de
Janeiro, Brazil
2. Universidad de la República
Regional
Norte,
unorte,
Universidad de la República
Regional Norte, Salto, Uruguay
3. Laboratory of Comparative and
Environmental Virology, LVCA,
Av. Brazil, 4365, Manguinhos,
Rio de Janeiro, Brazil E-mail:
[email protected]
Foodborne illness is an important
problem of global public health, leading
to a large number of hospitalizations
mainly due to acute gastroenteritis (AG).
Norovirus (NoV) is the most important
agent of outbreaks of AG and their
identification in foods is difficult due to
the complexity of the food matrix, the
presence of inhibitors, low levels of viral
contamination and genetic diversity of
these viruses. There is not currently a
single viral detection method validated
internationally suitable in foodstuffs.
In order to determine the efficiency
of a viral concentration method for
NoV by organic flocculation from
samples of red fruits (strawberries),
two different variables were evaluated
and compared: different elution buffer
(PBS 1X, pH 7.0 and Glycine 0.05M/
Tris-HCl 0.1M, pH 9.5); a Becker and
filter bag as containers to mix samples.
Strawberries from local commercial
sources were divided in 25 g samples.
Experiments on artificial contamination
with NoV GII.4 and murine norovirus
1 (MNV-1) were performed by using
flocculation with 1% skimmed milk.
The viral RNA was extracted from 140
µL of the concentrated sample with the
QIAamp viral RNA mini kit® (Qiagen).
For complementary DNA synthesis,
SuperScript® III Reverse Transcriptase
and random primers were used; the
viral detection was performed by real
time RT-PCR. The best recovery for
NoV GII.4 was achieved by combining
elution with Tris-glycine in filter bags
(3.6 to 43.3%). The recovery of MNV1 eluted with PBS buffer in Becker
presented the best recovery (14.7 to
50.5%), probably due to the presence
of pigments and pH value, as well as
inhibitors of enzymatic reactions. The
establishment of methods for viral
concentration from food matrices will
assist epidemiologic investigations
of outbreaks associated with NoV,
previously hampered by the lack of
appropriate methodologies, assisting
in mapping the routes of transmission
and demonstrating the importance
of these viruses in the occurrence
of foodborne outbreaks. Financial
support: POM LVCA, CNPq, APQ1/
FAPERJ.
EV1057 - ROTAVIRUS DETECTION IN
DIFFERENT AQUATIC ECOSYSTEMS
FROM THE METROPOLITAN AREA
OF BELEM, PARA, BRAZIL
Monteiro, J.S.V., Teixeira, D.M.,
Gurjão, T.C.M., Spada, P.K.P., Sá, L.L.C.,
Mascarenhas, J.D.P., Gabbay, Y.B.,
Sousa, M.S.
UFPA, Av. Bernardo Sayão, s/n.
Belém - Pará
123
BR 316 Km 7, Ananindeua - Pará
E-mail:
monteirojsv@hotmail.
com
Introduction: The concentration of
bacteria of the coliform group often
determines the microbiological quality
of water for consumption; however,
minimum standards of bacteria do
not indicate the absence of virus. The
enteric viruses are highly stable in
the environment while maintaining
their infectivity even after exposure
to treatment processes. These viruses
excel at etiology of waterborne diseases
such as acute gastroenteritis, especially
among children under five years of age
being the rotavirus (RV) considered
the major cause of these diseases.
Considering that the metropolitan
region of Belém, Pará State, has a great
influence of rivers, lakes and streams,
this emphasizes the importance of
researching RV in water samples from
different ecosystems. Methods: Water
samples were monthly collected from
six points of the metropolitan region of
Belém (Port of Açai, Port of Ver-o-Peso,
Tucunduba River, Black Water Lake
Canal, Bolonha Lake and UNA sewage),
from November 2008 to October
2010 and 24 samples were collected
from each point. The search of RV was
performed in duplicate by polymerase
chain reaction in real time using the
region of the NSP3 gene as target.
Results: Of the 144 water samples
analyzed 51.4% (n=74) were positive
for RV with no difference between the
frequencies of two years. The major and
minor events were observed in October
(n=12) and January (n=2), respectively.
The place with the higher number
of positive samples was the UNA
sewage with 75% (n = 18), followed by
Tucunduba River with 54.16% (n=13)
in addition to the Port of Ver-o-Peso
124
and Bolonha Lake with 50% (n = 12),
each. Conclusion: The high presence
of RV in important aquatic ecosystems
for the metropolitan region of Belém
alert to the need of monitoring viral
contamination of these sites in order to
improve water quality and consequently
the health of the population. Financial
support: PIBIC-UFPA/FAPESPA/CNPq,
PIBIC-IEC/FAPESPA/CNPq, Fundação
de Amparo a Pesquisa do Estado do
Pará - (FAPESPA).
EV1163 - KINETICS OF VIRAL
VIABILITY DECAY IN OYSTERS
CRASSOSTREA GIGAS SUBJECTED
TO DEPURATION UNDER UV
TREATMENT
Souza, D.S.M., Fongaro, G., Pilotto, M.R.,
Moresco, V., Delfino, N., Barardi, C.R.M.
Catarina, Laboratório de Virologia
Aplicada, MIP, Florianópolis, SC E-mail: [email protected]
Mollusks depuration using ultraviolet
(UV) disinfection can be employed
for their decontamination before
consumption. In the present study,
oysters remained 14 days in four
different places in the Florianópolis
Bay, SC, in order to be naturally
contaminated by a variety of
microorganisms and particles. The
strategy for oysters allocation was:
two sites approved by Brazilian
regulations for oyster’s cultivation (1
and 2) and two sites highly impacted
by pollution (3 and 4).The aim of this
study was to evaluate the interference
of a diversity of contamination on the
viral depuration of these oysters using
UV as disinfection method (43.85mJ/
cm2). Plaque forming unit (PFU)
assay was the method of choice for
viral infectivity evaluation. As positive
controls, some animals were artificially
contaminated with known amounts
of Human Adenovirus 2 (HAdV2) and
Murine Norovirus 1(MNV1) after 14
days in each selected environment.
All the oysters were distributed in
depuration tanks and analyzed after 96
and 168h of depuration. Three oyster/
time were collected, the tissue extracts
were produced and inoculated, in noncytotoxic dilutions, either in A549 or
in RAW cell monolayers for HAdV2
or MNV1 quantification respectively.
After 96h in all sites, HAdV did not
show significant reduction; after 168h,
reduction was of 1log (90%) on sites 1,
2 and 3 and 2 logs (99%) on site 4. MNV
reduction was of 3 logs (99.9%) after
96h on sites 1 and 3 and less than 1 log
on site 4. After 168h, MNV1 was not
detected in all the samples. The results
showing a higher disinfection of HAdV
in oysters from site 4 could be false
negatives since the great amount of
contaminants on the oyster’s meat can
inhibit cell infection. These previous
results allowed us to conclude that
HAdV was more resistant than MNV
to disinfection by UV on depuration
tanks. Further studies are necessary
to support these conclusions. Financial
support:
MAPA/CNPq/2008-2;
Universal 2009 MCT/CNPq; MCT/CNPq
CT-Agronegócio/MPA n º 25/2010 Formação de Recursos Humanos em
Pesca e Aquicultura
EV1168 - PRESENCE OF ADENOVIRUS
(ADV) IN SAMPLES OF HUMAN FECES
FROM PATIENTS IN THE REGION
OF THE SINOS RIVER WATERSHED,
SOUTHERN BRAZIL
Vetter, M.R., Luz, R.B., Staggemeier, R.,
Bianchi, E., Vecchia, A.D., Heinzelmann,
L.H., Rodrigues, M.T., Kluge, M., Fabres,
R.B., Solimann, M.C., Fontana, T.,
Pacheco, A.M., Fleck, J.D., Nascimento,
C.A., Spilki, F.R.
Universidade Feevale, Feevale, ERS239, 2755, Novo Hamburgo, RS,
CEP 93352-000 E-mail: michelev@
feevale.br
Enteric viruses are distributed
worldwide and are responsible for
different diseases that affect humans,
such as gastroenteritis, conjunctivitis,
pneumonia and hepatitis infections.
Outbreaks of viral gastroenteritis
have been reported, having been
the adenovirus (AdV) found in high
concentrations in feces of infected
individuals. The main route of
transmission of these pathogens
is the fecal-oral route, however, an
alternative route of transmission from
environmental samples, including,
drinking water, has been increasingly
considered a risk to public health.
The man is usually affected by virus
diseases of human origin, however,
may be an intermediate host of viral
agents of animal origin as Canine
AdV (CAV-1 and -2), Bovine AdV
(BAV) and Avian AdV (EDS-76). The
objective of this study was to identify
viral particles AdV and their species,
in samples of human feces. A total
of 150 feces samples were collected
during the winter 2011 and summer
of 2012, derived from patients aged
mixed in Esteio municipality. From the
extraction of viral DNA, viral detection
was performed by Polymerase Chain
Reaction in Real Time (qPCR) for AdV.
The results showed that 36% (55/150)
of samples were positive for EDS, 46%
(70/150) for CAV, 14% (22/150) for
BAV, 6% (9/150) for HAdV and 26%
(39/150) for AdV species is unknown.
The maximum amounts of DNA copies
/ ml for the EDS were 9.4 x 107 x 6.7
125
for 108 CAV, to BAV 7.6 x 107 to 5.33
x 107 HAdV and to AdV undetermined
species , 42 x 107. In addition, it was
possible to identify co-viral infection
in 45% (68/150) samples, and 21%
(32/150) had no positivity for any
AdV. In addition, the infection rate was
higher in the summer (72%), compared
to the winter (57%). The results
suggest a great spread of adenovirus
not restricted to host specificity, but
related to the means by which is shed
through the environment. Those
infections may have occurred, either by
consumption of contaminated water or
contact with other species carrying this
agent. Financial Support: FAPERGS,
CAPES, CNPq.
EV1169 - ADENOVIRUSES FROM
DIFFERENT ANIMAL SOURCES ON
TREATED WATER
Staggemeier, R., Bianchi, E., Vecchia,
A.D., Luz, R.B., Kluge, M., Fabres, R.B.,
Soliman, M.C., Pacheco, A.M., Fontana,
T., Heinzelmann, L.H., Rodrigues, M.T.,
Fleck, J.D., Nascimento, C.A., Spilki, F.R.
Universidade Feevale, Feevale, ERS239, 2755, Novo Hamburgo, RS, CEP
93352-000 E-mail: rstaggemeier@
gmail.com
Contamination of water by human
and animal wastes is routinely
monitored in Brazil using bacterial
indicators. However, the absence of
these indicators, namely Escherichia
coli, do not exclude the presence of
other pathogens such as viruses. The
ingestion of water contaminated by
these agents may induce diseases that
affect systems various. Among the viral
agents used for tracing the sources
or host species responsible for fecal
contamination of water, enteric viruses
are promising candidates since they
126
are host specific in most cases and high
resistant to conventional treatments
for drinking water. Adenoviruses (AdV)
are frequently found in environmental
samples, and their presence in water
may indicate contamination from
human or different animal sources since
they are species-specific. The objective
of this study was to detect these viral
agents and their species in samples of
treated water for public supply. A total
of 60 samples were collected monthly
from 10 Water Treatment Plants
(WTP), located along the Rio dos Sinos
watershed, from March to December
2011. The viral genomes were detected
using the qPCR technique (Polymerase
Chain Reaction in real time) and
species differentiation was made
using high resolution melting after
the amplification steps (qPCR-HRM).
The primers used were originally
designed and are capable of binding
to conserved regions from different
species of AdVs (human, canine,
bovine, swine, and avian). A total of
23.3% of the samples (14/60) were
positive for AdV avian, 8.3% (5/60)
for AdV canine and 11.6% (7/60) for
AdV species is unknown. There was cocontamination in 26.6% of the samples
(16/60) and 30% of samples (18/60)
were negative for any AdV. The results
confirm the spread of adenovirus
and its species in treated water at the
watershed level. In this sense, it may be
of fundamental importance to invest
in new technologies for water and
sewage treatment in order to reduce
the incidence of these agents and its
impacts on water quality. Financial
Support: FAPERGS, CAPES, CNPq.
EV1218 - VIRAL CONTAMINATION
OF SURFACES AND OBJECTS IN A
HOSPITAL FROM RIO GRANDE DO
SUL
dos Santos da Silva, V.S., Henrique de
Mello, M., Staggemeier, R., Fabres, R.,
Soliman, M., Bianchi, E., Rodrigues,
M.T., Spilki, F.R.
Universidade Feevale, FEEVALE, RS239, 2755 - CEP93352-000 - Novo
Hamburgo, RS E-mail: joseanesilva@
feevale.br
Viruses can invade the human body
through the conjunctiva, genital
tracts, intestinal or respiratory tracts.
Enteric viruses have been increasingly
recognized as causes of nosocomial
infections (NI), however specific
studies on viral pathogens on hospital
surfaces rarely occur. This study aimed
to detect by molecular methods Human
adenovirus (HAdV) and Rotavirus A
(RV-A) on hospital surfaces and to
establish a preliminary estimate of
the rate of occurrence of these viruses
on surfaces in hospitals. 32 samples
were collected at hospital clinics,
emergency room and surgical ward.
Each point was defined based on the
possibility of contact of the hands of
health workers (such as light switches,
table for medication preparation,
bench tops, computer mouse, sinks
for hands washing, handle, anesthesia
cart and support desks in surgical
room). The samples were subjected
to molecular biology methods, having
gone through the steps of extracting
nucleic acid, cDNA, PCR / qPCR and
electrophoresis. From the 32 samples
analyzed, 20/32 (62.5%) were positive
for HAdV by qPCR method. None of the
samples were positive for the presence
of RV-A. It was concluded that HAdV
can be considered a biological marker
of contamination of the hospital
environment, and highlights the
importance of hand washing and the
use of aseptic techniques and standard
operating procedure as a way to
prevent infection of a susceptible host
by this virus.
EV1313 - STABILITY OF HUMAN
ENTERIC VIRUSES IN SEAWATER
SAMPLES FROM MOLLUSKS DEPURATION TANKS COUPLED WITH
ULTRA-VIOLET IRRADIATION
Garcia, L.A.T., Corrêa, A.A., Souza,
D.S.M., Moresco, V., Kleemann, C.R.,
Barardi, C.R.M.
Catarina,
UFSC,
88040-970
Florianópolis, Santa Catarina, Brasil
Viruses have been associated with
episodes of illnesses related to
the consumption of contaminated
shellfish, and this risk may be reduced
by mollusk depuration. In this work,
the stability of human adenovirus,
murine norovirus and hepatitis A
virus in natural seawater in a closed
system depuration tank with and
without ultra-violet treatment was
investigated. Three hundred liters of
seawater was artificially seeded with
these viruses and disinfected using a
36 W lamp. Samples of 1 L of seawater
were collected at 24, 48, 72, 96 and 120
h, and viral particles were concentrated
by the skimmed milk flocculation
method. The viral decay was evaluated
by quantification of the genome copy
number by quantitative real time PCR
and quantitation of infectious viral
particles (except for hepatitis A virus)
in three independents assays. Based on
the molecular detection results, there
was a 5log10 and 3log10 reduction
in viral load for human adenovirus
and hepatitis A virus after 120 h of
ultra violet treatment, respectively.
127
For murine norovirus, a 4.5log10
reduction was observed at 72 h. Cell
culture results, similar to those of the
molecular detection assays, showed
that murine norovirus was not detected
after 72 h of treatment, while human
adenovirus remained infectious for up
to 72 h. Assays of viral stability without
ultra violet irradiation demonstrated a
progressive 1.5 to 2.5 log10 reduction
in the number of viral particles after
120 h of seawater recirculation for
the three viruses tested. In conclusion,
the ultra violet treatment effectively
reduced the number of viral particles
in seawater, and the natural decrease
in the concentration of each virus
could be due to seawater composition,
viral aggregation and the existence of
environmental factors such as ionic
strength and compounds naturally
found in seawater. Financial support:
CNPq Project 470808/2009-8 Edital
Universal/
CNPq/MAPA
Project
578200/2008-2
EV1349 - LONGITUDINAL SURVEY OF
HUMAN ADENOVIRUS ON TREATED
AND UNTREATED WATER FROM RIO
DOS SINOS, RIO GRANDE DO SUL,
BRAZIL
Rodrigues,
M.T.,
Vecchia,
A.D.,
Staggemeier, R., Bianchi, E., Fabres,
R.B., Heinzelmann, L., Bortoluzzi, M.,
Luz, R.B., Soliman, M.C., Fontana, T.,
Pacheco, A.M., Kluge, M., Fleck, J.D.,
Nascimento, C.A., Spilki, F.R.
UNIVERSIDADE FEEVALE, FEEVALE,
ERS-239, 2755 , Novo Hamburgo,
RS , CEP 93352-000 E-mail: manu@
feevale.br
Enteric viruses transmitted by fecaloral route, such as adenovirus (ADV)
are associated with various pathologies
such as acute gastroenteritis, especially
128
in children under four years old.
ADV viruses belong to the family
Adenoviridae, possessing a doublestranded DNA genome and are nonenveloped viruses. The present study
investigated the occurrence of human
ADV samples of water from the Rio dos
Sinos watershed, the major source of
public water supply for a population
of approximately 1.5 million people.
From July to December 2011, samples
were collected in 500 mL of water
treatment plants along seven points of
the Rio dos Sinos river and affluents.
A total of 78 samples were analyzed,
being 39 samples of untreated water
and 39 samples of treated water. The
samples were concentrated to isolate
the viral genome with a membrane
filtration system with negatively
charged, followed by extraction
and amplification of DNA by qPCR
(polymerase chain reaction in real
time). The viral genome was detected
in 76.9% (30/39) of treated water
samples and 74.3% (29/39) samples of
untreated water. In general, the number
of copies were elevated in both types of
samples for all points analyzed, which
the highest values reaching 7.02 x 109
copies of DNA per ml of treated water
and 3,77 x 1010 DNA copies per mL for
untreated water. The results show that
the presence of these viral agents may
overpass the water treatment systems.
Financial Support: CAPES, CNPQ,
FAPERGS, FEEVALE.
EV1354 - ONE YEAR MONITORING
OF NOROVIRUS AND ASTROVIRUS
IN WATER SUPPLY SYSTEM FROM
BELÉM, PARÁ, BRAZIL
Teixeira, D.M., Spada, P.K.P., Lima, I.C.G.,
Gurjão, T.C.M., Fernandes, J.S., Sá,
L.L.C., Sousa, M.S., Mascarenhas, J.D.P.,
Fumian, T.M., Medeiros, T.B., Gabbay,
Y.B.
1. SAVIR, Instituto Evandro Chagas,
IEC, Br 316, Km 7, Levilândia,
Ananindeua, PA SAMAM, Instituto
Evandro Chagas, IEC, Br 316, Km
7, Levilândia, Ananindeua, PA
NMT,
UFPA,
Generalíssimo
Deodoro, Umarizal, Belém, PA
Norovirus
(NoVs)
and
Human
Astrovirus (HAstV) are associated
with
gastroenteritis
outbreaks
worldwide and their presence in
aquatic environment, principally in
source waters, represent a great risk
to human health. The continuous
monitoring of these waters is extremely
important. This study aimed the
monitoring of a water supply system
composed for two lakes (Bolonha e
Água Preta) and a water treatment
plant (WTP), which are located in the
Utinga Environmental Park, Belém,
PA, responsible for water supply of
the Metropolitan Region of this city.
Sample collection occurred monthly,
from November 2010 to October
2011, in each lake and in the output
WTP. The viruses were concentrated
from 2L of each water samples by
adsorption-elution, followed of RNA
extraction by silica method. NoVs
detection was carried out using semi
nested RT-PCR and real time PCR. The
semi nested was performed using in
the first step the primers JV13I/JV12Y,
and in the second the pairs JV13I/GI
and JV12Y/NoroII-R specific for GI and
GII, respectively. Specific primers and
probes, targeting the ORF1-2 junction
region were used to detect NoV GI and
GII in the real time PCR. For HAstV
detection, the cDNA obtained after
reverse transcription was subjected
to conventional PCR using the primers
269/270. A total of 36 samples were
analyzed for NoVs and HAstVs. In semi
nested, NoVs was present in 25%
(9/36), all belonging to GI, except two
samples in which the co-circulation of
both genogroups was found. Only one
sample was classified as NoV GII by
real time, which was also detected by
semi nested. The nine NoVs positive
samples were obtained from lakes
Bolonha (n=4) and Água Preta (n=5).
HAstV were not found in any sample.
Our results showed the circulation of
NoVs in the two main lakes located in
Belém, indicating the existence of a
fecal contamination source in these
lakes. However, all samples from WTP’s
output were negative demonstrating
that the treatment applied has been
effective. Financial support: Fundação
de Amparo à Pesquisa do Estado do
Pará (FAPESPA), IEC/SVS/MS
EV1359 - ENVIRONMENTAL SURVEILLANCE OF ENTEROVIRUSES
FROM SEWAGE WATER IN RIO DE
JANEIRO: A PRELIMINARY ANALYSIS
Pereira, J.S.O., Costa, E.V., Silva, L.R.,
Silva, E.M., Da Silva, E.E.
Fundação
Oswaldo
Cruz/
Laboratório de Enterovírus, Fiocruz,
Av. Brasil, nº 4365, Pav. Hélio e Peggy
Pereira, sala B211, Manguinhos - RJ.
Human enteroviruses are primarily
transmitted via the fecal-oral route,
by direct contact with virus shed
from gastrointestinal tract of infected
individuals. Detection of these viruses
in river waters and sewage can be
an alternative method to monitor
circulating viruses in humans and
the environment. This study was
carried out to evaluate the presence of
129
enteroviruses in a sewage treatment
plant (ETE Alegria/CEDAE) located
in the city of Rio de Janeiro, Brazil.
From December 2011 to June 2012, 14
samples were collected weekly by Grab
Sample method and tested using three
concentration methods: Adsorption
by Chloride of Sodium, Two-phase
Separation and Silica Carrier. Following
virus isolation using RD and L20B
cells, isolates were identified and
typed by RT-PCR. Enteroviruses were
isolated from 14 (100%) of specimens.
A total of 7 out of 14 isolates were
positive for polioviruses (Sabin 2 = 6
isolates, Sabin 3 = 1 isolate) while the
others were non-polio enteroviruses.
Up to now, three out of 7 non-polio
enterovirus isolates were analyzed in
more detail by sequencing a fragment
of the gene coding for the viral protein,
VP1. The following enteroviruses
were identified: 1 Echovirus 6,
and 2 Echovirus 7. Environmental
surveillance has been used successfully
in monitoring enteric virus circulation
and is of crucial importance at the
final stages of the WHO global polio
eradication initiative. These results
show a continuous presence of Sabinrelated poliovirus and non-polio
enteroviruses in the analyzed area.
Financial support: CNPq, CGLAB/MS,
Fiocruz.
EV1385 - REMOVAL OF ADENOVIRUSES FROM ACTIVATED SLUDGE
USING ORGANIC AND INORGANIC
COMPOUNDS
Fabres, R.B., Fontana, T., Soliman, M.C.,
Kluge, M., Pacheco, A.M., Staggemeier,
R., Luz, R.B., Bortoluzzi, M., Bianchi, E.,
Fleck, J.D., Nascimento, C.A., Rodrigues,
M.T., Heinzelmann, L.S., Spilki, F.R.
Universidade Feevale, Feevale, ERS-
130
239, 2755 | Novo Hamburgo, RS |
CEP 93352-000 E-mail: rafafabres@
hotmail.com
The effluent treatment systems are
composed by the integration of methods
of treatment. The system is usually
divided into preliminary treatment,
primary, secondary and possibly
tertiary. The tertiary treatment is not
always used, can be accomplished by
physical and chemical processes such as
coagulation / flocculation / decantation
and is particularly good alternative for
the removal of heavy loads coliforms.
In this study we compared the
effects of removal of enteric viruses,
including nine coagulants, at 100
ppm and 1000 ppm each, aluminum
sulfate with and without iron, ferric
chloride,
polyaluminum
chloride
(inorganic) commonly used coagulants
in water treatment plants (WTP), and
commercial formulations of tannin,
WW ACQUAPOL®, ACQUAPOL® C1
18, ACQUAPOL® oF 18, ACQUAPOL®
T832, ACQUAPOL® 893/11 (organic
compounds). Enteric viruses have the
characteristic of being resistant to the
methods currently used in Brazil for
treating sewage, being of importance
use for monitoring water quality along
with the detection of total and fecal
coliforms. In the present study used
the adenovirus (ADV) gastroenteritiscausing and disease in humans. Samples
were collected from 50 L of sewage
soon after the preliminary treatment
and 50L of treated sewage in activated
sludge
(secondary
treatment).
Samples were collected in duplicate,
one in March in the year 2012 and
another collection in May of that year,
the sewage treatment plant in Canoas,
RS. Coagulation tests were performed
using a jar-test system. Then, its
RNA were extracted from which was
subjected to polymerase chain reaction
in real time (qPCR), using primers
to conserved regions of potential
alignment in the genome of the viral
species, corresponding to the hexon
protein gene of AdV , HAdVCf (VTB2Fw;
5'-GAGACGTACTTCAGCCTGAAT--3
')
and
HAdVCr-(
VTB2Rev;
5'-GATGAACCGCAGCGTCAA-3').
Of
54 samples analyzed, the viral copy
number diminished in 41 (76% of
total samples). The better results were
observed for the inorganic coagulant,
ferric chloride, which can remove
all of the enteric viruses. In the case
of organic coagulant was the most
effective ACQUAPOL® WW. Support:
CNPq, Fapergs, Seta S/A, Universidade
Feevale, Capes
EV1476 - EFFICACY OF AUTOMATED
COMPOSTING FOR REMOVAL OF
ADENOVIRUSES
FROM
SWINE
MANURE
Soliman, M.C., Fabres, R.B., Bortoluzzi,
M., Staggemeier, R., Kluge, M., Luz,
R.B., Schiavini, L., Bianchi, E., Vecchia,
A.D., Heinzelmann, L.S., Sá, M., Aita, C.,
Spilki, F.R.
1. Universidade Feevale, FEEVALE,
Rodovia RS 239, no. 2755, Vila
Nova, CEP: 93352-000 - Novo
Hamburgo, RS, Brazil
Maria, UFSM, Av. Roraima nº
1000 - Cidade Universitária, CEP:
97105-900, Santa Maria - RS
E-mail: mayra_soliman@hotmail.
com
The swine husbandry is a potentially
polluting activity to the environment
due to pollutants that may be contained
in their effluents. Aiming to reduce the
environmental impact, there are several
manure treatment systems, including
automated composting. However,
little is known about the efficiency
of this method for the removal of
microorganisms. Among the possible
microorganisms present in manure
are the adenoviruses (AdV), members
of the Adenoviridae family, consisting
of double-stranded DNA genome,
are often found. The purpose of this
work was to evaluate the efficiency of
automated composting system in the
elimination of AdV from swine manure.
The adenoviral species present on
swine manure were characterized
through the amplification by realtime PCR followed by a differential
step of high resolution melting.
(CanineAdV, AvianAdV, BovineAdV,
HumanAdV, and of course Porcine AdV
were detected and quantified before
and after compsoting. An automatic
composting unit was developed the
manure applied was from the swine
termination units and wood shavings as
substrate. The frequency of application
of the waste in the windrow and its
revolving occurred each five days.
Twelve samples were collected from
liquid waste before its addition to the
substrate and six samples the compost
(waste and wood shavings) for viral
analysis. The samples were diluted with
Minimum Essential Medium (MEM),
to be held the extraction of viral DNA
and the polymerase chain reaction
in Real Time (qPCR). The reaction
utilized primers Mastadeno AdV (F15'GCAGTGGTCGTACATGCACAT-3
',
5'-TCGGTGGTGACGTCGTGG
R1-3')
which allowed the detection of different
species AdV. These primers were
designed from the sequences of full
target viral genes, with the aid of the
Primer3 (http://frodo.wi.mit.edu/cgibin/primer3/primer3_www.cgi). From
131
the twelve samples analyzed before
composting, all were contaminated by
BAV, whereas the compost resulted
negative for the different species of AdV.
These results indicate the efficiency of
the process. Further experiments will
be conducted to assess the efficacy of
treatment allowing the use of slurry for
agronomic and avoiding contamination
of water sources.
EV1486 - DETECTION OF BK AND
JC POLYOMAVIRUSES BY NESTED-PCR FORM THE ENVIRONMENTAL
ISOLATES OF ACANTHAMOEBA SP
Comerlato, J., Arantes, T., Kulman, M.,
Caumo, K., Campos, F., Roehe, P.M.,
Franco, A.C.A.
Universidade Federal do Rio Grande
do Sul, UFRGS, Sarmento Leite,
500/208, Centro Porto Alegre, RS
The human polyomaviruses BK
and JC are double-stranded, naked
DNA viruses. They are transmitted
mainly through urine-oral route and
up to 80% of the human population
excretes one or both viruses in the
urine. Immunossuppression induces
virus replication and nephropathy,
one of the main causes of graft
rejection in renal transplant patients.
Acanthamoeba sp are free-living and
ubiquitous amoebae found in air, soil
and aquatic environments. They are
resistant to disinfection procedures
and responsible for the transmission
of different pathogens to humans
(e.g. Legionella sp and fungi). This
study aims to evaluate the presence
of BKV and JCV DNA in isolates of
Acanthamoeba sp obtained from
environmental samples. Fifty samples
of Acanthamoeba sp, isolated from
swimming pools and dust of hospital
132
were obtained and morphologically
characterized. The isolates were
submitted to DNA extraction and
subsequently detection of BKV and
JCV DNA by nested-PCR. The first PCR
targeted a DNA fragment shared by both
viruses. The JCV PCR was performed
with the following primer pairs: JLP16
(PF 5’- TAAAGCCTCCCCCCCAACAGAAA
-3’)
and
JLP15
(PR
5’ACAGTGTGGCCAGAATTCCACTACC
-3’), expected to give rise to a JCV
product that is 215 bp long. To
detect BKV, the primer pair BK6 (PF
5’CCAGGGGCAGCTCCCAAAAAG
-3’)
and
BK4
(PR
5’AGTAGATTTCCACAGGTTAGGTCCTC
-3’) was employed to give rise to
a 296 bp long amplicon. Of the 50
isolates analyzed, 28% (15/50)
amplified a fragment compatible
with BKV and 24% (12/50) with JCV
DNA. The presence of BKV and JCV
in Acanthamoeba sp may indicate
the ability of this microorganism to
promote the spread of viruses already
widely resistant in the environment as
human polyomaviruses. Finep, Capes,
CNPq
Posters Human Virology - HV
134
Human Virology: HV
October, 2012 – Foz do Iguaçu, Paraná, Brazill
HV677 - PRESENCE AND FULL-LENGTH GENOMIC SEQUENCE OF
HEPATITIS B VIRUS GENOTYPE E IN
A BRAZILIAN PATIENT
Alvarado-Mora, M.V., Botelho-Lima,
L.S., Santana, R.A., Sitnik, R., Mangueira,
C.P., Silva, A.O., Ceneviva, C., Duarte,
A.J., Carrilho, F.J., Pinho, J.R.
1. Gastroenterology,
School
of
Medicine, São Paulo University,
FMUSP, Rua Dr enéas de carvalho
aguiar, 500 São Paulo
2. Albert
Einstein
Diagnostic
medicine, HIAE, Liver Therapeutic
Specialized Center - CETEFI,
CETEFI, Clinical Laboratory,
Heart Hospital of São Paulo ,
HCOR, E-mail: monica.viviana@
usp.br
Introduction: Hepatitis B virus (HBV)
is estimated to cause chronic infections
in more than 350 million people
worldwide and death in one million
per year. Nine HBV genotypes (A-I)
have been described so far. Genotype
E (HBV/E) is largely distributed
in West Africa and has rarely been
found in other continents, except for
few cases in individuals with African
background. However, recent reports
found this genotype in a specific
community in Colombia and in India.
Objectives: The aim of this study was to
characterize the complete genome of
HBV genotype E infection in a Brazilian
patient. Methods: The patient is a man,
66 years old and was born in São Paulo,
Brazil. Amplification of the whole HBV
genome was performed with P1 and
P2 primers described previously. The
isolated complete genome (3200bp)
was aligned with other previously
reported complete genome sequences
(n=125) using MUSCLE software. The
Bayesian Markov chain Monte Carlo
simulation implemented in BEAST
v.1.5.4 was applied to obtain the
best possible estimates under both
relaxed uncorrelated lognormal and
exponential molecular clock and using
the model of nucleotide substitution
(GTR+G+I). Results: HBV complete
genome was successfully amplified.
The sequence was analyzed for all
reported mutations for antiviral
resistance and it did not show any of
them. After phylogenetic analysis, the
complete HBV genome sequence from
this patient grouped in a clade with a
sequence from Namibia and Argentina.
This is the first case of infection by
HBV genotype E in a Brazilian native
patient. We have recently shown that
genotype E was identified in some
Africans patients that are followed up in
Brazil by some clinicians. This patient
lives in Africa and comes to Brazil for
medical assistance. As HBV genotype
E has apparently spread recently in
Africa, it is may become a relevant
infectious agent in our country due to
the increasing of social and economical
relations between or countries. FAPESP
2011/52615-0, FFM.
HV678 - A NOVEL NUCLEOTIDE
INSERTION IN S GENE OF HEPATITIS
B VIRUS GENOTYPE A1 IN A
BRAZILIAN CHRONIC CARRIER
Alvarado-Mora, M.V., Botelho-Lima,
L.S., Santana, R.A., Sitnik, R., Ferreira,
P.A., Mangueira, C.L., Carrilho, F.J.,
Pinho, J.R.
1. Gastroenterology,
School
of
Medicine, University São Paulo ,
FMUSP, Av Dr Eneas de Carvalho,
500
2. Hospital Israelita Albert Einstein,
October 2012 Volume 17 (2) – Supplement 1 / Foz do Iguaçu, Paraná, Brazil - Posters - Human Virology: HV
HIAE, Federal University of São
Paulo, UNIFESP, E-mail: monica.
[email protected]
HBV is classified into nine genotypes
(A-I). In Brazil, genotype A is most
frequent, followed by D and F. North,
Northeast and Southeast regions have
a higher frequency of genotype A while
genotype D is the most frequent is in
the South region. The aim of this study
was determine the HBV genotype and
reported the mutations in the S/POL
region. The patient is female, 56 years
old and was born in Brazil. A fragment
of 1306bp partially comprising HBsAg
and polymerase coding regions (S/
POL) was amplified and sequenced.
Viral sequences were genotyped by
phylogenetic analysis using reference
sequences from GenBank (n=380). The
Bayesian Markov chain Monte Carlo
simulation implemented in BEAST
v.1.5.4 was applied to obtain the best
possible estimates and using the model
of nucleotide substitution (GTR+G+I).
Multiple alignment of partial HBV/S
gene (87 to 227 nt) comprising HBV/
A1 sequences from Brazil and other
countries, which are compared with
other sequences of HBV subgenotypes
previously reported. After completion
of the phylogenetic analysis, the genome
sequence of the patient was grouped
in a clade with four sequences from
Rondonia state. We have identified new
mutations in this case: an insertion of a
Serine before the position 115, just after
changing the nucleotide sequence from
Threonine to Asparagine in position
114. The origin of this variant HBsAg
was unclear but might occur naturally
due to lack of proof-reading activity of
rt domain. The major antigenic epitope
in the immunodominant loop is called
the α-determinant and is composed
of residues 124 to 147. This mutation
Human Virology: HV
135
probably does not affect the most
relevant antigenic regions of HBsAg,
particularly this α-determinant region,
where some mutations can interfere
with the recognition of HBsAg by
anti-HBs antibodies, especially those
contained induced by the commonly
used vaccines. FAPESP 2011/50562-0,
FMM, HCFMUSP.
HV680
SEROLOGIC
AND
MOLECULAR PROFILE OF PRO AND
ANTI-INFLAMMATORY CYTOKINES
IN PATIENTS INFECTED BY DENGUE
VIRUS
Azevedo, V.N., Feitosa, R.N., Azevedo,
R.S.S., Machado, L.F.A., Ishak, M.O.G.,
Vasconcelos, P. F.C., Ishak, R., Vallinoto,
A.C.R.
1. UNIVERSIDADE FEDERAL DO
PARÁ, UFPA, ICB, Laboratório de
Virologia, Rua Augusto Correa 1 Guamá - Belém - Pará
2. Instituto Evandro Chagas Laboratório de Arbovírus, IEC,
Rod BR 316, s/n km 7 - Levilândia
- Ananindeua - Pará E-mail: vna@
ufpa.br
The identification of cytokine profile of
individuals infected by dengue viruses
can be an important instrument to
detect patients with a tendency to
develop severe illness. In this study,
the quantification of cytokines serum
levels (IL-6, IL-8, and IL-10) was
performed among a group of patients
infected by dengue viruses, individuals
with clinical suspicion of infection by
dengue viruses – but with negative
laboratory diagnosis and a healthy
control group. We also investigated
the frequency of the polymorphism of
the genes IL-6 (-634 C/G), IL-8 (-353
A/T), and IL-10 (-1082 G/A), as well
136
Human Virology: HV
as the occurrence of the association
between the genetic variability and
the cytokine serum levels. The ELISA
test was used in order to detect
cytokine concentrations, while the
polymorphisms were investigated using
polymerase chain reaction, which was
followed by the use of the restriction
fragment length polymorphism (RFLP)
analysis. The lowest serum level of
IL-6 was identified on patients infected
with dengue as compared to the
control group and the highest levels
were described among the diseased
persons without dengue. The levels
of IL-8 and IL-10 were higher both in
the infected patients and non-infected
patients, but no exclusive profile of
these cytokines was associate to any
group. The genotypes GG (IL-6 -634
C/G), AT (IL-8 -353 A/T), and AA (IL-10
-1082 G/A) were the most prevalent in
all groups, but there was no statistical
differences among the genotype and
allelic frequencies. The cytokine
concentrations were not influenced by
the genetic polymorphism. Financial
Support: CNPq and UFPA
HV681 - HEPATITIS C VIRUS (HCV)
PATIENTS WITH HEPATOCELLULAR
CARCINOMA IN BRAZIL: GENOTYPES
DISTRIBUTION, CLINICAL IMPLICATIONS AND HCC-ASSOCIATED VIRAL
MUTATIONS
Moreira, J.P., Botelho-Lima, L.S.,
Paranaguá-Vezozzo, D., Kikuchi, L.O.,
Chagas, A., Alencar, R., Tani, C.M.,
Sumita, N.M., Alves, V.A.F., Pinho, J.R.R.,
Carrilho, F.J., Alvarado-Mora, M.V.
University of São Paulo School of
Medicine, FMUSP, Av. Dr. Enéas
Carvalho de Aguiar, 500, Cérquira
César, São Paulo, SP E-mail:
[email protected]
Hepatitis C virus (HCV), a major cause
of chronic liver disease, frequently
progresses to cirrhosis with increased
risk of hepatocellular carcinoma (HCC).
The present study was undertaken to
investigate the distribution pattern of
HCV genotypes, clinical implications
and HCC-associated virus mutations
in patients with HCV/HCC. This study
included 129 randomly selected
patients with HCV/HCC, who were
diagnosed with HCC between 2002 to
2012 at HCFMUSP, São Paulo, Brazil. A
fragment of 674bp partially comprising
the 5’UTR and Core regions was
amplified, sequenced and compared by
phylogenetic analysis with reference
sequences obtained from the GenBank
(n=318).
Bayesian
phylogenetic
analyses were conducted using the
MCMC
simulation
implemented
in BEAST v.1.5.3. Among 129 HCC
patients, 82.9% were Caucasians,
65.9% were male, mean age was
61.4 (29.8-80.0) years, AFP and ALT
means were 987.3ng/mL and 67.1U/L
respectively. The frequency of HCV
genotypes among HCC patients was:
1b (41.5%), 3a (30.8%), 1a (19.1%),
2b (4.2%), 2c (2.1%), 5a (1.1%) and
2a (1.1%). We determined HCV core
gene substitutions at residues 70 and
91 in patients infected with genotypes
1a and 1b. At position R70, 35.9%
had a Glutamine (Q) and at position
L91, 50.0% had a Methionine (M),
respectively. Subtypes 2c and 5a strains
were closely associated to strains from
Argentina and Africa, respectively.
AFP and ALT levels were elevated in
this HCC population. The frequency
of the different HCV genotypes was
similar to previous data from chronic
hepatitis C Brazilian patients. HCV
amino acid substitutions 70 and 91
in the core region affect the results of
combination therapies of interferon/
ribavirin and lead to progression to
HCC. A better understanding of these
risk factors leads to improved early
detection strategies and more-effective
therapies for patients with HCV-related
HCC. Both viral and host factors may
contribute to HCC risk in HCV patients
with chronic infection. FFM/HCFMUSP,
FAPESP2011/50562-0
HV682 - INFECTION BY HCV WITH A
NEW DELETION OF 69 AMINO ACIDS
AT THE MAIN HOMOTYPIC INTERACTION DOMAIN OF CORE GENE IN A
CHRONIC INFECTED PATIENT WITH
HEPATOCELLULAR CARCINOMA
Moreira,
J.P.,
Botelho-Lima,
L.,
Paranaguá-Vezozzo, D., Kikuchi, L.O.,
Chagas, A., Alencar, R., Tani, C.M.,
Carrilho, F.J., Pinho, J.R.R., AlvaradoMora, M.V.
School of Medicine, University of
São Paulo, FMUSP, Av. Dr. Enéas
Carvalho de Aguiar, 500 E-mail:
[email protected]
Hepatitis C virus (HCV) has a 9,600
nucleotides, sRNA(+) genome and
displays a high level of sequence
diversity. HCV core has been associated
in the development of hepatocellular
carcinoma (HCC). A Brazilian male
patient, 61-years old, Caucasian, and
with the following clinical assays
results (AFP 55.76 ng/mL; ALP 86
U/L; AST 75 U/L; ALT 101 U/L; GGT
159 U/L and Platelets count 1.04x105/
mm3) was diagnosed in 2009 with HCC
classified as BCLC-A stage. A fragment
of 674bp partially comprising the
5’UTR and core regions was amplified
and sequenced by automated DNA
sequencing. Genotyping was carried
out by using a phylogenetic analysis
using reference sequences. Patient
Human Virology: HV
137
was infected with HCV subtype 1a. The
amplified fragment covered the core
region between positions 250 to 674
nt of genome. A new deletion of 207
nucleotides, corresponding to 69 amino
acids at positions 49 to 116 in the core
protein was detected in the dominant
viral population. To our knowledge,
this deletion has not been reported
before. Many studies reported HCV
deletions in different isolated clones
in different regions of the HCV genome
but not as the major circulating viral
population in HCV infected patients.
Core contains a number of aminoacid
residues essential for assembly and
release of the viral particle. This
deletion partially included the aminoterminal hydrophilic portion (1aa115aa) of core protein responsible for
multimerization. This region contains
the main homotypic interaction
domain (82 -102 aa). Previous studies
revealed that numerous core residues
are essential for infectious virus
production, including a significant
number in the first 120 positions of
the protein but the first 75 N-terminal
residues of the C protein could generate
nucleocapsid-like particles (NLPs)
smaller in size. We can conclude that
many smaller viral particles were
circulating in this patient and this event
may be related to HCC development.
Support: FFM, HCFMUSP, FAPESP
2011/50562-0.
HV683 - STAGE, TREATMENT
APPROACH AND VIRAL GENOTYPE
IN
CHRONIC
HEPATITIS
B
PATIENTS WITH HEPATOCELLULAR
CARCINOMA (HCC) FOLLOWED UP
IN SÃO PAULO, BRAZIL
Botelho-Lima, L.S., Moreira, J.P.,
Paranaguá-Vezozzo, D., Kikuchi, L.,
Chagas, A., Alencar, R., ONO, S., Sumita,
138
Human Virology: HV
N., Pinho, J.R.R., Carrilho, F.J., AlvaradoMora, M.V.
School of Medicine, University of
Sao Paulo, FMUSP, Av.Dr. Eneas
de Carvalho Aguiar, 500 E-mail:
[email protected]
Hepatocellular carcinoma (HCC) is
a leading cause of cancer-related
death worldwide. Risk factors for
the development of HCC in patients
with chronic hepatitis B virus (HBV)
infection are being elucidated. Thirtyone HBV/HCC patients were enrolled
for this study. A fragment of 1,306 bp
(S/POL) was amplified, sequenced and
genotyped by phylogenetical analysis
using reference sequences. Among
HCC patients, 96.7% were cirrhotic
patients, 64.5% were Caucasians,
80.6% men and mean age was 56.5
years (range 39-85 years). The mean
values of clinical variables were: AFP:
2505.8ng/mL; ALP: 134U/L; AST:
58.9U/L ALT: 44.4U/L; GGT: 134U/L
and Platelets: 137.3mil/mm3. HCC
tumors were identified by: Screening
program–22.6%, Causal Finding–16.1%
and Symptoms–61.3%. From 7 cases
included within screening program,
28.5% presented an early stage HCC
(single nodule<2cm). The majority
of patients were BCLC early stage
(BCLC-A–54.8%) and applicability of
TACE and resection treatments was
more frequently: 38.7% and 22.5%,
respectively. Furthermore, tumors
in BCLC-B and BCLC-C stages were
detected in patients with symptoms.
The frequency of HBV genotypes
among HCC patients was A1 (41.6%),
C2 (16.6%), D3 (16.6%), A2 (8.3%),
F2a (8.3%) and D1 (8.3%). HBV/A1 and
male sex were more prevalent in these
patients. HBV/C2, commonly found in
Asia, was the second most prevalent
genotype. These results agreed with
the distribution of HBV genotypes
circulating in Brazil, where HBV/A1 is
the most prevalent, being predominant
in the Northern and Northeastern
states. Data demonstrate that the
majority of HCC patients are diagnoses
within symptoms stage that limits
the chance for precocious diagnosis
and effective therapy. However, the
screening program showed a positive
result for detection of very early tumors
in HBV patients. According to this
result, we concluded that is necessary
to invest in the adherence of patients
to the screening program. FAPESP
2011/50562-0, FFM and HCFMUSP.
HV684 - DETECTION OF A NOVEL
NOROVIRUS RECOMBINANT STRAIN
IN
AN
AFRICAN-DESCENDANT
COMMUNITY FROM THE AMAZON
REGION, BRAZIL, 2008
Fumian, T.M., Aragão, G.C., Mascarenhas,
J.D.P., Kaiano, J.H., Siqueira, J.A., Soares,
L., Linhares, A.C., Gabbay, Y.M.
Seção de Virologia, Instituto Evandro
Chagas. Rodovia BR316, , E-mail:
[email protected]
Norovirus, a major cause of acute
gastroenteritis outbreaks worldwide,
are constantly evolving. This ability is
reflected in the speed and efficiency
with which these viruses spread and
remain in human population. The
present study reports the detection of
a novel recombination event among
norovirus genotypes in Brazil in
the year of 2008. Initially the fecal
sample (QUI 38F1) was tested for
the presence of NoV antigen using
the RIDASCREEN® Norovirus 3rd
Generation enzyme immunoassay kit.
To confirm the immunoassay positive
result two RT-PCR methodologies were
used. Region B (ORF1) and region D
(ORF2) of NoV genome were amplified
by using specific primers and PCR
reaction conditions. The sample QUI
38F1 yielded positive results with the
three methodologies used. Amplicons
obtained were purified and directly
sequenced in both directions using
the Big Dye Terminator Reaction
Kit® (v. 3.1) and an ABI Prism 3130xl
DNA sequencer. Assignment of the
strain to specific NoV genotypes was
made according to the Genotyping
Tool available on line (Noronet)
and the phylogenetic analysis was
performed using MEGA version 5.05.
In order to investigate the possibility
of a recombination event in the sample
studied, ORF1/2 junction region was
amplified with primers Mon 431/432
and G2SKR. Phylogenetic analysis
carried out with partial polymerase
and capsid sequences resulted in
QUI 38F1 clustering within two
different genotypes, GII.7 (ORF1)
and GII.20 (ORF2), confirming the
results obtained with the genotyping
tool. Plot analysis revealed potential
recombination of QUI 38F1 within two
parental strains [GII.7 (Gwynedd) and
GII.20 (Leverkusen)] and identified the
breakpoint located at 60 nt upstream
the ORF1/2 overlap. The present study
revealed a novel NoV intergenotype
recombinant strain detected in a
relatively isolated, African-descendant
community living in Northern Brazil.
To our knowledge, this is the first
description of NoV intergenotype
GII.7/GII.20 recombinant strain. The
study was funded by the Foudation for
Research Support of the State of Pará
(Fundação de Amparo à Pesquisa do
Estado do Pará - Secretaria de Estado de
Desenvolvimento, Ciência e Tecnologia)
grant code MS/CNPQ/SECTAM –
Human Virology: HV
139
001/2006, agreement 032/2007, and
by Evandro Chagas Institute, Secretary
of Health Surveillance (IEC/SVS),
Ministry of Health, Brazil.
PAPILLOMAVIRUS TYPE 16 IN
CERVICAL ADENOCARCINOMA: CASE
REPORT
Buosi, A.S., Gomez, I.C., Souza, N.C.S.,
Tempaku, P.F., Carvalho, L.V., Caseiro,
M.M., Sa-Filho, D.J.
Centro
Universitário
Lusíada,
UNILUS, Rua: Oswaldo Cruz, 179,
Boqueirão, Santos - SP – Brazil,
11045-101 E-mail: deiasrf@gmail.
com
Background: Human Papillomavirus
(HPV) infection is a necessary cause
of cervical cancer, and is etiologically
associated with a subset of cancers of
the anus, oropharynx, penis, vagina,
and vulva. Several studies have
proposed an association between HPV
infection and oesophageal, laryngeal,
oropharyngeal, lung, urothelial, breast,
cervix and colon cancers. Objective:
To investigate the presence of Human
Papillomavirus (HPV) DNA in cases of
cervical adenocarcinoma. Material and
Methods: Four samples were obtained
from cases of cervical adenocarcinoma
diagnosed and treated at the Hospital
Guilherme Álvaro in Santos-São
Paulo, Brazil. DNA was extracted from
formalin fixed and paraffin-embedded
tumor tissues using QIAamp DNA
Mini Kit (Qiagen). The quality of DNA
extracted was verified by amplifying
the human CCR-5 gene. Detection of
HPV DNA was performed by PCR using
primers GP5 and GP6. HPV positive
and negative controls were performed.
The presence of DNA was verified
by agarose gel electrophoresis. The
140
Human Virology: HV
sequencing of HPV was performed
by ABI PRISM DyeTerminator. The
sequence was compared with Genbank
database. Results: The genomic DNA
from
paraffin-embedded
tumor
tissues presented 2 of 4 positive
amplification for the human CCR-5
gene. Cervical adenocarcinoma tested
showed positive result for HPV DNA
in one sample. The sequence analysis
revealed that strain belonged to HPV
type 16. Conclusion: Our results are
in agreement with some series and
showed evidence of HPV DNA in
cervical adenocarcinoma.
HV695 - EVALUATION OF CYTOMEGALOVIRUS AS IMPORTANT CAUSAL
AGENT IN THE MORBIMORTALITY IN
PATIENTS WITH HIV/AIDS, BELÉM,
PARÁ
Silva, D.F.L., Jr. Avelar, J.L.S., Santos,
T.V.R., Sousa, R.C.M., Arruda, L.M.F.,
Felipe, N.S., Moraes, M.M.
1. UNIVERSIDADE FEDERAL DO
PARÁ, UFPA, AV. AUGUSTO
CORREA S/N
2. INSTITUTO EVANDRO CHAGAS/
SVS/MS, IEC/SVS/MS, BR 316 KM
7 E-mail: dorotealobato@iec.
pa.gov.br
The Cytomegalovirus is considered
one of the main infectious agents
affecting humans, and also a
major cause of morbimortality in
immunocompromised patients. This
study aims to describe the epidemiology,
the clinical and laboratorial aspects
of the Cytomegalovirus as important
causal agent in the morbimortality
in the patients' HIV/AIDS. The
individuals were interviewed using
an epidemiological questionnaire,
review of the promptuary and
blood collection for diagnostic tests
serology and Real-Time PCR. It was
included 241 blood samples from
HIV-infected patients hospitalized in
HUJBB/Belém-Pa. The prevalence of
cytomegalovirus was 99.6% (IgG+).
Gastrointestinal
manifestations
were more frequent (68%) than the
ophthalmic manifestations (24.5%).
The positive results of real-time PCR
were higher than serology (55.6% RT
PCR positive against 2.1% IgM+). The
relation between time of treatment
with ART and RT PCR positivity showed
the highest rates of positivity in the
patients who didn’t realized treatment
(24.6%), those who realized the
treatment less than six months (32.8%)
and those who already realized HIV
treatment for over ten years (14.2%).
It was observed that the RT PCR
positivity was higher (55.2%) when
CD4+ levels were below 100/mm3,
with a reversal of this relation when
CD4+ levels were above 200/mm3,
when the negative results dominated
(38.6%). The CMV was prevalent in
the group of low socioeconomic level.
It was demonstraded superiority of
the RT PCR in relation to the serology
concerning early diagnosis of CMV
infection. Treatment with ART reduces
the incidence of CMV infection. In front
of this scenery and due the significant
morbidity
that
cytomegalovirus
imposes in HIV-positive patients,
it is important the elaboration of a
better screening of these individuals
in relation to the opportunistic CMV
disease. In patients with CD4+ levels
below 100/mm3, it is recommended to
conduct fundoscopy, serology and realtime PCR for the detection of HCMV.
HV698 - LONG CONTROL REGION
GENETIC VARIABILITY OF HPV
16 ISOLATES FROM PARAGUAYAN
WOMEN WITH DIFFERENT GRADE
OF CERVICAL LESION
Mendoza, L., Picconi, M.A.
Instituto de Investigaciones en
Ciencias de la Salud, UNA, IICS-UNA,
Rio de la Pla y La Gerenza Facultad
de Ciencias - Universidad de la
República, UDELAR, Montevideo,
Uruguay
Servicio
de
Virus
Oncogénicos, Malbrán, Malbrán,
Buenos Aires, Argentina E-mail:
[email protected]
Human papillomavirus type 16
(HPV 16) plays a cardinal role in the
pathogenesis of cervical cancer. HPV
16 has intratypic variants which show
different geographical distributions
and different oncogenic potentials.
This study aimed to characterize the
long control region (LCR) genetic
variability of HPV 16 isolates from
Paraguayan women. Sixty seven HPV
16 positive cervical samples were
studied, including 29 low-grade
squamous intraepithelial lesions, 29
high-grade squamous intraepithelial
lesions, 4 cervical cancer, and 5
samples from women with normal
cytology. Specimens were analyzed by
polymerase chain reaction-directed
sequencing of the LCR. Most variants
corresponded to the European
branch-E (82%). There were detected
8 HPV 16 variants; 28% GermanyE-G11, 46% G1-E, 3% G10, 1.5%
Brazil-B14-Asian-American-AA, 7.5%
India-IND8-AA, 1.5% Tanzania-T4African-Af1, 1.5% Navajo indian-AN12AA, 6% Amazonian Indian-AM6-AA
and 2 new isolates; 1.5% newPYa and
1.5% newPYb, with nucleotide changes
at A7752C and A7810T, respectively,
which were included in the E branch
by phylogenetic analysis. Furthermore,
Human Virology: HV
141
all non-E variants (18%) were
detected only in women with cervical
lesion, most of them with nucleotide
substitutions at binding sites of yin
yang 1 (YY1) and nuclear factor 1
(NF1) transcriptional factors. This
observation could partly explain the
differences in the pathogenic potential
of these variants. This is the first
report on HPV 16 variant distribution
and sequence variability in Paraguay.
The characterization of the LCR
contributes to better understand the
molecular epidemiology, geographical
relatedness and pathogenicity of HPV
16 infection.
HV700 - EXARCEBATION OF ASTHMA
IN PATIENTS WITH RESPIRATORY
VIRAL INFECTION
Silva, R.C., Mendes, G.S., Couceiro,
J.N.S.S., Pires, G.V., Valle, S.O.R., Abe,
A.T., França, A.T., Levy, S.A.P., Santos, N.
de Janeiro, UFRJ, Instituto de
Microbiologia Paulo de Góes,
UFRJ - R. Janeiro
Janeiro, UFRJ, Departamento
de Clínica Médica, Faculdade de
Medicina, UFRJ- R. Janeiro E-mail:
[email protected]
Asthma can be defined as a
chronic condition that results from
inflammation of the airways of the lungs.
The development of asthma emerges
from a complex interaction of genetic
predisposition and environmental
factors with viral infection likely
playing a significant role in the effect
of environment on asthma inception.
Over the last 20 years much effort has
been put into clarifying the role that
viral respiratory infections play in
142
Human Virology: HV
the eventual development of asthma.
Tests based on the amplification and
sequencing of the viral genome have
facilitated the understanding of the
association between viral infection
and asthma exacerbation. The aim
of this study was to determine the
rates of respiratory virus infections in
patients with exacerbation of asthma,
treated at the Immunology Service of
the HUCFF/UFRJ. Asthma inception or
exacerbation was defined as an abrupt
or progressive worsening of dyspnea,
wheezing, chest pain, cough or a
combination of those symptoms. A total
of 108 respiratory samples (nasal+oral
swabs combined) were obtained from
83 patients between 19 and 80 years of
age. The asthma attack was classified
by the attending physician as mild,
moderate or severe. Samples were
analyzed by real time PCR for virus
detection. Twenty-six samples (24%;
n = 108) were positive for at least one
virus: 8 single infection detected with
HBoV-2, 6 with HRV, 5 with HAdV, 2
with HCoV (1 229E and 1 HKU1), and
1 with HRSV, HMPV and KIPyV, each.
Additionally, co-infections with these
viruses were observed 2 samples. The
majority of patients with viral infection
(53.8%; 14/26) presented moderate/
severe episode of asthma with clinical
presentation of dyspnea (88.5%;
23/26), wheezing (84.6%; 22/26), and
cough (84.6%; 22/26), and duration
of symptoms of 7 to 15 days (80.8%;
21/26). These results suggest that in
the studied population, viral infections
may be associated with exacerbation
and/or worsening of asthma attacks.
Financial support: CNPq, CAPES,
FAPERJ.
HV708 - POLYOMAVIRUS DETECTION
IN SALIVA OF HIV-INFECTED
CHILDREN
Mendes, G.S., Robaina, T.F., Benati, F.J.,
Pena, G.A., Silva, R.C., Rojas, M., Otero,
R., Castro, G.F., Câmara, F.P., Santos, N.
UFRJ - R. Janeiro
de Janeiro, UFRJ, Faculdade
de Odontologia, UFRJ E-mail:
[email protected]
Human
Polyomaviruses
(HPyVs)
are DNA viruses members of the
Polyomaviridae
family.
Primary
infections generally occur early in
life, are typically subclinical and
followed by persistence of the virus
in the organism. Reactivation of HPyV
infection has been associated to disease
in immunocompromised individuals.
Despite of its growing importance, the
pathogenesis and natural history of
HPyVs infection remain unknown. We
aimed to detect the excretion of HPyV
in the saliva of HIV-infected children in
comparison to healthy control children
and evaluate the possible association
between viral infection and the stage of
immunodeficiency. The samples were
collected from patients attending the
School of Dentistry of the UFRJ. Saliva
was obtained from 60 HIV-infected
children ranging from 6 to 13 years of
age and 60 health children ranging from
7 to 12 years of age. Virus detection
and quantitation was done by real time
PCR assay. HPyVs were detected in 17
(28.3%) and 6 (10%) of HIV-infected
and control children, respectively. A
higher frequency of viral infection was
observed among HIV-infected children
(p = 0.011). Frequency of KIV infection
was significantly higher among
immunocompromised children (p =
0.02). No difference was observed for
BKV, JCV or WUV. The virus loads were
similar in both groups. HPyV was more
frequently detected among children
with severe immunosuppression (P
<0.001). However, no statisti¬cally
significant correlation between the
frequency of HPyV DNA detection and
the use of HAART was observed (p =
0.156). In present study, DNA of BKV,
JCV, WUV and KIV were detected in
saliva samples of both HIV-positive and
healthy control children, although the
frequency of infection was significantly
higher among the HIV-infected subjects.
To our knowledge this is the first report
of KIV and WUV in saliva samples.
These findings suggest that the saliva
may be a route of HPyV transmission
and the oral cavity could be a site of
virus replication and persistence.
HV713 - SHEDDING OF POLYOMAVIRUS IN THE SALIVA OF
IMMUNOCOMPETENT INDIVIDUALS
Mendes, G.S., Robaina, T.F., Benati, F.J.,
Pena, G.A., Silva, R.C., Rojas, M., Janini,
M.E.R., Câmara, F.P., Santos, N.
UFRJ - R. Janeiro
de Janeiro, UFRJ, Faculdade
de Odontologia, UFRJ E-mail:
[email protected]
The human polyomaviruses (HPyV)
are small, non-enveloped virions
with a double-stranded DNA genome,
members of the Polyomaviridae family.
These viruses establish persistent,
primarily asymptomatic, infections.
Although the excretion of HPyV in
Human Virology: HV
143
samples from immunocompetent
individuals has been described,
the significance of these infections
in such individuals has hardly
been characterized. The molecular
characteristics of HPyVs have been
thoroughly analyzed, however much is
unknown about their pathogenesis. It
has been suggested that for BKV and JCV
persistent infection occur in the urinary
tract and central nervous system,
respectively. Lymphoid tissue has been
shown to be the possibly reservoir for
WUV and KIV in immunocompetent
and immunosuppressed individuals.
The epidemiology pattern of HPyV
suggests that transmission can occur
by direct contact or aerosol. The aim
of this study was to investigate and
compare the frequencies of HPyVs in
the saliva of 291 healthy individuals.
The samples were analyzed by real
time PCR. A total of 71 samples (24.3
%) were positive for at least one of
virus: 12.7% (37) for WUV only, 7.2%
(21) for BKV only, 2.4% (7) for KIV only
and, 0.3% (1) for JCV only. Co-infection
of BKV and WUV were detected in 1.7%
(5) samples. The mean number of DNA
copies was high, particularly for WUV
and BKV, indicating active replication
of the viruses. Polyomavirus detection
was higher among individuals of
15 to 19 years of age (46.0%) and
≥50 years of age (33.3%). WUV was
more frequent in individuals of 15 to
19-year-old then decreased in older
age groups; BKV excretion on the other
hand, peaked in the third decade of
life remaining steady thereafter. KIV
was detected more often in subjects
≥50-year-old. These findings reinforce
the previous hypothesis that the saliva
may be a route of transmission of BKV
and the oral cavity could be a site of
virus replication. Moreover, the data
144
Human Virology: HV
also show that it might be true for JCV,
WUV and KIV as well.
HV717 - HUMAN PAPILLOMAVIRUS (HPV) INFECTION OF AMONG
SEXUAL PARTNERS
Rocha, W., Afonso, L., Pesca, L.,
Carestiato, F., Passos, M. Cavalcanti, S.
Lab Diagn Virologico, MIP, Instituto
Biomédico , UFF, Lab Diagn
Virologico, Rua Prof Ernani Melo
101, 321, Centro, Niterói 24210130 Setor de DST, UFF, DST, Campus
do Valonguinho E-mail: willker.
[email protected]
Human
papillomavirus
(HPV)
infections of the genital tract are the
most prevalent sexually transmitted
viruses
worldwide.
Oncogenic
HPV types cause pre-malignant
lesions that can progress to cervical
carcinoma. In the male genital tract,
most HPV infections are sub-clinical
and associated with a vicious circle
of treatment-reinfection of women.
Nevertheless, HPV epidemiological
pathways are still poorly understood.
The literature suggests that different
HPV types can be found among sexual
partners, due to site restriction. In our
study, we aimed to verify HPV infections
in female patients as well as in their
sexual partners, to test this hypothesis.
The HPV DNA prevalence in women
with Cervical Intraepithelial Neoplasia
(CIN) was 92.5% compared with 25%
for normal women, with a statistically
significant difference (p<0.001). In
male samples, the HPV DNA prevalence
in partners from CIN women was 50%
and for normal women partners, it was
17.5%. In the group of CIN women, we
observed that 20 couples had HPV DNA
in both partners. However, only 50% of
the couples shared the same HPV type.
In the group of normal women, only 6
couples were simultaneously infected
by HPV, and from them only 33.3%
had the same virus type. These results
may be attributed to differences in
local immunity and organization of
the genital epithelia of each sex. On
the other hand, female lesions may not
be the result of re-infection by sexual
partners, but rather a true recurrence
of a latent infection. Finally, such 40%
of agreement among all couples leads
us to suggest a redundant process of
infection and reinfection, perpetuating
HPV in the sexually active population.
HV718 - EFFECTIVENESS OF
STANDARD AND DOUBLE DOSE
OSELTAMIVIR AGAINST SYMPTOMS
AND VIRAL SHEDDING IN PATIENTS
WITH PANDEMIC 2009 INFLUENZA
A H1N1, NOT RESISTANT
Thomazelli, L.M., Oliveira, D.B.L.,
Macedo, P.V., Lotufo, J.P.B., Cunha, C.A.,
Neto, J.T., Durigon, E.L.
1. Instituto de Ciências Biomédicas
- Universidade de São Paulo, ICBUSP, Av Prof Lineu Prestes 1374,
São Paulo - SP
2. Hospital
Universitário
Universidade de São Paulo, HUUSP,
3. Serv. de Infectologia e Contr. de
Infec. Hosp. de Curitiba, SICIHCuritiba,
4. Núcleo de Pesq. em Geriatria Clín.
e Prevenção, NPGCP-SP, E-mail:
[email protected]
Of 199 patients screened within 48
hours from onset of symptoms of
influenza, thirty-seven patients aged
more than 5 years, showing rapid test
for influenza A antigen positive, fever
≥38̊C and at least one respiratory
symptom. They were randomized to
receive immediate treatment with
standard-dose (75 mg twice daily)
or double dose (150 mg twice a day)
of oseltamivir twice daily for 5 days
if adults. Pediatric patients received
equivalent doses suitable for children.
Nineteen patients received standard
dose and 18 double dose of oseltamivir.
We analyzed clinical nasopharyngeal
samples, obtained before and after
oseltamivir therapy, as well as the
questionnaire of signs and symptoms
filled in. We isolated the virus in
MDCK cell culture, tested for drug
resistance by a fluorescence-based
neuraminidase inhibition assay and
determined quantitative viral loads
by Real time reverse-transcriptase
polymerase chain reaction (qRT-PCR).
The median age was 22 years for
standard dose group SDG and 19 for
double dose group DDG (interquartile
range 6-47 and 7-53 respectively);
42.1% were male in the first group
and 50% in the second one. The most
common symptoms on the first day of
admission beyond fever were cough
(100% in both groups), rhinorrhoea
(84.2% SDG, 100% DDG), headache
(78.9% SDG, 83.3% DDG) and sore
throat (47.4% SDG, 55.6% DDG). By
day 5 after initiation of treatment
no patient had fever ≥38̊C, the main
symptoms were cough (52.6% SDG,
52.9% DDG), rhinorrhoea (52.6%
SDG, 29.4% DDG), sore throat (10.5%
SDG, 11.8% DDG) and headache (5.3%
SDG, 11.8% DDG). Of 37 samples from
the second visit, no one had virus
detectable by cell culture, 5/19 (26.3%
SDG) and 6/18 (33.3%DDG) had virus
detectable by qRT-PCR but in a very low
concentration. We found no statistically
significant differences in the reduction
Human Virology: HV
145
of viral shedding or time to clearance
of virus between the groups. Antiviralresistant viruses were not recovered.
Financial support: FUSP
HV719 - AN UPWARD TREND IN DNA
P16INK4A METHYLATION PATTERN
AND HPV INFECTION ACCORDING
TO THE SEVERITY OF THE CERVICAL
LESION
Moyses, N., Cavalcanti, S., Carestiato, F.,
Cordeiro, T.
Lab Diagn Virologico, MIP, Instituto
Biomédico, UFF, Rua Prof Ernani
Melo 101, 321, Centro, Niterói
24210-130 E-mail: natthymoyses@
gmail.com
High-risk
human
papillomavirus
(hr-HPV) infection is necessary but
not sufficient for cervical cancer
development. Recently, P16INK4A gene
silencing through hypermethylation
have been proposed as an important
cofactor in cervical carcinogenesis
due to its tumor suppressor function.
We aimed to investigate P16INK4A
methylation status in normal and
neoplastic epithelia and evaluate an
association with HPV infection and
genotype. This cross-sectional study
was performed with 141 cervical
samples from patients attending
at Hospital Moncorvo Filho, Rio de
Janeiro. HPV detection and genotyping
were performed through PCR and
P16INK4A methylation by nestedmethylation especific PCR (MSP). HPV
frequency was 62% (88/141). The
most common HPV were HPV16 (37%),
HPV18 (16.3%), HPV33/45 (15.2%).
An upward trend was observed
concerning P16INK4A methylation
and lesion degree: normal epithelia
(10.7%), low grade lesions (20%),
high grade (57.1%) and carcinoma
146
Human Virology: HV
(93.1%) (p<0.001). A multivariate
analysis was performed to evaluate an
association between methylation, age,
tobacco exposure, HPV infection and
genotyping. A correlation was found
concerning methylation with HPV
infection (p<0.0001), hr-HPV (p=0.01),
HSIL (p<0.0007) and malignant lesions
(p<0.0001). Since viral infection and
epigenetic alterations are related to
cervical carcinoma, we suggest that
P16INK4A methylation profile may be
thoroughly investigated as a biomarker
to identify patients at risk for cancer.
HV726 - INHIBITORY EFFECT
OF
BRAZILIAN
CYANOBACTERIA EXTRACTS AGAINST HERPES
SIMPLEX VIRUS TYPE 2
Oliveira, R.M., Carvalho, L.R., SantAnna,
C.L., Mendes, G.S., Tinga, A.C.C.,
Romanos, M.T.V.
1. Instituto de Microbiologia Paulo
de Góes - UFRJ, IMPG - UFRJ,
Bl. I, sala 064 ss - CCS-UFRJIlha do Fundão-Rio de Janeiro/
CEP21.941-590
2. Instituto de Botânica-Secretaria
do Meio Ambiente-São Paulo,
IBt-SMA-SP, Av. Dr. Miguel
Stéfano, 3687, Água Funda, São
Paulo/CEP 04301-902 E-mail:
[email protected]
Herpes simplex viruses (HSV)
infections are among the most common
diseases throughout the world.
The incidence and severity of HSVrelated pathologies have increased
recently and the illness is usually
more severe in immunocompromised
patients. Several drugs are currently
available for the treatment of HSV
infections such as acyclovir. However,
the emergence of drug-resistant
strains of HSV led to a search for
alternative antiherpetic agents that
have a wide range of efficacy without
serious adverse effects. Thus, new
anti-HSV drugs are urgently needed.
Cyanobacteria are prolific producers
of highly bioactive compounds, some
of them displaying interesting antiviral
activities. Cyanovirin-N is a protein
synthesized by Nostoc ellipsosporum,
which, besides inhibiting HIV and
influenza virus, blocks HSV-1 entry
into cells and prevents membrane
fusion mediated by HSV glycoproteins;
three Microcystis species showed
remarkable activity against influenza
virus and, estuarine cyanobacterial
extracts are active against HSV-1. In
this work, methanolic extracts of the
cyanobacterial species Phormidium
sp. CCIBt 1018, Merismopedia sp CCIBt
3048 and Geitlerinema unigranulatum
CCIBt 971 were evaluated against HSV2. Antiviral assays were performed on
Vero cell cultures, in the presence of the
cyanobacteria extracts at non-cytotoxic
concentrations. Percentage of inhibition
(PI), 50% effective concentration
(EC50) and selectivity index (SI) were
determined. Phormidium sp. showed
PI = 99.2%, with EC50 = 22 µg/mL and
SI superior to 22.8; G. unigranulatum,
PI = 99.8% with EC50 = 9 µg/mL and
SI = 50.5 and no activity was observed
when Merismopedia sp. was evaluated.
Our results are in agreement with the
ones displayed in literature about the
cyanobacterial extracts (compounds)
inhibitory activity against HSV. Bioactive
compounds identification studies will
be performed in order to elucidate
their structures and mechanism of
action. Financial Support: CAPES, CNPq
and FAPERJ.
HV733 - BETAHERPESVIRUS IN-
FECTIONS AFTER HEMATOPOIETIC
STEM CELL TRANSPLANTATION
(HSCT): DETECTION AND MONITORING OF ACTIVE INFECTIONS USING
ANTIGENEMIA ASSAY AND PLASMA
NESTED-PCR
Pancielli, P.N., Gullin, A.C., Oliveira, R.S.,
Vigorito, A.C., de Souza, C.A., Rossi,
C.L., Peres, R.M.B., Costa, S.C.B., Bonon,
S.H.A.
Department of Clin. Med. Faculty of
Medical Sciences/UNICAMP, FCM/
UNICAMP, UNICAMP, Campinas, SP,
Brazil Bone Marrow Transplant
Unit,
Hemocenter/UNICAMP,
HEMOCENTER/UNICAMP, UNICAMP,
Campinas, SP, Brazil Department of
Clinical Pathology, FCM/UNICAMP,
FCM/UNICAMP, UNICAMP, Campinas,
SP, Brazil E-mail: prinavas@fcm.
unicamp.br
Human Betaherperviruses (CMVH,
HHV-6 and HHV-7) cause frequent
complications
after
allogeneic
hematopoietic stem cell transplantation
(HSCT) and are the major cause of
opportunistic infections, increased
morbidity and mortality in patients
in these patients. To minimize the
possible clinical manifestations caused
by these herpesviruses in the posttransplant period, it is necessary to
use techniques for the rapid diagnosis
and early treatment. To standardize
the use of antigenemia (AGM) assays
in detecting active infection caused
by HHV-6 and HHV-7 in patients
undergoing
HSCT;
to
monitor
prospective
patients
undergoing
HSCT, using antigenemia assays and
nested PCR (N-PCR) to evaluate the
clinical impact caused by these viruses
and coinfection among them. Thirtyfive patients undergoing HSCT were
monitored weekly, from day 0 until
Human Virology: HV
147
day 100 in the post-transplantation
period, using antigenemia assays
and N-PCR for the detection of
active infections. HHV-6 and HHV-7
antigenemia assays were developed
in peripheral blood mononuclear cells
with the use of monoclonal antibodies
which are specific for these viruses
and peroxidase staining. Active HCMV
infection detection was performed
using a commercial kit (Iq Products) by
immunofluorescence staining. Using
N-PCR, twenty-six out of the 35 patients
monitored had active infections
caused by HCMV, HHV-6 and HHV-7,
respectively, in 26/35 (74.3%), 13/35
(37.14%) and 19/35 (54.3%) detected
by N-PCR and/or AGM. Active infections
detection using antigenemia assay
for HCMV, HHV-6, HHV-7 occurred,
respectively, in 16/35 (45.7%), 19/35
(54.3%) and 22/35 (62.8%). The
standardization and development of
the HHV-6 and HHV-7 antigenemia
assays appear to be effective in the
diagnosis of active infections caused
by these herpesviruses. However,
further studies are required to
establish the clinical impact, if any, of
immunomudalation due to HHV-6 and
HHV-7. Financial Support: FAPESP
HV742 - MOLECULAR CHARACTERIZATION
OF
INFLUENZA
VIRUSES COLLECTED FROM YOUNG
CHILDREN IN UBERLÂNDIA, BRAZIL
- FROM 2001 TO 2010: PRESENCE
OF REASSORTMENT BETWEEN 2002
STRAINS
Oliveira, T.F.M.S., Yokosawa, J., Motta,
F.C., Siqueira, M.M., Silveira, H.L.,
Chavéz, J.H., Queiróz, D.A.O.
1. Universidade
Federal
de
Uberlândia, UFU, Av. Amazonas,
2210,
Campus
Umuarama,
148
Human Virology: HV
Uberlândia/MG
2. Instituto Oswaldo Cruz , IOC, Av.
Brasil, 4365, Manguinhos, Rio
de Janeiro/RJ E-mail: thelmao@
umuarama.ufu.br
Influenza viruses are important
pathogens responsible for respiratory
disease worldwide and represent a
major threat in public health, causing
annual epidemics or even pandemics.
A study was conducted about the
influenza viruses that circulated
in Uberlândia city, in the state of
Minas Gerais, Midwestern Brazil,
during 2001-2010 seasons. The
purposes of the study were to detect
these pathogens in young children
presenting acute respiratory disease
(ARD), to characterize the strains
from the identified cases by analyzing
partial nucleotide sequences of HA and
NA genes (and to compare the strains
sequences with the vaccine sequences).
A total of 605 nasopharyngeal aspirates
were collected from children under
five years old presenting ARD and, by
immunofluorescence assay, 40 (6.6%)
samples showed to be reactive for
influenza viruses: 39 of type A and
one of type B. RT-PCR was carried
out to amplify segments of HA and
NA genes of these viruses. Sequence
analyses revealed that 41.0%, 17.9%
and 2.6% strains were characterized as
belonging to subtypes H3N2, H1N2 and
H1N1, respectively. Furthermore, the
receptor binding sites were preserved
in all isolates, although all of them
contained variations in the antigenic
sites. Substitutions of specific amino
acids residues for sialic acid binding
were observed in all 2001-2007 strains.
Moreover, extra potential N-linked
glycosylation sites were identified
in two H3 strains. Some amino acid
substitutions that were observed in the
neuraminidase gene were not related
to the antigenicity. All the H3 isolates
detected in the 2001-2003 period were
different from the vaccine strain A/
Moscow/10/99 that was given in the
same period. Constant surveillance of
antigenic variants over the years has
become vital in order to detect new
strains and to determine the need to
add them to the flu vaccine in order to
reduce the burden that these strains
may cause in public health.
HV744 - COMPARISON OF DIFFERENT
EPIDEMIOLOGICAL METHODS OF
DETECTION OF DENGUE VECTOR IN
DIVINOPOLIS, MG
Taranto, M.R., dos Santos, M., de Souza,
J.P., Andrade, A.C.D.S.P., Camargos,
V.N., Alves, S.N., dos Santos, L.L., de
Magalhães, J.T., Oliveira, C.D.L., Taranto,
A.G., Magalhães, J.C., Kroon, E.G.,
Drumond, B.P., Figueiredo, L.B., Ferreira,
J.M.S.
João del Rei, UFSJ, Rua Sebastião
Gonçalves Coelho, 400 Chanadour
- Divinópolis MG
Gerais , UFMG, Av. Antônio Carlos,
6.627 Pampulha -Belo Horizonte
MG
3. Universidade
Federal
de
Juiz de Fora, UFJF, Rua José
Universitário São Pedro - Juiz de
Fora MG
João del Rei, UFSJ, Rodovia MG
443 Ouro Branco MG E-mail:
[email protected]
Dengue is principal arboviruses that
affecting humans, becoming a major
public health problem. Minas Gerais
(MG) is one of the Brazilian States with
high endemicity. Divinopolis, a city of
MG State, has already notified 131 cases
of the disease in 2012, being among the
30th cities with the largest number of
cases. The main goal of this work was
to carry out an epidemiological study of
the vector in Divinopolis, and compare
these results with data from the Survey
Quick Index of Aedes aegypti (LIRAa).
The methodology used in this work is
different from that used by LIRAa. The
last uses the source present in existing
breeding sites in the city to determine
the rate of infestation by the vector
of dengue, four times in year. In this
study, we used traps containing wood
vanes manufactured and placed in 44
places, that were distributed in six
regions in the city (North, Northeast,
Southeast, Southwest, West and
Central). Subsequently, eggs of each
area were counted and the pallets
were submersed in water containing
fish meal for 6 days, and these stages
L3 and L4 were identified in A. aegypti
or A. albopictus. Following, the number
of larvae was compared to the number
of foci in the same period collected
by LIRAa. In the Northeast, in the
month of January/2012, 358 larvae
were collected in this study and 65
sources of dengue were detected by
LIRAa, numbers considered high.
In contrast, in the Southeast, in the
month of June/2011, larvae were not
detected and only three source in the
data obtained by LIRAa. These findings
were expected since the January/2012
was a rainy season, while June/2011
was a month that had low temperatures
(10 °C) and low rainfall. Thus, this
study provides contributions to
epidemiological studies of dengue and
Human Virology: HV
149
alert to the intensification of preventive
and educational especially in regions
with a high incidence of the vector.
FAPEMIG, CNPq, CAPES, UFSJ
HV746 - DIFFERENTIAL DIAGNOSIS
OF FEBRILE ILLNESSES MEASLES,
RUBELLA, DENGUE AND PARVOVIRUS B19 IN CENTRAL BRAZIL
Argolo, A.F.L.T., Paula, D.S., Ferreira,
V.A.M., Silva, M.M.J., Finotti, A., Silva,
V.L., Ramos, C.H., Martelli, C.M.T.
1. LABORATORIO
DE
SAUDE
PUBLICA
DR.
GIOVANNI
CYSNEIROS,
LACEN-GO,
AV.
CONTORNO, Nº3556. JD. BELA
VISTA. GOIANIA-GO
GOIÁS, UFG, RUA 235 - SETOR
UNIVERSITARIO - CEP: 74605050
- GOIÂNIA - GOIÁS - BRASIL
E-mail:
angelafarmaceutica@
hotmail.com
The incidence of measles and rubella
has been decreasing exponentially in
Brazil since the implementation of
mass vaccination campaigns in the
last two decades. No autochthonous
measles or rubella case was registered
by national surveillance program ever
since 2000 and 2009 respectively. In
contrast, there is an increasing trend
of dengue cases among in adults and
children countrywide. The diagnosis
of febrile rash illnesses may be difficult
and laboratory tests are essential to
confirm the etiologic diagnosis and for
surveillance purpose. A cross-sectional
study was conducted to detect the
frequency of IgM antibodies against
dengue virus and parvovirus B19
among serum samples from suspected
clinical diagnosis of measles or rubella
sent to Public Health Laboratory
150
Human Virology: HV
of Goias, LACEN-GO in Central
Brazil in 2011. The epidemiological
surveillance included data of social
characteristics, vaccination status and
symptoms. The inclusion criteria were:
(1) serum samples from patients who
fulfilled the criteria of case according
to Health Ministry (febrile rash illness
with other specific symptoms); (2)
samples collected within 28 days
of the onset of the symptoms; (3)
samples previously tested with IgM
negative or indeterminate results
for measles and rubella. All samples
were stored at -20°C. IgM antibodies
against dengue were tested by enzyme
linked immunosorbent assay (ELISA)
(PanBio). If samples were IgM negative
for dengue virus they were also tested
for specifc IgM antibodies against
parvovirus B19 (Biotrin). SPSS 17.0
(SPSS Inc., Chicago, IL) software was
used for data analyses. Of 263 samples
sent to LACEN-GO, 127 were eligible
for the study. Of participants 44.1%
were younger than 1 year of age and
43.2% were female. Acute infection by
dengue virus and parvovirus B19 were
confirmed in 23.6% (95%CI 16.3%31.3%) and 7.1% (95%CI 3.1%-11.7%)
febrile cases respectively. Among 4
indeterminate rubella cases 3 were
confirmed by dengue virus infection
(according national epidemiological
protocol). Laboratory tests provide
invaluable contribution for the
differential diagnosis of febrile rash in
different epidemiological settings. This
is the first evidence of parvovirus B19
circulation in Central Brazil. Financial
support: Secretaria de Saúde do Estado
de Goiás
HV747 - IDENTIFICATION OF AEDES
AEGYPTI AND AEDES ALBOPICTUS LARVAE IN THE CITY OF BELO
HORIZONTE, STATE OF MINAS
GERAIS, IN 2011
Miranda, D.P.J.
Gerais, Secretaria Municipal, E-mail:
[email protected]
Dengue virus is the most important
human arboviral pathogen worldwide.
World Health Organization estimates
that over 40% of the population is at
risk of acquiring dengue and there may
50 million of dengue infection every
year. There are two important vectors
of Dengue virus (DENV) throughout
the world: Aedes aegypti and Aedes
albopictus. In Brazil outbreaks of
dengue has been associated with
the presence of Aedes aegypti, due
to its high capacity to spread and
adapt to human environment. The
introduction of Aedes albopictus in
Brazil in 1986 is especially worrying
because it can readily transmit major
arthropod –borne viruses such as
Dengue virus and Chikungunya virus.
Belo Horizonte, the capital of Minas
Gerais, has been suffering from dengue
outbreaks since 1996. Two major
outbreaks have already occurred in
Belo Horizonte: one in 1998 with
approximately 80.000 cases and
another one in 2010 with 57.000 cases.
This study aimed to identify larvae of
Aedes aegypti and Aedes albopictus
from oviposition traps displayed in all
nine administrative districts of Belo
Horizonte city in 2011. To perform
this study, oviposition traps were
displayed in residential area of Belo
Horizonte, for a period of one week,
during four times of the year: January,
April, July and October of 2011. The
eggs from ovitraps were counted and
subsequently hatched in laboratory
control conditions. After eclosion, each
larva was identified into specie by
morphological characteristics. A total
of 68.827 larvae were identified. 66.423
(97%) was identified as Aedes aegypti
and 2.404 (3%) as Aedes albopictus.
The results show that Aedes aegypti is
more prevalent than Aedes albopictus.
This implicates Aedes aegypti as the
major vector responsible for dengue in
Belo Horizonte. However, the presence
of Aedes albopictus increases the risk
of transmission of dengue. Therefore,
entolomological surveillance must be
taken to provide data for control plans
of these two vectors.
HV749 - GENETIC VARIABILITY OF
HEPATITIS B VIRUS AND HEPATITIS C
VIRUS IN CHRONIC CARRIERS WITH
AND WITHOUT HEPATOCELLULAR
CARCINOMA
Araujo, O.C., Barros, J.J.F., Ó, K.M.R.,
Nabuco, L.C., Moraes, M.T.B., VillelaNogueira, C.A., Araujo, N.M.
1. Laboratório
de
Virologia
Molecular, Fundação Oswaldo
Cruz., Fiocruz, Av. Brasil, 4365 Manguinhos, Rio de Janeiro.
2. Hepatologia Hosp. Universitário
Clementino Fraga Filho, HUCFF,
Rua Rodolpho Paulo Rocco, 255
Cidade Universitária Ilha do
Fundão Rio de Janeiro E-mail:
[email protected]
Hepatocellular carcinoma (HCC) is
globally the fifth most common cancer.
The major risk factors for developing
HCC are chronic hepatitis B virus (HBV)
and hepatitis C virus (HCV) infections.
Several mutations in these viruses
have been previously associated to
hepatocarcinogenesis. The purpose of
this study is to assess the prevalence
of HBV subgenotypes, core promoter
Human Virology: HV
151
(CP) and pre-S mutations, as well as,
HCV genotypes and core mutations
in chronic carriers with and without
HCC. At this moment, 80 patients with
chronic HBV infection (15 with HCC)
and 58 patients with chronic HCV
infection (35 with HCC) referred from
HUCFF were enrolled in the study. HBVDNA and HCV-RNA were extracted from
serum samples and amplified by PCR
and RT-PCR, respectively. Nucleotide
sequences were determined and
phylogenetic analysis was conducted
using MEGA version 4.1. HBV
subgenotypes frequencies in HCC
patients were 90% for A1 (9/10) and
10% A2 (1/10), whereas in patients
without HCC the distribution was 60%
A1 (21/35), 17% A2 (6/35), 3% D1
(1/35), 6% D6 (2/35), 3% D7 (1/35),
6% F2 (2/35) and 6% F4 (2/35). The
HBV CP mutations A1762T, G1764A,
pre-S F141L mutation and pre-S
deletions, were found in 60% (3/5),
60% (3/5), 14% (1/7), and 13% (1/8),
respectively. In HBV chronic carriers
without HCC, these mutations were
detected in 37% (10/27), 41% (11/27),
13% (4/30), 9% (3/33). Among HCV
chronic carriers with HCC, genotypes
frequencies were 39% for 1a (12/31),
36% 1b (11/31) 26% 3a (8/31),
whereas in patients without HCC were
33% 1a (6/18), 56% 1b (10/18) and
11% 3a (2/18). In the HCV 1b genotype
isolates, R70Q and L91M were found
in 36% (4/11) and in 73% (8/11) of
HCC patients, and in 50% (5/10) and
in 100% (10/10) of patients without
HCC, respectively. Our preliminary
results have shown a higher frequency
of mutations, previously associated to
hepatocarcinogenesis, in HBV chronic
carriers with HCC. However, among
HCV chronic carriers from our study,
this association could not be observed.
152
Human Virology: HV
HV751 - DIAGNOSIS OF DENGUE
VIRUS INFECTION BY DETECTION
OF SPECIFIC IMMUNOGLOBULIN A
Morais, V.M.S., Coêlho, M.R.C.D., Silva,
V.G., Cordeiro, M.T.
1. Universidade
Federal
de
Pernambuco, UFPE, Av. Prof.
50670-901
2. Centro de Pesquisas Aggeu
Magalhães , CPqAM, Av. Prof.
50670-901 E-mail: vivi.martha@
hotmail.com
Dengue diagnosis by detection
of immunoglobulin M (IgM) has
limitations when used in endemic
regions, because in case of secondary
infection the levels of IgM are lower,
sometimes undetectable. Detection
of immunoglobulin A (IgA) could be a
parameter in the early confirmation
of dengue, especially in secondary
infections, since it occurs in higher
levels in these cases. We performed
in house ELISA for IgA detection in
serum samples of 78 dengue patients,
previously confirmed by IgM and/or
RT-PCR. The assay was performed as
follows: Microtiterplate was coated
with 100µL/well (1µg/100µL) of antihuman IgA diluted in carbonate buffer
pH 9.6; blocking was done with 4%
bovine albumin in PBS pH 7.2; 50µL
of serum diluted 1/100 was added in
duplicate, after 1h incubation at 37ºC,
50µL of DENV antigen was added
and incubated 1h at 37ºC. Positive e
negative controls were used. Washing
was done using PBS-T; 25µL/well of
anti-dengue conjugate was added.
Tetramethylbenzidine (100µL/well)
was used as substrate, followed by the
addition of stopping solution. Optical
density was read at 450 nm and results
were calculated by dividing the mean
absorbance of test-samples by the
mean absorbance of negative control,
samples with results >2 was reactive. Of
the 78 individuals, 43 were classified as
primary infection and 35 as secondary
infections. IgA was detected in 16.3%
(7/43) of the primary infection and
65.7% (23/35) of the secondary
infections. It was found a sensitivity
of 45.2% and specificity of 87.5%.
Positive and negative predictive values
were 93.3% and 29.2%, respectively.
The results have shown that using both,
IgM and IgA antibody detection may
be very useful and could help in the
interpretation of results in the acute
disease phase, and also in the early
detection of dengue cases, allowing
the adoption of preventive measures to
prevent the occurrence of severe cases.
Financial Support: CAPES
HV752 - STANDARDIZATION OF
REAL-TIME PCR TO DIAGNOSE
HUMAN CYTOMEGALOVIRUS REACTIVATION AND DETERMINE CUTOFF
FOR PREEMPTIVE TREATMENT IN
RECEPTORS OF HEMATOPOIETIC
STEM CELLS
Peres, R.M.B., Bonon, S.H.A., Costa,
C.R.C., Andrade, P.D., Franco, J.C.,
Alves, W.R., Costa, S.R.C., Vigorito, A.C.,
Aranha, F.J.P., Souza, C.A., Costa, S.C.B.
Department of Clinical Medicine
- UNICAMP, SP, Brazil, UNICAMP,
BRAZIL Bone Marrow Transplant
Unit, Hemocenter - UNICAMP,
SP,Brazil, UNICAMP, Brazil E-mail:
[email protected]
Hematopoietic
stem
cell
transplantation (HSCT) is an important
therapeutic tool for treating malignant
and non-malignant disorders, and
the human cytomegalovirus (CMV)
reactivation occurs in 50-90% of
allogeneic
transplant
recipients.
Monitoring of its reactivation is critical
for HSCT recipients. While detection
of CMV antigenemia is still widely
used for monitoring CMV infection
and guide preemptive therapy in
patients at risk of developing CMV
disease, the quantification of CMV
DNA in blood by PCR is emerging as an
alternative to the antigenemia assay
and may soon become the standard
for the surveillance of CMV infection
in allogeneic HSCT recipients, because
it presents advantages over the
antigenemia assay. Aim: standardization
of real-time PCR to diagnose CMV
reactivation and determine cutoff for
preemptive treatment in receptors
of HSC. The reference standard curve
for calibration of CMV copy numbers
was constructed inserting the US17
amplicon into a plasmid, using a
cloning strategy, and propagated in
competent cells. For this construct,
plasmid DNA was purified on columns,
DNA concentration was determined
by measuring using a NanoDrop ND1000 spectrophotometer and the
corresponding copy number was then
calculated. The construct was serially
diluted in water within a range of 102
to 107 copies/μl. Receiver operating
characteristic (ROC) plot analysis
will be performing to determine a
threshold value of the CMV DNA load
by real-time PCR using antigenemia
as gold standard. Between 11/2010
and 06/2012 a total of 499 samples
was included in the study. The samples
came from 35 HSCT recipients. Of
these, only 11 (31,5%) had positive
antigenemia. Real-time PCR is still
Human Virology: HV
153
being made. The quantification of CMV
DNA load using real-time PCR appears
to be applicable to the clinical practice
and an optimal cutoff value for guiding
timely preemptive therapy should be
clinically validated in future studies.
Financial support: FAPESP
HV753 - DETECTION OF PAPILLOMAVIRUS 16 AND ITS VARIANTS IN
A REGION OF HIGH INCIDENCE OF
PENILE CANCER IN BRAZIL
Silvestre, R.V.D., Lopes, B.P.T., Fonseca,
A.G., Rahal, P., Mota. M.T.O., Mello,
W.A.
1. Instituto Evandro Chagas SVS/
MS, IEC, BR 316 Km 07, Levilândia
- Ananindeua - Pará.
2. Hospital Ophir Loyola, HOL, Av.
Gov. Magalhães Barata, 992.
Belém - Pará
UNESP, Rua Cristóvão Colombo,
2265, Jardim Nazareth - São
José do Rio Preto - SP. E-mail:
[email protected]
Introduction. Emerging evidence
suggest that penile cancer is in part
associated to HPV infection. Laboratory
data indicate that HPV DNA is found
in until 48% of all penile tumors, and
most of these cases correspond to HPV
16. This is a rare tumor location with a
global incidence about 0.2% however,
is relatively high in developing
countries as Brazil where men posses
2.1% of chance to develop this cancer,
mainly among the inhabitants in
the North of the country. Material
and Methods. Eighty-two paraffin
embedded biopsies from patients with
histological diagnosis of Squamous
penis carcinoma, from a reference
154
Human Virology: HV
hospital of cancer in the city of BelemPara were submitted to DNA extraction
using “QIAamp FFPE”. HPV specific
detection was done using the “INNo
Lipa HPV” that is able to detect 26
HPV types. The HPV 16 variants were
assessed using a primer sets that is able
to amplify a 211 bp fragment of HPV
16 E6 gene and was amplified with the
same DNA used to HPV identification.
The amplified fragments of E6 were
directly sequenced and compared with
sequences contained in the Gene Bank,
the accession No. K02718. The positive
samples of HPV 16 variants obtained
were classified according Chopjitt et
al (2009) using the BioEdit sequence
analyzer. Results. From the total 82
samples analyzed, 52 (63.41%) were
positive for any HPV type and 14
(26.9%) were positive for HPV 16.
The alignment of amplified sequences
from E6 gene of HPV 16 revealed
12 samples with 100% homology to
the European prototype, one sample
was identified as Asian/American
G and another samples determined
as AF2 variant with substitutions in
the positions T109C, G132T, C143G
and G145T. Conclusion As observed
worldwide, our findings suggest HPV
16 as the viral type most frequently
associated with cancer of the penis in
our region. Parallel to these findings,
the European HPV 16 variants appear
to be more commonly associated with
penile cancer than other non-European
variants in our study.
HV754 - A SOPHISTICATED GIANT:
ACANTHAMOEBA
POLYPHAGA
MIMIVIRUS IS ABLE TO REPLICATE
IN HUMAN PBMCS AFTER IFN
ALPHA TREATMENT DESPITE THE
INDUCTION OF EXPRESSION OF THE
ISGS MXA, OAS AND 6/16 MRNAS
Silva, L.C.F., Dornas, F.P., Almeida, G.M.,
Oliveira, D.B., Kroon, E.G., Ferreira,
P.C.P., Abrahão, J.S.
6627 - Pampulha - Cep; 31270-901
- BELO HORIZONTE - MG E-mail:
[email protected]
Acanthamoeba polyphaga mimivirus
(APMV), the largest virus known,
belongs to Mimiviridae family. APMV
was first isolated few years ago from
a water cooler, in an English hospital
during a pneumonia outbreak.
Although free-living amoebas are the
APMV hosts, recent studies showed that
this virus is able to replicate in some
murine and human phagocytes. Since
APMV is considered a hypothetical
human pathogen, this work aimed
to investigate if human PBMCs are
permissive to APMV replication and
the interaction of APMV with the
interferon system in peripheral blood
mononuclear cells (PBMCs) human.
Therefore, human PBMCs were grown in
6 well plates (500.000 cells/well), were
treated with 1000 U of IFN alpha for 18
hours and subsequently infected with
APMV (M.O.Is: 1, 10 and 50) or directly
infected with APMV, without treatment
with IFN alpha. Then, infected PBMCs
monolayers and supernatants were
collected and submitted to titration in
amoeba culture and to Real-Time PCR
to APMV helicase gene amplification.
In addition, Real-Time PCR to verify
the expression of mRNAs of interferon
stimulated
genes
(ISGs)
were
performed. Real-Time PCR and titration
results showed that APMV is able to
replicate in human PBMCs, even after
induction with IFN alpha. Although
the presence of the antiviral state was
confirmed in treated cells by real time
PCR for ISGs (OAS, 6-16 and MxA), this
fact did not showed any influence in
virus yield. Analysis of gene expression
showed low expression of IFNs mRNAs
in PBMCs infected only with APMV,
untreated with IFNs. Taken together,
our results suggest that APMV is able to
replicate in human PBMCs, even after
induction with IFN alpha . The large
genome of APMV seems to be able to
encode genes involved in mechanisms
that allows the virus do not stimulate
the IFN system. This fact reinforce the
theory that mimivirus may be a human
pathogen, and could be a reflection of
an interaction between the ancestors
of APMV and vertebrate hosts.
HV764 - ROTAVIRUS INFECTIONS
IN AMERINDIAN CHILDREN FROM
WESTERN BRAZIL: CHARACTERIZATION OF UNCOMMON HUMAN
G8P[6] GENOTYPE WITH SIMILARITY TO BOVINE AND BAT STRAINS
Luchs, A., Cilli, A., Morillo, S.G.,
Timenetsky, M.C.S.T.
Instituto Adolfo Lutz, IAL, Av Dr
Arnaldo, 355 Centro de Virologia
Cerqueira Cesar 01246-902 E-mail:
[email protected]
Gastroenteritis is the leading problem
in indian communities. During national
rotavirus (RV) surveillance 20082011 G2P[4], G3P[8] and G8P[6]
genotypes were detected in children
≤3 years from indigenous villages in
western Brazil. The aim of this work
was to carry out sequence analysis of
the two outer capsid proteins (VP4
and VP7) in order to obtain further
information on the genetic relationship
between human and animal RV. This
retrospective study was conducted
with
convenient
surveillance
specimens. RV were detected using
Human Virology: HV
155
ELISA, PAGE, and genotyped by RT-PCR.
The genetic relationship was analyzed
by sequencing of selected strains: two
G2 (IAL-RN191, IAL-RN71094), one
G8 (IAL-RN376), two P[6] (IAL-RN376,
IAL-RN377), and one G3 (IAL-R2758).
IAL G2 RV sequences showed 79%78.6% similarity compared to animal
representative strains. IAL-RN376
G8 sequence shares a clade with
bovine and human strains, displaying
highest nucleotide identity to African
human strains (DRC86-98%, DRC8897.9%), following by the African
bovine NGRBg8 strain (95.1%). IALRN376 and IAL-RN377 P[6] sequences
showed the highest identity to human
R330 strain (99.6%). In addition,
IAL P[6] sequences were also closely
related to an African fruit bat RV strain
(KE4852/07) (94.6%). IAL-R2758 G3
sequence share the highest nucleotide
similarity to human strains (98.6-99%),
however, demonstrated moderateto-low nucleotide identity to animal
strains
(90-70.5%),
highlighting
two feline strains: Cat2 (89.5%) and
BA222 (90%). Taking the risks, this
study suggest that a reassortment
between bovine RV G8 and bat RV P[6]
could be occurred in animal host(s)
preceding the transmission to human;
nevertheless, also suggest that humans
could serve as a RV reservoir, resulting
in anthropozoonotic transmission.
In
indigenous
population,
an
anthropozoonotic transmission is
probably fairly frequent once the
inhabitants live in close contact with
animals together with poor hygienic
conditions. Sponsor:PPG-PLSP-CCDSES/SP
HV767 - SEROPREVALENCE FOR
HANTAVIRUS IN RURAL WORKERS
FROM CORURIPE COUNTY, STATE OF
156
Human Virology: HV
ALAGOAS
Barros, P.A., Medeiros, N.P.T., dos Santos
Júnior, J.A., Santos, F.M., Feitosa, R.C.S.,
Melo, D.M., Solano, W.N., Tenório,
J.B.A., Lima, M.C., Figueiredo, L.T.M.,
Borges, A.A.
1. Federal University of Alagoas,
UFAL, Praça Afrânio Jorge s/n,
57010-020 / Maceió - Alagoas
2. Central Laboratory of Public
Health of Alagoas , LACEN-AL, Av.
Marechal Castelo Branco, 1773
- Jatiúca, 57030-340, Maceió Alagoas
3. Faculty of Medicine of Ribeirão
Preto, USP, FMRP - USP, Av.
Bandeirantes, 3900 - Monte
Alegre - CEP: 14049-900 Ribeirão
Preto/SP
E-mail:
patricia_
[email protected]
The genus Hantavirus belongs to family
Bunyaviridae and includes rodentborne viruses that are transmitted to
humans by inhalation of infectious
aerosols from infected rodent excreta,
and may cause lethally illness. Since
1993, in Brazil, the hantavirus
cardiopulmonary syndrome (HCPS)
has been reported in an increasing
number of municipalities and regions
previously considered free for
hantavirus circulation. Nevertheless,
in the Northeast region of Brazil data
about HCPS is very scanty, and is
absent for the state of Alagoas. In the
last decades, the landscape from region
of south of this state - where is located
the Coruripe county – underwent
intense alteration because of the
sugar cane agroindustry development.
These environmental disturbances
may alter the frequency of human
contact with hantaviral hosts, such as
Necromys lasiurus and Oligoryzomys
nigripis, and therefore, could result in
the emergence of human hantavirus
infections. In this sense, the aim of this
study was investigate the prevalence
of IgG antibodies to hantavirus in rural
workers from a Plant in the Coruripe
county, conducing a serological survey.
Sera of 704 volunteers healthy rural
workers were collected and used to
detect IgG antibodies against N protein
of Araraquara hantavirus (rN ARAV),
by enzyme immunoassay (ELISA). The
positive samples were then tittered.
The prevalence of IgG anti-rN ARAV
antibodies were 10.51% (74/704),
with titers from 200 to 6,400. Of these,
57 (77.02%) volunteers positive for
IgG attested that had never worked
out of state of Alagoas. Our findings
demonstrate serological evidence
of past infections with hantavirus
in human from the state of Alagoas,
and suggest that there is hantavirus
circulation in this state from Northeast
Region, Brazil. Financial support: CNPq,
CAPES, FAPEAL
HV769 - NO ASSOCIATION BETWEEN
HUMAN CERVICAL LESION AND
POLYMORPHISMS IN MDM2 AND
WAF1 GENE
Amaral, C.M.M., Gurgel, A.P.A.D.,
Freitas, A.C.
UNIVERSIDADE
FEDERAL
DE PERNAMBUCO, UFPE, AV.
PROFESSOR MORAES REGO, 1235
- CIDADE UNIVERSITÁRIA E-mail:
[email protected]
The product of Double Minute-2 gene,
protein MDM2, is a major negative
regulator of p53. Polymorphisms in
the MDM2 promoter region enhancer
the levels of MDM2 transcription.
Regulated by p53, the WAF1 gene plays
tumor suppressor function by arrest the
cell cycle in G1 phase. Polymorphisms
in the WAF1 3’UTR region may cause
instability of the mRNA and disrupt the
cell cycle. Thereby, polymorphisms in
the p53 pathway genes may deregulate
the cell cycle and has been associated
with the development of many
tumor types. Thus, the present study
was undertaken to investigate the
possible association of polymorphic
variants of MDM2 at promoter T309G
(rs2278744) and WAF1 at 3’UTR region
C590T (rs1059234), with increased
susceptibility for cervical lesion in
women infected with HPV. The study
subjects were Brazilian women HPV
infected presenting cervical lesions and
women HPV infected without cervical
lesions. Detection and typing of HPV
were performed by polymerase chain
reaction. Genotyping of MDM2 T309G
and WAF1 C590T polymorphisms were
performed from peripheral blood DNA
and cervical smear f by polymerase
chain reaction-restriction fragment
length polymorphism (PCR-RFLP). The
control group was patients positive for
HPV without cervical lesions. There
was no significant difference in the
distribution of the allele and genotype
variants in women without cervical
lesions and with cervical lesions for
MDM2 and WAF1 polymorphisms. We
didn’t observed an increased risk of
cervical lesions associated with MDM2
T309G (p=0.09; OR, 2.02; 95% CI, 0.864.77) or WAF1 C590T (p= 0.84; OR,
1.12; CI, 0.48-2.71). Although MDM2
and WAF1 polymorphisms have been
found to be associated with tumor
development, our findings suggested
that MDM2 T309G and WAF1 C590T
are not correlated with increased of
susceptibility to cervical lesions in
patients from Northeastern Brazil.
Human Virology: HV
157
HV773 - MODULATION OF THE HOST
IMMUNE RESPONSES BY DIFFERENT
STRAINS OF VACCINIA VIRUS
Freitas, D., Lorena. F., Chinália, L.A.,
Gomes, J.A.S., Barbosa-Stancioli, E.F.,
da Fonseca, F.G.
- MG CEP 31270-901 E-mail:
[email protected]
Fears that Variola virus could be used
as an agent of bioterrorism up surged a
decade ago, boosting scientific studies
looking at the immune mechanisms
underlying the protection generated
by the administration of Vaccinia virus
(VACV). Although such studies have
provided large amounts of data on
the immunological memory following
exposure to the vaccine, the same
cannot be said about the understanding
of immune responses elicited during
the onset of an acute orthopoxvirus’
infection. We have evaluated aspects
of the cellular immune responses in
humans naturally infected by VACV
during zoonotic outbreaks taking place
in Brazil. Ex-vivo experiments using
PBMCs from infected and uninfected
individuals revealed a marked virusinduced modulation in macrophages, B
and T-CD4+ cell responses. To further
look into that we infected BALB/c mice
with different VACV zoonotic isolates:
the more virulent GuaraniP1 (GP1)
and the avirulent Passatempo (PSTV)
strains. Ex-vivo analysis of virusstimulated splenocytes from mice
infected with the virulent GP1 revealed
CD14+ and B cell modulated responses,
similarly to results obtained in the
human studies. On the other hand,
animals infected with PSTV showed
few signals of virus-induced immune
158
Human Virology: HV
modulation. Likewise, replicative
vaccine strains, such as Lister,
showed some modulation toward
macrophage and B cell responses in
mice-infected experiments, whereas
the non-replicative MVA showed no
signs of immune modulation. Animals
infected with the WR strain showed
intense signals of immune modulation
directed toward CD14+, T CD4+ and
B cell responses. Taken together, our
results revealed a tendency in which
Th2 responses are modulated during
acute VACV infections.
HV774 - MOLECULAR CHARACTERIZATION OF HUMAN PARVOVIRUS B19
STRAINS CIRCULATING IN NITEROI/
RJ-BRAZIL
Pereira, R.F.A., Cubel Garcia, R.C.N.,
Azevedo, K.M.L., Setubal, S., Siqueira,
M.A.M.T., Castro, T.X., Oliveira, S.A.
1. Hospital Universitário Antônio
Pedro, DIP/HUAP, rua Marques
do Paran s/n
2. Universidade do Grande Rio,
UNIGRANRIO, rua Prof. Jose de
Souza Herdy, 1160 - Duque de
Caxias/RJ
3. Universidade
Federal
Fluminense, IB/UFF, R. Ernani
Melo, 101. Niteroi/RJ
4. Fundação Oswaldo Cruz Departamento de Virologia,
FIOCRUZ, Avenida Brasil, 4365 Rio de Janeiro E-mail: re.freire@
uol.com.br
Erythrovirus infection is generally
acute
and
self-limiting
in
imunocompetent people. However, in
immunocompromissed
individuals
(HIV-infected patients) the infection
is not readily cleared and its long
persistence leads to chronic anemia.
The infectious erythema or fifth
disease is an acute rash illness that
occurs mainly in children between
5-14 years of age and it is believed that
the rash of erythema infectiosum is the
result of the interaction between the
virus and the host immune response,
since its development is related to the
presence of IgM antibodies in serum.
Recent studies have shown many more
genetic variations among erythrovirus
than previously thought, and human
strains has been classified in genotype
1(B-19), genotype 2 (A6) and genotype
3 (3a-V9 and 3b-D91.1). The aim of
this study was to determinate the
genotypes of B19V in patients with
erythema infectiosum and in HIVinfected patients attending at Hospital
Universitário Antônio Pedro (HUAP/
UFF). These samples were collected
during outbreaks of erythema
infectiosum in Niterói. B19V DNA was
extracted from serum samples using
QIAamp DNA Blood Mini Kit (QIAGEN)
and the nPCR assay was performed
using a set of primers P12 /P16 and
P13/P16 that amplifies a partial
VP1/VP2 region of the B19 genome.
Sequencing reactions were performed
using the Big Dye Terminator v3.1 Cycle
Sequencing kit (Applied Biosystems®,
CA, US). A total of 21 samples (5 HIVpatients and 16 erythema infectiosum
cases) were analysed. The samples
from HIV-patients were collected
during an outbreak of erythema
infectiosum in Niteroi (2005-2006).
After sequencing we found that four of
the five samples of HIV- patients were
genotype 1 and one were genotype 3b.
All samples of erythema infectiosum
cases were genotype 1. This is the first
description of genotype 3b in Rio de
Janeiro state. These results represents
a further contribution to the study
of B19 variants in Brazil. Finnancial
support: MCT/CNPq: 14/2008 Edital
Universal (Processo 471618/2008-0)/
FAPERJ
HV775 - MOLECULAR CHARACTERIZATION OF PARAINFLUENZAVIRUS
1, 2 AND 3, IN CHILDREN FROM SÃO
PAULO, CITY, FROM 1995 TO 2006
Perini, A.P., Sacramento, P., Oliveira,
D.B.L., Giglio, A., Vieira, S., Stewein, K.,
Durigon, E.L., Botosso, V.F.
1. Instituto Butantan, IB, Av Vital
Brasil
2. Instituto de Ciências Biomédicas
da USP, ICB-USP,
3. Hospital Universitário da USP,
HU-USP, E-mail: priscila.perini@
usp.br
Introduction: Human parainfluenza
viruses (HPIVs) are important cause
of lower respiratory tract infections
in infants, young children and
immunocompromised patients, and
have a worldwide distribution. There
are 5 virus distributed in two distinct
genus: Respirovirus (HPIVI-1 and
HPIV-3) and Rubulavirus (HPIV-2,
HPIVI-4A and HIVI-4B). In paediatric
population the characteristic illness
associated with HPIV-1 and -2 is
laryngotracheobronchitis , but it can also
be responsible for upper respiratory
tract infection and pharyngitis,
whereas HPIV-3 is also associated
with pneumonia and bronchiolitis.
The diagnosis cannot be based only in
clinical signals and symptoms, because
other respiratory pathogens can cause
similar syndromes. Rapid laboratorial
diagnosis can impact positively in the
patient care and treatment. Objectives:
Human Virology: HV
159
The aim of this study was to carry
out the molecular analysis of the
fragment of the HN gene. Methods:
Nasopharyngeal aspirates from 2152
infants and children under five years
old, hospitalized at the University of
São Paulo Hospital (HU-USP) with acute
respiratory illness were collected from
1995 to 2006. The detection of HPIV 1,
2 and 3 were performed by multiplex
RT-PCR using specific primers to HN
gene, labeled with FAM. The fragment
of HN gene was amplified, sequenced
and, the molecular analysis was carried
out. Results and discussion: A total of
6% (n=135) samples were positive for
one of the HPIV, and the HPIV-3 was
the most prevalent virus during all
years studied, corresponding to 80%
(108/135) of positive cases, followed
by HPIV-1 with 15% (20/135) and
HPIV-2 7% (10/135). The positivity
among the years studied ranged from
1% (1/195) in 1996 to 18% (24/154)
in 1999. Most mutations observed
were silent in all PIVs, however, some
amino acids alterations in conserved
areas, verified in PIV-3 and PIV-2, and
alterations in N-glicosilation sites were
observed. Some of these alterations
were related with changes in the
function of neuraminidas
HV777 - FREQUENCY OF HCV-RNA
IN SERUM AND SALIVA ANTI-HCV
POSITIVE PATIENTS
Simões, C.A., Coêlho, M.R.C.D., Morais,
V.M.S., Silva, J.L.A., Correia, A.L., Castro,
J.F.L.
UNIVERSIDADE
FEDERAL
DE
PERNAMBUCO,
UFPE,
RUA
PROFESSOR MORAES REGO ,1235
- CIDADE UIVERSITÁRIA E-mail:
[email protected]
Although the transmission of hepatitis
160
Human Virology: HV
C virus (HCV) by parenteral exposure
is well documented in the literature,
around 40% of infected patients don’t
have history of exposure in this way.
Therefore, several studies have been
conducted to establish other possible
transmission routes. Detection of
HCV in saliva could be could be
an inexpensive and noninvasive
parameter detection and monitoring of
hepatitis C. The aim of this study was
to investigate the presence of HCVRNA in serum and saliva of patients
anti-HCV positive. A questionary was
administered to each patient and the
reports of complications due to viral
infection and genotype were obtained
from medical records. Saliva and serum
were collected from 54 patients for
RNA extraction, performed according
to the instructions of Qiamp viral RNA
isolation kit (Qiagen). The molecular
confirmation of HCV was performed the
synthesis of complementary DNA. Then
was applied a nested-PCR to the 5NCR
region, containing the first reaction
primers 939 and 209 and second
reaction primers 940 and 211. The
study population was 54% of females
and 46% of male. Blood transfusion
was reported in 38% the cases and
22% of patients didn’t report history of
parenteral exposure. The HCV-RNA was
detected in 82% (41/50) of anti-HCV
positive patients, the genotypes were
identified in 70.7% (29/41). Among
the different genotypes, type 1 was
found in 72.4% (21/29) and type 3 in
27.6% (8/29) of the patients. The HCVRNA wasn’t detected in any sample of
saliva analyzed. The divergent results
in the literature about the presence of
HCV-RNA in saliva may be explained
by the different techniques used for
collection, storage, and manipulation
of the sample, diagnostic methods and
especially the separation of cells saliva,
reflecting the need for more studies
that may contribute to determining the
true role of saliva in the transmission
of HCV. Financial support: CNPq.
Universal 2009
HV778
DISTRIBUTION
OF
HEPATITIS B VIRUS SUBGENOTYPE
F2A IN SÃO PAULO, BRAZIL
Alvarado-Mora, M.V., Botelho-Lima, L.,
Santana, R., Noble, C., Sitnik, R., Ferreira,
P.A., Mangueira, C.L.P., Carrilho, F.,
Pinho, J.R.R.
1. School of Medicine, University of
São Paulo, São Paulo, FMUSP, Av
Dr. Eneas de Carvalho Aguiar, 500
2. Albert
Einstein
Medicine, HIAE,
Einstein, 627/701
Diagnostic
Av. Albert
3. Federal University of São Paulo,
UNIFESP, Rua Sena Madureira,
1500 E-mail: monica.viviana@
usp.br
HBV genotype F is primarily found in
indigenous populations from South
America and is classified in four
subgenotypes (F1 to F4), some of them
further divided in subgenotypes, such
as F1a, F1b, F2a and F2b. Subgenotype
F2a is the most common in Brazil among
genotype F cases. The aim of this study
was to characterize HBV genotype F2a
circulating in 16 patients from São
Paulo, Brazil. Samples were collected
between 2006 and 2012 and sent to
the Clinical Laboratory of Hospital
Israelita Albert Einstein. A fragment
of 1306bp partially comprising
HBsAg and DNA polymerase coding
regions was amplified and sequenced.
Viral sequences were genotyped by
phylogenetic analysis using reference
sequences from GenBank (n=206),
including 76 classified as subgenotype
F2a. Bayesian Markov chain Monte
Carlo simulation implemented in
BEAST v.1.5.4 was applied to obtain the
best possible estimates using the model
of nucleotide substitutions GTR+G+I.
HBV F2a sequences of patients from
São Paulo grouped in two clusters with
other sequences from Brazil previously
described. One cluster included six
sequences with another one also
obtained in Brazil and was closer to
other branch containing two other
new sequences and 4 other sequences
from Rondônia. The other cluster of
six sequences grouped with another
sequence from Brazil and was close to
a group with other Brazilian sequences
(including one from the present study).
Another new sequence from this study
grouped isolated from all the others.
Since most sequences subgenotype F2a
are from Brazil and it is not reported
from which state each sequence was
obtained, it was not possible to infer its
routes of spread, The spreading and the
dynamic of subgenotype F2a in Brazil
needs the study of a higher number of
samples from different regions as it is
present through in almost all Brazilian
populations studied so far. Supported
by IIRS-SBIBAE, FAPESP 2011/505620, CNPq, FFM and HCFMUSP.
HV782 - CO-CIRCULATION OF
DIFFERENT ARBOVIRUSES DURING
DENGUE OUTBREAK IN SINOP, MT
Vieira, C.J.S.P., Siqueira, C.E.H., Silva,
D.J.F., Barreto, E.S., Carvalho, C.P.T.,
Colombo, T.E., Mondini, A., Nogueira,
M.L., Bronzoni, R.V.M.
1. Universidade Federal de Mato
Grosso, UFMT-Sinop, Avenida
Alexandre Ferronato, 1200 Sinop,
Human Virology: HV
161
MT
2. Pronto Atendimento Municipal,
PAM-Sinop, Avenida das Itaubas,
2795 Sinop, MT
José do Rio Preto, FAMERP, Av.
Brigadeiro Faria Lima, 5416 São
José do Rio Preto, SP
UNESP - Araraquara, Rod.
Araraquara-Jaú Km 1 Araraquara,
SP E-mail: carlajulia15@hotmail.
com
Arboviruses are frequently associated
with outbreaks in humans and represent
a serious public health problem.
Among the Brazilian arboviruses,
Dengue virus (DENV), Yellow Fever
virus (YFV), Rocio virus, Saint Louis
Encephalitis virus (SLEV), Mayaro
virus (MAYV) and Oropouche virus are
responsible for most of human cases.
All these arboviruses usually produce
undistinguishable
acute
febrile
illness, especially in the acute phase of
infection. In this study we investigated
the presence of arboviruses in sera of
180 patients presenting acute febrile
illness, during a dengue outbreak in
Sinop, state of Mato Grosso. The DuplexRT-PCR was performed for Flavivirus
and Alphavirus genus detection
followed by a Multiplex-Nested-PCR,
using species-specific primers. The
molecular analysis showed that 38
samples were positive to DENV-1, 2
to DENV-4, 1 to SLEV, and 6 to MAYV.
These results indicated that during
DENV outbreak, different arboviruses
co-circulated causing human disease.
Thus, it is necessary to have an efficient
surveillance system to control the
dissemination of these arboviruses
in the population. Financial Support:
162
Human Virology: HV
CNPq
HV785 - MAYARO VIRUS INFECTION
DURING DENGUE OUTBREAK IN
SINOP, MT
Silva, D.J.F., Siqueira, C.E.H., Vieira,
C.J.S.P., Barreto, E.S., Carvalho, C.P.T.,
Colombo, T.E., Mondini, A., Nogueira,
M.L., Bronzoni, R.V.M.
Grosso, UFMT-Sinop, Avenida
Alexandre Ferronato, 1200 Sinop,
MT
2. Pronto Atendimento Municipal,
PAM-Sinop, Avenida das Itaubas,
2795 Sinop, MT
Brigadeiro Faria Lima, 5416 São
José do Rio Preto, SP
UNESP - Araraquara, Rod.
Araraquara-Jaú Km 1 Araraquara,
SP E-mail: david_snp@hotmail.
com
Mayaro fever is a dengue-like viral
disease caused by Mayaro virus (MAYV)
(genus Alphavirus; family Togaviridae).
Clinical cases and virus isolation have
been reported only from South America,
where MAYV circulates in an enzootic
sylvatic cycle involving tree-canopydwelling Haemagogus spp. mosquitoes
as vectors and nonhuman primates
as natural hosts. The symptoms of
Mayaro fever include fever, headache,
myalgia, cutaneous rash, and arthralgia.
Although the death rate is low, the
morbidity caused by the disease,
specially the temporary incapacitating
arthralgia, could cause an important
social, economic and public health
impact. In this study we investigated
the presence of arboviruses in sera of
180 patients presenting acute febrile
illness, during a dengue outbreak in
Sinop, state of Mato Grosso. The DuplexRT-PCR was performed for Flavivirus
and Alphavirus genus detection
followed by a Multiplex-Nested-PCR,
using species-specific primers. MAYV
infection was confirmed in 4 cases
by RT-PCR followed by nucleotide
sequencing. The patients with Mayaro
fever presented mainly fever, headache,
diffuse body pain and retro-orbital
pain. Mayaro fever is important to
public health in rural populations, with
an increasing incidence of human cases
in the Amazonian basin. It is the first
time that Mayaro fever is diagnosed
in the state of Mato Grosso, in a city of
Sinop, located in the north section of
the state in a geographical transition
zone between savannah and rainforest.
Because Dengue fever is endemic in
Sinop, and the fact that the Mayaro
fever is not well known outside MAYVendemic regions, the MAYV infections
are misdiagnosed. Ever since that
some authors consider the possibility
of urbanization of the disease, since
experimental studies have shown that
the virus could infect Aedes aegypti,
the occurrence of this virus in Sinop
could originate urban outbreaks of
Mayaro fever. Financial Support: CNPq
HV793 - DENGUE VIRUS IN
SALVADOR, BAHIA: DETECTION AND
SEROTYPING
Pinho, A.C.O., Sardi, S.I., Paula, F.L.,
Peixoto, I.B., Welby-Borges, M.,
Brandão, C.J.F., Bandeira, A.C., Kroon,
E.G., Campos, G.S.
1. Centro de Ciências da Saúde,
CCS, Universidade Federal do
Recôncavo da Bahia, Santo
Antônio de Jesus, Bahia;
2. Laboratório
de
Virologia,
Instituto de Ciências da Saúde,
ICS, Av Reitor Miguel Calmon, sn,
ICS-UFBA, Canela, CEP:40110903, Salvador
3. Instituto de Ciências Biológicas,
ICB, Universidade Federal de
Minas Gerais, Belo Horizonte,
Minas Gerais.
4. Hospital Aliança, HA, Salvador,
Bahia E-mail: ary-cop@hotmail.
com
Dengue is a human viral disease
transmitted by mosquitoes of the
genus Aedes. Infection by Dengue Virus
(DENV) constitutes a serious public
health problem in tropical countries
including Brazil. DENV, member of the
family Flaviviridae, genus Flavivirus,
is a positive single-stranded RNA, an
enveloped virus with four antigenic
serotypes: DENV 1, DENV 2, DENV 3
and DENV 4. Infection by DENV causes
a disease whose spectrum ranges
from clinically asymptomatic to severe
clinical forms (hemorrhagic dengue).
The objective of this study was to
determine the serotypes of DENV in
hospitalized patients (n = 256), with
clinical suspects of dengue during
2011-2012 in Salvador, Bahia. The
viral RNA was extracted from serum
samples with QIAamp Viral RNA Mini
kits (QIAGEN, USA) for detection and
serotyping of DENV by RT-PCR (Reverse
Transcriptase - Polymerase Chain
Reaction) and nested-PCR, respectively.
RT-PCR was performed using primers
D1 and D2 which amplify the region
corresponding to the junction C/prM
of the genome. This result showed
that from the total samples (n=256),
100 (39%) were positive for DENV.
Human Virology: HV
163
Each DNA product (fragment of 511
bp) from RT-PCR was next submitted
to nested-PCR using serotype-specific
primers (TS1-TS4). This result revealed
the presence of DENV 2 in 17/100
(17%) samples, DENV 3 in 4/100 (4%)
samples and DENV 4 in 79/100 (79%).
These findings showed the presence
of three serotypes in the population,
with high occurrence of DENV 4. The
co-circulation of different serotypes
could increase the probability of the
most lethal clinical manifestation:
hemorrhagic
dengue.
Financial
support: PRONEX-DENGUE CNPq and
Fundação de Amparo à Pesquisa do
Estado da Bahia (FAPESB)
HV796 - STUDY AND CHARACTERIZATION OF MOLECULAR HUMAN
ROTAVIRUS IN CHILDREN IN
SALVADOR, BAHIA
Peixoto, I.B., Sardi, S.I., Pinho, A.C.O.,
Paula, F.L., Welby-Borges, M., Brandão,
C.J.F., Bandeira, A.C., Campos, G.S.
1. Centro de Ciências da Saúde,
Universidade Federal do Recônca,
CCS, Universidade Federal do
Recôncavo, Santo Antônio de
Jesus, Bahia
2. Hospital Aliança, HA, Salvador,
Bahia
3. Laboratório
de
Virologia,
Instituto de Ciências da Saúde,
ICS-UFBA, Av. Reitor Miguel
Calmon s/n – Vale do Canela.
CEP 40.110-100 Salvador, Bahia
E-mail:
isabelapeixoto@gmail.
com
Acute diarrhea is responsible for
high morbidity and mortality in the
population. It can be associated to virus,
bacteria or parasite. Among viruses,
164
Human Virology: HV
Human Rotavírus (HRV), member
of Reoviridae family, is commonly
associated to gastroenteritis in children.
The viral infection is characterized by
vomits, diarrhea and fever with large
amounts of viral particles excreted
in the faeces. The objective of this
study was to analyze the fecal samples
during an outbreak of gastroenteritis
in children under 5 years from April to
July in 2010 in Salvador, Bahia. A total
71 fecal samples were obtained from
patients hospitalized with symptom
of acute gastroenteritis. The samples
were submitted to HRV detection
by
immunoenzymatic
technique
ELISA (RIDASCREEN ® Rotavirus,
R-Biopharm),
RT-PCR
(Reverse
Transcription
Polymerase
Chain
Reaction) and RNA electrophoretic
analysis by SDS-PAGE (Sodium
Dodecyl Sulfate Polyacrylamide Gel
Electrophoresis). The samples analyzed
by ELISA, 78% (n=56) were positive
for HRV. From these ELISA positive
samples, the viral RNA was extracted
(QIAmp viral RNA kit, QIAGEN) and
submitted to RT-PCR using primers
specific Beg9 and End9 (VP7 gene).
Subsequently, the cDNA product
(1062pb) from RT-PCR was submitted
to nested-PCR using serotype-specific
primers (RVG9, aAT8, aBT1, aCT2,
aDT4, aET3, aFT9), that identified
the genotypes G1, G2, G3 and G9. The
electropherotype of the genomic RNA
analized by SDS-PAGE 7,5% showed
that all samples were classified as
group A. The highest incidence of
HRV infection was found in infants
under two years of age (60%, 34/56).
Regarding the prevalence of HRV, it was
observed a higher positive sample in
June (33%, 19/56). Concluding, these
findings indicate the greater incidence
of HRV group A and the circulation of
four different genotypes in children
under two years old in Salvador,
Bahia. Financial support: Fundação de
Amparo à Pesquisa do Estado da Bahia
(FAPESB)
HV802 - RT-SEMI-NESTED-PCR AS
COMPARED TO VIRAL ISOLATION IN
MOSQUITO CELL CULTURE FOR THE
DIAGNOSIS OF DENGUE VIRUS: AN
ACCURACY STUDY
Vale-Alves, N.B., Andriolo, R.B., Silva,
E.V.P., Carvalho, V.L., Casseb, S.M.M.,
Nunes, B.T.D., Vasconcelos, P.F.C., Cruz,
A.C.R.
1. Universidade do Estado do Pará,
UEPA, Rua Perebebuí, nº 2623;
Bairro: Marco; CEP: 66087-670 Belém - PA.
2. Instituto
Evandro
Chagas,
IEC, Rodovia BR-316 km 7
s/n - Levilândia - 67030-000
- Ananindeua / Pará / Brasil
Dengue virus (DENV -Flaviviridae,
Flavivirus) is transmitted by Aedes
mosquitoes and is recognized to cause
an infectious disease that affects over
100 countries, where more than 2.5
billion of people worldwide are living
in risk areas. Therefore, it is considered
as an important public health concern.
Virus isolation in cell culture is assumed
the gold standard technique for DENV
detection and serotyping, which it is
complex and takes 7-14 days long for a
result. Facing this issue, it is encouraged
the research for reliable, alternative
and faster methods. Hence, we aimed
to evaluate the Lanciotti-described RTSemi-Nested-PCR and compare it to the
gold standard technique. For such, we
analyzed 872 samples (blood, sera, and
viscera) of dengue-suspected patients
admitted at Instituto Evandro Chagas
(Ananindeua, Para, Brazil) throughout
January to December 2011. According
to the cell culture virus isolation, the
real prevalence was of the 18.1%. RTSemi-Nested-PCR correctly identified
the serotypes (DENV-1, DENV-2 and
DENV-4) and revealed a high specificity
of 0.93 (confidence intervals-CI95%, 0.91, 0.95), and an acceptable
sensitivity of 0.75 (CI 95%, 0.67, 0.82).
Positive (0.71) and negative (0.94)
predictive values were considered as
good and high, respectively. Moreover,
the test was associated with a high
positive likelihood ratio (11.2) and
a moderated negative likelihood
ratio (0.27). Therefore, these data
demonstrate an excellent diagnosis
accuracy of the RT-Semi-NestedPCR. Thus, taken all data together, we
suggest RT-Semi-Nested-PCR as an
easy (non-complex), fast (~10 hours)
and reliable molecular method with
good predictive values (specific and
sensible) and should be considered
alternatively to virus isolation method
for DENV detection. Additional studies
considering other epidemiological and
methodological circumstances would
be helpful to confirm these findings in
order to increase the external validity
of the individual studies when analyzed
together.
HV808 - GENETIC DIVERSITY
OF ROTAVIRUS NSP4 GENE IN
TRIÂNGULO
MINEIRO
REGION,
BRAZIL, FROM 2005 TO 2011
Dulgheroff, A.C.B., Figueiredo, E.F.,
Moura, L.M.S., Bueno, L.M.T., Silva,
G.A.V., Gouvea, V., Naveca, F.G.,
Domingues, A.L.S.
1. Universidade
Federal
do
Triângulo Mineiro, UFTM, Av.Frei
Human Virology: HV
165
Paulino, 30 - Bairro Abadia,
Uberaba-MG.
2. Instituto Leônidas e Maria Deane
Fiocruz - Amazônia, ILMD/
Fiocruz, Universidade Federal
do Rio de Janeiro, UFRJ, E-mail:
[email protected]
Rotavirus A (RVA) is the main cause of
acute diarrhea in children worldwide
and a vaccine was recently introduced
in the Brazilian vaccination program.
The virus genome is composed by
11 segments of double-strand RNA.
Viral particle is formed by a triple
capsid; the outer layer is composed
of VP7 and VP4 proteins, which are
used for a dual classification system,
defining G and P types, respectively.
Recently, a new classification system
based on characterization of 11
segments of RVA was proposed. Nonstructural protein 4 (NSP4) is involved
in viral morphogenesis and have an
enterotoxin-like activity; until now,
14 NSP4 genotypes (E1-E14) are
described. The aim of this study was
to investigate the genetic diversity
of NSP4 gene from RVA detected in
children in the Triangulo Mineiro
region, Brazil, period 2005 to 2011.
Twenty-one rotavirus samples were
selected, submitted to nucleic acid
extraction, NSP4 amplification by
RT-PCR, followed by sequencing and
phylogenetic analysis. Three distinct
NSP4 genotypes could be recognized:
six samples clustered with NSP4
genotype E1 and were found to be
associated with G1P[8], G9P[8] and
G12P[8], other 14 samples fell into
genotype E2 and were associated with
G2P[4], G8P[4], G3Pnon-typed (NT)
and one sample was genotyped as E3
associated with G12PNT. Samples from
genotypes E1 and E2 split equally into
166
Human Virology: HV
three distinct clusters; one sample of
genotype E2 clustered closely with
cow RVA from India and the specimen
of genotype E3 grouped with a feline
rotavirus. Alignment of deduced
amino acid (aa) sequences of NSP4
genes with prototype strains Wa, DS-1
and Au-1 reveals some aa changes,
including the region of the cytotoxic
peptide. This study provides valuable
information for understanding the
evolution of RVA strains and subsides
for further studies, since NSP4 has also
been discussed as a target for vaccine
development.
Financial
support:
FUNEPU;CAPES;FAPEMIG.
HV809 - EPIDEMIOLOGIC PROFILE
OF
PATIENTS
ATTENDED
IN
EVANDRO CHAGAS INSTITUTE WHIT
MUMPS VIRUS INFECTION
Brasil-Costa, I., Polaro, A.A., Souza,
W.T., Monteiro, T.A.F.
Rodovia BR-316 Km07 - Levilândia,
Ananindeua, Pará E-mail: igorcosta@
iec.pa.gov.br
Mumps virus (MuV), the causative agent
of mumps, infects mainly the parotid
glands and is transmitted by droplet
spread. Approximately, one third of all
MuV infections are asymptomatic. The
most common symptoms are salivary
gland swelling, headache, vomiting,
fever, and neck rigidity. Despite being a
vaccine-preventable disease, outbreaks
continue to occur worldwide. The
trivalent
measles-mumps-rubella
(MMR) vaccine is available to all healthy
children between 12 and 15 months of
age. In Brazil, a second dose is offered
at the age of 4 to 6 years for adequate
protection. However, the national
vaccination coverage is not full and wild
virus continues to circulate. A survey of
clinical and epidemiological study was
conducted at Evandro Chagas Institute,
PA, with 149 patients from 2003 to
June 2012, to search for the presence
of IgM antibodies against MuV [States
of PA (91); RJ (51); and BA (2)]. The
association between seropositivity
and patient’s information was tested
by multiple logistic regression and
Fisher’s exact tests using the program
BioEstat 5.0. Statistical significance
was considered when p<0.05. The
results showed that cough, swelling,
and anorexia were present twice more
often in positive patients, however with
no statistical significance (p>0.05).
Males group had more positive results
(62,5%; p= 0.0478). PA state had
more positive cases compared with RJ
(p=0.0107); 28.5% (26/91) and 9.8%
(5/51) of patients from PA and RJ
states were positive, respectively, and
only one patient was positive from BA
state. In PA, the high rate of positivity
could be related with lower vaccination
coverage (97%-1st dose and 81%-2nd
dose) compared with data from RJ
(99.5%-1st dose and 83%-2nd dose).
Our data suggest that the increase in
vaccination coverage can reduce the
circulation of wild MuV preventing
outbreaks. It was also found that the
profile of patients with MuV infection
were mainly male patients presenting
symptoms like swelling, cough and
anorexia.
HV810 - NOROVIRUS INFECTION IN
CHILDREN WITH GASTROENTERITIS
Paula, F.L., Sardi, S.I., Pinho, A.C.O.,
Peixoto, I.B., Welby-Borges, M.,
Brandão, C.J.F., Bandeira, A., Silva, L.R.,
Campos, G.S.
1. Universidade
Federal
do
Recôncavo da Bahia, UFRB,
Av.Carlos Amaral, 1015 - Santo
Antônio de Jesus - BA. CEP:
44.570-000
2. Universidade Federal da Bahia,
UFBA, Av. Reitor Miguel Calmon
s/n - Salvador - BA. CEP 40.110100
3. Hospital Aliança, HA, Av Juracy
Magalhães Jr, 2096 - Salvador
- BA. CEP 41920-900 E-mail:
[email protected]
Gastroenteritis is a public health
problem worldwide, affecting on
average each year 700 million children
under 5 years old, observed especially
in developing countries. Among the
viruses that cause gastrointestinal
infections, there is the Norovirus
(NoV), found in sporadic cases and
identified more recently in most
outbreaks of acute gastroenteritis
worldwide, accounting on average for
80 to 90% of cases. The transmission
occurs mainly through food or water
contamination and the illness is
characterized by a sudden onset of
nausea, vomiting, abdominal pain,
diarrhea and occasionally, low-grade
fever. The NoV classification is based
according to their genetic variants
in five genogroups (G), of which 3
(GI, GII and GIV) are known to cause
infections in humans. Among these, the
genogroup GII is regarded as the most
prevalent in the world, and genotype
4 accounts for approximately 80% of
all infections. Our work confirms that
in Brazil, the molecular epidemiology
of a NoV shows the predominance
the GII.4 strain. From two hundred
thirty-three stool samples collected
from children under 5 years old with
Human Virology: HV
167
gastroenteritis, NoV infection was
detected in 60.08% (140/233), using
a
commercial
immunoenzymatic
assay (RIDASCREEN® Norovirus 3rd
Generation R-Biopharm, Germany).
These samples were from patients at
the Aliança Hospital in Salvador, Bahia,
during an outbreak from March-June
2009. The median age of them was 1
year and 8 months; with predominance
of male gender (56.17%). From the total
of positive stool samples, forty-seven
(n=47) were submitted to Reverse
Transcription-Polymerase
chain
reaction using the specific primers
CAL-32 and MO3-N (first pair) and
primers JV-12 and ACAL-36 (second
pair) and all samples were positive for
this virus. Among these were randomly
selected, 28 samples which were
subjected to genome sequencing. After
the analysis on NCBI / BLAST, we found
that all samples exhibited a high degree
of similarity with GII.4 strains (9599% homology). Financial support:
Fundação de Amparo à Pesquisa do
Estado da Bahia – FAPESB
HV817 - INHIBITORY EFFECT OF A
FLAVONOID FROM KALANCHOE DAIGREMONTIANA AGAINST HERPES
SIMPLEX VIRUSES, IN VITRO
Saraiva, G.F., Gomes-Ürményi, F.G.,
Cavalcanti, J.F., Costa, S.S., Romanos,
M.T.V.
Universidade
Federal
do
Rio de Janeiro, UFRJ, E-mail:
[email protected]
this threat for human health, no specific
168
Human Virology: HV
DENV infection. Since the first report
chromatography
[P15,
µg/mL (maximum concentration
(51 KDa) and P17 (60 KDa) and 85%
P6 presented an ED50 value of 1.43 µg/
mL and P7 of 0.79 µg/mL and selective
treatment.
HV820 - INHIBITORY ACTIVITY
OF DL-GALACTANS FROM CRIPTONEMIA SEMINERVIS MARINE
ALGA ON DENGUE VIRUS TYPE 1, IN
MOSQUITO CELL
Cavalcanti, J.F., Colodi, F., Ferreira, L.G.,
Mendes, G.S., Noseda, M.D., Duarte,
M.E.R., Romanos, M.T.V.
de Janeiro, UFRJ, Universidade
Federal do Paraná, UFPR, E-mail:
[email protected]
this threat for human health, no specific
DENV infection. Since the first report
chromatography
[P15,
μg/mL (maximum concentration
(51 KDa) and P17 (60 KDa) and 85%
P6 presented an ED50 value of 1.43 μg/
mL and P7 of 0.79 μg/mL and selective
treatment.
HV824 - MONITORING THE CIRCULATION OF DENGUE VIRUS IN AEDES
AEGYPTI AND AEDES ALBOPICTUS
BY OVITRAPS’ INSTALLATION IN
OURO BRANCO AND OURO PRETO,
MINAS GERAIS
Cecilio, S.G., Magalhães, J.C., Diniz,
J.S., Ferreira, J.M.S., Magalhães, C.L.B.,
Tótola, A.H.
Universidade Federal de São João del
Rei, UFSJ, Campus Alto Paraopeba UFSJ Rodovia MG 443, Km 07 Ouro
Branco - MG CEP 36420-00 E-mail:
[email protected]
Dengue is considered one of the most
important arthropod borne viral
diseases in Brazil. The disease is caused
by 4 serotypes of dengue virus, which
are transmitted to humans by vectors
from genus Aedes. The fast expansion
of these insects has been favored by
Human Virology: HV
169
modern urbanization. Ouro Branco and
Ouro Preto/MG are endemic in some
periods of the year, which indicates a
possible adaptation of vectors. In 2011,
48 cases were reported in Ouro Branco
(44 autochthonous and 4 imported),
and 2 imported cases were reported
in Ouro Preto. In 2012, 9 cases have
been notified in Ouro Branco (6
autochthonous, 3 imported). In both
cities, programs to prevent outbreaks
of the disease were put in place. The
success of such programs depend on
the early detection of cases or evidences
to recommend control procedures and
prevent further occurrences. Ovitraps
with wood vanes and a mosquitoattractive infusion were installed in
32 public schools, 16 of each city of
the study. The evaluation was done
monthly between September/2011
and May/2012 and the epidemiological
data, obtained from the City Health
Department, was monitored. Mosquito
eggs were counted and hatched in water
with fish meal at 26,5°C. The larvae
were reared to the fourth stadium and
identified according to the species, A.
Aegypti or A. Albopictus, and stored
at -80°C. From the 445 eggs collected,
274 larvae came to the fourth stadium;
62% of the larvae from Ouro Branco
were identified as A. albopictus and
85,5% of larvae from Ouro Preto were
identified as A. aegypti. The larvae
were macerated with Biomixer tissue
homogenizer and RNA was extracted
with RNeasy QIAGEN Kit®. RNA
was quantified by NanoVue Plus®.
Reverse Transcription PCR and Real
Time PCR were performed to detect
the viral RNA. No positive samples
were detected, which is consistent
with epidemiological data. It can be
inferred that the control of zoonosis
and environmental care in these cities
170
Human Virology: HV
interfere positively in the circulation of
these vectors.
HV831 - OCCULT HBV INFECTION IN
INSTITUTIONALIZED INDIVIDUALS
IN THE STATE OF GOIAS, BRAZIL.
Moraes, T.C., Castro, I.A., Cunha, M.P.,
Souza, M.D., Fiaccadori, F.S., Cardoso,
D.D.P.
IPTSP/Universidade Federal de
Goiás, IPTSP/UFG, Rua 235, s/n.
Setor Universitário CEP 74605050
Hepatitis B (HBV) is a major public
problem because of the high number
of affected individuals and the
complication resulting from the
acute and chronic forms of infection.
Although the diagnosis of HBV infection
is made based on the detection
of the serological marker HBsAg,
recent studies have demonstrated
the presence of viral DNA, detected
by molecular techniques, in patients
with isolated serological profile for
anti HBc. This profile corresponds to
a particular form of chronic hepatitis
B with undetectable HBsAg marker,
which has been classified as Occult HBV
infection. In order to determine the rate
of HBV infection in an institutionalized
population, presenting with psychiatric
and neurological disorders, in the state
of Goias, 334 blood samples were
collected and an overall prevalence of
HBV infection of 14.1% was observed,
with an index of isolated positivity
for anti-HBc marker of 5.4%. In this
context, this study aimed to survey
the DNA/HBV in samples with isolated
anti-HBc and HBsAg positive profiles.
Twenty-one samples were subjected
to viral DNA extraction, followed by
genomic amplification by polymerase
chain reaction (PCR), for identification
of DNA/HBV. Of the total isolated antiHBc samples, 17/19 (89.5%) were
positive for HBV-DNA. Among the
samples with HBsAg positive profile,
2/3 (66.7%) were positive for viral DNA.
These results highlight the importance
of the molecular techniques to confirm
HBV infection. Further studies are
being conducted in the lab in order to
elucidate the HBV infection status of
these individuals. Financial support:
CNPq
HV832 - MOLECULAR ANALYSIS
OF VP4, VP7 AND NSP4 GENES
OF ROTAVIRUS G1 CIRCULATING IN BELÉM AND MARITUBA,
PAR&AACUTE;, BRAZIL
Soares, L.S., Mascarenhas, J.D.P., Farias,
Y.N., Maestri, R.P., Oliveira, A.S.L.,
Guerra, S.F.S., Santos, F.S., Menezes,
E.M.F.C., Linhares, A.C.,
FUNDAÇÃO
OSWALDO
CRUZ,
FIOCRUZ, INSTITUTO EVANDRO
CHAGAS, IEC, BR 316, Km 07, s/n
Rotaviruses are major viral agents of
acute gastroenteritis and responsible
for 36% of hospitalization for diarrhea
among children less than five years
of age, resulting in 453.000 deaths
annually, mostly in developing
countries. Rotavirus is a member of
Reoviridae family, and its genome
consists of 11 double-stranded RNA
(dsRNA) which encode 12 proteins.
G1 rotavirus is commonly detected
in epidemiological investigations,
occurring under different prevalence
rates. The aim of this study was to
analyze the VP4, VP7 and NSP4 diversity
genetic of G1 rotavirus circulating in
Belém and Marituba, Pará, Brazil, from
1982 to 2008. We selected 83 samples
previously characterized as G1 type
and submitted to RT-PCR. It was
possible amplification for 63 (75.9%)
specimens. Lineages 1 (8/63, 12.7%),
2 (29/63, 46.0%), 3 (18/63, 28.6%)
and 9 (8/63, 12.7%) of VP7 gene were
detected. The sublineages 2E and
3A were co-predominant detected
in 57.1% (36/63) of samples. Three
amino acid substitutions (97 [D�E],
147 [S�N] and 218 [I�V]) were
observed in VP7 antigenic regions (A,
B and C) in samples of lineages 1, 2 and
9. All samples showed P[8] specificity
for VP4 gene and lineages 2 (21/63,
33.3%) and 3 (42/63, 66.7%) were
detected. Two substitutions (35 [I�V]
and 38 [S�G]) occurred in antigenic
region of VP4 gene. For NSP4 gene,
all samples belonged to E1 type.
Phylogenetic analysis of NSP4 gene
revealed that occurred changes in
amino acid positions 16 (S�P) and 34
(L�P) in all samples and 9 specimens
displayed amino acid substitution
in NSP4 toxicity residue (aa 131).
Conclusion: This study allowed us to
broaden our understanding about
genetic diversity and circulation of
G1 variants and corroborating the
high heterogeneity of this genotype.
Financial Support: CNPq, IEC/SVS/MS.
HV833 - VIROLOGICAL AND HOST
FACTORS INVOLVED IN INCREASED
DISEASE SEVERITY IN HUMAN RHINOVIRUS INFECTIONS IN YOUNG
CHILDREN.
Costa, L.F., Queiróz, D.A.O., Silveira,
H.L., Neto, M.B., Ueira-Vieira, C., Paula,
N.T., Dias, E.H.V., Tolardo, A.L., Oliveira,
T.F.M.S., Freitas, G.R.O., Chávez, J.H.,
Yokosawa, J.,
1. Universidade
Federal
de
Uberlândia, UFU, Av. Pará, 1720,
Bloco 4C – Campus Umuarama;
Human Virology: HV
171
Uberlândia, MG, CEP 38400-902
2. Instituto de Ciências Biomédicas,
ICBIM, Av. Pará, 1720, Bloco 4C –
Campus Umuarama; Uberlândia,
MG, CEP 38400-902
3. Hospital de Clínicas, HC, Av.
Pará, 1720, Campus Umuarama;
4. Instituto
de
Genética
e
Bioquímica, INGEB, Av. Pará, 1720,
Bloco 2E – Campus Umuarama;
Although human rhinoviruses (HRV)
are well-known pathogens that cause
the common cold, recently they have
been investigated in severe respiratory
infections. Whether or not disease
severity in children younger than
five years old may be increased by
infection with a second virus or by
host comorbidities was investigated.
We have tested 434 nasopharyngeal
aspirates from children presenting
acute respiratory disease for presence
of HRV, respiratory syncytial virus
(RSV), influenza virus, parainfluenza
virus (PIV), adenovirus and human
metapneumovirus. HRV was detected
in 181 (41.7%) samples: as the only
agent in 107 and with a second
virus in 74 samples. Moderate-tosevere symptoms were observed in
31.8% (34/107) cases of single HRV
infection and in 48.6% (36/74) of
co-infections (p=0.0297). These data
suggested that presence of a second
virus may slightly increase disease
severity in HRV infections. Indeed,
multivariable analyses showed that
co-infections were associated with
lower respiratory tract infections
(LRTI) (χ2=33.96; p=0.026) and
factors of disease severity (χ2=84.05;
172
Human Virology: HV
p=0.000); However, canonical analyses
showed that PIV and especially RSV
played a role in the association. In
regard to underlying risk conditions,
prematurity, congenital heart diseases
and
non-infections
respiratory
diseases showed to be associated with
disease severity (p=0.0022, <0.0001,
and 0.0017, respectively). Although
increased severity frequency was
higher in children < 12 months old
(p<0.05), when considered as the only
risk factor the association was not
observed (p=0.0731). In conclusion,
co-infections with RSV or with PIV,
prematurity, congenital heart diseases,
and non-infections respiratory diseases
have shown to play important role in
disease severity in infections caused
by HRV.
HV834 - IMPACT OF THE EMERGENCE
AND RE-EMERGENCE OF DIFFERENT
DENGUE VIRUS SEROTYPES IN THE
STATE OF RIO DE JANEIRO
Heringer, M., Nogueira, R.M.R., Filippis,
A.M.B., Lima, M.R.Q., Simões, J.B.S.,
Santis, B.G.
Brasil, 4365, Manguinhos, 21045900, Rio de Janeiro, Brazil E-mail:
[email protected]
The state of Rio de Janeiro has been
of great epidemiological importance
for introduction and spread of
dengue viruses (DENV) and over
the last 26 years, was marked by
extensive epidemics. The existence of
a continuous program of virological
surveillance aims to detect and monitor
the circulation of DENV serotypes
in the state, where DENV-1, DENV-2,
DENV-3 and DENV-4 co-circulate. Given
the limited options for prevention and
control of epidemics, it has been shown
that laboratory diagnosis plays an
important role in the Epidemiological
Surveillance System, by continuous
monitoring infections and confirming
new cases. In this study a total of 1,621
dengue suspected cases, received by
the Laboratory of Flavivirus / Regional
Reference Laboratory of the IOC /
FIOCRUZ, were analyzed from January
2010 to December 2011. Using three
methods, virus isolation in cell culture,
RT-PCR and MAC-ELISA, 738 cases
(45.5%) of all cases studied were
confirmed. The MACELISA confirmed
31.69% (309/975) of the cases, the
RT-PCR confirmed with 35.34%
(527/1,491) and 20.8% of 1,173
sera inoculated into C6/36 cells were
isolated. DENV-2 was the prevalent
serotype in 2010. During 2011, DENV2 was displaced by DENV-1 and the
first DENV-4 cases were isolated in the
city of Niterói. Our analysis have shown
that DENV-1 cases had more chance of
increased severity (OR = 1.06 / 95% CI
0.49 to 2.29 / p <0001), moreover, the
severe forms were more frequent on
children 15 years old and under (OR
= 1.8 / 95% CI = 1 to 3.22 / p <0001).
From the total of fatal cases confirmed
(n = 47), 51.6% were due to secondary
infections. Fatal cases were more
frequent in children 15 years old and
under in 2011in comparison to 2010.
The emergence of DENV-1 in 2011 led
to the occurrence of fatal and severe
cases in children 15 years old and
under and, despite the re-emergence of
DENV-1 in 2011, DENV-4 was isolated
and spread in the State, causing an
epidemic in 2012. Financial support:
FAPERJ, CNPq, CAPES and FIOCRUZ.
HV835 - TWENTY FIVE YEARS OF
DENGUE: THE EXPERIENCE OF A
REGIONAL REFERENCE LABORA-
TORY
dos Santos, F.B., de Filippis, A.M.B.,
Lima, M.R.Q., Nogueira, F.B., Faria,
N.R.C., Simões, J.B.S., Sampaio, S.A.,
Nunes, P.C.G., de Santis, B., Heringer,
M., Lima, D.C., Nogueira, R.M.R.
Oswaldo Cruz Institute, IOC/
FIOCRUZ,
Av.
Brasil,
4365,
Manguinhos, Rio de Janeiro E-mail:
[email protected]
During the past 25 years high dengue
activity in Brazil has been evidenced by
the large number of cases, in almost all
states of the country. DENV-1, DENV2 and DENV-3 were introduced in Rio
de Janeiro, in 1986, 1990 and 2000,
respectively. DENV-4 has recently
been isolated after 28 years of its first
isolation. The introduction of DENV2 in 1990 caused the first DHF/DSS
cases. The introduction of DENV-3 led
to severe epidemics in 2002 with the
largest number of cases, DHF cases
and deaths. In 2007–2008, the country
experienced the most severe dengue
epidemic in terms of morbidity and
mortality and a higher incidence of
severe cases in children. From March
1986 to December 2011, 47,346
dengue suspected cases were received
in the Laboratory and 41,614 were
submitted to one or more routine
diagnosis techniques available. A total
of 32,374 cases (77.8%) were tested
by MAC-ELISA, 25,037 (60.2%) were
submitted to virus isolation, 181
cases (0.4%) to HI test and 829 cases
(2.0%) to IgG-ELISA. The RT-PCR was
performed in 7,441 cases (17.9%) and
the NS1 ELISA in 1,124 cases (2,7%).
Cases confirmation were 59.7% and
58% in the DENV-1 epidemic occurred
in 1986 and 1987, respectively; 52.8%
in the DENV-3 epidemic occurred in
2002, 52% in the DENV-2 epidemic in
Human Virology: HV
173
2008 and 47% and 45% in the DENV-1
occurred in 2010 and 2011, respectively.
In the last 25 years, virus isolation
and RT-PCR identified the infecting
serotype in a total of 4,990 dengue
cases. Genomic analysis performed on
DENV-2 identified the introduction of a
distinct lineage of the Asian/American
genotype. In 2009 and 2010, DENV1 re-emerged and, this serotype has
been prevalent in many States of Brazil.
The phylogeny has also demonstrated
the introduction of distinct lineages
of DENV-1. Our experience has shown
that the implementation of new
laboratorial techniques over the years
constituted an important tool for the
disease surveillance in Brazil. Financial
support: FAPERJ, CNPq, CAPES and
FIOCRUZ
HV839 - CO-INFECTION OF DENGUE
VIRUS BY SEROTYPES 1 AND 4 IN
PATIENT FROM SÃO JOSÉ; DO RIO
PRETO, SÃO PAULO
Vedovello, D., Colombo, T., Mondini, A.,
Nogueira, M.L.
José do Rio Preto , FAMERP,
Avenida Brigadeiro Faria Lima,
5416 - Vila São Pedro, São José do
Rio Preto,
2. Universidade Estadual de São
Paulo, UNESP, Rua Cristóvão
Colombo,
2265
Jardim
Nazareth São José do Rio Preto
Universidade Estadual de São
Paulo - Araraquara, UNESP
Araraquara, Rodovia Araraquara
- Jaú Km 1, Araraquara, SP E-mail:
[email protected]
Dengue is a major public health problem
in tropical and subtropical countries.
There are four immunologically related
174
Human Virology: HV
serotypes phylogenetically classified
into genotypes. Historically, the State
of Sao Paulo, Brazil, has been suffering
dengue outbreaks since 1990. The
natural co-infection with different
serotypes of dengue virus has been
observed for many years. We report
one case of DENV-1 and DENV-4 coinfection in human serum detected
by molecular tests in the sample RP/
BR/2012 isolate in São José do Rio
Preto, São Paulo, Brazil, on 12 March
2012. The diagnosis was performed by
EIA for NS1 protein and did not detect
the coinfection, subsequently identified
by molecular method. We sequence a
fragment of 365 bp (from DENV-1) and
252 bp (from DENV-4) representing the
sequences encoding the capside gene
(C ). This sequences and sequences of
DENV 1 and 4 published in genbank
were used to alignment of aminoacids
and for phylogenetic reconstruction.
The phylogenetic analysis indicated
that they are classified as genotype
5 and 2 for DENV-1 and DENV-4,
respectively. In terms of aminoacids,
no significant change was identified in
DENV-1 C protein and three aminoacid
substitutions were identificated in
DENV-4: F5L (Phenylalanine to Leucine
in fifth position), R10F (Arginine to
Phenylalanine in tenth position), S14F
(Serine to Phenylalanine in fourteenth
position). The F5L substitution are
present only in recent Brazilians
strains and can be considered a genetic
signature of Brazilian isolates and
needs further investigation. Our report
demonstrates that co-infection with
diferents serotypes of dengue virus can
occur naturally and emphasizes the
importance of the molecular method
in the dengue diagnosis once the EIA
for NS1 protein can not identify the
presence of two serotypes in a single
sampl
HV844 - EXPRESSION LEVELS OF
VIRAL SENSOR RIG-I AND ITS REGULATORS RNF125 AND TRIM25 IN HIV
INFECTED ADULTS AND CHILDREN
Britto, A.M.A., Amoedo, N.D., Pezzuto,
P., Afonso, A.O., Martinez, A.M.B.,
Silveira, J., Sion, F.S., Machado, E.S.,
Soares, M.A., Giannini, A.L.M.
1. UNIVERSIDADE FEDERAL DO RIO
DE JANEIRO, UFRJ, Departamento
de Genética; CCS; Bloco A; 2º
andar; sala 66; CEP: 21941-590;
RJ
2. Instituto de Bioquímica Médica
Universidade Federal de Rio
Grande, FURG,
3. Faculdade de Medicina Hospital
Universitário Gaffrée e Guinle,
HUGG, Instituto de Puericultura
e Pediatria Martagão Gesteira,
IPPMG
UFRJ,
E-mail:
[email protected]
RIG-I is a citoplasmatic viral sensor
that recognizes viral RNA genome.
Its activation culminates in IFN type I
production. TRIM25 and RNF125 are
ubiquitin ligases that regulate RIG-I.
Ubiquitination via TRIM25 is necessary
to activate innate response pathway.
On the other hand, ubiquitination by
RNF125 signals RIG-I degradation via
proteassome. Since it is known that
RNF125 inhibits HIV-1 replication; that
RIG-I recognizes HIV-1 RNA and that
IFN type I regulation is important in
control/progression of HIV-1 infection
we decided to analyze the expression
levels of RIG-I, RNF125 and TRIM25
in healthy donors and in HIV infected
individuals assessing their progression
levels. We performed qRT-PCR from
PBMC of adults: 14 non-infected (NI)
adults, 10 progressors (P), 10 nonprogressors (NP); and children: 8
healthy, 8 progressors and 4 nonprogressors. The adult and children
groups were analyzed separately.
We found that all 3 genes were
more expressed in healthy controls,
independent of age. Furthermore, we
found that RIG-I was more expressed
in P than in NP patients. Moreover
we could see an evident relationship
among the pattern expression of the
three genes studied within NI and P
groups. In NI, RNF125 was at least
2 fold more expressed than TRIM25
and RIG-I. In P group, RIG-I was the
more expressed gene and RNF125
and TRIM25 were equally expressed.
NP and NI had the same expression
pattern. We concluded that not only
one gene is important to control AIDS
progression but the balance among
them. Furthermore RNF125 seems
to be essential to maintain the innate
signalling off when it is not necessary in
NI groups and NP. Finally we concluded
that RIG-I seems to be important in
AIDS progression since is up regulated
in P individuals and could then help to
predict disease progression.
HV848 - ASYMPTOMATIC GASTROENTERIC VIRUS SHEDDING AMONG
CHILDREN THAT ATTEND A DAY
CARE CENTER IN GOIÂNIA-GO,
BRAZIL
Mendanha, D.M., Santos, H.C.P., Borges,
V.H.B., Fiaccadori, F.S., Cardoso, D.D.P.,
Souza, M.D.
Universidade Federal de Goiás, UFG,
Rua 235, Setor Universitário, CEP
74605-050. Goiânia - Goiás - Brasil
Gastroenteric viruses are important
etiological agents of childhood acute
Human Virology: HV
175
gastroenteritis. These agents are
transmitted by the fecal-oral route, and
by person-to-person spread, although
environmental contamination could
contribute to viral dissemination, with
outbreaks frequently occurring in
closed institutions such as hospitals,
cruise ships and day care centers. Viral
excretion by asymptomatic individuals
has been reported. In this study,
children that attend a day care center in
Goiânia, Goiás, Brazil, were monitored
for gastroenteric virus detection for a
period of three years. For this study,
90 fecal samples, collected from RVAvaccinated children, were screened
for human adenoviruses (HAdVs)
using antigen detection kits, by ELISA,
polyacrylamide gel electrophoresis
(PAGE) and a rapid cromatography test
for group A rotavirus (RVA) detection.
Sixty-seven samples were also tested
for caliciviruses-CVs (norovirusesNoVs and sapoviruses-SaVs) by RTPCR, using primers targeting the C
region of the viral capsid. Two of 90
fecal samples tested positive for RVA
by all three screening tests and third
sample, collected from a different
child, was positive for adenovirus
by both tests used. Five of the 67
(7.5%) samples were positive for CVs.
Molecular characterization identified
one RVA-positive sample as genotype
G2 (VP7 gene) and genotype P[4]
(VP4 gene), and the other positive
sample remained un-typeable by RTPCR. Among the samples screened
for CVs, four were positive for GII
NoVs and one was positive for SaVs.
Our data shows RVA, HAdVs, and CVs
circulation among asymptomatic RVAvaccinated children that attend a day
care center in Brazil, reinforcing the
potential for viral spread in such close
settings. Ongoing studies, focusing on
176
Human Virology: HV
genomic sequencing and phylogenetic
analysis are being conducted in order
to better evaluate the epidemiology
of gastroenteric viruses among day
care children, and also the possible
role of asymptomatic children in viral
dissemination.
HV850 - SCREENING FOR HUMAN PARECHOVIRUS IN LYMPHOID TISSUES
OF THE UPPER RESPIRATORY TRACT
de Souza Luna, L.K., Proença-Modena,
J.L., Paula, F.E., Valera, F.C.P., Tamashiro,
E., Anselmo-Lima, W.T., Arruda, E.
1. Centro de Pesquisa em Virologia FMRP - USP, CPV - FMRP - USP, Av.
Bandeirantes, 3900 - Vila Monte
Alegre. 14049-900, Ribeirão
Preto - SP
2. Oftalmologia,Otorrinolaringolo
gia,Cirurg da Cabeça e Pescoço,
HCFMRP - USP, Av. Bandeirantes,
3900 - Vila Monte Alegre. 14049900, Ribeirão Preto - SP E-mail:
[email protected]
Chronic adenotonsillitis (CAT) is a
persistent hypertrophy of tonsils and
adenoids, of poorly understood etiology.
Several viruses are frequently detected
in palatine tonsils and adenoids from
patients with chronic adenotonsillitis
who undergo tonsillectomy. However,
the viral panels used for screening by
PCR do not include human parechovirus
(HPeV), an emerging picornavirus
related with a broad spectrum of
diseases, similar to those caused by
enteroviruses. In this preliminary study,
the presence of HPeV was assessed in
respiratory samples and palatine tonsil
and adenoid tissue fragments collected
from patients with CAT at the University
of Sao Paulo Hospital in Ribeirao Preto,
Brazil. A total of 104 samples were
tested for HPeV with a two-step realtime RT-PCR assay for the conserved
5'UTR, of which 82 samples (25 nasal
swabs, 22 nasopharyngeal washes,
18 palatine tonsils and 17 adenoid
fragments) were from 27 patients with
CAT, and 22 samples (5 nasal swabs,
5 nasopharyngeal washes, 6 palatine
tonsils and 6 adenoid fragments) from
6 patients without CAT as control
group. Samples were collected from
November 2011 to July 2012 and were
previously tested by real-time PCR for
a panel of common respiratory viruses.
Patients in the study were aged from
1.8 to 13 years. HPeV was detected
in 2 samples (1 palatine tonsil and
1 adenoid fragment), both from one
patient in the control group (2 yearold female). Further investigation shall
be done in a larger number of patients
in order to determine the role of HPeV
infection in lymphoid tissues in the
upper respiratory tract. Financial
support: FAPESP, CNPq.
HV851 - IMPACT OF THE EMERGENCE
AND RE-EMERGENCE OF DIFFERENT
DENGUE VIRUS SEROTYPES IN THE
STATE OF RIO DE JANEIRO
Heringer, M., Nogueira, R.M.R., De
Filippis, A.M.B., Lima, M.R.Q., Simões,
J.B.S., De Santis, B., Sampaio, S.A.,
Santos, F.B.S.
Brasil, 4365, Manguinhos, 21045900, Rio de Janeiro, Brazil E-mail:
[email protected]
The state of Rio de Janeiro has been
of great epidemiological importance
for introduction and spread of
dengue viruses (DENV) and over
the last 26 years, was marked by
extensive epidemics. The existence of
a continuous program of virological
surveillance aims to detect and monitor
the circulation of DENV serotypes
in the state, where DENV-1, DENV-2,
DENV-3 and DENV-4 co-circulate. Given
the limited options for prevention and
control of epidemics, it has been shown
that laboratory diagnosis plays an
important role in the Epidemiological
Surveillance System, by continuous
monitoring infections and confirming
new cases. In this study a total of 1,621
dengue suspected cases, received by
the Laboratory of Flavivirus / Regional
Reference Laboratory of the IOC /
FIOCRUZ, were analyzed from January
2010 to December 2011. Using three
methods, virus isolation in cell culture,
RT-PCR and MAC-ELISA, 738 cases
(45.5%) of all cases studied were
confirmed. The MAC-ELISA confirmed
31.69% (309/975) of the cases, the
RT-PCR confirmed with 35.34%
(527/1,491) and 20.8% of 1,173
sera inoculated into C6/36 cells were
isolated. DENV-2 was the prevalent
serotype in 2010. During 2011, DENV2 was displaced by DENV-1 and the
first DENV-4 cases were isolated in the
city of Niterói. Our analysis have shown
that DENV-1 cases had more chance of
increased severity (OR = 1.06 / 95% CI
0.49 to 2.29 / p <0001), moreover, the
severe forms were more frequent on
children 15 years old and under (OR
= 1.8 / 95% CI = 1 to 3.22 / p <0001).
From the total of fatal cases confirmed
(n = 47), 51.6% were due to secondary
infections. Fatal cases were more
frequent in children 15 years old and
under in 2011in comparison to 2010.
The emergence of DENV-1 in 2011 led
to the occurrence of fatal and severe
cases in children 15 years old and
under and, despite the re-emergence of
DENV-1 in 2011, DENV-4 was isolated
and spread in the State, causing an
Human Virology: HV
177
epidemic in 2012. Financial support:
FAPERJ, CNPq, CAPES and FIOCRUZ.
HV853 - IMMUNOLOGICAL PROFILE
AND RISK FACTORS ASSOCIATED TO
POSSIBLE OCCUPATIONAL INFECTIONS BY VACCINIA VIRUS
Costa, G.B., Ribeiro, K.C., Figueiredo,
P.O., Leal, L.L.D., Alves, P.A., Ferreira,
P.C.P., Abrahão, J.S., Kroon, E.G.,
Trindade, G.S.
Laboratório de Vírus, Departamento
de Microbioliga, ICB-UFMG, UFMG,
Avenida Antônio Carlos, 6627,
Pampulha, Belo Horizonte, Minas
Gerais E-mail: [email protected]
Despite the eradication of smallpox,
the orthopoxviruses (OPV) are still a
source of concern due to the possible
use of Variola virus as a biological
weapon, as well as the increase in
outbreaks of zoonotic infections
caused by OPV in several regions
of the world, as by Vaccinia virus
(VACV) in Brazil. VACV is the primary
component of the smallpox vaccine
and infections with this virus can
occur after direct contact with animals
naturally infected, individuals recently
vaccinated or through exposure to the
virus in laboratories, as described in
Brazil and other countries of the world.
The aim of this study was to investigate
the immunological profile of workers
in a research laboratory, seeking to
evaluate the humoral immunity and
the risk factors involved in possible
occupational infections by VACV. We
conducted an epidemiological survey
and collected 50 samples of human
sera. A total of 18 (36%) men and
32 (64%) women are part the study
population, mostly students, beyond
teachers and technical assistants, aged
19 to 70 years. It was found that 34
178
Human Virology: HV
(68%) individuals have direct contact
with OPV contaminated material
and 29 (58%) forgot, at some time
of experimentation, using protective
equipment required. Still, 18% suffered
an accident with any contaminated
material, and of these, 8% had signs
and symptoms, and 16% have a history
of vaccination, where only 8% have the
vaccine take. After diagnosis, it was
found that 9 (18%) individuals have
anti-OPV neutralizing antibodies. In
Brazil, after the smallpox eradication,
the vaccination that confers immunity
to OPV was discontinued and even
researchers and health professionals
are not vaccinated. Therefore, it is
important to conduct studies of this
nature due to the widespread risk
that poxvirus offer to human health,
as well as its dispersion in laboratory
and hospital environments. Financial
support: CNPq, FAPEMIG.
HV857 - SEROLOGICAL PROFILE OF
EPSTEIN-BARR VIRUS INFECTION
IN PATIENTS SUSPECTED OF INFECTIOUS MONONUCLEOSIS IN BELÉM,
PARÁ, BRAZIL
Barros, P.R., Polaro, A.A., Costa, M.R.M.,
Souza, W.T., Monteiro, T.A.F., BrasilCosta, I.
1. Instituto
Evandro
Chagas,
IEC, Rodovia BR-316 Km07 Levilândia, Ananindeua, Pará
2. Fundação de Amparo à Pesquisa
do Estado do Pará, FAPESPA,
Travessa Nove de Janeiro nº
1686 E-mail: prodriguesbarros@
gmail.com
Epstein-Barr virus (EBV) infection
is the major cause of Infectious
Mononucleosis (IM), a self- limited
syndrome, characterized by the
main symptoms: fever, sore throat,
lymphadenopathy and pharyngitis.
Clinical manifestations may vary
according to the immune status of
patients infected; therefore, a definitive
diagnosis should rely on the assessment
of specific markers for EBV infection.
The most common serological markers
sought to confirm EBV infection in IM
are IgM to VCA antigens; however these
markers are insufficient to designate
an EBV etiology either a primary
infection or reactivation. Were tested
20 sera from patients suspicious of IM
from Belém, Pará, previously known
to be IgM VCA antibody-positive by
ELISA, using Immunoblot IgG kit for
the following antigens: EBNA-1, p18,
p23, BZLF1, p138 and p54. The results
were analyzed according to respective
symptoms: fever, fatigue, sore throat,
lymphadenopathy,
exanthema,
tonsilitis and hepatosplenomegaly.
Statistical analyses were performed
with BioEstat 5.0 software, using
fisher’s exact test and kappa test. The
main symptoms found were: fever
(65%), lymphadenopathy (45%) and
exanthema (20%). In patients aged
4 years or more, only one EBNA-1negative result was found, since most
children become infected by EBV
between 3 years of age. EBNA-1 is
the most studied marker to define
past EBV infection, although 5% of
infected patients have a lack of antiEBNA-1 response. To assess the
absence of EBNA-1, p18 was used as
a complementary result (excellent
replicability).
The
sore
throat
symptom, related to the primary site
of EBV infection, was diagnosed only
in patients non-reactive to p18 and
EBNA-1 (p=0,0035), past markers for
EBV infection. Results from the present
study suggest that assessment of
serological markers for EBV infection
by Immunoblot assay appears to be
useful to differentiate between primary
infection and reactivation; in addition,
sore throat was found to be a major
sign of primary EBV infection.
HV859 - SEROEPIDEMIOLOGY OF
HEPATITIS B VIRUS INFECTION IN
AN INSTITUTIONALIZED POPULATION OF THE STATE OF GOIÁS
Moraes, T.C., Cunha, M.P., Castro, Í.A.,
Souza, M.D., Cardoso, D.D.P., Fiaccadori,
F.S.
Instituto de Patologia Tropical e
Saúde Pública, IPTSP, Rua 235 - s/n Setor Universitário - CEP: 74605050
- Goiânia - Goiás - Brasil E-mail:
[email protected]
The hepatitis B virus (HBV) remains
as an important health problem
worldwide, transmitted mainly by
vertical, parenteral and sexual routes.
In this context, institutionalized
individuals with mental problems
constitute a risk group for acquisition
of HBV infection due to their low
awareness of the risks of viral infection,
and because of their prolonged stay in
mental institutions. In this sense, this
study aimed to determine the HBV
infection rate in an institutionalized
population with psychiatric disorders
and neurological state, located in the
State of Goiás, Brazil. Blood samples
were collected from 334 participants,
and analyzed for the presence of
serological markers of HBV infection
(HBsAg, anti-HBsAg, anti-HBc) by
enzyme-linked immunosorbent assay,
using commercial kits. The overall
prevalence of HBV infection was
14.1% (47/334), with a rate of occult
HBV infection of 5.4% (18/334).
Higher infection rates were observed
Human Virology: HV
179
among adult males (36-59 years-old),
the elderly (>60 years-old), and also
among those individuals that had been
institutionalized for over 10 years
(p<0.05), when compared to the rest of
the population. Only 3.9% (13/334) of
the population had serologic evidence
of previous vaccination against
hepatitis B virus and susceptibility to
HBV, characterized by the absence of
any serological marker, was observed
in 82% of the population. These results
demonstrate a high prevalence of
HBV infection in the institutionalized
population, with a significant rate of
occult infection. The data emphasize
the importance of adopting preventive
measures, with emphasis on HBV
vaccination, to control HBV infection in
risk groups such as the institutionalized
population.
HV866 - LOW PREVALENCE OF
SUBTYPE B VARIANT (B’-GWGR)
IN THE SOUTHERMOST REGION OF
BRAZIL
Maletich, D., de Medeiros, R., Leite, T.F.,
Guimarães, M.L., Gräf, T., Pinto, R.A.,
Almeida, S.
1. Centro
de
Desenvolvimento
Científico e Tecnológico, CDCTFEPPS, Porto Alegre, RS, Brazil
2. Pós-Graduação em Genética
e Biologia Molecular UFRGS,
PPGBM-UFRGS, Porto Alegre, RS,
Brazil
3. Laboratório de AIDS & Imunologia
Molecular FIOCRUZ, FIOCRUZ,
Rio de Janeiro, RJ, Brazil
4. Departamento de Microbiologia,
Imunologia e Parasitologia, UFSC,
Florianópolis, SC, Brazil E-mail:
[email protected]
180
Human Virology: HV
The vast majority of HIV-1 subtype
B (HIV-1B) sequences present the
GPGR motif at the tip of the v3 loop.
Epidemiological studies have detected
a subtype B variant, which presents the
GWGR signature (B'-GWGR), in about
17 to 50% of infections in Southeast
Brazil, although the prevalence of B'GWGR in the southernmost region is not
yet clear. So, the present study aimed to
trace the temporal trends of this variant
in two cities of the Southernmost
region of Brazil. Blood samples from
189 HIV-1 treatment-naive patients
were obtained at two distinct time
periods in the city of Porto Alegre
(PoA), Rio Grande do Sul State: 1998
and 2005-2008. In addition, 98 blood
samples were collected between 2004
and 2008, in the city of Florianopolis
(FL), capital of Santa Catarina State.
Phylogenetic analysis of a 302-base
pair fragment of the env gene (C2-V3
region) revealed the presence of 46
(51%) samples of non-B subtypes and
46 (50%) HIV-1B samples from 1998 in
PoA, distributed as follows: B'-GWGR
(9%), B-GPGR (20.5%) and GXGX
(20.5%). In the second studied period,
subtype B was detected in 32 samples
(33%) and subtype B signatures were
as follows: B'-GWGR (6.2%), B-GPGR
(14.4%) and GXGX (12.4%). In FL
subtype B accounted for 33% of HIV
infections and the HIV-1B signatures
were distributed as follows: B'-GWGR
(11.2%), GPGR (7%) and GXGX (14.3%).
HIV-1B molecular signatures were
distributed equally within PoA (1998
and 2005-2008) and between PoA and
FL. No association could be established
between HIV-1B molecular signatures
and a specific exposure category in the
PoA epidemic. However, the B'-GWGR
seems to be related to homosexuals
in FL (p=0.012). Southernmost HIV-
1 subtype B epidemic seems to be
different from that established in the
Southern region of Brazil. Our results
suggest lower prevalence maintenance
of B'-GWGR along the time in PoA
and no association between exposure
category and B signatures, however
an association between B'-GWGR and
homosexuals was verified in FL.
HV868 - ASEPTIC MENINGITIS BY
COXSACKIEVIRUS B5 IN NEONATE
Vieira, H.R., Machado, B.C., Sousa, C.A.,
Russo, D.H., Timenetsky, M.C.S.T., Lo,
D.S., Ibidi, S.M., Gilio, A.E., Carmona,
R.C.C.
1. University Hospital of USP, HU/
USP, Av. Prof Lineu Prestes, 2565,
Cid. Universitária São Paulo, SP,
05508-900
2. Adolfo Lutz Institute, IAL, Av.
Dr. Arnaldo, 355 São Paulo CEP:
01246-000 E-mail: helorv@
yahoo.com.br
Aseptic meningitis (AM) is considered
one of the most common infectious
diseases in children, especially
those less than 5 years old. Neonatal
infections from enteroviruses are
fairly common, associated with a wide
spectrum of signs and symptoms,
which range from a non-specific
febrile illness to potentially fatal
multisystem disease. Enterovirus
genus (EV) is a RNA virus that belongs
to the family Picornaviridae, and was
originally classified as Poliovirus,
Coxsackievirus A and B, Echovirus and
Enterovirus, based on their associated
pathogenicity. Coxsackievirus B5 (CVB5) belongs to HEV-B species and is
one of the most predominant serotypes
in humans being frequently associated
with sporadic cases of neurological
diseases and epidemics of AM. The aim
of this study was to describe the case
of a previously healthy neonate with
AM evolution by CV-B5. The newborn
patient, male, was taken by his mother
to the University Hospital of São Paulo
searching for medical care with a history
of sneezing, dry cough and fever. From
the history reported and the clinical
examination, the patient was initially
diagnosed with suspected AM and
the cerebrospinal fluid (CSF) material
was sent to the Adolfo Lutz Institute
– Virology Center, Enteric Diseases
Laboratory. CSF was inoculated in
RD and HEp-2 cell cultures. Viruses
isolated were identified by Reverse
Transcription
Polymerase
Chain
Reaction and Sequencing and CV-B5
was detected. Especially in neonates
and infants, CV-B5 has a more serious
evolution, affecting the central
nervous system and muscles, causing
complications such as AM. Several
publications have reported neonates
with symptomatic EV infection on day
1 of life, indicating that the infection
must have been acquired antenatally
- either transplacentally or potentially
via ascending infection. This case
demonstrates the importance of CV-B5
circulation in the population and the
early etiologic diagnosis of meningitis
to search for the specific etiological
treatment.
HV869 - ASSAY OF PCR AND CLONING
STANDARDIZATION OF HUMAN RESPIRATORY SYNCYTIAL VIRUS GENES
Gomes, D.E., Teixeira, T.S.P., Paiola,
L.C.C.C., Araújo, G.C., Cornélio, M.L.,
Fossey, M.A., Souza, F.P.
Estadual Paulista , UNESP, R.
Cristóvão Colombo, 2265. Jd
Human Virology: HV
181
(hRSV) is the major agent of respiratory
infections in children, like bronchiolitis
and pneumonia causing deaths around
the globe. Its genome is a single strand
RNA, with genome organization NS1NS2-N-P-M-SH-G-F-M2.1-M2.2-L.
Several works have been made focusing
in diagnosis, inhibitor for the virus, how
viral proteins interact with themselves,
and so on. However, it is necessary,
firstly, to obtain these proteins using
DNA recombinant technology. Thus,
the aim of this project includes the
standardization of PCR amplification
and cloning of hRSV genes. The
standardization of PCR was performed
testing four different DNA Polymerases
(taq DNA Polymerase (Recombinant)
–
Fermentas®;
High
Fidelity
DNA
Polymerase
(Recombinant)
–
Fermentas®;
Biotools
DNA
Polymerase – Biotools®; JumpStart
Sigma®)
following
manufacture
recommendations. The cloning of the
fragments was developed in vector
pCR-XL-TOPO (Invitrogen®) and subcloning standardization into expression
plasmid pET28a - Novagen® and
into two-hybrid plasmids (pGBKT7
and pGADT7 - Clontech®) were
standardized with objective to
avaliable proteins for interaction tests.
In result, it was successfully obtained
amplicons of genes NS1 (438bp),
NS2 (393bp), P (744bp), M (789bp),
Complete G (912bp), G endodomain
(135), G ectodomain (700), F1
(1131bp), F2 (360bp), M2-1 (603bp)
and M2-2 (288bp) and better results
was obtained with taq DNA Polymerase
(Recombinant) – Fermentas® and
JumpStart Sigma®. The cloning of NS1,
NS2, P, M, G1, G2, F1, F2, M2.1 and M2.2,
genes on pCR-XL-TOPO vector was
182
Human Virology: HV
obtained with success. Into pET28a
were subcloned the genes NS1, M2.1,
M2.2, P, G1 and G2 and into pGBKT7
and pGADT7 were subcloned the
genes NS1, G1, G2, M, M2.1, M2.2 and P.
Once PCR and cloning standardization
is an important and difficult step in
diagnosis and protein expression
works, the protocols we developed will
help the groups who works with hRSV
to outcome this first steps.
HV872 - EVALUATION OF CROSS-REACTIVITY IN ENZYME-LINKED
IMMUNOABSORBENT ASSAY (ELISA)
TO EPSTEIN-BARR VIRUS (EBV),
CYTOMEGALOVIRUS (CMV) AND
HUMAN HERPESVIRUS 6 (HHV-6)
Brasil-Costa, I., Barros, P.R., SousaJunior, E.C., Silva, D.F.L., Oliveira, D.S.,
Polaro, A.A., Monteiro, T.A.F.
1. Instituto
Evandro
Chagas,
IEC, Rodovia BR-316 Km07 Levilândia, Ananindeua, Pará
2. Fundação de Amparo à Pesquisa
do Estado do Pará, FAPESPA,
Travessa Nove de Janeiro nº 1686,
Belém, Pará E-mail: igorcosta@
iec.pa.gov.br
Viruses EBV, CMV and HHV-6, herpes
family, are spread worldwide and persist
in the host organism in a latent state,
infecting mainly leukocytes. Besides the
structure and biology of these viruses,
the epidemiology, transmission and
clinical manifestations of symptomatic
infections are similar, coursing
mainly with fever, sore throat and
lymphadenopathy. In addition, HHV6 produces rash as major clinical
manifestation. The similarity between
the three viruses may lead to diagnostic
difficulties in relation to the specificity
of routine serological tests. It is
important to assess the seropositivity
for the virus and compare it with the
patient's symptoms. Were selected 20
serum samples, which were positive by
IgM ELISA anti-VCA of EBV (BALF4),
and tested by IgM ELISA to CMV and
HHV-6, both made from viral lysates.
To evaluate the possibility of crossreactivity was tested in silico homology
to the antigenic sites (AS) of the protein
BALF4 with the AS of the proteins
encoded by HHV-6 and CMV. To
research described AS, predict possible
AS and test homologies were used
immune epitope database, hlapred
and Geneious Pro 4.8.5 softwares,
respectively. Statistical analyses were
performed with BioEstat 5.0 program,
using fisher’s exact test. Patients were
aged between 10 months and 54 years,
average of 17 years. Were found more
positive for HHV-6 (90%) than for
CMV (35%) (p<0.001). There was no
relationship between positive tests
with symptoms (p>0.05). Homology
was found between proteins BALF4 of
EBV, U39 of HHV-6 and gpB of CMV. The
homologue AS in BALF4 (ID:114667)
and U39 (ID:166736) have been well
described, while in gpB had to be
inferred. There was greater similarity
between AS of EBV and HHV-6. Although
these viruses have a structure, biology,
transmission path and epidemiology
similar, which favors the co-infection,
the possibility of false-positive routine
test exists due to the similarity between
the AS targets of the immune response
against these viruses.
HV875 - ASSOCIATION BETWEEN
POLYMORPHISMS IN THE ORGANIC
CATIONS TRANSPORTER OCT-1 AND
THE FAILURE TO ANTIRETROVIRAL
THERAPY AGAINST HIV.
Dias, J.Z.C., Arruda, M.B., Brindeiro, R.,
Tanuri, A., Cardoso, C.C., Aguiar, R.S.
UNIVERSIDADE FEDERAL DO RIO
DE JANEIRO, UFRJ, Av. Carlos Chagas
Filho, 373, Ilha do Fundão - Cidade
Universitária/RJ E-mail: juzdias@
gmail.com
The HIV is a pandemic infection with
approximately 33,4 million people
infected. A total of 6,65 million patients
are undergoing HAART (Highly Active
Antiretroviral Therapy) and about
10-20% of these patients do not
reach therapeutic success, mostly due
to the emergence of drug resistant
viruses but also because of the host
inter-individual variations in drug
absorption, activation and metabolism.
Since
therapeutic
success
is
dependent on the maintenance of drug
intracellular levels, the cellular drug
transporters are important candidates
for pharmacogenetic studies. In this
study we investigate the association
between 40 polymorphisms in
the genes SLC22A1, SLC22A2 and
SLC22A3 and ABCB1, coding for the
Organic Cation Transporters 1-3 and
P-Glycoprotein and antiretroviral
failure. For this purpose, we have
conducted a case-control study
including 219 HIV+ individuals (117
cases of HAART failure and 120
controls for whom first-line therapy
successfully reduced HIV-1 viral loads
to undetectable levels) selected from
the cities of Curitiba and Porto Alegre,
in the Brazilian south, and treated for
at least 6 months. Genotyping was
performed using SNaPshot and the
TaqMan OpenArray Platform (Life
Technologies). Comparisons between
cases and controls were performed
by logistic regression models with
adjustment for the covariates age and
therapy scheme. Our results showed
Human Virology: HV
183
an association between the deletion
of a single nucleotide in codon 420 of
the SLC22A1 gene (rs3516751) and
increased risk of therapeutic failure
among heterozygotes (adjusted OR
= 2.86; 95% IC 1.47-5.55; p=0.001).
We were unable to define allele dose
effects due to the complete lack of
homozygotes for this deletion. This is
the first study designed to investigate
the pharmacogenetics of HAART
effectiveness in Brazil. Our data
describe a clear association between
variations in SLC22A1 and HAART
failure, suggesting that studies of host
genetics are crucial to predict better
treatment for HIV+ patients. Financial
support: CNPq and FAPERJ.
HV877 - EPIDEMIOLOGICAL STUDY
OF
ADENOVIRUS
ASSOCIATED
WITH ACUTE DIARRHEAL DISEASE,
DURING 2007-2010, IN MINAS
GERAIS, BRAZIL
Reis, T.A.V., Valle, D.A., Júnior, A.N.P.,
Pires, D.O., Portes, S.A.R., Rose, T.L.,
Leite, J.P.G., Rosa e Silva, M.L.
1. Universidade
Federal
de
Juiz de Fora, UFJF, R. José
Lourenço Kelmer, s/n, Campus
Universitário-S. Pedro - Juiz de
Fora,MG
2. Instituto Oswaldo Cruz , IOC
- FIOCRUZ, Av. Brasil, 4365 Manguinhos - Rio de Janeiro, RJ.
Acute diarrheal disease (ADD) is an
important cause of child morbidity
and mortality in developing countries.
Among viruses, enteric adenoviruses
are one of the most important etiologic
agents of ADD. Human adenoviruses
(HAdV) are classified into seven species
(A-G) and 54 serotypes. Among them,
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Human Virology: HV
serotypes 40 and 41, both of the species
F, are the most commonly associated
with ADD. Given the importance of
this disease in developing countries
and the large number of cases without
an etiologic agent defined was
performed this study in Juiz de Fora,
Minas Gerais. Between January 2007
and December 2010 were analyzed
395 diarrheic stools samples. HAdV
was detected by PCR and molecular
sequencing and phylogenetic analysis
of partial sequences of the hexon gene.
For statistical analyzes was used SPSS
program, adopting a value of p <0.05
as significant. The HAdV prevalence
was 10.9% (43/395). There was
no significant correlation between
the origin of the sample (hospital
X ambulatory) and the occurrence
of infection (p=0.152), as well as,
in relation to gender of the infected
(p=0,393). The majority of positive
cases was detected in children up to 24
months of age, showing a significative
correlation between the age of the
infected individuals and the occurrence
of infection (p=0,007). In most cases of
infection (36/43), HAdV was the only
virus detected, but it has been observed
some cases of co-infection with RV
(5/43) and NoV (2/43). Phylogenetic
analysis of partial sequences of the
hexon gene from 35 positive samples
revealed that all samples clustered
with HAdV species F, serotypes 41,
confirming the association of enteric
HAdV (EHAdV) with ADD. This survey
revealed the presence and circulation
of these viruses in Juiz de Fora, MG,
in this period as well as its important
role in the genesis of ADD, reaveling
a great number of diahrreal cases
which normally remains unidentified.
Financial Support: CAPES, CNPq,
FAPEMIG and Propesq-UFJF
HV879 - PREVALENCE OF ANTI-ORTHOPOXVIRUS
ANTIBODIES
IN HUMANS IN THE ABSENCE OF
BOVINE VACCINIA OUTBREAKS
Figueiredo, P.O., Silva-Fernandes A.T.,
Alves, P.A., Braga, E.M., Abrahão, J.S.,
Kroon, E.G., Trindade, G.S.
6627, Campus Pampulha, CEP:
31270-901. Belo Horizonte, Minas
E-mail: polianaofigueiredo@yahoo.
com.br
The Vaccinia virus (VACV) circulates
in Brazil since the 60s. Infections
by this virus affects both cattle and
humans and all human cases reported
to date are always associated with
outbreaks in cattle. In order to analyze
retrospectively the immune status
against Orthopoxvirus of populations
of the Amazon and southeast regions
of the country, samples were analyzed
from Mantena and Terra Nova do Norte,
belonging to the states of Minas Gerais
and Mato Grosso, respectively. Sera
samples were collected in 1995 and
1996, dates prior to the notification of
outbreaks in these regions. The samples
were subjected to IgG ELISA and PRNT
for the detection of anti-Orthopoxvirus
antibodies. Furthermore, molecular
tests were performed by real-time PCR
for the vgf gene aiming to detect viral
DNA on the sera samples. Of the 70
samples tested belonging to the Amazon
region, 27% were IgG positive and from
those, 27% of positive samples are
from people who were not vaccinated
against smallpox, or were born after
1977, closing date of vaccination in
Brazil. It was found that 6% of the
vaccinated population was positive by
PRNT, indicating the efficiency of the
immunity generated by the smallpox
vaccine. From Mantena rural area, 62
samples were analyzed, with 11% IgG
seropositivity and of these, 29% of
people are not vaccinated. The PRNT
revealed 15% of seropositivity and
all positives are vaccinated patients.
The molecular test was able to detect
viral DNA in some samples thus
suggesting a possible DNAemia. The
detection of Orthopoxvirus antibodies
in unvaccinated individuals indicates
a possible silent circulation of VACV
in human populations, and seems to
be not related to outbreaks of bovine
VACV. This data is reinforced by the
detection of viral DNA in some samples.
Thus, these results support the need
for more research on the subject,
especially as regards the identification
of potential animal reservoirs and new
forms of disease transmission.
HV881 - PLASMA LIPIDOMIC
EXPRESSION SIGNATURE DISTINGUISHES HEPATITIS C-RELATED
HEPATOCELLULAR CARCINOMA AND
LIVER CIRRHOSIS
Passos, A.M., Lo Turco, E.G., Ferraz,
M.L.C.G., Matos, C.A.L., Silva, I.S.S.,
Parise, E.R., Pilau, E.J., Gozzo, F.C.,
Granato, C.F.H.
Universidade
de
Campinas,
UNICAMP, Rua Josué de Castro,
Campinas-SP
CEP
13083-970
UNIFESP, Rua Pedro de Toledo, 15o
andar, São Paulo-SP CEP 04039-032
Hepatitis C (HC) is a major cause of
hepatocellular carcinoma (HCC). Late
diagnosis of HCC represents the main
factor for the poor survival of patients.
The most widely used biomarker for
Human Virology: HV
185
HCC, alpha-fetoprotein (AFP), has
poor sensitivity and specificity and
has recently been removed from the
American Association for the Study of
Liver Diseases (AASLD) guidelines for
HCC management. Thus, identification
of sensitive and specific biomarkers
for HCC diagnosis is an urgent need.
In the present study, plasma lipid
patterns of patients with HC-HCC and
HC-liver cirrhosis (LC) were assessed
by
performing
matrix-assisted
laser desorption/ionization mass
spectrometry (MALDI-MS). Plasma
samples of 25 patients with HC-HCC
and 15 patients with HC-LC were
evaluated. Lipids were extracted from
plasma using the Bligh-Dyer protocol.
The extracts were subjected to MALDIMS. Data matrix was exported for
univariate and multivariate analysis.
A total of 2205 ions were initially
identified and 7 m/z signals were
highlighted as the most important
lipids for the discrimination of patients
with HC-HCC. The specific lipidomic
expression signature generated allows
an overall predictive accuracy of 93% of
HC-HCC and HC-LC. All 7 peaks showed
more than 4-fold change in HC-HCC (P <
0.01). The 7-peak algorithm was able to
distinguish the 2 groups at a sensitivity
of 96% and a specificity of 87%. MALDIMS specific peaks signature accurately
distinguished patients with HC-HCC
from those with HC-LC. The results
indicate the potential of this technique
and the selected peaks to improve the
surveillance of HCC in patients with
HC-LC.
HV883 - HUMAN PAPILLOMAVIRUS
TYPES 6 AND 11 E6 VARIANTS FROM
LARYNGEAL PAPILLOMATOSIS IN
BRAZILIAN PATIENTS
Matos, R.P.A., Bonfim, C.M., Mansur,
186
Human Virology: HV
I.M., Bittar, C., Nogueira, R.L., Kupper,
D.S., Valera, F.C.P., Nogueira, M.L., Villa,
L.L., Sichero, L., Rahal, P., Calmon, M.F.
São Pedro - São José do Rio Pre
UNESP, Rua Cristovão Colombo,
2265 - Jardim Nazareth - São José
do Rio Preto - SP
3. Instituto de Câncer do Estado de
São Paulo, ICESP, Av Dr. Arnaldo,
251- Cerqueira César -SP
4. Faculdade de Medicina de Ribeirão
Preto, Universidade de São Paulo,
USP, Avenida Bandeirantes, 3900
- Monte Alegre - Ribeirão Preto SP. E-mail: renatapram@hotmail.
com
Recurrent respiratory papillomatosis
(RRP) is a disease characterized by
benign neoplasms and can occur
anywhere within the upper respiratory
tract, but the most common lesion
site is the larynx. This disease has
a bimodal age distribution which
forms the basis of its classification as
juvenile or adult. The main etiological
agent of RRP is Human Papillomavirus
virus (HPV), a group of DNA virus
of which over 150 types has been
identified. HPV-6 and 11 are the most
common types found in RRP. HPV
stimulates proliferation of the mucosal
epithelium leading to the development
of papillomas. E7 and E6 oncoproteins
from high-risk HPVs induce cell
immortalization by interfering in cell
cycle regulation. However, the role of
E6 and E7 proteins from low risk HPVs
are not fully understand as yet and
E6 functions might also be related to
the clinical course of the RRP. In this
study we analyzed genetic variability
of the E6 gene in samples of laryngeal
papillomas. The complete coding region
of E6 was cloned and sequenced in 25
samples: 18 isolates of HPV-6 (72%)
and 7 isolates of HPV-11 (28%). A total
of three different E6 genomic variants
were identified among HPV-6 isolates.
Only one identified variant showed
an amino-acid change corresponding
to 1.3% of the E6 protein. Within the
7 HPV-11 isolates, we identified one
genomic variant and two synonymous
mutations were detected in this variant.
Phylogenetic trees were constructed
for both HPV-6 and HPV-11 variants.
These enclosed sequences obtained
in this study in addition to sequences
obtained from GeneBank, including the
reference sequences of each genotype.
In both phylogenetic trees sequences
from Brazil did not group together.
We further observed that overall the
sequences did not cluster according
to their geographical origin neither or
the anatomic site of infection. Our data
reinforce the hypothesis that HPV-6 and
HPV-11 variants are not geographically
restricted as observed in HPV-16 and
HPV-18. Financial Support: CAPES,
FAPESP
HV888 - INVESTIGATION OF DENGUE
VIRUS IN JUIZ DE FORA, MINAS
GERAIS, BRAZIL
Sacchetto, L., Botelho, J., Lima, G.A.D.,
Rezende, I.M., Carvalho, C.M., Veiga, R.,
Fernandes, G.C., Mendonça, A.E., Rosa
e Silva, M.L., Kroon, E.G., Drumond, B.P.
1. Universidade Federal de Juiz de
Fora, UFJF, Rua José Lourenço
Kelmer,Campus
Universitário
Bairro São Pedro/CEP: 36036900,JF
2. Centro de Pesquisa da Santa Casa
de Misericórdia, , Av. Rio Branco,
n° 3353 - Centro Juiz de Fora CEP
36.021-630
3. Dep. de Vigilância Epidemiológica
e
Ambiental
PMJF,
DVEA,
- MG CEP 31270-901 E-mail:
[email protected]
Dengue virus (DENV)(Flavivirus,
Flaviviridae) is the most important
arbovirus worldwide, being transmitted
by Aedes sp. mosquitoes. DENV exists
as four different serotypes: DENV-1 to
DENV-4. The epidemiological scenario
in Brazil is characterized by the cocirculation of four DENV serotypes and
the country has the higher number of
DENV in Latin America. Minas Gerais
state usually present high number
of dengue cases and Juiz de Fora,
located at Zona da Mata Mineira, had
great epidemics of DENV in last years
with 11,582 notified cases from 2010
to 2012. The aim of this work was to
perform a prospective study of DENV
in patients with clinical suspect of
DENV infection and naturally infected
larvae of A. aegypti obtained in Juiz
de Fora, MG. Biological samples were
obtained from January to April/2012.
Serum samples and pools of larvae
(containing up to 50) were used for
total RNA extraction. Total RNA was
used for cDNA synthesis followed by
nested-PCR to detect DENV. From 9
serum samples one was positive for
DENV-2, by PCR. This same serum
sample was IgG and IgM reactive when
tested by Dengue IgG/IgM Test Bioeasy.
Using this later test, five other PCR
negative serum samples, were IgG/IgM
non-reactive. From 56 pools of larvae,
Human Virology: HV
187
two were positive for DENV-1. Pools of
mosquito collected in the same period
are also going to be tested. The obtained
amplicons are going to be sequenced
to determine the viral genotype and
perform further phylogenetic analysis.
This is the first report of DENV
surveillance in Juiz de Fora, pointing
to co-circulation of DENV-1 and DENV2 during the last epidemic period.
Financial support: FAPEMIG, CNPq,
CAPES, UFJF, PROPESQ/UFJF.
HV889 - LIPIDOMIC FINGERPRINTING OF CANCEROUS AND
NONMALIGNANT
TISSUES
IN
HEPATITIS C-RELATED HEPATOCELLULAR CARCINOMA: A PROOF
OF CONCEPT STUDY
Passos, A.M., Lo Turco, E.G., Ferraz,
M.L.C.G., Matos, C.A.L., Silva, I.S.S.,
Parise, E.R., Pilau, E.J., Gozzo, F.C.,
Granato, C.F.H.
Paulo, UNIFESP, Rua Pedro de
Toledo 781, 15o andar, São PauloSP CEP 04039-032
2. Universidade
de
Campinas,
UNICAMP, Rua Josué de Castro,
Campinas-SP CEP 13083-970
Hepatocellular carcinoma (HCC) is an
aggressive liver cancer but biomarkers
that can predict natural history of
malignant progression are lacking.
Lipidomic profiling using matrixassisted laser desorption/ionization
time of flight mass spectrometry
(MALDI-TOF
MS)
enables
the
identification of biomarkers for
cancer. The present study explored
the lipidome-wide patterns of HCC to
identify lipids that could distinguish
cancerous and nonmalignant tissues.
188
Human Virology: HV
Tumor and adjacent nontumor tissues
were obtained from a patient with
hepatitis C-related HCC. Samples
were pulverized in liquid nitrogen
before lipids were extracted using
the Bligh-Dyer protocol. The extracts
were subjected to MALDI-MS analysis
in 4 replicates each. Data matrix
was exported for univariate and
multivariate analysis. A total of 322 ions
were initially identified and 10 m/z
signals were highlighted as the most
important lipids for the discrimination
of cancerous and nonmalignant tissues
in HCC. All 10 ions presented more
than 5-fold change in tumor tissue
and were significant at P<0.01. Partial
least square discriminant analysis
(PLS-DA) could distinctly resolve
the 2 groups of replicates assessed.
Five peaks were increased in tumor
tissue and 5 were decreased. The
main classes of the distinguishing
lipids
were
Glycerophosphates
[GP10],
Glycerophosphocholines
[GP01],
Glycerophosphoserines
[GP03], Acidic glycosphingolipids
[SP06],
Glycerophosphoinositols
[GP06], Flavonoids [PK12], Neutral
glycosphingolipids
[SP05],
and
Triradylglycerols [GL03]. This proof-ofprinciple study suggests that MALDIMS lipidomic fingerprinting may be
a powerful tool for the identification
of biomarkers for HCC. In conclusion,
these data demonstrated that the
lipid fingerprinting of cancerous and
nonmalignant tissues in HCC was able
to select a number of lipids that should
be functionally investigated in the
pathogenesis of the disease. MALDI-MS
could successfully distinguish tumor
and adjacent nontumor tissues in HCC.
HV890 - TEMPORAL PATTERN OF
ROTAVIRUS
PREVALENCE
AND
DIARRHEA HOSPITALIZATION, 2005
TO 2011, IN JUIZ DE FORA, MG
Assis, A.S.F., Valle, D.A., Nicolau, R.S.,
Cruz, L.T., Junior, A.N.P., Tibiriça, S.H.C.,
Rosa E Silva, M.L.
Universidade Federal de Juiz
de Fora, UFJF, R. José Lourenço
Kelmer, s/n, Campus Universitário,
S. Pedro, Juiz de Fora, MG E-mail:
[email protected]
Rotaviruses (RV) are associated with
acute diarrheal disease (ADD) on many
occasions become severe, leading
many children to hospitalization and
death. The high prevalence associated
with the fact that only hygienic and
sanitary actions would not be enough
to decrease the prevalence of ADD
caused by RV, pointed to the need of
development of the effective vaccine.
In March 2006, the Health Ministry
of Brazil has included Rotarix®
vaccine in National Immunization
Program (PNI), with goal of to provide
protection against severe disease, thus
reducing the number of hospitalization
and deaths. The aim of this study
was investigate the association
of RV disease with official data of
hospitalization by ADD. In that way, 705
fecal diarrheic samples, obtained from
January 2005 to December 2011, in
Juiz de Fora, MG were analyzed. RV was
detected after acid nucleic extraction
by polyacrylamide gel electrophoresis.
The rates of hospitalization for ADD
were obtained through Ministry of
Health's Hospitalization Data System.
In 2005, period preceding introduction
of the vaccine in PNI, was observed a
higher prevalence of RV associated with
major number of hospitalizations for
ADD, probably indicating more severe
cases of ADD caused by RV. In 2006, year
of introduction of Rotarix® in PNI, the
highest number of hospitalizations was
observed. A total of 60 cases occurred
in July and 39 cases in August. Beside
this, the prevalence of RV was 44,44%
(n=20/45) and 34,21% (n=13/38) in
July and August, respectively. On the
other hand, since 2007, it has not been
observed an association between the
number of hospital admissions and
the RV prevalence, fact that may be
related to milder cases of the disease.
The lack of association between
the RV prevalence and number of
hospitalizations by ADD after the
introduction of Rotarix® vaccine
may indicate the likely effectiveness
of vaccination in preventing serious
cases of ADD caused by RV. Financial
support: CAPES, CNPq, FAPEMIG and
Propesq-UFJF
HV892
ELECTROPHORETIC
PROFILES OF G- AND P- TYPES OF
ROTAVIRUS DETECTED IN SÃO
PAULO STATE, BRAZIL
Fernandes, A.M., Luchs, A., Vieira, H.R.,
Vilanova, B.C., Timenetsky, M.C.S.T.,
Carmona, R.C.C.
Instituto Adolfo Lutz , IAL, Av. Dr.
Arnaldo, 355 - São Paulo- SP
Group-A rotavirus (RV-A) is the
most important pathogen of acute
gastroenteritis in infants and young
children. In developing countries,
more than 125 million cases of RV-A
infection have been estimated to occur
annually. Analysis of mobility of the
11 dsRNA genomic segments of the
by polyacrylamide gel electrophoresis
(PAGE) yields a characteristic for each
strain and has often been used as an
indicator of the RV-A diversity. The
aim of present study was to determine
the frequency of RV-A infection and
to detect of the diversity of dsRNA
Human Virology: HV
189
genomic electrophoretic pattern from
patients with acute diarrhea in the
state of São Paulo, from January 2011
to May 2012. Stool samples from 1,277
patients were tested by ELISA, PAGE
and multiplex Nested PCR for detection
of RV-A. A total of 140 (11.0%) stool
samples were RV-A positive: in infant
children <1 year of age (36.4%;
51/140), children 1-5 years (57.1%;
80/140), children 6-15 years (3.6%;
5/140), and adults > 15 years (2.1%;
3/140). A total of 106 (75.7%;
106/140) samples were confirmed by
PAGE and the dsRNA migration pattern
were visualized in 93 samples. Based on
migration pattern of dsRNA segments
(10 and 11), two distinct groups of
electrophoretypes were identified:
83 long profile strains (89.2%) and
11 short profile strains (11.8%). RV-A
with long profile were correlated with
G3P[8], G9P[8], G12P[8], G3P[3], and
G12P[9] genotypes; whereas short
profile RV-A were correlated with
G2P[4], G3P[6] and G12P[6] types. The
long profiles showed a high frequency
during the studied period. Recent
studies have demonstrated a reduction
rate of RV-detection. In addition, there
is a need for further detailed analysis
of the migration patterns of the 11
dsRNA segments of RV-A, which would
have important contributions to the
knowledge of the RV-A epidemiology
and the evaluation of vaccine programs.
HV899 - PHYLOGENY OF DENGUE
VIRUS TYPE 1 ISOLATED FROM
FIELD-CAUGHT
VECTORS
AND
HUMANS REVEALS DIFFERENT
LINEAGES OF THE AMERICAN/
AFRICAN GENOTYPE
Castro, M.G., Nogueira, F.B., Nogueira,
R.M.R., Ferreira, A.A., Faria, N.R.C.,
Santos, C.M.R., Nunes, P.C.G., Lourenco
190
Human Virology: HV
de Oliveira, R., dos Santos, F.B.,
OSWALDO
CRUZ
INSTITUTE,
IOC/FIOCRUZ, AV. BRASIL, 4365
MANGUINHOS RIO DE JANEIRO
In Brazil, dengue became a major
public health problem after DENV-1
introduction in 1986 in Rio de Janeiro
(RJ) and in 2009, this serotype reemerged causing major epidemics in
the country. Since then, a virological
and entomological program was
established for monitoring dengue
viruses (DENV) in human sera and
vectors and it has constituted an
important tool for dengue epidemiology
and vector-virus-host interactions
studies. Aedes aegypti specimens
collected in 1986 (n=120) and in 2001
(n=2,434) in Nova Iguacu (RJ) and
collected in Boa Vista (RR) in 2010
(n=3,705) were pooled and analyzed.
Six DENV-1 isolates (three from Ae.
aegypti and three from humans) were
available. Vector macerates were
submitted to conventional RT-PCR and
virus isolation. For viral quantification,
the RNA from original Ae. aegypti
individually macerated was submitted
to Real Time qRT-PCR. Sequencing
of E gene (1,485 nucleotides) was
performed. DENV-1 was identified by
virus isolation and RT-PCR during the
1986, 2001 and 2010 entomological
surveillances performed in RJ and
Roraima (RR) and the Real Time qRTPCR detected 1.6x104 copies/mL of
DENV-1 in the macerate of a single
Ae. aegypti female naturally infected.
The phylogeny demonstrated that
DENV-1 isolated from both vector
and humans belong to genotype V
(Americas/Africa), although the cocirculation of two distinct lineages
(lineages II and III) was detected.
A higher sequence divergence was
observed between lineages II and III,
and most amino acid substitutions
were observed on domain III from E
protein. Moreover, some residues were
exclusive to some lineages, and may
be predicted to be differentiating the
three lineages. The use of molecular
techniques combined to virus isolation
showed to be important approaches
for the surveillance and molecular
characterization studies of DENV from
field-caught vectors. Financial support:
CNPq,
FAPERJ,
PAPES/FIOCRUZ,
FIOCRUZ
HV900 - DETECTION OF FLAVIVIRUS
IN THE MOSQUITO VECTOR AEDES
AEGYPTI COLLECTED IN AN URBAN
CITY OF MANAUS, AMAZONAS,
BRAZIL
de Matos, A.K.M., Ramasawmy, R.,
Itapirema, E.F., Dias, J.L., Fonseca, I.S.,
Roque, R.A., Barbosa, M.G.V., Mourão,
M.P.G., Ázara, T.M.F., Degener, C., Geier,
Eiras, A.E., de Figueiredo, R.M.P.
1. Universidade Nilton Lins, UNL,
Av. Professor Nilton Lins, 3259.
Parque das Laranjeiras,CEP:
69058-030 Manaus/AM
2. Fundação de Medicina Tropical
Doutor Heitor Vieira Dourado,
FMT-HVD, Av. Pedro Teixeira,
25. Dom Pedro,CEP: 69040-00
Manaus/AM,Brasil.
3. Universidade do Estado do
Amazonas, UEA, Av. Djalma
Batista, 3578 - Flores CEP 69050010 - Manaus/AM
6627 - Pampulha, CEP: 31270901 - Belo Horizonte/MG, Brasil
5. Universität Regensburg , UR,
Universitätsstraße 31 93,053
mil Regensburg Deutschland
/ Alemanha E-mail: karol_
[email protected]
Arboviruses are viruses transmitted to
human and/or animals by arthropods
that can cause infections. Arboviruses
are mainly present in tropical countries
due to the hot and humid climate
which are favorable conditions for
proliferation of the mosquito vector
Aedes aegypti. Among the arboviruses,
Flavivirus is of great epidemiological
importance as it causes diseases in
humans and / or animals. We aimed
to identify flaviviruses in pools of A.
aegypti collected in the District of
Cidade Nova, Manaus / Amazonas
state, Brazil. From December 2008
through June 2010, 2,389 females of A.
Aegypti were collected in BG-Sentinel
traps and distributed in 726 pools.
All pools were macerated and kept
in Trizol ® at -80°C until RNA was
extracted. cDNA was obtained using the
technique of Reverse TranscriptasePolymerase Chain Reaction. 574 Pools
containing 1-8 mosquitoes were
studied. RNA quality was verified in
2% agarose gels and cDNA quality
was tested by amplifying the genes
nicotinamide adenine dinucleotide
dehydrogenase subunit 4 (ND4) and
Cytochrome c oxidase II (COII) of the
A. Aegypti. For the identification of
the genus Flavivirus, primers FL200R
and FL100F encompassing the NS5
region were used for PCR. Sera from
patients with infection of dengue virus
were used as positive controls. Region
encompassing the C-prM genes of
dengue virus was also amplified using
the primers D1 and D2. Molecular
nucleotide sequencing reaction. Of
Human Virology: HV
191
the 574 pools, 19 were positive with
the primers FL100F and FL200R,
and 15 pools were positive with the
primers D1 and D2. Eleven pools were
nucleotide sequenced by D1 and D2
primers. All were either of genotypes
I or II of DENV-4. Studies of molecular
epidemiology of DENV provide us a
basis for understanding how these
viruses evolve genetically in nature.
HV904 - INVESTIGATION OF
CULICIDAE AND DENGUE VIRUS
IN MONTES CLAROS, NORTHERN
MINAS GERAIS, BRAZIL
Rezende, I.M., Lima, G.A.D., Sacchetto,
L., Botelho, J., Silva, A.C., Rodrigues,
R.A., Silva, M.C., Kroon, E.G., Borges,
M.A.Z., Drumond, B.P.
1. Universidade
Federal
de
Juiz de Fora, UFJF, Rua José
Universitário,
São
Pedro.
CEP:36036-900
2. Universidade
Estadual
de
Montes Claros, UNIMONTES,
Av. Dr. Rui Braga, s/nº, Campus
Universitário. Vila Mauricéia.
CEP:39401-089
6627, Pampulha. Belo Horizonte
- MG, CEP: 31270-901 E-mail:
[email protected]
Dengue virus (DENV) (Flavivirus,
Flaviviridae)
occurs
as
four
antigenically distinct but genetically
related viruses named DENV-1 to -4.
DENV is transmitted by species from
Aedes genus (Culicidae), mainly A.
aegypti. DENV-1 to -4 circulate in
Brazil and the country has the higher
number of DENV in Latin America.
192
Human Virology: HV
Minas Gerais state usually present
high number of dengue cases and
Montes Claros is among the five cities
presenting the higher number of
dengue cases in this state. The aim of
this work was to perform a prospective
study of Culicidae and DENV in Montes
Claros. Mosquitos were collected
from February to April of 2012 at
UNIMONTES-MG. Adults were collected
using Shannon traps and larvae were
collected from a transient rainwater
pool. Larvae were maintained in
BOD incubator until they emerge and
mosquitoes were identified based on
morphological characters. Pools of
mosquitos (up to 20) were macerated
and total RNA was extracted and used
for cDNA synthesis followed by nestedPCR to detect DENV. A total of 4210
mosquitoes were collected, including
A. scapularis, A. aegypti, A. albopictus,
Psorophora albigenu, P. ferox, P. cilliata,
P. lanei and Limatus durhamii. The
great majority of mosquitoes were A.
scapularis (>4000). From 257 pools, 47
were initially tested for the presence of
DENV and one pool of A. aegypti was
positive for DENV-1. The other pools
are being tested for DENV and the
obtained amplicons are going to be
sequenced. Although there is no report
of DENV infecting A.scapularis, it is
known that different species of Aedes
sp are related to DENV transmission.
Additionally, A.scapularis has been
implicated in Rocio virus transmission
and is a competent vector for yellow
fever virus. Moreover, these results
demonstrate the presence of naturally
infected mosquitos with DENV-1
during the last epidemic period in
Montes Claros, North region of Minas
Gerais. Financial support: FAPEMIG,
CNPq, CAPES, UFJF, PROPESQ/UFJF
HV905 - SEROLOGIC SURVEY FOR
ARBOVIRUS IN HUMAN POPULATION OF THE ACRE STATE
Chiang, J.O., Chagas, L.L., Martins, L.C.,
Rodrigues, S.G., Vasconcelos. P.F.C.,
Instituto Evandro Chagas, IEC, BR316 Km 07 - Levilândia - Ananindeua/
Pará E-mail: janniferchiang@iec.
pa.gov.br
In the literature, little information
about arboviruses circulation in the
state of Acre, Amazon region was
available. The most recent scientific
studies related to the circulation of
arboviruses in this state occurred
in 2004 in the rural municipality of
Acrelândia. More recently, between
the years 2004-2006, were identified
sporadic cases of Oropouche Virus.
This state has favorable conditions for
the maintenance and spread of these
viruses, and this study performed
a serologic survey for arboviruses
among febrile humans of the Acre
state between the years 2011 – 2012.
In this period, 382 serum samples
were collected in patients with fever
and symptoms suggestive of dengue
fever. For the serologic survey, the
hemagglutination inhibition (HI) test
was used for 19 arboviruses, belonging
the viral genera as follows; Alphavirus
(EEE, WEE, Mayaro and Mucambo);
Orthobunyavirus (Guaroa, Tacaiuma,
Maguari, Caraparu, Oropouche and
Catu) and flavivirus (Yellow fever
wild and vaccine strains, Ilheus, Saint
Louisencephalitis, Rocio, dengue 1,
dengue 2, dengue 3 and dengue 4), while
the MAC-ELISA for IgM capture antibody
was used for dengue and yellow fever
viruses. By HI, 289 (75,6%) serum
samples were positive and 93 (24,4%)
negative. Among positive samples, 267
(92,4%) were to flaviviruses, 55 (19%)
for orthobunyaviruses and 62 (21,5%)
for alphaviruses. It is important to
emphasize that each sample can be
positive for a single or more viruses
from different genera. By ELISA test,
just 284 serum samples were tested,
and 56 (19,7%) positive for dengue
virus, no positive sample was obtained
for yellow fever virus. The results of this
study, showed the active circulation of
the dengue virus in the Acre State and
of other arboviruses in specially for
those of the flavivirus genus. Financial
support: IEC/CNPQ
HV913 - RAPID IMMUNOCHROMATOGRAPHIC (IC) TESTS FOR
EARLY DIAGNOSIS OF DENGUE
VIRUS INFECTION: A PRELIMINARY
ANALYSIS
Lima, M.R.Q., Nogueira, R.M.R., de
Filippis, A.M.B., de Sousa, C.S., dos
Santos, F.B.
Oswaldo Cruz Institute , IOC,
Avenida Brasil, 4365, Manguinhos,
Rio de Janeiro - RJ - Brasil E-mail:
[email protected]
Dengue is associated with explosive
urban epidemics and has become a
major public health problem in many
tropical developing countries. The
laboratory diagnosis of dengue can be
carried out using several approaches,
however sensitive and specific assays
useful to diagnosis in the early stage
of fever are desirable. The NS1 protein,
a highly conserved and secreted
glycoprotein, is a candidate protein
for rapid diagnosis of dengue. Several
studies have compared IC technologies
with the reference test for dengue. We
aimed to evaluate the potential use of 3
commercial rapid IC assays in a panel of
120 serum samples, from the collection
of the Flavivirus Laboratory. The NS1
Human Virology: HV
193
Ag Strip, Early Rapid and SD Bioline
are disposables tests using lateral flow.
Both NS1 Ag strip and the Rapid Early
showed similar sensitivity (66.1%) to
confirm dengue cases. A lower sensitive
was observed the SD Bioline 52.3%.
The detection rate by the NS1 Ag Strip
and the Early Rapid were the same
(60%) in the presence of anti-DENV
IgM, while the SD Bioline was 55%.
In this study, the presence or absence
of IgM did not influence detection by
all kits. However, specificities were
100%, in all assays, based on the
analysis of sera of healthy individuals
and individuals negative for dengue.
No cross-reactivity was observed
for the Rapid Early and SD Bioline,
nevertheless, the NS1 Ag Strip showed
cross-reactivity with one yellow fever
vaccine. No differences were observed
by the tests NS1 antigen in confirming
primary and secondary infections. In
this report, theresults indicate that
the commercially available IC tests
have overall low sensitive and high
specificity for early and rapid diagnosis
of dengue virus infection. It maybe
appropriate for use as a screening
test consired to other techniques such
as RT-PCR in acute samples and/or
MAC-ELISA in convalescent samples
in routine laboratory and surveillance
of dengue disease in public health
services. Financial support: FAPERJ,
CNPq, CAPES, FIOCRUZ
HV921 - THE FIRST CASE OF DENGUE
SEROTYPE 4 IN CEARÁ, BRAZIL
Ramalho, I.L.C., Perdigão, A.C.B., Lima,
E.G., Roriz, M.L.F.S., Melo, M.E.L.,
Teixeira, F.M., Burgoa, J.S.V., Silva, L.B.,
Dantas, C.A., Silva, L.A.B., Sousa, A.S.S.,
Holanda, S.G.S., Escócia, K.N.F., Araújo,
F.M.C.
194
Human Virology: HV
1. Laboratório Central de Saúde
Pública do Ceará, LACEN-CE, Av.
Barão de Studart, 2405 - Aldeota
CEP:60120-002
2. Secretaria de Saúde do Ceará,
SESA-CE, Av. Almirante Barroso,
600 - Centro Rede Nordeste de
Biotecnolgia - UECE, RENORBIOUECE, Av. Paranjana, 1.700 Campus do Itaperi - 60740-000
Fortaleza/CE
E-mail:
izabel.
[email protected]
Dengue virus serotype-4 was first
reported in Brazil in a restricted
outbreak in Rio Branco, Roraima, 1981.
More than 20 years after, it was isolated
in patients living in the North region of
the country and since then it started
to spread to other regions. The State
of Ceará reports dengue transmission
since 1996, with circulation of DENV1, DENV-2 and DENV-3 until 2011,
year that showed the largest dengue
epidemic of the State, with the
predominance of DENV-1. In order
to detect early the entry of the new
serotype, the virological surveillance
was implemented. Whole blood or
serum samples obtained from acute ill
patients were inoculated into C6/36
cells. Isolated strains were identified
by indirect immunofluorescence assay
using monoclonal antibodies to the four
serotypes. Despite DENV-1 epidemic, it
was possible to isolate, in March, the
first case of DENV-4 in a patient with
fatal outcome, who lived in Morada
Nova, a city 161 Km far away from
the capital, Fortaleza. Another case of
classical DENV-4 with full recovery,
was isolated in December, from a
patient living in Fortaleza. This year,
from January to June, 38.852 dengue
cases were serologically confirmed
from 152/184 (82.6%) counties of the
State with the majority of the cases
from the capital with 30.437 (78.3%),
followed by the city of Maracanaú with
1.195 cases (3%). In relation to severe
dengue, 194 cases were reported
with 54 DHF and 140 dengue with
complications; 16 patients evolved to
death, 11 from the capital and five from
the other counties. The predominant
serotype at the current year is DENV-4
with 95.5% of the isolated. The spread
of serotype 4 keeps happening through
the cities, what is concern about a
severe epidemic for the next year. The
vector control should be implemented
to avoid a major epidemic and the
patients care should be improved
to avoid fatal outcomes. Financial
support: FUNASA
HV923 - HEPATITIS B VIRUS
INFECTION IN A POPULATION OF
RECYCLABLE WASTE COLLECTORS,
CENTRAL BRAZIL
Marinho, T.A., Lopes, C.L.R., Teles, S.A.,
Matos, M.A.D., Carneiro, M.A.S., Silva,
A.M.C., Reis, N.R.S., Kozlowski, A.G.,
Andrade, A.A., Martins, R.M.B.
1. Instituto de Patologia Tropical e
Saúde Pública, IPTSP/UFG, Caixa
Postal 131, CEP: 74.605-050,
Goiânia-GO
2. Faculdade de Enfermagem, FEN/
UFG, Caixa Postal 131, GoiâniaGO E-mail: enftamiris@hotmail.
com
Hepatitis B virus (HBV) remains a major
cause of liver disease worldwide despite
vaccination programs implemented
over the last decade. Worldwide, it
is estimated that 2 billion people are
infected with HBV and that more than
350 million are chronically infected.
Patients with chronic hepatitis B are at
risk for developing liver cirrhosis and
hepatocellular carcinoma. Based on
sequence divergence of 8% or greater
in the entire genome, HBV has been
classified into 10 genotypes (A-J), which
have a distinct geographic distribution.
Recyclable waste collectors have
a lifestyle that is characterized by
unfavorable social, cultural, and
environmental factors. There is
currently very little data on HBV
infection in this population. Therefore,
the aim of the present study was to
investigate the HBV prevalence, risk
factors and genotypes in a population
of recyclable waste collectors in
Central Brazil. A cross-sectional survey
was carried out with 431 individuals
who were recruited in all 15 recycling
cooperatives/associations in Goiania
city, Goias state, Central Brazil. All
individuals were interviewed, and
their serum samples were tested
for the presence of HBV serological
markers. HBsAg-positive samples
were tested for HBV DNA by nested
PCR and were genotyped by restriction
analysis. The overall HBV prevalence
infection was 12.8% (95% CI: 9.816.2). A multivariate analysis of risk
factors showed that age >30 years and
illicit drug use were independently
associated with HBV infection. HBV
DNA was detected in 2/3 HBsAgpositive samples, in which genotypes
D and F were identified. These findings
confirm that recyclable waste collectors
are at high risk for hepatitis B infection
and highlight the importance of having
a public health policy that addresses
this population. Financial support:
CNPq
HV926 - PHYLOGENETIC ANALYSIS
OF DIFFERENT LINEAGES OF
Human Virology: HV
195
DENGUE VIRUS IN BRAZIL
Drumond, B.P., Mondini, A., Schmid,
D.J., Bosch, I. , Nogueira, M.L.
José do Rio Preto , FAMERP,
Laboratório de Pesquisa em
Virologia,FAMERP, São Paulo,
Brazil.
2. Universidade
Estadual
de
São Paulo, UNESP, Lab de
Saúde Pública. F. de Ciências
Farmacêuticas.
Araraquara.SP
Brazil
3. Universidade Federal de Juiz
de Fora, UFJF, Laboratório de
Virologia, ICB - UFJF , Juiz de Fora,
MG, Brasil
4. Cumming School of Veterinary
Medicine, , Department of
Biomedical Sciences. Tufts - North
Grafton, MA, USA. Massachusetts
5. Institute of Technology, MIT,
Genome Resources in Dengue
Consortium.
Cambridge,
Massachusetts,
USA
E-mail:
[email protected]
Dengue virus (DENV) (Flavivirus,
Flaviridae) comprises four genetically
and antigenically distinct serotypes,
named DENV-1 to DENV-4. Brazil is the
country with the highest number of
dengue cases occurring in the Americas.
DENV-1 was the most predominant
virus in Brazil in the 80’s when it was
replaced by DENV-2 in the 90´s, which
was subsequently replaced by DENV3, in 2000. The four DENV serotypes
circulate in Brazil nowadays. Here, we
sequenced the genome of 12 isolates
of DENV-2 obtained from patients
with dengue fever, from São José do
Rio Preto, São Paulo, Brazil, in 2008.
196
Human Virology: HV
These sequences and other genome
sequences from DENV-2 viruses were
used to perform phylogenetic and
molecular evolutionary analyses.
Phylogenetic analysis demonstrated
that all Brazilian DENV-2 isolates
were clustered within the American/
Asian genotype being subdivided into
three lineages. Analysis of the deduced
polyprotein
sequences
revealed
that some amino acids substitutions
distinguish those lineages. Four isolates
had identical genome sequences. The
three lineages of Brazilian DENV-2
grouped strains isolated in different
periods: (i) 2007 to 2010, (ii) 2000 to
2006 and (iii) 1990 to 2000. Based on
the analysis of envelope gene sequence,
those lineages were estimated to be
introduced in the country in (i) 20032006, (ii) 1998–99 and (iii) 1988-90.
Lineage iii did not contain viruses
isolated after 2000, what could be
a result of surveillance sampling
methods or represent a lineage
replacement event, by the two other
newer lineages. Finally, the sources
of exogenous viruses were countries
from Latin America, reinforcing the
need of genotype surveillance in order
to detect and trace virus populations
that are circulating in Brazil and
Latin America, what is especially
important in a scenario of circulation
of different DENV serotypes in this
region. Finnancial support: FAPESP,
CNPq, INCT-DENGUE, PROPESQ/UFJF,
FAPEMIG, CAPES
HV930 - HEPATITIS A VIRUS (HAV)
TRANSMISSION BY BLOOD TRANSFUSION
Amado, L.A., de Paula, V.S., Brito, S.M.,
Oliveira, J.M.O., Motta, I.J.F., Pinto,
M.A., da Silva, S.G.C.
Av. Brasil 4365, Manguinhos, Rio
de Janeiro-RJ Instituto Nacional do
Cancer, INCA, E-mail: l_amado@ioc.
fiocruz.br
Hepatitis A infection is the most cause
of acute hepatitis and is primarily
spread by the fecal-oral route. Viremia
appears 1 to 2 weeks before the onset
of clinical symptoms, consequently,
although a rare occurrence, parenteral
transmission of HAV via blood during
viremia is possible and has been
associated with transfusion of blood.
In this report we present a serological
and molecular tracing of transfusiontransmitted HAV by a single blood
collection. A 39-year-old asymptomatic
male volunteer made a whole-blood
donation for the Hemotherapy Service
of the National Institute of Cancer
(INCA), RJ. Twenty days later, the donor
became jaundiced and an acute HAV
infection was confirmed by detection
of IgM anti-HAV and hepatic functions
altered. Two patients, who received
this donor hemocomponents, were
identified. 1) A 27-year-old male with
acute leukemia received platelets, two
days after the donation. This patient
did not developed clinical symptoms
of hepatitis and anti-HAV IgM was
not detected. 2) A 39-year-old male
received the red blood cells. This
patient was a chronic hepatitis C and
was immunosuppressed after bone
marrow transplantation. Ten days
post-transfusion, he developed clinical
symptoms of acute hepatitis and died
twenty-five days after with fulminant
hepatitis. Retrospectively, the sera
samples collected from the donor at the
donation day and from the two patients
collected thirty days post-transfusion
are tested for HAV-RNA detection by
RT-PCR. HAV RNA was identified in
all sera samples evaluated. Analyzed
sequences of the HAV RNA revealed
that the virus of the three patients were
identical, according to the sequences of
the region VP1/2A and complete VP1 of
the HAV. These results demonstrated a
rarely event of transfusion-transmitted
HAV evidenced by molecular and
serologic tracing, suggesting that
additional blood screening tests
should be implemented for ensuring
product safety to specific groups
of immunocompromised patients.
Fomento:IOC/Fiocruz
HV931 - DETECTION OF NOROVIRUS,
SAPOVIRUS AND ASTROVIRUS IN
PATIENTES WITH ACUTE GASTROENTERITIS, FROM FEDERAL
DISTRICT (BRAZIL)
Silva, F.G., Anjos, K., Nagata, T., Silva,
P.A., Lima, L.M.P.
1. Universidade Católica de Brasília,
UCB, EPCT QS 01 - Águas Claras 71966-700
2. Universidade de Brasília, UnB,
Campus Darcy Ribeiro - Asa
Norte - 70910-900 E-mail: fabio_
[email protected]
Annually 2 billion cases associated with
acute gastroenteritis occur worldwide.
Since 1994, the Brazilian Ministry of
Health, organizes and coordinates
the monitoring of acute diarrheal
disease, the sentinel surveillance
system known as MDDA, in order to
detect early outbreaks. The program
calls for culture for bacterial isolation
and identification, search of intestinal
parasites and detection of rotavirus
by ELISA. When all these results are
negative, the case remains without a
proper diagnosis, clinically defined as
viral infection. A total of 145 diarrheal
Human Virology: HV
197
samples negative for rotavirus, from
MDDA program between 2006 and
2011 were tested. These samples
were subjected to a multiplex RT-PCR
for detection of astrovirus, norovirus
GI, norovirus GII and sapovirus.
After extraction of genetic material,
RNA was subjected to a RT-PCR, on
a subsequent step the cDNA was
carried to a multiplex PCR, and finally,
using agarose gel electrophoresis, the
amplified fragments were separated
according to their molecular weight,
making it possible to differentiate
pathogen which was present in the
sample. Among the 145 samples tested,
12 samples were positive (8.3%). In
this positive group, 9 (75%) were
norovirus GII, 2 (17%) were sapovirus
and 1 (8%) astrovirus. The age group
most affected was of individuals under
12 years old and higher prevalence
occurred during the drought period.
We found that the peak of infection
occurred in July extending to September,
months that are within the period of
drought. It was possible to prove the
movement of the three pathogens in
this study, which reinforces the need
for further investigation of different
gastroenteric viruses in Federal District
than Rotavirus, also shows that the
multiplex PCR used in this study might
be suitable to be used in laboratorial
routine practice. Financial support:
Universidade Catolica de Brasilia and
LACEN/SES/DF
HV932 - DEVELOPMENT OF A
REAL-TIME PCR- BASED SYTEM FOR
RAPID DETECTION AND QUANTIFICATION OF HEPATITIS DELTA VIRUS
(HDV) IN THE WESTERN AMAZON
REGION, BRAZIL.
Botelho, L.F.S., Santos, A.O., Borzacov,
L.M.P., Silva, A.S., Salcedo, J.M.V., Vieira,
198
Human Virology: HV
D.S.
1. Research Center for Tropical
Medicine , CEPEM, Porto Velho,
Rondônia,
Brazil
Tropical
Pathology Research Institute,
IPEPATRO, Porto Velho, Rondônia,
Brazil
2. Fundação Universidade Federal
de Rondônia, UNIR, Porto Velho,
Rondônia, Brazil
3. Fundação Oswaldo Cruz de
Rondônia, FIOCRUZ Rondônia,
Porto Velho, Rondônia, Brazil
E-mail: luan_botelho@hotmail.
com
Hepatitis delta is a serious infectious
disease that causes severe inflammation
in hepatocytes and rapidly progressive
and caused by Hepatitis Delta Virus
(HDV).HDV is classified as HBV subvirus
satellite because it is unable to infect in
the absence of HBV,it requires HBsAg.
The HDV RNA genome is of very small
circular approximately 1700nt and
negative polarity.The western region of
the Brazilian Amazon area considered
highly endemic for HDV.We selected
35 patients from Rondônia and Acre
with anti-HDV positive.In quantitative
RT-PCR kit was used TaqMan PCR
Master Mix with probes labeled with
FAM/TAMRA primers that amplify a
fragment corresponding to HDAg-L.
For standardization of quantitative PCR
two standard curves were constructed
using serial dilutions of the cloned
fragment linearized with EcoRI and
another transcribed into RNA with
T7 RNA polymerase, both produced
"in house". Thereafter dilutions were
subjected to quantitative RT-PCR
to obtain the standard curves,the
following tests in triplicate intraassay and inter-assay four times in
consecutive days. Standard curves were
produced and showed a detection limit
of 1.9 million to 19 copies/mL for the
linearized and 8.4 million to 84 copies/
mL for the RNA transcript.We generated
two linear regression curves relating
the number of copies/mL x Ct,with a
coefficient of determination R2=0.93
and slope -3.3(p <.05) and R2=0.99
and slope -2.9(p <.05) of efficiency
linearized and transcribed into the
RNA,respectively.The reproducibility of
the test was observed by the coefficient
of variation produced by each dilution
tested and the specificity of the test
by not obtaining a signal in any of the
negative controls added in each race.A
molecular approach described in this
study is important and immediate
impact on public health, particularly
for chronic patients in the region
favoring confirmation of the diagnosis
as well as the orientation therapy to
be instituted. Financial support:UNIR/
FIOCRUZ/CNPq/IPEPATRO/CEPEM
HV934 - OCCULT HEPATITIS B VIRUS
INFECTION IN HIV-1-POSITIVE,
TREATMENT-NAIVE PATIENTS IN
CENTRAL BRAZIL
Oliveira, M.P., Matos, M.A.D., Lemes,
P.S., Pimentel K.N., Del-Rios, N.H.A.,
Carneiro, M.A.S., Lago, B.V., Mello,
F.C.A., Gomes S.A., Martins, R.M.B.
Saúde Pública, IPTSP/UFG, Caixa
Postal 131, CEP: 74.605-050,
Goiânia-GO
2. Instituto Oswaldo Cruz, IOC/
FIOCRUZ, Rio Janeiro, RJ E-mail:
[email protected]
Two billion people have been infected
with hepatitis B virus (HBV) and
more than 400 million are chronically
carriers. Worldwide, there may be 3
to 6 million HIV-infected people living
with chronic HBV. HIV-HBV coinfection
increases the morbidity and mortality
beyond those caused by either
infection alone. People coinfected with
HIV have higher levels of hepatitis B
viremia, have faster progression to
chronic hepatitis B, and have a higher
risk of cirrhosis and hepatocellular
carcinoma. Occult HBV infection, a
peculiar form of chronic infection,
is characterized by the presence of
HBV DNA in serum and/or liver in
HBsAg-negative individuals. Occult
HBV infection is relevant in different
clinical contexts and its prevalence
varies from 0% to 89% among HIVinfected patients. This study aimed to
assess the prevalence of HBV occult
infection in HIV-infected, treatmentnaive patients in Goiania-Goias, Central
Brazil. HBV genotypes were also
investigated. A cross-sectional study
was conduct among HIV-infected,
treatment-naive patients attended at a
reference hospital in Goiania city. The
participants (n=505) were tested for
serological markers of HBV infection.
HBV DNA was detected by a seminested PCR and quantified using the
real-time PCR TaqMan technology.
The amplicons were genotyped by
nucleotide sequencing. Of 73 anti-HBc/
anti-HBs reactive samples, 7 were HBV
DNA-positive. Among the 26 anti-HBc
only reactive samples, 9 was HBV DNApositive. These results were confirmed
by repeating both the DNA extraction
and amplification procedures, thereby
establishing the occult HBV infection
rate at 16.2%. As expected, low HBV
DNA levels were found in these patients
(mean: 6.59 x 102 copies/mL). HBV
genotypes A (62.5%, A1), F (25%,
F2) and D (12.5%, D2 and D3) were
Human Virology: HV
199
identified. These findings revealed a
high prevalence of occult HBV infection
and the predominance of genotype A
(A1) in HIV-infected, treatment-naive
patients in Central Brazil. Financial
support: FAPEG
HV935 - INCIDENCE AND CLINICAL
ASPECTS OF CONGENITAL CYTOMEGALORIVUS (CMV) INFECTION IN
THE ILHEUS, BA
Marin, L.J., Cardoso E.S.C., Jesus, B.L.S.,
Françoso, M.F.S., Santana, J.G., Júnior,
H.M.C., Raiol, M.R.
Universidade Estadual de Santa
Cruz , UESC, Rodovia Ilhéus Itabuna,
km 16, Salobrinho, Ilhéus, Bahia,
cep 45662900 E-mail: lajumarin@
hotmail.com
The cytomegalovirus (CMV) infection
is highly prevalent in humans and
it is associated with lower levels of
education and incomes. The virus
can be vertically transmitted to the
newborn by different ways. The CMV
has been recognized as a major cause
of congenital infection (prevalence
between 0.2 to 3%). In addition, it
has been demonstrated that 90%
of congenital infected infants have
no symptoms at delivery, but it has
been shown that they can present
later disorders, including neurologic
problems and hearing loss. In this
study, we investigated CMV infection
in infants delivered at Santa Helena
Hospital of Ilhéus, Bahia. In addition,
we pretend to verify clinical aspects
of CMV infection and to observe
neurological disorders for four years.
This study will be conducted from
between July 2010 to December 2013.
The study was approved by the UESC
Ethics Committee. The mothers or
responsible of all newborns are asking
200
Human Virology: HV
to sign a written informed consent.
Saliva and urine samples have been
collected at delivery. The samples
have been analyzed by nested PCR
and the positives will be confirmed
by another PCR using new samples
of saliva and urine obtained until 3
weeks of life. Sociodemographic and
clinical data have been obtained by
standardized questionnaire. Until
now, it was possible collect samples
of 1.825 newborns and we had 18
positive result (incidence 1%). These
children will be analyzed until 4 years
to verify possible late disorders and
hearing loss. We consider that it is
necessary to carry out continuous
studies in order to define the main
risk factors for congenital infection.
These studies could help to define best
program in public health to prevent
vertical transmission. Besides that, the
identification of a congenitally infected
newborn infant soon after birth is
very helpful to early intervention
in order to avoid or minimize the
potential involvement of the central
nervous system, sensorineural hearing
loss, developmental delay, or motor
abnormalities
HV939 - CORRELATION OF RS
12979860 AND RS8099917 IL28B
POLYMORPHISMS IN BRAZILIAN
PATIENTS WITH HEPATITIS C TO
PREDICT TREATMENT RESPONSE
Sitnik, R., Oyakawa, L., Muto, N.H.,
Santana, R.A.F., Castro, V.F.D., Ramos,
O.P.S., Dastoli, G.T.F., Petroni, R.C.,
Rodrigues, J.N.M., Souza, J.M.A.,
Oliveira, V.M., Carvalho, F.P., Pinho,
J.R.R.
Hospital Israelita Albert Einstein,
HIAE, Av. Albert Einstein, 627 E-mail:
[email protected]
Genetic variation in the IL28B gene
region on chromosome 19 was
identified for predicting sustained
viral response of patients chronically
infected with genotype 1 HCV infection.
The most strongly associated single
nucleotide polymorphism (SNP) for
treatment response, rs12979860, has
also been shown to be significantly
associated with spontaneous hepatitis
C clearance. In addition, other SNPs
were described, such as rs8099917.
In order to evaluate the combined
genotyping of IL28 polymorphisms
rs12979860 and rs8099917 in our
population, we performed genotyping
of the two sites using TaqMan custom
designed probes (Applied Biosystems)
on an ABI7500 instrument and the ABI
TaqMan allelic discrimination kit from
Applied Biosystems. We analyzed 538
samples from HCV patients of a private
hospital in São Paulo during the period
from December 2010 to April 2012. The
results of both SNPs were compared to
find a correlation of better and worse
response according to the genotype. We
found an agreement of 65,2% between
the results (25,3% better response,
35,5% intermediate and 4,5% worse
response), and 34,7% discordant
results. The discordant results were
composed by 3,3% totally divergent
results and 31,4% heterozygous result
for one SNP (21,9% CT for rs12979860
and 9,5% GT for rs8099917). The
heterozygous patients for rs12979860
had 21,7% rs8099917 TT genotype
(worse response) and 0,2% rs8099917
GG genotype (better response).
According to literature, patients with
heterozygous genotype of rs12979860
benefit from further determination of
rs8099917. Based on this finding and the
high rate of rs12979860 heterozygous
patients with discordant results in our
cohort, despite the good correlation of
the studied SNPs (65,2%), we suggest
that the combined determination of
both SNPs will improve treatment
decisions in a considerable number
of Brazilian chronic HCV patients.
Financial support: Hospital Israelita
Albert Einstein
HV947 - DENGUE VIRUS TYPE 1
GENOTYPE V WAS CIRCULATING IN
RIBEIRÃO PRETO, SÃO PAULO, IN
2011
Soares, A.M., Amarilla, A.A., Aquino,
V.H.
Faculdade de Ciências Farmacêuticas
de Ribeirão Preto, FCFRP-USP, Av. do
Café, s/nº. - Campus Universitário
- Ribeirão Preto - SP - 14040-903
Dengue is the most important mosquitoborne viral disease worldwide. Dengue
virus (DENV) is a member of the genus
Flavivirus and family Flaviviridae.
DENV include four antigenically related
viruses named dengue virus type 1 to 4
(DENV 1-4). Each of the four serotypes
is able to cause disease with a full
spectrum of clinical manifestation.
DENV-1 is composed of five genotypes:
genotype I is represented by viruses
of Taiwan and Thailand; genotype II
is represented by viruses of Thailand;
genotype III is represented by strains
of Malaysia; genotype IV is represented
by viruses of Southeast Asia, South
Pacific and Australia and genotype V
is represented by viruses of America,
Africa and Southeast Asia. Considering
that Ribeirão Preto is an endemic city
for dengue, it is of great importance
to monitor the circulating viruses,
which would allow the identification
of new serotypes or subtypes that
could be related to most severe cases
Human Virology: HV
201
of the disease. Phylogenetic analysis
also allows a better assessment of the
pattern of migration and evolution
of these viruses. In this study, four
dengue viruses isolated in Ribeirão
Preto in 2011 and confirmed by
real-time RT-PCR and by indirect
immunofluorescence were included.
The partial gene sequence of the
nonstructural protein 5 (NS5) was
amplified by RT-PCR, purified and
sequenced. Sequences were analyzed
and aligned with the BioEdit and the
MEGA 5.05 software. Based on the
alignment, a phylogenetic tree was
constructed using the Neighbor-joining
method. The phylogenetic analysis
showed that DENV-1 isolated in
Ribeirão Preto correspond to genotype
V of DENV-1 and are closely related to
other Brazilian DENV-1 isolates.
HV961 - HIV-1 MOLECULAR EPIDEMIOLOGY IN URUGUAIANA/RS:
MONITORING THE DISPERSION OF
SUBTYPE C IN A BRAZIL-ARGENTINA
BORDER TOWN
Gräf, T., Junqueira, D.M., Tamayo, A.,
Almeida, S.E.M., Pinto, A.R.
Catarina, UFSC, Florianópolis/SC
- 88040-900 - Brasil
2. Fundação Estadual de Produção
e Pesquisa em Saúde, FEPPS, Av.
Ipiranga, 5400, 3º Andar - Porto
Alegre/RS - CEP: 90610-000
3. Serviço
de
DST/aids
de
Uruguaiana, , E-mail: akograf@
yahoo.com.br
The epidemic of HIV/AIDS in Brazil
faces its worst scenario in Southern
region, where the highest AIDS
incidences and mortality rates due
202
Human Virology: HV
to HIV-1 infections are reported.
Furthermore, while in most of the
regions of Brazil subtype B is dominant,
in the capital cities of Southern region
HIV-1 subtype C is highly prevalent
with evidences of a slow dispersion
through vicinities countries (e.g.,
Argentina and Uruguay). In the view
of subtype C epidemic expansion, the
current study investigated the HIV-1
molecular features in Uruguaiana, a
Brazilian border city with Argentina.
Blood samples from 26 HIV-1 infected
patients from the local reference
hospital were collected in FTA card.
After sample processing, nestedPCR amplification and sequencing
of the HIV-1 gp120-C2/V3 region
were performed. Sequences were
aligned using Mussel and phylogenetic
analyzes were performed by Maximum
Likelihood algorithm with GTR+G+I
nucleotide substitution model. HIV1 subtype C was observed in 9 (43%)
patients, subtype B in 11 (52%)
and subtype D, a rare HIV-1 form in
Brazilian epidemic, in 1 (5%). Patients
were mainly infected by heterosexual
intercourse and no correlation between
subtypes and exposure categories
were observed. The results presented
here show a significant prevalence
of subtype C in the border of Brazil
and Argentina, a geographical region
without previous studies in HIV-1
molecular epidemiology. Uruguaiana
is roughly located halfway between
Porto Alegre and Buenos Aires and is
an important monitoring point of the
Brazilian and Argentinian epidemics.
In conclusion, the partial results
presented herein indicate a significant
expansion of subtype C epidemic in a
border city among Brazil and Argentina
with possible influences to the HIV-1
epidemic in South America. Financial
support: FAPESC
HV967 - RISK BEHAVIORS AND PREVALENCE OF HTLV-1 INFECTION IN
FEMALE SEX WORKERS IN GOIÂNIA–
GOIÁS, BRAZIL
Souza, D.H., Martins, R.M.B., Matos,
M.D., Marinho, T., Diniz, F., Teles, S.A.,
Araujo, L.A., Vicente, A.C., Otsuki, K.,
Carneiro, M.A.S.
1. INSTITUTO
DE
PATOLOGIA
TROPICAL PUBLICA / UFG, IPTSP/
UFG, AV. UNIVERSITÁRIA S/N, S.
UNIVERSITÁRIO
2. Faculdade
de
Enfermagem,
Universidade Federal de Goiás,
FEN/UFG, S. Universitário 3. Instituto Oswaldo Cruz, Fiocruz,
IOC, Rio Janeiro, RJ, Brazil E-mail:
[email protected]
Human T-cell lymphotropic virus type 1
(HTLV-1) is a retrovirus associated with
T cell leukemia/lymphoma in adults
(ATL) and tropical spastic paraparesis
(TSP) or HTLV-1 associated myelopathy
(HAM / TSP). HTLV-1 transmission
occurs by sexual, parenteral and
vertical paths. Female sex workers are
a vulnerable population to parenteral
and sexually transmitted infections
since they have high risk behaviors
such as illicit drug use and unprotected
sex. This study aimed to determine the
HTLV-1 infection prevalence and risk
behaviors in a population of female sex
workers (FSW) in Goiânia city, Goiás,
Central Brazil, using the respondent
driven sampling methodology. From
May 2009 and June 2010, 402 FSW were
interviewed about demographic and
risk characteristics for HTLV infection.
Blood samples were collected from all
females and were tested by enzyme-
for the presence of HTLV antibodies
(anti-HTLV1/2). Reactive samples were
tested for confirmation by polymerase
chain reaction (PCR). PCR products
were purified and directly sequenced.
Nucleotide sequences obtained were
subjected to phylogenetic analysis.
The mean age of the study population
was 27.5 years (SD: 9.1 years). Most
(67.1%) were single and 47.3% had 10
to 12 years of formal education. One
third of female sex workers reported
illicit drug use (34.1%), thought only
2.7% reported injecting drug use,
51.9% had more than seven sexual
partners in the last week and 36.3%
did not use condom with their steady
sexual partners. Some women reported
to recruit their clients in more than
one type of venue, being nightclubs
(41%), bars (27.7%) and streets (25%)
predominant. Three of the 402 samples
were found to be positive by ELISA
and, when subjected to the detection of
HTLV-DNA for the tax and LTR regions,
only one was positive for HTLV-1,
resulting in a prevalence of 0.25%.
(CI 95%: 0.0-1.6). The virus isolate
was classified as Transcontinental
subgroup of the HTLV-1 Cosmopolitan
subtype. Although the findings of this
study showed high frequencies of risk
behaviors, a low prevalence of HTLV-1
was found among female sex workers
in Goiânia city.
HV970 - PREVALENCE OF OCCULT
HEPATITIS B VIRUS INFECTION IN
HIV PATIENTS
Silva, J.L.A., Cahú, G.G.O.M., de Deus,
D.M.V., Coêlho, M.R.C.D.
Universidade
Federal
de
Pernambuco, UFPE, Av. Prof. Moraes
Rego, 1235 - Cidade Universitária,
Human Virology: HV
203
Recife - PE - CEP: 50670-901 E-mail:
[email protected]
The hepatitis B virus (HBV) coinfection
in human immunodeficiency virus
(HIV) patients is common, because these
viruses present similar transmission
routes. Therefore, the liver disease has
become the main cause of morbidity
and mortality in HIV/HBV coinfected
patients with more prolonged survival,
due to advancement of highly active
anti-retroviral therapy. The occult HBV
infection is defined as the presence
of HBV-DNA in the liver, followed by
detectable or undetectable HBV-DNA in
serum, of HBsAg negative individuals
with viral load usually very low (<200
IU/mL). The aim of the study was to
investigate the prevalence of occult HBV
infection in HIV patients attended at
Infectious Diseases Outpatient Clinic at
the Clinics Hospital, Federal University
of Pernambuco, Recife, Brazil. The
serological markers HBsAg, anti-HBc
total and anti-HBs were screened by
enzyme immunoassays kits (Bio-Rad
Laboratories, France) and for detection
and quantification of HBV viral load in
the plasma was used a real time PCR
kit (Qiagen, Germany). Ninety patients
were analyzed, 43 (48%) were female
and 47 (52%) were male, mean age
36.76 ± 10.65 years. The anti-HBc total
was identified in 20.11% (19/90), of
which 31.58% (6/19) had only this
marker, 63.16% (12/19) exhibited antiHBs and 5.26% (1/19) had the HBsAg
positive. Others 27.77% (25/90) had
only the anti-HBs, suggesting the
vaccination history for hepatitis B. It
was emphasized that 50% (45/90)
of patients did not show any of the
serological markers evaluated and they
were considered susceptible to virus
infection. The HBV-DNA was detected
in 25.56% (23/90), of which 95.65%
204
Human Virology: HV
(22/23) in HBsAg negative patients.
Among these cases, 50% (11/22) had
high viral load (4.447.725 ± 9.055.188
IU/mL) and the others it were not
quantified (<3.000 IU/mL). Thus, only
the HBsAg negative patients with low
viral load were considered as cases
of occult HBV infection, revealing a
prevalence of 12.22% (11/90), of which
was similar than reported in the states
of Rio de Janeiro and São Paulo (5%
to 14%). Besides helping the clinical
management of HIV/HBV coinfected
patients, the presented data had a
negative impact on the transmission
of the virus, especially in cases of
negative serology with the presence of
HBV-DNA. Financial support: National
Council for Scientific and Technological
Development (CNPq).
HV978 - DEVELOPMENT OF DIAGNOSTIC TEST FOR THE DETECTION
AND QUANTIFICATION OF HUMAN
HERPESVIRUS 1 AND 2 IN CSF FOR
REAL TIME PCR
Oliveira. D.B., Almeida, G.M.F., Botelho,
L.M., Abrahão, J.S., Trindade, G.S.,
Ferreira P.C.P., Kroon, E.G.
6627 E-mail: danilobretas@yahoo.
com.br
Meningitis is a disease with worldwide
distribution, affecting people in all
parts of the globe. The main etiologic
agents of this disease are viruses,
bacteria and fungus. Viral infections
are the main causes of infection in the
central nervous system (CNS) around
the world. In Brazil, on average, there
are reported probable viral meningitis
11,500 cases / year. However, for
most cases there is no identification
of the agent. Human Herpesvirus 1
(HHV-1) and Human Herpesvirus 2
(HHV-2) are responsible for about
2% of cases of acute viral meningitis.
Meningitis by HHV-1 and HHV-2 cause
more neurological complications than
most other viral meningitis, since
about 30% of all patients develop
CNS complications. The objective of
this work is to use real-time PCR for
development of a diagnostic test for
detection and quantification of HHV 1
and 2 in cerebrospinal fluid (CSF) of
patients with clinical suspect of viral
meningitis. Primers and a probe were
designed for the viral polymerase gene,
inside a region with 100% genetic
similarity between HHV 1 and HHV 2.
The viral isolate HHV-1 EK and HHV-2
(ATCC VR 590) , were used for initial
primer tests, and also for fragment
amplification, which was used for
cloning in plasmid pGEM ®-T Easy.
After obtaining this control plasmid it
was sequenced and used to measure
the efficiency of the reaction. To test
the analytical sensitivity a matrix that
mimics the CSF with known amounts
of viral load was used. The reaction
used in this test showed high efficiency
(114%). When an analytical sensitivity
test was performed, we observed
that the test is very sensitive with a
detection limit up to 1 PFU/µl. So this
test shows potential for use in the
health system, with the aim of reducing
mortality and morbidity of meningitis
caused by HHV1 and HHV-2.
HV979 - PANDEMIC INFLUENZA A
H1N1 2009 VIRUS INFECTION IN
PREGNANCY IN CEARÁ
Perdigão, A.C.B., Araújo, F.M.C., Melo,
M.E.L., Ramalho, I.L.C., Lemos, D.R.Q.,
Barroso, M.I.C., Vilar, D.C.L.F., Justino,
L.G., Sousa, A.S.S., Silva, L.A.B., Guedes,
M.I.F., Oliveira, D.M.
1. Rede Nordeste de Biotecnolgia
- UECE, RENORBIO-UECE, Av.
Paranjana, 1.700 - Campus do
Itaperi - 60740-000 Fortaleza/CE
CEP:60120-002
600 - Centro Universidade Federal
do Ceará - Medicina Cariri, UFCCariri, E-mail: carolbperdigao@
gmail.com
Influenza season in Ceará occurs in the
first semester of the year. It is known that
during influenza epidemics, pregnancy
constitute a high-risk group related to
morbidity, with an increased risk of
adverse outcomes (eg, spontaneous
abortion, preterm delivery). This study
aimed to describe the epidemiological
and clinical features of the pregnant
women with Influenza A/H1N1/09pdm
virus infection in Fortaleza, Ceará
from January to June 2012. Of 518
individuals with an influenza-like
illness that had nasopharyngeal
swab collected, 154 were pregnant
women. From those, 53(34.4%) had
laboratory confirmation for Influenza
A/H1N1/09pdm virus infection by
qRT-PCR, 15(28.3%) were outpatients
and 38(71.7%) were hospitalized.
Of the 53 patients, 05(9.4%) were
in the first, 20(37.7%) were in the
second, 25(47.2%) were in the third
trimester of pregnancy and 3(5.7%)
unknown. The age pregnant women
ranged from 14 to 37 years old, mean
24.8 years. The virus was detected
in 11 cities with predominance in
the capital (67.9%). Only 12(22.6%)
pregnant women had been previously
Human Virology: HV
205
vaccinated against influenza. The most
prevalent symptoms were fever and
cough, 41(77.3%), dyspnea, 38(71.1%)
and coryza, 34(64.2%). Seven patients
had some co-morbidity associated
such as asthma, smoking, lung disease,
hypertension and diabetes. One case,
a new sample collected 7 days after
treatment with oseltamivir continued
positive, what might be a resistant
strain. Four women were admitted to
intensive care unit, with one maternal
death and a stillbirth. The pregnant
woman who died presented with
respiratory insufficiency, kidney and
liver failure due to virus infection. While
they were hospitalized, five women
underwent preterm cesarian delivery.
Two children were born infected with
H1N1 virus, characterizing vertical
transmission. The results of this
study corroborate the findings in the
literature where complications such as
premature birth, stillbirth and death
in pregnant women were reported.
Financial support: FUNCAP, FUNASA
HV983 - HPV GENOTYPES DISTRIBUTION IN MEN WITH PENILE CANCER
FROM TWO DIFFERENT GEOGRAPHIC AREAS IN BRAZIL
Mota, M.T.O., Camilo, H.P., Bonilha, J.L.,
Rosa, B.M., Soares, F.A., Arruda, J.G.F.,
Fonseca, A.G., Rahal, P.
1. São Paulo State University,
IBILCE (UNESP), 2265, Cristóvão
Colombo street. São José do Rio
Preto, SP, Brazil. 15054-000
2. School of Medicine of São José do
Rio Preto, FAMERP, Brigadeiro
Faria Lima Avenue. São José do
Rio Preto, SP, Brazil. 15090-000
3. A.C. Camargo Hospital, , 109, Prof.
Antônio Prudente Street. São
206
Human Virology: HV
Paulo, SP, Brazil. 01509-010
4. State University of Campinas,
CIPED (UNICAMP), 126, Tessalia
Vieira de Camargo street.
Campinas, SP, Brazil. 13083-887
5. Evandro Chagas Institute, ECI
(SVS/MS), Km 7 of BR-316
highway. Ananindeua, PA, Brazil.
67030-000
6. Ophir Loyola Hospital, OLH, 992,
Magalhães Barata avenue. Belém
do Pará, PA, Brazil. 66060-281
Human
papillomavirus
(HPV)
infections are associated to different
genitourinary tumors. Until now about
120 HPV genotypes had been described
infecting the urogenital area, classified
as high or lower risk based on their
association with tumor development.
Squamous cell cancer of the penis
(SCCP) is a rare and understudied
tumor which, in the last years, had
been associated to HPV infections. It
were studied 99 SCCP samples from
patients of two different geographic
areas, whose population have distinct
socio-economic patterns. One group is
from São Paulo (SP group, 39 samples)
the richest state of the country and the
other group is from Pará (PA group,
60 samples), a state which has lower
socioeconomic indicators. The mean
age is 64.7 for SP group and 60.9 for
PA group. The prevalence of HPV in
single or mixed infections is higher
in PA group (81.67%) than in SP
group (64.10%), as well as the rate of
multiple infections, mainly with high
risk genotypes (43.3% and 23.1%
respectively). It was found a diverse
set of HPV genotypes in the PA group
(HPV6, 11, 16, 18, 33, 52, 53, 58 and 68)
while in SP only three genotypes were
found (HPV6, 11 and 16). The tumor
stage was also observed. Patients in
PA group show a more advanced stage.
While in SP group only 12.8% of the
tumors are in the stage IIIa, IIIb or
IV, in PA group 48.3% of the tumors
are in these stages, indicating a worst
prognostic. These data suggests that
much more HPV genotypes, mainly
high risk genotypes, are circulating in
Pará than in São Paulo and they may
contribute to the higher prevalence and
worst prognostic of SCCP in the Pará.
The differences in the HPV genotype
circulation between populations from
São Paulo and Pará prompts for more
studies about HPV circulation among
different populations. These data may
be useful in development of guidelines
for the implementation of more
efficient and less expensive vaccination
programs. Financial support: FAPESP.
HV994 - THE USE OF SALIVA
SAMPLES FOR DETECTION OF CYTOMEGALOVIRUS (CMV) DNA FOR
NEONATAL SCREENING OF CONGENITAL INFECTION
Santana, J.G., Oliveira, J.S., Souza,
G.O.S., Almeida, L.S., Jesus, B.L.S.,
Cardoso, E.S.C., Marin, L.J.
Universidade Estadual de Santa
Cruz, UESC, Campus Soane Nazaré
de Andrade, km 16 Rodovia IlhéusItabuna E-mail: jgs.biomed@yahoo.
com.br
The CMV congenital infection is a
important problem of public health
in the world. Identification of a
congenitally infected newborn infant
soon after birth before hospital
discharge is very helpful in planning to
appropriate early diagnosis, follow up
and intervention regard the potential
involvement of sensorial hearing
loss, motor abnormalities and other
problems. The aim of this study is
evaluated the usefulness of saliva
from newborns infants as a sample of
neonatal screening of congenital CMV
infection as compared with urine when
both are processed by PCR. Newborn
with any gestational age that was
possible to collect saliva and/or urine
sample within 3 weeks of life were
enrolled in a public maternity of Ilheus
city, Bahia, from July of 2011 until
now and was approved by the UESC
Human Research Ethics Committee
and written informed consent was
obtained from the mothers. Infants for
whom the presence of viral DNA was
confirmed by PCR in at least two saliva
or urine samples obtained before 3
weeks of birth were considered with
CMV congenital infection. Urine and
saliva samples were attempted to
be obtained from the 750 infants,
when the CMV DNA were detected
by PCR, the presence of the virus was
confirmed in follow up urine and saliva
samples. It was not possible to obtain a
urine sample from 489 of 750 (65,2%)
newborns because difficulties such
contamination with meconium, a delay
in establishing abundant diuresis,
urine leackage from the colleting
bags, perineal cutaneous irritation
and excessive infant handling. Among
750 infants successfully screened for
CMV, 10 (1,33%) were congenitally
infected, all asymptomatic until here.
Comparison of the proportions of CMV
excretion did not show a difference
when the PCR was applied only in
saliva samples, 6/489 (1,23%) and
in both samples, saliva and urine,
4/261 (1,53%). We conclude that in a
screening of CMV infected infants, the
easily obtained saliva samples are as
useful as urine to identify DNA of CMV
Human Virology: HV
207
by PCR.
HV1000 - ANALYSIS OF THE EPSTEIN-BARR VIRUS LATENT MEMBRANE
PROTEIN 1 (LMP1) COMPLETE
GENE SEQUENCE IN EBV+ HODGKIN
S LYMPHOMA AND DESCRIPTION
OF AFRICAN-RELATED NEW LMP1
VARIANTS CIRCULATING IN BRAZIL
Garcia, A.C., Guimarães, A.P., Guiretti,
D., Stefanoff, C.G., Hassan, R.,
Instituto Nacional de Câncer, INCA,
Praça Cruz Vermelha, 23 - Centro
- 20230-130 - Rio de Janeiro - RJ
Epstein-Barr virus (EBV) is a
transforming
herpesvirus.
LMP1
codifies the viral oncoprotein, being
a very variable gene with potential
selection of oncogenic variants.
Hodgkin's lymphoma (HL) is associated
with EBV in ~50% in Brazilian S.E
region, expressing LMP1 in all cases.
LMP1 molecular variability has not
been completely understood. We
aimed to perform an analysis of the
complete LMP1 sequence, including
coding and promoter regions from HLassociated virus and non neoplastic
controls (NNC). LMP1 gene (1624pb)
was analyzed from 27 EBV+ HL and 4
NNC. EBV was detected by EBER-ISH.
Sequences obtained in an ABI3130xl
sequencer were submitted to phylogeny
analysis with Mega4 software.
Reference sequences: B95.8, Med+/, China 1/2, CAO (Asian), Alaska, Raji
(African) and A, B, C and D (European)
variants. Phylogenetic reconstruction
of the promoter identified B95.8 and
D/CAO variants as distinct lineages.
Within B95.8 clade, 48% of sequences
clustered around B95.8 and 45%
around Raji (2 samples with outside
position). In the coding region, 93.5%
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Human Virology: HV
sequences clustered around B95.8,
including 3 Med+/-, 2 B-like, 3 B95.8/Alike, 6 Raji-like sequences and a distinct
branch containing 14 sequences more
similar to each other than to the others
in the clade. Two sequences clustered
together with Asian variants. The
14 unclassified sequences exhibited
consistent aminoacid replacement at
E2D, H3L, L25I, V43I, D46N, S57A, I63V,
I124V(unique), L126F, M129I, L151I,
I152L(u), G212S, H213N, E214Q. Most
sequences (12/14) exhibited Rajilike promoter. We have extended the
picture of LMP1 molecular variability,
describing for the first time the entire
LMP1 gene in Brazilian EBV isolates,
the circulation of African-related
variants outside Africa, and a potential
new LMP1 variant. The role of immune
escape in the shaping of LMP1
variability in Brazil, and the oncogenic
role of these variants deserve further
studies. Financial Support: INCT para
Controle do Câncer, INCA, CNPq/
CAPES.
HV1002 - CIRCULATION OF DENGUE
VIRUS SEROTYPES 1 AND 4 IN MATO
GROSSO, BRAZIL IN 2012
Zuchi, N., Heinen, L.B.S., Santos, M.A.M.,
Viniski, A.E., Kinoshita, S., Mussi, A.D.,
Dezengrini, R.
Grosso, UFMT, Av. Fernando
Correa da Costa, n.2367 Bairro
Boa Esperança Cuiabá, MT
2. MT-Laboratório, MT-Laboratório,
Rua Thogo da Silva Pereira,
n.63, Centro, Cuiabá, MT E-mail:
[email protected]
Dengue virus (DENV) is an arthropodborne Flavivirus transmitted mainly
by Aedes Aegypti. In the past decade,
approximately 70% of dengue cases
in the Americas occurred in Brazil.
The aim of this study was to verify
the serotype frequency of DENV in
the serum of 600 patients with acute
febrile illness in Mato Grosso (MT) by
virus isolation in C6/36 cells followed
by indirect immunofluorescence assay
using serotype-specific monoclonal
antibodies against dengue and yellow
fever viruses. Our preliminary results
have shown 125 positive samples:
30.3% (112/369) for DENV-4 in Cuiabá
(80/112), Várzea Grande (23/112),
Poconé (4/112), Acorizal (2/112),
Nossa Senhora do Livramento (2/112)
and Nobres (1/112), and 3.5%
(13/369) for DENV-1 in Sinop (6/13),
Sorriso (4/13), Pontes e Lacerda
(1/13), Tangará da Serra (1/13) and
Várzea Grande (1/13). Among positive
patients, 98.4% and 1.6% are residents
in urban and rural areas, respectively.
There was no gender predominance
(50.4% [63/125] female and 49.6%
[62/125] male) however age correlation
was observed, being dengue fever
more frequent between13-25 years (y)
(0-12y 12% [15/125]; 13-25y 41.6%
[52/125]; 26-40y, 21.6% [27/125];
41-60y, 20.8% [26/125] and >61, 4%
[5/125]). Clinical manifestation of
dengue included hyperthermia 74.4%
(93/125), myalgia 66.4% (83/125),
headache 61.6% (77/125), retroorbital pain 37.6% (47/125) and
petechiae 24% (30/125). Previous
history of similar disease was present
in 17.6% (22/125). According to
occupation, 33.7% (30/89) were
children/students, 30.8% (24/78)
general services workers, 60% (6/10)
college level professionals, 33.3%
(4/12) health care professionals, 35.7%
(5/14) commerce employee, 40%
(4/10) retired, 28.5% (4/14) public
employee, 18.1% (2/11) housewives,
33.3% (2/6) informal workers and
60% (3/5) unemployed. DENV-4 was
identified in MT for the first time in
2012, but the origin of the isolates
is unknown. As 66.1% (244/369) of
samples from clinically characteristic
acute febrile patients were negative for
DENV, further studies are necessary
to identify other arboviruses possibly
circulating in Mato Grosso. * Project
supported by Conselho Nacional
Tecnológico - CNPq.
HV1003 - THE INFLUENCE OF HLA
CLASS I PROFILE ON AIDS PROGRESSION OF HIV-INFECTED PATIENTS
FROM THE SOUTHERNMOST STATE
OF BRAZIL
Matte, M.C.C., Medeiros, R.M., Santos,
B.R., Simon, D., Jobim, M., Chies, J.A.B.,
Almeida, S.E.M.
e Pesquisa em Saúde, FEPPS, Av.
Ipiranga, 5400
Grande do Sul, UFRGS, Av. Bento
Gonçalves, 9500
3. Hospital
Nossa
Senhora
Conceição, GHC, Av. Francisco
Trein, 596
4. Universidade Luterana do Brasil,
ULBRA, Avenida Farroupilha,
8001
5. Hospital de Clínicas de Porto
Alegre, HCPA, Rua Ramiro
Barcelos, 2350
6. Universidade Feevale, , RS-239,
2755 E-mail: mcristina.matte@
gmail.com
Human Virology: HV
209
Variations in immune response genes
have been investigated to explain the
heterogeneity observed in the clinical
course of HIV-1 infection. HLA class I
molecules, such as HLA-A and HLA-B,
and non-classical HLA class I genes,
as HLA- G seems to play an important
role on the modulation of HIV disease
progression. The main objective of this
study was to investigate the influence
of HLA-B alleles, HLA-A*03, HLA-A*11
and 14bp insertion/deletion and +342
G/C HLA-G polymorphisms on aids
progression of HIV-infected patients
from southernmost state of Brazil.
From 3,300 medical records of HIV+
patients reviewed retrospectively
in Infectology Service of Nossa
Senhora Conceição Hospital between
January and July of 2011, 98 patients
with well defined criteria of clinical
progression to AIDS were included
in the study (21 rapid progressors,
29 long-term nonprogressors and 48
cronic progressors). HLA-B alleles,
HLA-A*03 and HLA*11, and HLA-G
polymorpphism were identified using
molecular techniques. To evaluate the
influence of genotypes and alleles in
the AIDS progression statistical tests
as Kaplan-Meier curves and log-rank
test and multivariate Cox regression
analyses were performed. Despite the
admixture observed in Porto Alegre
population, a higher frequency of
African-derived individuals were found
in LTNP group (p=0.010). Our results
support the role of HLA-B homozygote
genotype on rapid aids progression
(p=0.006). Also, we suggest an influence
of HLA-G on aids progression, since a
lower median time to aids was observed
for the alleles associated with a higher
expression of the molecule (C and del
alleles). None of the classical genetic
factors described in the literature
210
Human Virology: HV
as markers for rapid (HLA-B*53
e HLA-B*35) or slow (HLA-B*57,
HLA-B*27) progression to aids showed
an independent influence on this group.
The identification of genetic markers
in different populations with different
ethnical origin could contribute for
a better comprehension of the HIV-1
pathogenesis.
HV1006 - MAYARO, DENGUE 1 AND 4
IN PATIENTS WITH ACUTE FEBRILE
ILLNESS IN MATO GROSSO, BRAZIL
Zuchi, N., Santos, M.A.M., Mondini, A.,
Nogueira, M.L., Dezengrini, R.
Correa da Costa, n.2367 Bairro
Boa Esperança Cuiabá, MT
Brigadeiro Faria Lima, 5416, Vila
São Pedro São José do Rio Preto,
SP
3. Universidade Estadual Paulista
Julio de Mesquita Filho, UNESPAraraquara, Rod. AraraquaraJaú Km 1, Bairro Machados,
Araraquara, SP
4. MT-Laboratório, MT-Laboratório,
Rua Thogo da Silva Pereira,
n.63, Centro, Cuiabá, MT E-mail:
[email protected]
Arthropod-borne viruses are frequent in
tropical areas, representing a significant
problem for human health. Mayaro
(MAYV) is an Alphavirus belonging
to Togaviridae family, transmitted by
Haemagogus janthinomys and Aedes
aegypti mosquitoes. MAYV is enzootic
in tropical South America, where it is
maintained in sylvatic cycles involving
wild primates and birds, and it is
endemic in the Amazon region, being
reported during human outbreaks in
Tocantins, Goiás, Mato Grosso do Sul and
Pará. Patients with acute febrile illness
usually are diagnosed clinically with
dengue in Mato Grosso. We collected
600 serum samples from patients
with acute febrile illness suspected of
having dengue infection between the
period of January and July 2012. A total
of 35 samples were tested by multiplex
RT-PCR with genera-specific primers
(Alphavirus, 433 bp; Flavivirus, 988
bp) followed by multiplex semi-nested
RT-PCR for dengue virus (DENV)
serotypes (DENV-1, 472 bp; DENV-2,
316 bp; DENV-3, 659 bp and DENV4, 222 bp), other Flavivirus (YFV, 253
bp; WNV, 195 bp and SLEV 232 bp)
and Alphavirus (MAYV, 270 bp; AURAV
98 bp; EEEV, 124 bp; WEEV, 208 bp
and VEEV, 400 bp). Amplicons were
submitted to sequencing to confirm
virus specificity. We found 3/35
positive samples for Mayaro virus in
Várzea Grande and Nossa Senhora
do Livramento; 3/35 for DENV-1 in
Cuiabá and Poconé and, 24/35 for
DENV-4 in Cuiabá, Várzea Grande,
Pontes e Lacerda, Cáceres and rural
area of Poconé. MAYV generally causes
a mild febrile illness clinically mistaken
with dengue fever, with a short viremic
period and recovery in 3 to 5 days.
These aspects, combined with absence
of routine laboratorial diagnose in Mato
Grosso state may contribute to the lack
of detection of MAYV circulation. This
is the first description of this agent in
neighboring cities of Cuiabá. Further
studies are necessary to understand
the magnitude of Mayaro infection in
Mato Grosso and the possibility of cocirculation with other arboviruses.
Project suported by Conselho Nacional
Tecnológico - CNPq.
HV1007 - VIROLOGICAL FEATURES
OF HEPATITIS DELTA AND B VIRUSES
AMONG PATIENTS WITH CHRONIC
INFECTION FROM RONDÔNIA STATE
Vieira, D.S., Gouvea, G., Ferreira, A.C.,
Botelho, L.F., Santos, A.O., Pinho, J.R.,
Salcedo, J.M.
1. FUNDAÇÃO OSWALDO CRUZ
RONDONIA, FIOCRUZ RO, RUA DA
BEIRA, 7671, LAGOA
2. CENTRO DE PESQUISA EM
MEDICINA TROPICAL DE RO,
CEPEM, AV. GUAPORE, 215, LAGOA
3. LABORATÓRIO
DE
GASTROENTEROLOGIA
E
HEPATOLOGIA,
USP,
E-mail:
[email protected]
BACKGROUND AND AIMS – In the
Brazilian Amazon delta hepatitis is a
serious public health problem with
prevalence exceeding 85% in patients
with chronic hepatitis. The state of
Rondônia in the western Brazilian
Amazon is considered highly endemic
for hepatitis B and Delta, but little is
know about genetic diversity of B and
D viruses circulating in this region.
The aim of this study was characterize
HDV and HBV genotypes in HBV/
HDV chronic hepatitis cases from
Rondonia state. METHODS – Serum
samples isolated from seventeen antiHDV positive patients were included
in this study. HBV genotypes were
characterized by phylogenetic analysis
of a 1306 bp fragment partially
comprising surface and polymerase
genes and HDV genotypes by analysis
of a 403 bp fragment comprising part of
the L-HDAg. RESULTS – HDV RNA was
detected in all samples and genotype
Human Virology: HV
211
3 was identified among then. Fourteen
also showed HBV DNA detectable and
HBV genotypes were characterized
in 13; among these samples HBV
subgenotypes A1 (38%), D2 (8%),
D3 (46%) and F2a (8%) were found.
.CONCLUSION – This study confirm that
HDV genotype 3 is the most prevalent
genotype in Amazon region and it is not
associated to a specific HBV genotype.
The genotypic characterization of
HBV and HDV shown in this study
is important for understanding the
evolution of chronic patients in the
region favoring the elucidation related
to disease progression. Financial
support: FIOCRUZ RO, SUS.
HV1009 - INVESTIGATION OF NOROVIRUSES IN FECAL SAMPLES
RECOVERED OF CHILDREN WITH
ACUTE GASTROENTERITIS FROM
MANAUS, AMAZONAS IN 2010
Costa, S.T.P., Fumian, T.M., Lima,
I.C.G., Lucena, M.S.S., Reymão, T.K.A.,
Silva, L.D., Soares, L.S., Mascarenhas,
J.D.P., Linhares, A.C., Gabbay, Y.B.
1. UNIVERSIDADE DO ESTADO DO
PARÁ, UEPA, Trav. Perebebuí,
2623 - Marco - 66087-670 - Belém
/ Pará / Brasil
2. INSTITUTO EVANDRO CHAGAS,
s/n - Levilândia - 67030-000
- Ananindeua / Pará / Brasil
E-mail:
costasamya@hotmail.
com
Acute gastroenteritis (AGE) is a
worldwide public health problem.
Nowadays, the noroviruses (NoVs)
are recognized as the major cause
of outbreaks of nonbacterial AGE in
humans, commonly reported in closed
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communities such as hospitals, schools,
and cruise ships. The genome of NoVs
is composed of a non-enveloped capsid
with a single-stranded, positive-sense
RNA that encoding three open reading
frames (ORF-1, -2 and -3). The goal of
this study was to investigate the role of
NoV in cases of AGE and determine the
circulating genotypes in children under
10 years old, from Manaus in the year of
2010. Two methods were used for NoVs
detection: the enzyme immunoassay
(EIA) and the reverse transcriptasepolymerase chain reaction (RT-PCR)
using primers specific for B region
(polymerase). Positive samples by RTPCR were sequenced using primers
that target a partial region (D) of the
genome that encodes the main capsid
protein. Amplicons obtained by RTPCR were purified and sequenced
using the Big Dye Terminator Reaction
Kit® (v. 3.1) and the ABI Prism 3130xl
DNA sequence. Sequences were edited
and aligned using the Bioedit and the
phylogenetic analysis was performed
using MEGA version 5.05. A total of 171
stool samples were sent by the LACEN of
Manaus to the Evandro Chagas Institute
in 2010. NoVs was detected in 39.2%
of the samples being 34.5% by EIA and
28.6% by RT-PCR. Of the 12 positive
strains sequenced, GII.4 variant 2010
was the most prevalent, found in 91.7%
(11/12) of the cases and GII.7 (8.3%) in
one sample. These results corroborate
with other obtained in different places,
like in Rio de Janeiro where NoVs was
detected in 35.1% of the cases and
the most prevalent genotype was the
GII.4 variant 2010. Thus, detection
methods associated with molecular
characterization important tools to
contribute with the establishment of
a continuous genotype surveillance
of NoVs in Brazil, which may help
in the future in the formulation of a
possible vaccine. Financial Support:
Instituto Evandro Chagas, Secretaria
de Vigilância em Saúde, Ministério da
Saúde.
HV1022 - ANALYSIS OF ANTIVIRAL
ACTIVITY OF THE HEMOLYMPH
FROM LEPIDOPTERA (MEGALOPYGIDAE)
Carvalho, N.D., Giovani, D.N.S., Moraes,
R.H.P., Mendonça, R.M.Z., Mendonça,
R.Z.
Instituto Butantan, IBU, Av Vital
Brasil, 1500. São Paulo/SP. Brasil
Several studies have shown the
presence of active properties in the
hemolymph of arthropods, some
of which are of interest for the
development of new pharmacological
drugs. However, relatively little data
are available on molecules from insects
with antiviral activities. In this study, the
effects of supplementation of infected
culture with hemolymph from larvae
of Lepidoptera were investigated.
Citotoxicity and genotoxicity were
evaluated and no adverse effects were
observed in culture after hemolymph
addition (up to 5%). The effect of
hemolymph on virus growth was
measured on confluent monolayers
of infected cells with measles virus,
influenza virus (H1N1) (enveloped
virus) and picornavirus (non enveloped
virus). The cultures were observed
daily for evidence of cytopathic
effect. The analyses of the viral titer
demonstrated that the addition of 1%
of hemolymph decreased significantly
(p=0.002) the virus titer. The antiviral
protein responsible for this activity
was isolated and purified by gel
filtration chromatography using a gel
filtration column system (Superdex
75) and further fractionated using
a Resource-Q ion exchange column
system. Experiments with the purified
protein led to a 32-fold reduction
in influenza virus production, 64fold reduction in measles virus
production and a 256-fold reduction in
picornavirus production. Heating and
freezing seem to have no influence over
its antiviral activity. Also, the protein
does not display virucidal activity
and does not act on receptors on the
cell membrane. The observations
suggest an intracellular mechanism
of action and that the protein may act
as a constitutive agent that affects the
innate antiviral immune response.
Financial support: FAPESP 10/524346
HV1023 - RECOMBINANT ANTIGEN-BASED ELISA FOR DETECTION OF
ANTI-DENGUE IGM FROM BLOOD
SAMPLES COLLECTED IN FILTER-PAPER
Cursino, A.E., Andrade, K.R., Rocha,
E.S.O., Vilela, A.P.P., Figueiredo, L.B.,
Oliveira, J.G., Marinho, P.E.S., Bonjardim,
C.A., Ferreira, P.C.P., Kroon, E.G.
1. Fundação Oswaldo Cruz, CPqRR/
Fiocruz, Avenida Augusto de
Lima, 1715, Barro Preto, BH,
Minas Gerais
Gerais, UFMG, Avenida Antônio
Horizonte, Minas Gerais E-mail:
[email protected]
Dengue fever is an infection carried by
mosquitoes and caused by any of four
related Dengue virus (DENV) serotypes
belonging to the family Flaviviridae.
The clinical manifestations range from
Human Virology: HV
213
asymptomatic to severe characterized
by hemorrhagic fever and shock
syndrome. The indirect enzyme
is one of the most used serological
tests for dengue diagnosis whereby
immunoglobulin M for acute phase
or immunoglobulin G for phase
convalescent. The objective of this
study was to detect IgM anti-dengue
in blood samples collected on filterpaper obtained from peripheral blood
using an ELISA based on recombinant
antigens and compare its results to
a commercial kit. For this study 48
blood samples were collected from
suspected dengue infected patients
from Caratinga (MG), between 6º and
87º days after the onset of symptoms in
the period from November 2010 to July
2011. Samples of whole blood collected
on filter-paper were submitted to a
recombinant protein IgM-ELISA and
serum samples were tested with a
commercial Kit (IgM ELISA anti-dengue
Human do Brasil). The ELISA plates
were coated with DENV recombinant
proteins, and blood samples collected
on paper-filter were diluted in PBS-T
(Phosphate buffered saline + 0,05% of
Tween 20) and BSA 0,1%. Reading of the
ELISA was made at 450nm. Forty-three
(89.6%) samples were concordant
in both methods, 7 positive and 36
negative. Only 5 (10.4%) samples
were discordant, IgM-ELISA sensitivity
was 87.5% and specificity was 92.5%.
Therefore the ELISA performed with
samples collected on paper-filter has
a good correspondence between the
results from serum samples. Thus, the
ELISA based on dengue recombinant
proteins showed be efficient for
dengue diagnosis. Financial support:
CNPq, CAPES, DECIT/MS, FAPEMIG e
PRONEX-Dengue and INCT-Dengue.
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Human Virology: HV
HV1026 - VALIDATION OF REAL
TIME PCR FOR HTLV-1 PROVIRAL
LOAD IN PERIPHERAL BLOOD MONONUCLEAR CELLS
Rosadas, C., Cabral-Castro, M.J., Peralta,
J.M., Puccioni-Sohler, M.
de Janeiro, UFRJ, Rua Professor
Rodolpho Paulo Rocco 255, 3 andar,
Serviço de Patologia Clínica E-mail:
[email protected]
HTLV-1 infection remains asymptomatic
in the majority of infected individuals.
However it may cause a neurological
chronic disorder called as HTLV-1associated myelopathy (HAM/TSP).
The laboratory diagnosis is based
on immunoassays such as ELISA and
Western blot. These techniques have
some disadvantages in indeterminate
results, immunosuppressed patients
and in neonatal infections. In such
cases, the molecular biology techniques
appear as an alternative. The real-time
quantitative PCR (qPCR) quantify the
proviral load, which may help to assess
disease progression. Although qPCR
has a high sensitivity and specificity
it is still an in house technique. Thus
prior validation is essential before the
implementation of this technique on
laboratorial routine. The aim of the
study is to validate a TaqMan qPCR
assay for HTLV-1 proviral load detection
in peripheral blood mononuclear cells
based on a conserved region of tax
gene. DNA of TARL-2 cells were used to
prepare the standard curve. The range
of the TARL-2 standard was 5x104 to
5 copies of virus/rxn. The samples
were evaluated in triplicate on two
consecutive days. To evaluate the limit
of detection a sample containing only
one copy of the target gene was added
and analyzed in triplicate. Nineteen
patient samples reactive for HTLV-1 by
ELISA and one negative sample were
included in the study. To evaluate the
intra- and inter-assay variation one
sample was tested 20 times in two
consecutive days. All positive samples
presented gene amplification. The
negative sample did not presented
amplification of tax gene, but presented
amplification of the reference gene .The
limit of detection was 1 copy/rxn. The
qPCR efficiency, slope and correlation
coefficients (r2) were all acceptable
(presenting at least 98,58%, -3,298 and
0,993, respectively). The assay gave
coefficient of variation for the Ct values
of less than 1,53% and 1,93% for intra
and inter assay, respectively. This assay
is able to reliably quantify proviral
load. Financial support: FAPERJ
HV1030 - OVEREXPRESSION OF
ANXA1 IN PENILE CARCINOMAS
POSITIVE FOR HIGH RISK HPVS
Calmon, M.F., Mota, M.T.O., Candido,
N.M., Girol, A.P., Mendiburu, C.F.,
Thomé, J.A., Rosa, B.M., Soares, F.A.,
Oliani, S., Villa, L.L., Vassallo, J., Rahal, P.
1. Instituto de Biociências, Letras e
Ciências Exatas/UNESP, IBILCE/
UNESP, Rua Cristovão Colombo,
2265- São José do Rio Preto, São
Paulo
2. Laboratório
Inst.
Anatomia
Patológica e Citopatologia IAPC,
Brigadeiro Faria Lima, 5416. Vila
São Pedro. CEP: 15090-000. S. J.
do Rio Pr
3. Hospital A.C. Camargo., , Rua
Professor Antônio Prudente, 211.
CEP: 01509-010. São Paulo-SP.
4. Departamento de Radiologia e
Oncologia Básica/USP, USP, Av. Dr.
Arnaldo, 255. CEP: 01246-903.
São Paulo-SP. E-mail: macal131@
gmail.com
The incidence of penile cancer varies
between populations but is rare in
developed nations. Penile cancer is
associated with a number of established
risk factors and associated diseases
including phimosis with chronic
inflammation, human papillomavirus
(HPV) infection, poor hygiene and
smoking. In penile carcinomas the
most common HPV types are HPV 16
and HPV 18 being that HPV 16 is most
prevalent in North America, Europe,
South America and India. HPV 18 is
the second most common HPV type
identified in squamous cell carcinoma
samples, but it has low prevalence.
HPV contributes to tumorigenesis
predominantly through the action of
viral oncoproteins E6 and E7, which
regulate the expression of known genes
like p53 and pRb. The objective of this
study was to identify genes related to
penile carcinoma. The detection of HPV
infection was analyzed in 47 penile
squamous cell carcinoma samples.
HPV DNA was detected in 48.9% of
penile squamous cell carcinoma cases.
High-risk HPV infections were present
in 42.5% of cases and low-risk HPV
infections were detected in 6.4% of
penile squamous cell carcinomas. The
RaSH approach identified differential
expression of Annexin A1 (ANXA1),
p16, RPL6, PBEF1 and KIAA1033
in high-risk HPV positive penile
carcinoma; ANXA1 and p16 were
overexpressed in tumoral cells. ANXA1
and p16 proteins were significantly
more expressed in the cells from HPVpositive penile carcinoma as compared
to HPV-negative tumors (p<0.001). We
Human Virology: HV
215
suggested the p16 could be a marker
for penile carcinoma, confirming
the diagnosis of malignant penile
lesions infected with high risk HPVs.
Overexpression of ANXA1, which has
anti-inflammatory, antipyretic and antihyperalgesic activities and is associated
with various physiological processes
including cellular differentiation, cell
proliferation and signal transduction,
was demonstrated in penile squamous
cell carcinoma samples and its protein
expression is strongly associated with
high risk HPV infection. Financial
support: FAPESP
HV1031 - DETECTION AND GENOTYPING OF SAPOVIRUS IN FECAL
SPECIMENS OF CHILDREN WITH
MANAUS - AM, BRAZIL
Reymão, T.K.A., Costa, S.T.P., Lucena,
M.S.S., Silva, L.D., Hernandez, J.M.,
Siqueira, J.A.M., Portal, T.M., Soares,
L.S., Fumian, T.M., Oliveira, D.S.,
Mascarenhas, J.D.P., Linhares, A.C.,
Gabbay, Y.B.
INSTITUTO EVANDRO CHAGAS,
IEC, Rodovia Br 316 Km 07
Levilândia, Ananideua-PA E-mail:
[email protected]
Sapovirus (SaV) is an important
pathogen of acute gastroenteritis
(AGE) in humans. As member of
the Caliciviridae family, this nonenveloped virus has a single-stranded
positive-sense RNA genome of
approximately 7.5 kb. SaV strains can
be divided into five genogroups (G)
of which GI, GII, GIV, and GV infect
humans, and can be further divided
into many genotypes. In general, this
pathogen is associated with sporadic
cases of AGE in young children and
elderly people. It is also related with
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outbreaks in day care centers, nursing
homes and hospitals. Symptoms
include diarrhea with watery stools,
vomiting, and fever. The transmission
occurs by fecal-oral route, by aerosol
and by consumption of contaminated
food or water. Fecal samples collected
from January/2010 to May/2011,
of children with AGE, previously
negative for rotavirus and norovirus,
were tested for the presence of SaV.
It was used Reverse TranscriptionPolymerase Chain Reaction (RT-PCR),
with the primers 289/290, specific
for human calicivirus. The products
obtained were visualized in an agarose
gel and samples that showed specific
amplicons of 331 bp were considered
positives. The sequencing of positive
samples was performed using primers
289/290 (polymerase region) and
SLV5317/5749 (capsid region) with
Big Dye Terminator Kit and the results
compared with sequences registered in
the GenBank. SaV was detected in 6/131
samples tested (4.5%). Two samples
were sequenced and classified as GI.2
and GI.1. The positivity rate detected
in this study (4.5%) was similar to the
ones obtained in researches conducted
in Belém-PA (4.9%) and Australia
(4.1%), in the years of 2010 and 2006,
respectively, however lower than the
registered in India (10.2%) in 2008.
This is the first report concerning
the detection and characterization of
SaV in Manaus-AM. Considering few
studies conducted in Brazil relating to
SaV, further researches are required
to elucidate the real epidemiological
importance of this pathogen.
HV1033 - SCREENING OF ANTIVIRAL
ACTIVITY OF ORGANISMS FROM THE
MARINE ENVIRONMENT AGAINST
THE BOVINE VIRAL DIARRHEA
VIRUS, SURROGATE MODEL FOR
THE HEPATITIS C VIRUS
Bastos, J.C.S., Kohn, L.K., Padilla, M.A.,
Berlinck, R.G.S., Fantinatti-Garboggini,
F., Arns, C.W.
1. Universidade de São Paulo, USP São Carlos, Avenida Trabalhador
são-carlense, nº 400, Bairro
Centro. CEP 13566-590 - São
Carlos
2. Universidade
Estadual
de
Campinas, Unicamp, Cidade
Universitária "Zeferino Vaz"
Distrito de Barão Geraldo 13083970 Campinas E-mail: jusantiago_
[email protected]
The hepatitis C virus (HCV) causes
chronic hepatitis, which can progress
to liver cirrhosis and hepatocellular
carcinoma. There is no vaccine available
and treatment has limited effectiveness.
The marine environment is a promising
source for new antiviral drugs, because
of its wealth and diversity, it has been
little explored. This study evaluated the
antiviral activity of 400 extracts from
invertebrates and microorganisms
from the marine environment against
bovine viral diarrhea virus (BVDV), a
surrogate model for HCV. For this, we
performed the evaluation of antiviral
activity of each extract (50 µg/mL),
through its ability to inhibit citopathic
effect against to BVDV virus (100
TCID50/50 µL) after 72 hours of
incubation. The results were obtained
through the observation of citopathic
effect and the inhibition percentage
was calculated through the MTT assay.
The extracts that showed protection
percentage greater than or equal to
80% were considered potentially
active, and selected for further tests.
400 extracts were tested, and the results
showed that 5 % were active against
BVDV. For the extracts obtained from
fungi, 4.2 % were active, from bacterial
6,9% were active , from ascidian 0
% were active, and from sponge 4.8
%.were active. The active extracts will
be evaluated on which phase of the
viral replicative cycle they operate. For
this, different tests will be performed.
In conclusion, the organisms of the
marine environment represents a
source of compounds with potential
antiviral activity and, against the BVDV,
the extracts obtained from bacterial
showed the best results, generating
the greatest amount of active extracts.
Financial support: Capes
HV1035 - AN OVERVIEW OF DENGUE
FATAL CASES DURING 25 YEARS BY
A REGIONAL REFERENCE LABORATORY
de Santis, B., dos Santos, F.B., de Filippis,
A.M.B., Lima, M.R.Q., Faria, N.R.C.,
Simões, J.B.S., Sampaio, S.A., Nunes,
P.C.G., Heringer, M., Nogueira, R.M.R.
Instituto Oswaldo Cruz, IOC, Av
Brasil, 4365 E-mail: bianca_santis@
yahoo.com.br
In Brazil, dengue became a public
health problem after the DENV-1
introduction in 1986, by the State of Rio
de Janeiro. Here, we aimed to perform
a retrospective analysis on dengue fatal
cases occurred in the past 25 years,
received in the Flavivirus Laboratory,
IOC/FIOCRUZ, Regional Reference
Laboratory for the Brazilian Ministry
of Health. Cases records were analyzed
and all data, such as patients´ personal
information, signs and symptoms,
sample collection were input on the
Laboratory´s Database. Cases were
submitted to the different techniques
according to its availability over at that
Human Virology: HV
217
time. From April 1986 to December
2010, a total of 688 suspected dengue
fatal cases were analyzed and 27.9%
(192/688) was confirmed. Cases were
received from 14 Brazilian States,
however 76.9% were from Rio de
Janeiro. Cases were more frequently
received and confirmed from February
to May. No differences were observed
on cases confirmed on males (51.6%)
or females (48.4%), however children
≤15 years old were the most affected
(29.1%, 49/168 of the fatal cases
confirmed). Fifty percent of the deaths
occurred on defervescence. Commonly
observed signs and symptoms in the
confirmed cases included fever 87.2%,
myalgia (52.4%), headache (47.5%),
vomiting (46.3%) and prostration
(43.9%). Shock was observed in
30.5% of the cases, non-characterized
bleeding in 29.3%, low platelet count
in 23.8%, hypotension in 20.1% and
abdominal pain in 16.5%. DENV-2 was
identified in 60.2% of the cases. On 2002
and 2009, most of the fatal outcomes
were due to primary infections (71%
and 57%, respectively), however in
2007 and 2008, fatal cases were due to
secondary infections (61% and 69%,
respectively). The data obtained is this
analysis may help the elucidate the role
of dengue on fatal outcomes during
distinct epidemiological scenarios,
where different serotypes were
introduced, emerged and re-emerged
in an endemic country such as Brazil.
Financial support: FAPERJ, CNPq,
CAPES and FIOCRUZ
HV1038 - PREVALENCE AND QUANTIFICATION OF THE HEPATITIS C
VIRUS (HCV) IN PATIENTS FROM
BLUMENAU-SC
Herkenhoff, M.E., Ferreira, P.S.,
Rodakiewicz, S.M., Gaulke, R., Branco,
218
Human Virology: HV
L.C., Pitlovanciv, A.K., Remualdo, V.R.
1. Universidade do Estado de Santa
Catarina, CAV - UDESC, Av Luiz de
Camões, 2090 - Conta Dinheiro,
Lages-SC. CEP: 88.520-000,
2. Genolab, Genolab, Rua Floriano
peixoto, 425 - Centro-BlumenauSC. CEP: 89010-500 E-mail:
[email protected]
Hepatitis C is an infectious disease
affecting primarily the liver, caused
by the hepatitis C virus (HCV). The
virus is a small, enveloped, singlestranded, positive-sense RNA virus.
It is a member of the hepacivirus
genus in the family Flaviviridae. The
infection is often asymptomatic, but
chronic infection can lead to scarring
of the liver and ultimately to cirrhosis,
which is generally apparent after many
years. HCV is spread primarily by
blood-to-blood contact associated with
intravenous drug use, poorly sterilized
medical equipment and transfusions.
An estimated 130–170 million people
worldwide are infected with hepatitis C.
Many infected people suffered chronic
hepatitis from that time onwards and
urgently needed treatment and care.
The virus is transmitted by exposure
to infectious blood or body fluids such
as semen and vaginal fluids, while viral
DNA has been detected in the saliva,
tears, and urine of chronic carriers.
Besides genotype, quantitative analysis
of HCV infection is extensively used
for monitoring disease progression
and treatment. Real-time PCR has
engendered wide acceptance for
quantification of hepatitis B virus (HCV)
DNA in the blood due to its improved
rapidity, sensitivity, reproducibility,
and reduced contamination. The
aim of this study was to determine
the HCV prevalence in a population
in Blumenau, Santa Catarina state,
southern Brazil, in January/2010 to
July/2010. We obtained a sample of
172 individuals, which was used the
blood serum to estimate the prevalence
and the number of viral copies. We
have used the Real-time PCR method
to indentify the virus and estimate the
number of viral copies in each positive
individual. After that, we have made
a statistical analysis using the Z-test
with 5% of significance. This study
has showed a prevalence of 54,65%
on the total sample, 51% on the male
population and 59,72% on the female
population. The number of viral copies
was showed an average of 8270676,824
copies/ml in the male population and
7520585,837 copies/ml in the female
population, there is no statically
difference in those two populations,
male and female, determined by
the Z-test (5% of significance). In
conclusion, our study have showed a
significant prevalence by this virus in
the population that was studied, and
that are not a difference on the number
of copies between the male and the
female population. Financial Support:
Genolab
HV1039 - LABORATORY INVESTIGATION FOR FLAVIVIRUSES IN
PATIENTS WITH FEBRILE ACUTE
SYNDROME
Martins, L.C., Oliveira, C.F., Chiang, J.O.,
Rodrigues, S.G., Ferreira, M.S., Carvalho,
V.L., Vasconcelos, P.F.C.
Instituto
Evandro
Chagas,
IEC, BR 316, Km 7 s/n Bairro
Levilândia- Ananindeua-PA E-mail:
[email protected]
Introduction: In the family Flaviviridae,
only the Flavivirus genus has significance
in arbovirology, where more than 50%
the viruses of this genus have been
associated with disease in humans. In
infection by arboviruses the fever is
the principle clinical manifestations
observed. Objective: Laboratorial
investigation for arboviruses of the
genus Flavivirus in patients with acute
febrile syndrome, residents in the
municipality of Parauapebas, state of
Pará, from August 2010 to March 2011.
Material and methods: Were obtained
159 sera samples during this period.
For the detection of total antibodies
and IgM antibodies was used the
Hemagglutination Inhibition (IH) test
and immunoenzymatic assay (ELISA)
respectively for 8 Flaviviruses (Yellow
Fever virus - YFV, Dengue virus - DENV
(serotypes 1, 2, 3, 4), Saint Louis
encephalitis virus - SLEV, Rocio virus
- ROCV and Ilheus virus - ILHV); 100
blood samples of patients with up five
days after the onset of clinical symptoms
were inoculated for attempts of viral
isolation in cell line clone C6/36 (Aedes
albopictus). Results: Of the 100 blood
samples tested were isolated 12 (12%)
of DENV-1, one (1%) of the DENV-2 and
87 (87%) were negative. Of the 159
sera tested by HI, 45 samples (28.3%)
were negative, and 114 (71.7%) were
positive for the viruses of the genus
Flavivirus. The 114 positive samples by
HI were tested by ELISA for DENV and
YFV, with 82 (72%) positive samples for
DENV and 32 negative. The 32 negative
samples were tested by ELISA for other
flaviviruses, where only one (3.1%) was
positive for SLEV. Conclusion: A high
prevalence of antibodies to flaviviruses
were detected in febrile patients living
in the municipality of Parauapebas-PA,
however most of the infections were
caused by DENV and also the possibly
circulation of the SLEV. Financial
supports: CNPQ/IEC/VALE
Human Virology: HV
219
HV1040 - IDENTIFICATION OF
CALOMYS FECUNDUS AS THE TRULY
RODENT RESERVOIR OF LAGUNA
NEGRA HANTAVIRUS IN NORTH
WESTERN ARGENTINA
Pini, N., Garcia, J., Sen, C., Calderón, G.,
González Ittig, R., Gardenal, N., Jayat, P.,
Ripoll, C., Levis, S.
1. INEVH “Dr. Julio I. Maiztegui", ,
Monteagudo 2510, Pergamino,
Argentina
2. Cátedra
de
Genética
de
Poblaciones y Evolución, UNC,
Córdoba, Argentina
3. Laboratorio de Investigaciones
Ecológicas de las Yungas,,
LIEY, San Miguel de Tucumán,
Argentina
4. Dirección
Provincial
de
Programas Sanitarios, , San
Salvador de Jujuy E-mail:
[email protected]
Laguna Negra virus (LNV) was
first identified as etiologic agent of
Hantavirus Pulmonary Syndrome
(HPS) in Paraguay in 1997, and was
isolated from Calomys laucha rodents.
In 2000, LNV was found in Argentina
and Bolivia from human and rodent
(Calomys callosus) samples and in
2012 in Brazil (human and Calomys
callidus samples). LNV sequences
obtained from HPS cases and rodent
samples from North Western Argentina
(Jujuy province) were compared with
LNV sequences from other regions;
we also performed the molecular
identification of rodent reservoirs
trapped in the same area in 2000 and
2011. Human samples with a positive
IgM serology and rodent samples with
positive IgG by ELISA using Maciel
virus antigen were analysed to detect
220
Human Virology: HV
viral RNA. Mitochondrial DNA was
used to confirm morphological field
identification of rodents captured in
2011 and 2000 (previously identified
as C. callosus); all positive rodents
corresponded to C. fecundus. Viral RNA
was amplified by nested RT-PCR with
specific primers for S (447 bp) and
M (513 bp) segments. Phylogenetic
analysis was carried out by Neighbor
Joining method and bootstrap support
calculated, as implemented in MEGA
5.0. For the S fragment, all argentine
strains showed differences < 1%
(nucleotides) and were identical in
aminoacids. Jujuy strains showed less
than 4% difference when compared
with reference strain (Paraguay) and
Brazil strain (Bolivia strains were not
available at GenBank). At present, LNV
was identified in four different reservoir
species of the Calomys complex: C.
laucha (Paraguay), C. callosus (Bolivia),
C. callidus (Brazil) and C. fecundus
(Argentina); only molecular DNA
mitochondrial analysis could determine
the proper identification of this cryptic
species. However, they are infected
with variants of the same hantavirus.
It is not common that a hantavirus
infects different rodent species, and
we agree with the hypothesis that LNV
had infected a common ancestor to
the genus. Financial support: Fondos
concursables Focanlis 2010
CYTOMEGALOVIRUS (HCMV) IN
GLIOBLASTOMA
MULTIFORME
(GBM)
Januário, C.S., Figueiredo, E.G., Correa,
C., Cabrera, H.N., Schimdt, M., Teixeira,
M.J., Silva, M.C.C.
1. Universidade Federal do ABC,
UFABC, Rua Santa Adélia, 166.
Bairro Bangu. Santo André - SP Brasil . CEP 09.210-170
2. Hospital
das
Clínicas
da
Faculdade de Medicina da USP,
FMUSP, Av. Dr. Enéas de Carvalho
Aguiar, 255 - Cerqueira César 05403-000 - São Paulo E-mail:
[email protected]
The Human Cytomegalovirus (HCMV) is
an ubiquitous infectious agent, present
in almost 90% of the world's population.
In healthy individuals, the infection is
normally asymptomatic, however, in
cases of immunosuppression the virus
can be a life threatening agent. Recent
findings suggested a relationship
between HCMV and cancer. The virus
has been detected in different cancer
types, specially in glioblastomas, the
most malignant kind of glial tumors.
The studies of the HCMV role in tumor
progression are rapidly advancing and
additional work needs to be done to
confirm the viral presence in tumors
and identify possible viral proteins
involved in malignity. The aim of
this work was to detect the HCMV
genome in tumor biopsies obtained
from glioblastoma patients at the
Hospital das Clinicas of Sao Paulo,
Brazil. We employed three different
techniques: conventional PCR (cPCR),
real time quantitative PCR (qRTPCR) and semi-nested PCR to detect
HCMV in peripheral blood and lesions
of glioblastomas patients. The viral
genome was only successfully detect
by semi-nested PCR using primers
for the variable region of the viral
glycoprotein gB. Using a HCMV-BAC
the detection limit of the techniques
was 9 copies/ul in semi-nested
PCR, 6800 copies/ul in cPCR and 20
copies/ul in qRT-PCR, indicating that
semi-nested PCR is a more sensitive
technique in this case. At the moment
20 glioblastomas samples were tested
and 9(45%) were positive for the gB
region. Four (44%) of the 9 positive
patients had also detectable HCMV
in their peripheral blood. Sequencing
analysis of the gB demonstrated the
presence of different gB genotypes in
the tumors and we are analyzing the gB
genotypes in peripheral blood of the
patients. We are currently performing
experiments in order to verify the viral
gene expression in the glioblastomas
samples. Our results confirm the
presence of HCMV in glioblastomas
indicating a possible relationship of
the virus with tumor malignity.
HV1046 - CHARACTERIZATION OF
GENOTYPES AND MUTATIONS IN
HEPATITIS B VIRUS RESISTANT TO
ANTIVIRAL DRUGS IN PATIENTS
WITH HBV IN THE STATE OF
AMAZONAS, BRAZIL
Galvão, R.S., Braga, W.S.M., Castilho,
M.C., Vasconcelos, H.L., Rocha. J.M.,
Silva, S.S., Oliveira, C.M.C.
1. Fundação de Medicina Tropical
-Heitor Vieira Dourado, FMTHVD, Av. Pedro Teixeira, nº 25 Bairro D. Pedro CEP 69.040-000
Manaus Amazonas
Amazonas, UEA, Av. Pedro
Teixeira, nº 25 - Bairro Dom
Pedro CEP 69.040-000 Manaus
Amazonas E-mail: rsgalvao@
hotmail.com
Introduction: The Amazonian region
is characterized as one with the
highest incidence of hepatitis B
(HBV) worldwide. In the Amazonas
State, the gutters regions of the rivers
Jurua, Purus and midst Solimões
Human Virology: HV
221
are considered to have the highest
hepatitis B endemicity. Besides these
areas being known for their highest
endemicity, very little is known about
the virological and clinical information
for providing adequate treatment. The
genotypic characterization and the
search for mutations associated with
resistance to therapy are important
for the monitoring of viral dynamics
and treatment. This study aims to
characterize the HBV genotype as
well as the mutations associated
with primary resistance to therapy
from patients with HBV in the state
of Amazonas. Methodology: Blood
samples from 77 treatment-naive HBV
patients with viral load > 250UI/mL
(COBAS TaqMam Hepatatis B virus)
were collected. The PreS and S regions
of the surface gene were amplified
by polymerase chain reaction (PCR)
and nucleotide sequenced in the ABI
3130 xl automatic sequencer (Applied
Biosystems). The sequences were
edited and aligned using a BioEdit
Sequence Alignment Editor, version
7.1. The phylogenetic relationship
of the S gene fragment sequences
was determined using Molecular
Evolutionary
Genetics
Analysis
(MEGA), version 5.19. The Tamura–
Nei algorithm was used, employing
the neighbor–joining method. The
phylogenetic groups were evaluated
by the bootstrap test (1,000 bootstrap
replicates). Mutation analysis was
performed using the web site www.
hepseq.org/public/web_front_main.
php and hivdb.stanford.edu. /hbv/
hbvseq/deselopment/hbvseq.html.
Results: Of the 77 samples analyzed,
53 (68,8%) were positive for HBV
DNA. The following genotypes were
obtained in 44 HBV DNA samples
analyzed: 21(47,7%) A, 7 (15,9%) D,
222
Human Virology: HV
6(13,6) F, 3(6,8%) C and 1(13,6) G.
One mutation S202G was observed in
the reverse transcriptase region. This
mutation is related to resistance to
antiviral Entecavir in a patient with
HBV genotype D undergoing treatment
since 3 years. Conclusion: The HBV
genotype A was prevalent in this study
population confirming other studies
performed in the region. Of interest,
the detected mutation S202G resistant
to the use of Entecavir suggests that it
is important to sequence the RT region
for providing adequate treatment.
Financial support: CNPq, DN DST, Aids
e HV/MS, CAPES
HV1049 - PREVALENCE AND QUANTIFICATION OF THE HEPATITIS B
VIRUS (HBV) IN PATIENTS FROM
BLUMENAU-SC
Herkenhoff, M.E., Rodakiewicz, S.M.,
Ferreira, P.S., Gaulke, R., Branco, L.C.,
Pitlovanciv, A.K., Remualdo, V.R.
1. Genolab, Genolab, Rua floriano
peixoto, 425 - Centro-Blumenau
cep: 89010-500
2. Universidade do Estado de
Santa Catarina, UDESC, Av. Luis
de Camões, 2090, Bairro Conta
Dinheiro,
Lages/SC
E-mail:
[email protected]
Hepatitis B virus (HBV) infection is
a public health problem worldwide.
It is estimated that approximately
3 billion people have been exposed
to HBV, of whom more than 350
million are chronically infected.
The virus, a species of the genus
Orthohepadnavirus, which is likewise
a part of the Hepadnaviridae family
of viruses, is a major cause of chronic
liver disease worldwide. The virus is
transmitted by exposure to infectious
blood or body fluids such as semen and
vaginal fluids, while viral DNA has been
detected in the saliva, tears, and urine
of chronic carriers. Real-time PCR
has engendered wide acceptance for
quantification of hepatitis B virus (HBV)
DNA in the blood due to its improved
rapidity, sensitivity, reproducibility,
and reduced contamination. The
aim of this study was to determine
the HBV prevalence in a population
in Blumenau, Santa Catarina state,
southern Brazil, in January/2010 to
July/2010. We obtained a sample of
158 individuals, which was used the
blood serum to estimate the prevalence
and the number of viral copies. We
have used the Real-time PCR method
to indentify the virus and estimate the
number of viral copies in each positive
individual. After that, we have made a
statistical analysis using the student
test with 5% of significance. This study
has showed a prevalence of 24,5% on
the total sample, 29,35% on the male
population and 18,19% on the female
population. The number of viral copies
was showed an average of 25016079,22
copies/ml in the male population and
2870525,583 copies/ml in the female
population, there is no statically
difference in those two population,
male and female, determined by the
student test (5% of significance). In
conclusion, our study have showed a
significant prevalence by this virus in
the population that was studied, and
that are not a difference on the number
of copies between the male and the
female population. Financial Support:
Genolab
HV1052 - GENOTYPIC RESISTANCE
PROFILE OF HUMAN INFLUENZA A
VIRUS TO NEURAMINIDASE INHIBITORS IN CITIES OF NORTH AND
NORTHEAST REGIONS OF BRAZIL
DURING 2012 SEASON
Pacheco de Pinho, R.A., Santos, M.C.,
Barbagelata, L.S., Ferreira, J.A., Ferreira,
D.L., Silvestre, R.V.D., Filizzola, E.M.A.,
Sousa Junior, E. C., Ferreira, L.E.A.,
Medeiros, R., Mello, W.A.
1. Evandro Chagas Institute, IEC,
Highway BR-316 km 7 s/n Levilândia - Ananindeua / Pará /
Brazil
2. Nucleus of Tropical MedicineUFPA, Belém, PA, Brazil, NMT/
UFPA, Avenue Generalíssimo
Deodoro, 92 - Umarizal Belém/ Pará/ Brazil E-mail:
[email protected]
Introduction. Influenza virus causes
millions of deaths and hospitalizations
worldwide every year. In Sporadic
infections, annually epidemics or
pandemics, antiviral drugs become
the principal means for managing
the disease specially neuraminidase
inhibitors (NAI). However due to the
viral genetic evolution some viral
strains acquire point mutations in the
Neuraminidase (NA) codifying gene that
drive some amino acid substitutions
leading to drug resistance. Objectives.
Describe the occurrence of mutations
in NA encoding gene from Influenza A
virus, which may be related to (NAI)
resistance in circulating strains at the
North and Northeast of Brazil in the
period between January and June 2012.
Material and Methods. From the period
described 1204 samples were selected
based on the clinical symptoms of
acute respiratory infection (ARI) with
up to five days of evolution. Viral
RNA was purified and RT-PCR was
performed with specific primers to
amplify NA gene sequence of Influenza
Human Virology: HV
223
A (H3N2 and H1N1 pdm). RT-PCR
fragments were direct sequenced in
the ABI3130XL (Applied Biosystems).
Results. We had 151 positive samples
to Influenza A, 100 (66.2%) for
(H1N1pdm) and 51 (33.8%) for
(H3N2). Partially analyzed Nucleotides
sequences from 18 samples of the
subtype N1 and 11 N2 demonstrate
in two samples N1 from Fortaleza and
Manaus the substitution His275Tyr that
confers high Oseltamivir resistance,
low sensitivity to Peramivir and still
sensible to Zanamivir. Genotyping
of N2 samples showed the following
amino acid substitutions: Asn329Thr,
Ser334Ile, His347Gln, Ser367Asn and
Lys369Thr that were not associated to
NAI resistance. Conclusion. The 2009
pandemic period alert us for Influenza
monitoring through surveillance of
circulating strains and mutations in
the viral genome that can be associated
with resistance to antiviral drugs.
These are the first reported cases of
Influenza A virus circulation H1N1pdm
resistant to oseltamivir in the North and
Northeast. Financial support: National
Council of Scientific and Technological
Development - CNPq.
HV1054 - LCR GENETIC VARIABILITY
AND PHYLOGENY OF LOW-RISK HPV
IN LARYNGEAL PAPILLOMATOSIS
Bonfim, C.M., Sichero,L., Nogueira, R.L.,
Mansur, I.M., Matos, R.P.A., Kupper,
D.S., Valera, F.C.P., Nogueira, M.L., Villa,
L.L., Rahal, P.
1. Faculty of Medicine of Ribeirao
Preto, Sao Paulo University, USP,
Bandeirantes
Avenue,
3900
- Monte Alegre 14049-900 Ribeirão Preto - SP.
2. Sao Paulo State University ,
224
Human Virology: HV
Unesp/Ibilce, Cristovão Colombo and substitutions of nucleotide in this
Street, 2265, São José do Rio genomic region. A total of six genomic
Preto - SP, Brazil 15054-000
variants were identified. Phylogenetic
analysis of the LCR complete genomic
3. Faculty of Medicine of Sao Jose region of 14 Brazilian HPV-6 isolates, in
do Rio Preto, Famerp, Brigadeiro addition to 77 Slovenian and 12 African
Faria Lima Avenue, 5416 - 15090- HPV-6 isolates previously described,
000 São José do Rio Preto- SP
and the HPV-6a, HPV-6vc and the
4. Brazil Center of Translational HPV-6b prototype reference isolates
Research in Cancer, ICESP, E-mail: revealed a tree with two separated
clusters. The sequences of this study
[email protected]
were grouped in branches into nonRecurrent respiratory papillomatosis prototypic closely related HPV-6a and
(RRP) is a benign disease associated HPV-6vc-related genomic variants. We
with Human Papillomavirus (HPV) will further analyse the transcriptional
infection, particularly low-risk HPV activity of these samples in order to
types 6 and 11, and is characterized by verify if changes in this highly variable
the formation of papillomas mainly in region are involved in regulating the
the larynx. Replication and transcription expression of genes involved in cell
of HPVs depend on the interaction of cycle control and differentiation.
host cell and viral transcription factors
with the non-coding region of the virus HV1059 - HTRA1 EXPRESSION IN
called Long Control Region (LCR). This HIGH-RISK HPV-POSITIVE PENILE
region encloses diverse cis-regulatory TUMOR SAMPLES AND CELL LINES
elements central for its activity. It
has been predicted that nucleotide Stuqui, B., Termini, L., Sichero, L., Villa,
substitutions in the LCR region L.L., Soares, F.A., Rahal, P., Calmon, M.F.
leads to differences in the malignant 1. UNESP, São José do Rio Preto,
potential among HPV variants. This
IBILCE/UNESP, Rua Cristóvão
study aims to expand the knowledge
Colombo, 2265, São José do Rio
concerning genetic variability of HPV
Preto/SP
types commonly found in laryngeal
papillomas, and to further identify the 2. Center for Translational Research
in Oncology , ICESP, Av. Dr.
impact of nucleotide variations in the
Arnaldo, 251 - Cerqueira César LCR region upon HPV transcriptional
São Paulo A.C. Camargo Hospital,
activity. HPV detection was conducted
Department
of
Pathologic
by PCR using the PGMY09/11 primer
Anatomy
,
A.C.Camargo
Hospital,
system followed by Restriction
Rua Prof. Antônio Prudente, 109,
Fragment
Length
Polymorphism
Liberdade, São Paulo
(RFLP) genotyping. We analyzed 11
biopsy specimens of juvenile laryngeal 3. National Institute of Science and
papillomatosis and 9 of adult laryngeal
Technology - HPV Institute, INCT
papillomatosis. HPV-6 was found in 14
HPV, E-mail: bru.stuqui@gmail.
(70%) samples and HPV-11 in 6 (40%)
com
samples. Cloning and sequencing of
the HPV-6 LCR showed insertions The Human Papillomavirus is the
most prevalent virus among sexually
transmitted infections and it is
associated with various malignancies.
The E6 and E7 viral oncoproteins of
high-risk HPVs are the main responsible
for cell homeostasis alteration
and immortalization. One of the
mechanisms used in cell transformation
by E6 protein is the interaction of its
carboxy-terminal domain, known as
PDZ, with PDZs domains presents
in some cellular proteins, triggering
them to degradation. A protein that is
associated with various pathological
conditions and has PDZ domain is
the protease HTRA1. The aim of this
study was to evaluate the HTRA1 gene
expression in HPV-positive penile
tumor samples and high-risk HPV
positive cell lines. The HTRA1 was
selected from a microarray where it was
observed low expression of this gene in
cell line positive for HPV 16 (HF698).
In our laboratory, we evaluated HTRA1
expression by qPCR in high-risk
HPV positive penile tumor samples
compared to normal samples, where
we observed low HTRA1expression
in 70% of the samples analysed. After
gene validation in tumor samples, we
evaluated the HTRA1 mRNA expression
in high-risk HPV-positive cell lines:
HF698, SiHa, CasKi and HF698
transfected
with
pCMV6/HTRA1
expression vector. The gene presented
low expression in HF698 cell line like
previous microarray results. However,
when the HF698 line was transfected
with pCMV6/HTRA1 vector this gene
expression was significantly increased.
The lines SiHa and CasKi also showed
low expression for this gene. The HTRA1
protein expression in transfected cell
line with pCMV6/HTRA1 vector was
confirmed by Western blotting. These
results suggest that HTRA1 expression
Human Virology: HV
225
is low in high-risk HPV positive cells.
The HTRA1 overexpression effect
will still be measured in HF698 cell
line by viability, cell proliferation and
apoptosis assays. Financial Support:
CAPES, FAPESP
HV1075 - COMPARATIVE GENOMIC
ANALYSIS BETWEEN AFRICAN AND
BRAZILIAN HBV ISOLATES: THE
ROLE OF AFRICAN COUNTRIES IN
THE DISSEMINATION OF HBV/A/A1
IN BRAZIL
Lago, B.V., Valente, F.L., Soares,C.C.,
Spitz, N.T.D., Mello, F.C.A., Gomes, S.A.
1. Fundação Oswaldo Cruz, FIOCRUZ,
de Janeiro - CEP: 21040-360
2. Ministério da Saúde - República
de Angola, , E-mail: barbaravl@
ioc.fiocruz.br
It is estimated that 2 billion people have
been infected with hepatitis B virus
(HBV) worldwide and more than 400
million people are at risk of developing
cirrhosis and hepatocellular carcinoma
due to chronic infection. Based on a
genomic sequence divergence in the
entire genome exceeding 7,5%, HBV
strains have been classified into 8
genotypes, denoted A (HBV/A) to H
(HBV/H). It have been established that
HBV/A1 is one of the most prevalent
genotype in Africa, especially in
southern and eastern coast. In previous
studies we have shown that HBV/A1 is
the main genotype circulating in Brazil.
Studies conducted in isolated AfroBrazilian communities demonstrated
that these communities have almost
exclusively HBV/A1, suggesting that
it was introduced by the slave trade.
The aim of this study is to compare
HBV/A1 isolates from different African
226
Human Virology: HV
regions with Brazilian isolates in order
to investigate, throughout genetic
identity, which African countries
have contributed to dissemination
of HBV/A1 in Brazil. A comparison
among samples from African countries
and Brazilian isolates may help to
establish possible routes of HBV/A1
spread. For this purpose, 50 samples,
previously classified as HBV/A1 by
RFLP analysis, from different Brazilian
regions were selected. Until this
moment, 20 samples were amplified
for HBV complete genome by PCR
assay. Fourteen HBV/A1 samples
were successfully sequenced for HBV
entire genome directly from PCR
products. Sequences were compared
with HBV/A1 samples available in
GenBank/NCBI. Phylogenetic analysis
demonstrated that Brazilian sequences
are more closely related to Asian/East
African sequences than with sequences
from other African regions (genetic
distance values: 0,02 versus 0,03).
These preliminary results suggest that
HBV infected slaves brought to Brazil
came mostly from the East African
coast during the slave trade. However,
further studies are necessary to confirm
this hypotesis and to estimate which
African countries have contributed
to the spread of HBV/A1 in Brazil.
Financial support: IOC/FIOCRUZ
HV1076 - HEPATITIS B AND DELTA
VIRUS PREVALENCE AMONG BLOOD
DONORS IN LUANDA, ANGOLA
Savassi-Ribas, F., Borges, L.F., Varella,
R.B., Gomes, S.A., Soares, C.C.
Av. Brasil, 4365, Manguinhos - Rio
de Janeiro-RJ - CEP: 21040-360
Janeiro, UFRJ, E-mail: savassi@
ioc.fiocruz.br
Hepatitis delta virus (HDV) is associated
with hepatitis B virus (HBV) infection
and is frequently related to more severe
disease than that due to the underlying
HBV monoinfection. HDV is a subviral
pathogen of humans, a satellite of
HBV that depends on the envelope
protein of HBV for its assembly and
propagation. HDV is spread in the same
way as HBV, mainly through parenteral
exposure. The virus is highly endemic
in Mediterranean coutries, Middle East,
Central Africa and northern parts of
South America. Worldwide, more than
350 million people are considered to
have chronic HBV infection, and 15 – 20
million of these individuals are thought
to be coinfected or superinfected with
HDV. Although Africa is considered
an endemic region for this infection,
for many countries, there is no
available data in the literature. The
aim of this study was to investigate
seroprevalence of HDV in blood donors
from Luanda, Angola. A total of 213
samples were submitted to HBsAg
detection. All HBsAg reactive samples
were subsequently tested for the
detection of anti-HDV and, finally, antiHDV reactive samples were tested for
HDAg detection. Serological tests were
performed by ELISA. HBsAg presence
was detected in 19.2% (41/213)
of blood donors samples. Nine out
41 (22%) HBsAg positive were also
reactive to anti-HDV, 34.1% (14/41)
were anti-HDV negative and 43.9%
(18/41) were in the gray zone. All
positive and gray zone samples were
tested for HDAg and this antigen was
found in 2 samples, suggesting that
these individuals are actively infected.
Both HDAg samples were in the gray
zone in ELISA for anti-HDV detection.
Molecular analysis of the HDV positive
samples are in progress. Our results
shows a still high prevalence of HBV
in Angolan general population and
also describes a high seroprevalence
of HDV among these HBsAg carriers.
Studies about HDV epidemiology in
sub-Saharan countries are scarce,
these data contributes for a better
understanding of HDV circulation in
African continent. Financial support:
CNPq, Fiocruz
HV1084 - DENV-2 DETECTION IN
UMBILICAL CORD FROM A DENGUE
FATAL CASE
Nunes, P.C.G., de Filippis, A.M.B., Soares,
A.C., dos Santos, F.B., Pelajo-Machado,
M., Oliveira, B.C.E.P.D., Nogueira, R.M.R.
INSTITUTO OSWALDO CRUZ, IOC, AV.
BRASIL, 4365, MANGUINHOS E-mail:
[email protected]
In Brazil more than seven million cases
of dengue have been reported and
currently the four serotypes circulate
in the country. Although dengue is
endemic in several regions of Brazil,
there are few reports of dengue
infection in pregnant women and the
consequences for the fetus. This study
reports a fatal outcome due to dengue
resulting in maternal and fetal death. In
November of 2010 a pregnant woman
aged 23 years old, was admitted to the
maternity of Miguel Couto Municipal
Hospital, in Rio de Janeiro complaining
of abdominal pain, vomiting and
diarrhea, fainting and inaudible fetal
heartbeat. The ultrasound revealed a
single fetus with no active movements,
little or absente amniotic fluid and
placenta with reduced dimensions.
The fetus was 32/33 weeks presenting
placental abruption with apparently
early detachment. During the caesarean
Human Virology: HV
227
there was a large extravasation of
blood in cavity, deterioration of
clinical picture, hypothermia, anuria,
hemodynamic instability, mydriasis,
absent reflexes and death. Tissues
of spleen, placenta and umbilical
cord were paraffin-embedded to
investigate dengue virus. The RNA
was extracted from sections of 5 um
and RNA extraction was performed
using the kit Purelink FFPE RNA
Isolation Invitrogen. Real time PCR
and RT-PCR was performed. DENV-2
was detected by both methods only in
the umbilical cord. Considering that
dengue infection during pregnancy
may represent a risk to mother and
concept measures of epidemiological
surveillance and virological diagnosis
should be implemented to this specific
group especially in dengue endemic
areas. Financial Support: CNPq and
FAPERJ
HV1087 - MOLECULAR TRACKING
OF
LAMIVUDINE
RESISTANCE
MUTATIONS IN HBV SUBPOPULATION FROM HIV/HBV COINFECTED
PATIENTS BY PYROSEQUENCING
Spitz, N.T.D., Lago, B.V., Moraes, M.T.B.,
Gomes, S.A., Soares, C.C.
de Janeiro - CEP: 21040-360 E-mail:
[email protected]
Due to similar routes of transmission,
human immunodeficiency virus-1
(HIV-1) and hepatitis B virus (HBV)
coinfection remains one of the
most frequent comorbidities with
a significant impact related to liver
disease. Approximately 10% of
the HIV-infected population has
concurrent chronic HBV. Both can lead
to chronic disease, and neither can
228
Human Virology: HV
be eradicated with the use of current
therapies.
Reverse-transcriptase
(RT) is an important enzyme for the
replication of both viruses and for
this reason it is used as a target in
antiretroviral therapy. Lamivudine
(LAM) is a nucleoside analogue which
inhibits RT and is used in treatment
against HBV and HIV. However, its
clinical benefit has been compromised
by the emergence of resistant viral
strains carrying specific mutations
in HBV and HIV RT genes. In HBV, the
primary LAM-resistance mutation
(rtM204V/I) affects viral replication
and
compensatory
mutations
(rtL180M, rtV173L) that partially
restore replication efficiency are often
co-selected. The aim of this study is
to evaluate by pyrosequencing, the
incidence of LAM resistance mutations
in both viruses. At the moment, only was
made the evaluation of HBV mutation
and 26 samples were analyzed.
rtM204V/I were successfully analyzed
in 21 samples. Mixed population (wild
type -wt- and mutant strains) was
found in 81% (17/21) of samples. Only
rt204V mutant population was found
in 14.3% (3/21) and in 4.7% (1/21)
of samples only wt strains were found.
Compensatory mutation rtL180M was
investigated in 14 samples and only
wt strains were found. RtV173L was
studied in 13 samples. Only wt strains
were found in 85% (11/13) and mixed
population was found in 15% (2/13)
of the samples. Pyrosequencing is a
very sensitive technique that may be
useful in detecting and quantifying
subpopulations of resistant viruses,
that may be not be detected by other
methods. It can be useful in predicting
the appearance of mutant strains that
can derail the treatment, improving the
outcome of therapy. Financial support:
Fiocruz, CNPq/PIBIC
HV1091 - SEROPREVALENCE OF
HEPATITIS DELTA VIRUS (HDV)
AMONG HEPATITIS B VIRUS (HBV)
CHRONIC CARRIERS FROM CAMPO
GRANDE- MATO GROSSO DO SUL,
CENTRAL BRAZIL.
Savassi-Ribas, F., Lindenberg, A.S.C.,
Freitas, S.Z., Gomes, S.A., Motta-Castro,
A.R.C., Soares, C.C.
Av. Brasil, 4365, Manguinhos - Rio
de Janeiro-RJ - CEP: 21040-360
2. Universidade Federal do Mato
Grosso do Sul, UFMS, E-mail:
[email protected]
Hepatitis delta virus (HDV) is a subviral
pathogen of humans, a satellite of
hepatitis B virus (HBV) that induces
severe acute and chronic liver diseases.
This agent depends on the envelope
protein of HBV for its assembly and
propagation, so it can only infect an
individual who has coexistent HBV,
either after simultaneous transmission
of the two viruses or via superinfection
of an established HBV carrier. HDV
infection has a worldwide distribution
and it is estimated that 15 – 20 million
individuals are anti-HDV positive.
This virus is highly endemic in
Mediterranean countries, the Middle
East, Central Africa and northern
parts of South America. The Brazilian
Amazon is known as an endemic area
for this infection and it represents
a significant public health problem,
but few studies have assessed its
prevalence in non-Amazonian regions
in the country. The Amazon River
region is located in the northern part of
Brazil and includes areas of 7 Brazilian
states (Acre, Amazonas, Amapá, Pará,
Rondônia, Roraima and Mato Grosso).
The aim of this study was to evaluate
the seroprevalence of HDV among
HBsAg chronic carriers from Campo
Grande - MS, West Central region,
located outside of the Brazilian Amazon
Basin. A total of 125 samples from
HBV chronic carriers (HBsAg positive)
were enrolled in this study. All samples
were submitted to anti-HDV detection
by ELISA and reactive samples were
subsequently submitted to HDAg
detection. Antibodies anti-HDV were
found in 4.8% (6/125) of the samples,
and HDAg was found in only one
sample, suggesting that this individual
is actively infected. Molecular analysis
are in progress. These data reflects
HDV circulation outside the Brazilian
Amazon Basin and highlights the
importance of epidemiological studies
in different Brazilian regions. Clinical
and epidemiological studies are still
needed to clarify the presence and the
role of HDV infection in Brazil. Financial
support: CNPq, Fiocruz
HV1093 - INFLUENCE OF POLYMORPHISMS IN GENES OF THE INNATE
IMMUNE RESPONSE IN THE COURSE
OF HIV-1 INFECTION
de Medeiros, R., Matte, M.C.C.,
Mirandolli, T.B., Araújo, L.A.L., Lunge,
V.R. , Melo, M.G., Almeida, S.E.M.,
Chies, J.A.B.
Grande do Sul, UFRGS,
e Pesquisa em Saúde - RS, FEPPSRS,
3. Grupo Hospitalar Nossa Senhora
da Conceção Porto Alegre - RS,
Human Virology: HV
229
GHC - RS,
4. Universidade Luterana do Brasil RS, ULBRA - RS,
5. Universidade Feevale, Feevale,
E-mail: medeirosrubia@gmail.
com
Variations in innate immune response
genes have been associated with
different AIDS progression. In HIV
infection, TLRs (Toll-like receptors)
recognizes molecules of the virus
present inside the infected cell; ie TLR7
and TLR8 recognizes the viral RNA,
and TLR9 the proviral DNA. This study
investigated the influence of TLR7,
TLR8 and TLR9 polymorphisms in the
course of HIV-1 infection in patients
from Southernmost Brazil. From 3.300
medical records of HIV+ patients, 98
individuals were defined as rapids,
slow and chronics AIDS progressors.
TLR7(Gln11Leu),
TLR8(Met1Val)
and TLR9(T-1237C and G1635A)
polymorphisms were determined by
PCR and restriction enzymes cleavages.
To evaluate the influence of genotypes
in the progression to AIDS Survival
Analysis tests and Cox regression
corrected by the presence of protective
alleles CCR5del32 and HLA-B27/57
were performed. Kaplan-Meier curves
to polymorphism -1237T/C in TLR9
revealed a significant association
between C allele carriers and longer
time (10 years) to AIDS progression
when compared with T allele carriers
(6 years). Moreover, this association
was also reproduced in the multivariate
Cox regression (0.616 Hz, 95% CI
0.379 to 1.003, p <0.05), including age
and ethnicity as variables. However,
adjusting the model to the presence
of the protective alleles CCR5del32
and HLA-B27/57 the association
230
Human Virology: HV
was lost. For the polymorphism
+1635G/A in TLR9, when analyzing
the variation within ethnic groups,
statistically significant association of
allele A (1.966 HZ, 95% CI 1.052 3.674,
p<0.05) with rapid AIDS progression
was observed in European-derived
patients. No significant results for
TLR7 and TLR8 polymorphism and
AIDS progression were shown. Our
data highlights a relationship between
the investigated polymorphisms in
TLR9 gene and progression to AIDS.
In addition, the results suggests the
genetic background is important in
HIV-1 infection, although several
genes and variants responsible by this
behavior remain to be identified.
HV1095 - EVIDENCE OF MULTIPLE
INTRODUCTIONS OF HIV-1 SUBTYPE
C IN RIO DE JANEIRO
Delatorre, E., Couto-Fernandez, J.C.,
Guimarães, M.L., Pilotto, J.H., Morgado,
M.G., Bello, G.
Oswaldo Cruz, IOC - FIOCRUZ, Av.
Brasil, 4365. Manguinhos - Rio de
Janeiro - RJ - Brasil. CEP: 21040-360
Subtype C is the most prevalent HIV1 clade worldwide and has spread
very efficiently in the southern states
of Brazil. Phylogeographic studies
indicate that the subtype C epidemic
in southern Brazil was initiated by the
introduction of a single founder virus
probably through Parana at some time
point between 1960 and 1980. Recent
studies demonstrate an increasing
prevalence of this subtype in the
southeast and central-west Brazilian
regions. Little is known, however,
about the genetic characteristics and
spatial dynamics of subtype C viruses
circulating in those regions. The
objective of this study was to trace the
origin of the HIV-1 subtype C viruses
circulating in the state of Rio de Janeiro.
Thirty HIV-1subtype C samples isolated
in this sate between 2003 and 2011
were compared with subtype C pol
sequences of Brazilian and African origin
previously described. Phylogenetic
and phylogeographic analyses were
conducted using Maximum-likelihood
and Bayesian methods. These analyses
reveal that there have been multiple
independent introductions of the HIV1 subtype C clade in Rio de Janeiro,
as signified by the presence of more
than 20 phylogenetically distinct
lineages. Most (>90%) subtype C
viruses circulating in Rio de Janeiro
originated in the southern Brazilian
states. Five independent subtype C
lineages, however, probably originated
from southern and eastern African
countries. We also find evidence that
a few subtype C lineages were locally
disseminated throughout the state.
These results indicate a massive
influx of HIV-1 subtype C strains from
southern states into Rio de Janeiro
and also demonstrate the importance
of Rio de Janeiro as a route of entry of
new viral strains from Africa to Brazil.
Finantial support: CAPES.
HV1099 - DETECTION OF DENGUE
VIRUS OF SEROTYPE 4 AND
GENOTYPE I IN AEDES AEGYPTI
FROM MANAUS CITY, BRAZIL
Dias, J.P., Silva, C., Patricia, L., Costa,
C.A., Luz, S.L.B., Figueiredo, M.L.G.
1. Instituto Nacional de Pesquisas
da Amazônia, INPA, Av. André
Araújo, 2936, Aleixo Manaus –
AM. CEP: 69060-001
/ Fundação Osvaldo Cruz, ILMDFIOCRUZ, Rua Teresina, 476.
Adrianópolis. Manaus – AM. CEP:
69.057-070 E-mail: jonaidias@
yahoo.com.br
Dengue epidemics have been reported
in Brazil since 1985. In Manaus, a large
city in the Brazilian Amazonic region,
dengue is hyperendemic with all 4
serotypes simoultaneously causing
human disease and an increase of DHF/
DSS cases as well as the number of
fatalities. Two dengue serotype 4 virus
(genotypes I and II) have been found
in Manaus in the last 5 years. Herein,
we report for the first time in Brazil,
dengue serotype 4 virus of genotype
I infecting a mosquito. This study was
part of a dengue virus surveillance
performed in Tancredo Neves, District
of Manaus, 2008-2010, where 3240
mosquitoes were captured, identified
and grouped into 324 pools. RNA
extracts of mosquito pools were tested
by a Reverse Transcription-Polymerase
Chain Reaction (RT-PCR), followed by a
Nested PCR, assays for detection and
identification of flaviviruses. Flavivirus
amplicons of putative dengue serotype
4 were obtained from 1 pool of Aedes
aegypti. The nucleotide sequence of
the amplicon, with ~980 pb showed
it was dengue serotype 4 of genotype
I, a virus previously only found in Asia
but described in Manaus since 2008.
This virus was probably introduced
into Manaus as a consequence of the
extensive trading of Manaus city with
Asian countries and our results show
that it has been transmited by Aedes
aegypti.
HV1103 - IMMUNOGENICITY DETERMINATION OF THE INFLUENZA
VIRUS VACCINE/2011 IN THE POPU-
Human Virology: HV
231
LATION OVER 60 YEARS IN MACEIÓ
Cruz, M.E.M., Sa, J.P.O., Laureano,
P.G.M.R., Meira, R.A.
1. Universidade
Estadual
de
Ciências da Saúde de Alagoas,
Uncisal, Rua Doutor Jorge de
Lima, 113 - Trapiche da Barra 57010-300 - Maceió/AL
2. Universidade Federal de Alagoas,
UFAL, Av. Lourival Melo Mota, s/n,
Cidade Universitária - Maceió
- AL, CEP:57072-900 E-mail:
[email protected]
The influenza virus is the cause of a
high respiratory infection that can
lead to complications, especially in the
elderly, target of an annual campaign of
vaccination. An epidemiologicalserum
study was conducted to determine the
prevalence of influenza antibodies of the
circulating serotypes between elderly
patients surveyed in 2011, in Maceió,
considering two cultures of the type
A and one of the type B, components
of the current vaccine. The objectives
of this study are determine the
immunogenicity of the new influenza
vaccine; monitor the circulation of
new strains of the virus; notify the
appropriate epidemiological service.
179 blood samples were collected
during the pre and post-vaccination
period, which have been processed by
the hemagglutination inhibition test
for influenza A/California/7/2009
(H1N1), A/Perth/16/2009 (H3N2)
and B/Brisbane/60/2008, influenza
vaccine/2011
components.
The
presence or absence of antibodies was
analyzed in terms of seroconversion,
title stationary and negative title.
High rates of seropositivity have been
observed for cultures of influenza
A H1N1 (83%), H3N2 (71%) and
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Human Virology: HV
influenza B strain studied (68%) in the
period pre-vaccination. The positive
values were considered for dilution
above 1:40. The negative ones for
dilution below and including 1:40.
179 samples analyzed, the Influenza
Vaccine/2011 stationary title was
high in the study population, in any of
the three components of the vaccine
cultures. It was noted, therefore, that
the influenza A/California/7/2009
(H1N1), A/Perth/16/2009 (H3N2) and
B/Brisbane/60/2008 had already been
circulating in the population before
administration of the vaccine. Brazil
has continental dimensions and great
variety of climate, which influences
the respiratory viral outbreaks. It is
necessary to evaluate a change at the
time of vaccination in Maceió or even in
Northeast of Brazil. Financial support:
Lacen/AL.
HV1104 - DETECTION OF ADENOVIRUS IN HOSPITALIZED CHILDREN
WITH
ACUTE
RESPIRATORY
INFECTION IN THE STATE OF PARA,
BRAZIL
Magalhães, R.M.R., Ferreira, J.A., Silva,
A.K., Santos, M.C., Mello, W.A., Sousa,
M.S.
UFPA, Av. Bernardo Sayão, s/n.
Belém - Pará
BR 316, Km 7. Ananingeua Pará E-mail: rebeccablack_15@
hotmail.com
Introduction:
Acute
respiratory
infections (ARI) remain a major cause
of morbidity and mortality worldwide
especially in children under five years
old and in less developed regions such as
north of Brazil. As viruses are important
etiologic agents of such infections, it is
necessary to monitor these pathogens
such as adenovirus (AdV). The objective
of this study is to detect adenovirus
in children hospitalized with acute
respiratory infection in Pará. Material
and methods: From July 2009 to June
2010 were randomly selected, with a
sampling error of 5%, 178 samples of
nasopharyngeal aspirate or combined
oral plus nasal swab collected from
children aged zero to five years old,
hospitalized with respiratory infection
without pandemic H1N1 influenza
virus. The study of AdV was performed
on samples from 21 districts of the Pará
State by polymerase chain reaction
in real time, followed by sequencing
of positive samples. Results: In total,
18.54% (n = 33) of samples were
positive with no prominence in any
age group. The positive samples
represented 100% (n = 2) of the cases
referred from Parauapebas, 31.25% (n
= 5) of the Ananindeua and 19.55% (n
= 26) of Belém. During the months of
February to May 2010 were identified
61% (n = 20) of positive cases, more
frequently (30.3%, n = 11) in March
2010, however, no difference (p =
0.6663) between the frequencies of
July to December 2009 (21.31%, n =
13) and January to June 2010 (17.39%,
n = 20). Among the positive samples
were identified AdV-3, AdV-5 and
AdV-6. Conclusion: In Pará State, the
AdV were present in approximately
20% of children aged zero to five
years admitted with acute respiratory
infection without pandemic H1N1
influenza virus, demonstrating the
importance of monitoring this virus in
order to prevent outbreaks of unusual
types. Financial support: Fellowship
Program for Scientific Initiation of
UFPA (PIBIC-UFPA/FAPESPA/CNPq).
HV1105 - HYPERENDEMIC CIRCULATION OF DENGUE VIRUS IN SAO JOSE
DO RIO PRETO IN THE PAST TWO
YEARS
Colombo, T.E., Mondini, A., Vedovello,
D., Biselli, J.M., Favaro, E.A., Nunes,
S.H.P., Ferraz-Cury, A.A., Oliveira, F.H.,
Bernades, E.R.G., Polli, C.W.R., Reis,
A.F.N., Nogueira, M.L.
Júlio de Mesquita Filho, UNESP,
Rua Cristóvão Colombo, 2265,
Jardim Nazareth. São José do Rio
Preto
São Pedro, São José do Rio Preto,
SP
3. Secretaria de Saúde de São José
do Rio Preto, SP, , E-mail: taty_ec@
hotmail.com
Dengue is the most common arboviral
infection worldwide and it is caused
by four distinct serotypes (DENV
1-4). Sao Jose do Rio Preto (SJRP)
has been presenting a hyperendemic
circulation of DENV since 2008, when
three serotypes started circulating
in the city. This is a report of DENV
transmission in SJRP from October
2010 to June 2012. We used serum
samples of suspected and confirmed
DENV patients provided by the Health
Secretariat to profile DENV circulation.
The viral surveillance was performed
using Multiplex RT-PCR with Flavivirus
generic primers based on the nonstructural protein (NS5), followed by
Nested assays with species-specific
primers for the identification of DENV
1-4. We examined 46 samples in
2010, 431 in 2011 and 76 until June
Human Virology: HV
233
2012. There were 378 cases officially
confirmed in SJRP from October 2010
to June 2012. Our PCR results showed
that DENV-1 was the main circulating
serotype in both years. We examined
551 samples and 383 were positive for
DENV (341 were positive for DENV1, 21 for DENV-2, 20 for DENV-4). A
co-infection of DENV-1 with DENV-4
was detected in one patient. DENV1 was the first and only serotype to
cause autochthonous DENV cases in
SJRP from 1990 to 1998. Only in 2008,
DENV-1 was detected again in SJRP. It
was also the main serotype in the 2011
outbreak. DENV-2 was introduced in
the city in 1998 and DENV-4 in 2011.
Thus, we show that three different
serotypes of dengue have been detected
in the city in different proportions
and the impact of this hyperendemic
circulation should be further evaluated
for epidemiological purposes.
HV1109 - HPV 6 IN ONE CASE OF
INVASIVE
CERVICAL
CANCER:
ANALYSIS OF BIOMARKERS
Amaro Filho, S.M., Golub, J.E., Levi, J.E.,
Andrade, C.V., Russomano, F., Tristão,
A., Pires, A., Nuovo, G.J., Nicol, A.F.
1. Fundação Oswaldo Cruz, Fiocruz,
Av. Brasil, 4.365 - Manguinhos,
Rio de Janeiro, Brazil
2. Johns Hopkins University, JHU,
Baltimore, EUA
3. São Paulo University , USP, Av. Dr
Enéas Carvalho Aguiar, 470 São
Paulo, Brazil
4. Instituto Fernandes Figueira
Institute , IFF, Niterói, Brazil
5. OHI University Hospitals,
Columbus, OH, USA
,
234
Human Virology: HV
6. Laboratório Fonte de Medicina
Diagnóstica, FONTEMD, E-mail:
[email protected]
Single low-risk HPV infections in highgrade lesions are rare. Some studies
have reported low-risk HPV DNA in
high-grade lesions but they are often
co-infected with high-risk HPV types.
HPV E6 and E7 viral oncoproteins can
form specific complexes with tumor
suppressor proteins that are capable of
changing mechanisms of the cell cycle,
modifying the expression of cellular
proteins. p16INK4a, Ki-67, and more
recently MCM-2 proteins have been
associated with tumor aggressiveness,
showing overexpression in highgrade lesions and invasive cervical
cancer (ICC). Conversely, p53 has often
been associated with low expression
in ICC. The goal of this study is to
report an unusual case where a single
infection with low-risk HPV (type 6) is
observed in an invasive cervical cancer
sample and to analyze the expression
of p16, Ki-67, MCM-2 and p53 in
this rare case. One invasive cancer
specimen was analyzed by means of
immunohistochemistry for p16, Ki-67,
MCM-2 and p53. HPV DNA was detected
by PCR following the genotyping by
sequencing. Additionally, INNO-LiPA
and PapilloCheck Kit were used in
order to confirm the single HPV type
infection. The single HPV 6 infection
was confirmed by the three techniques:
automatic sequencer, INNO-LiPA and
PapilloCheck. The markers related to
proliferation, Ki-67 and MCM-2, were
overexpressed with a mean of 65% and
35% positive cells per field, respectively.
The literature has reported high p16
positivity but low-level expression
of p53 in ICC. Curiously, in this case
p16 was reported negative, while p53
was overexpressed showing a mean
greater than 90% of positive cells per
field. Consideration should be given to
alternate pathways leading to virally
induced carcinogenesis. Other factors
such as polymorphic or epigenetic
events may play a role in an association
with cervical cancer. LIPMED - IOC –
Fiocruz; CAPES; Fogarty International
Center/US – NIH. Pós-Graduação em
Biologia Celular e Molecular – IOC –
Fiocruz.
HV1116 - ISOLATION OF DENGUE
4 IN SAO JOSE DO RIO PRETO, SÃO
PAULO, BRAZIL
Colombo, T.E., Mondini, A., Vedovello,
D., Biselli, J.M., Favaro, E.A., Nogueira,
M.L.
Júlio de Mesquita Filho , UNESP,
Rua Cristóvão Colombo, 2265,
Jardim Nazareth. São José do Rio
Preto
José do Rio Preto , FAMERP, Av.
São Pedro, São José do Rio Preto,
SP E-mail: [email protected]
DENV-4 had a brief circulation in
Brazil in 1982 in the Northwestern
region of Brazilian Amazon in a
focal epidemic. No further cases of
infection had been registered in the
country until 2008, when the virus
was detected in three patients, who
had no international traveling history,
in Manaus. DENV-4 reemerged in the
country in 2010 in the municipalities
of Boa Vista and Cantá in Roraima
State, spread to different geographic
regions of Brazil. In the present work
about DENV-4 transmission in SJRP
from 2009 to 2012, we used serum
samples of suspected and confirmed
DENV patients provided by the Health
Secretariat to profile DENV circulation.
The viral surveillance was performed
with Multiplex RT-PCR using Flavivirus
generic primers based on nonstructural protein (NS5), followed by
Nested assays with species-specific
primers and cultured cells of Aedes
albopictus for the identification and
confirmation of DENV-4. We examined
383 positive samples for DENV and 20
were positive for DENV-4. Due to the
availability issues, only 14 have been
subjected to specific RT-PCR reaction
for the envelope gene for DENV-4.
Fragments were purified from PCR
mixtures and sequenced using the
BigDye v3.1 (Applied Biosystens, USA)
in a ABI3130 automatic sequencer. The
nucleotide sequences were analyzed
using the DS Gene 2.0 Software and
were confirmed as envelope gene for
DENV-4. The reemergence of DENV4 should be a concern for health
authorities since there are evidences
that the replacement of a dominant
circulating genotype is associated with
the rising of a previously rare lineage.
HV1121 - IN VITRO ANTI-ROTAVIRUS
ACTIVITY ASSESSMENT OF CELASTRACEAE FAMILY CONSTITUENTS
AND EXTRACTS USING MA-104
CELLS
Vieira Filho, S.A., Barcelos, L.G., Marçal,
E.C., Assenço, R.A., Lanna, M.C.S.,
Magalhães, C.L.B., Magalhães, J.C.,
Silva, G.D.F., Silva, F.C.
1. Universidade
Federal
de
universitário Morro do Cruzeiro,
s/n, cep 35.400-000, Ouro Preto,
MG
Human Virology: HV
235
6627, Pampulha, Belo Horizonte,
Minas Gerais
del Rey, UFSJ, Praça Frei Orlando,
170, Centro - São João Del Rei MG, 36307-352 E-mail: bibo@
ef.ufop.br
Rotavirus targets mainly children and
infants from developing countries. It It
accounts for 30-50% cases of diarrhea
and about 1.9 million deaths annually
worldwide. Celastraceae family extracts
and constituents were evaluated due to
their medical potential as an infusion
using MA-104 cells. Austroplenckia
populnea and Maytenus gonoclada
ethanolic and ethyl-acetate extracts
of leaves branches and roots were
assessed, in vitro, for their toxicity and
antiviral activity. Studies associated
their biological activity with triterpenes
which was confirmed to be present
in all tested extracts. Cytotoxicity was
assessed using MTT method; thus CC50
was estimated as the concentration
capable of reducing the cell viability
in 50%. Extracts toxicity ranged from
25 to 152,7 µg/mL. Crude extracts
dilutions, below CC50 threshold,
were used for the antiviral evaluation.
MTT was also used to determine cell
viability. All experiments were done
in triplicate and the viral infection
happened prior to the extract exposure.
96 well plates were incubated at 37°C
in a 5% humidified CO2 chamber for 72
hours. Results analysis expressed the
concentration capable to inhibit 50%
of viral induced death (CE50) these
values ranged from 1701 to 24,74 µg/
mL. All extracts, when tested in higher
concentrations, presented activity
against the Rotavirus. To determine
whether they were safe or not we used
236
Human Virology: HV
SI, which is calculated as it follows:
CC50/CE50. Only 3 extracts presented
values above 1, being the highest
1,59. . In this study we could assert
that three of the tested compounds
have potential to serve as antiviral
drugs, new experiments could try to
isolate molecules from this extracts in
search of a more active and less toxic
substance.
HV1123 - COMPARATIVE ANALYSIS
OF BIOMARKERS IN DETECTING
ADVANCED STAGES OF INVASIVE
CERVICAL CANCER
Amaro Filho, S.M., Golub, J.E., Nuovo,
G.J., Cunha, C.B., Levi, J.E., Villa, L.L.,
Andrade, C.V., Russomano, F., Tristão,
A., Pires, A., Pirmez, C., Nicol, A.F.
2. Johns Hopkins University, JHU,
3. Instituto Fernandes Figueira, IFF,
4. Ohio Medical University, OMU,
5. Laboratório Fonte Medicina
Diagnóstica, , Instituto do HPV,
INCTHPV, Universidade de São
Paulo, USP, E-mail: sergioafilho@
gmail.com
Cell cycle protein expression plays
an important role in the pathology
and clinical diagnosis of cervical
cancer. However, few studies have
attempted to correlate the use of
these biomarkers with the clinical
progression of the tumor. Objective: to
analyze the expression of Ki-67, MCM2, p53 and p16 in cervical cancer and
to search for a differential expression
that can assist in the assessment
of clinical tumor staging according
to FIGO classification. Methods:
two blocks of Tissue Micro-Array
containing 87 cervical samples from
patients with invasive cervical cancer
(ICC) and 43 controls were analyzed.
Sociodemographic, behavioral and
clinical characteristics of patients were
obtained from the medical records.
HPV DNA detection was done by PCR
and in situ hybridization. By means of
immunohistochemistry the markers
were analyzed. Statistical analysis was
performed by STATA 10.1. Results:
a strong association (p<0.005) in
advanced tumor stages (III and IV) was
observed in women over 55 years, with
more than four pregnancies and without
school education. The prevalence of
HPV DNA by PCR in the ICC was 94.3%.
The most prevalent types found in the
ICC were HPV16 (67.5%), followed by
HPV33 (12.0%) and HPV35 (3.6%).
An increase (p<0.05) expression of
Ki-67, MCM-2, p53 and p16 was found
in ICC compared to controls. Ki-67
and p16 showed strong expression
in advanced disease, FIGO III and IV
(p=0.008 and p=0.023, respectively).
There was no association between the
expression of p53 and MCM-2 with the
tumor staging. Women with HPV16
tended to be younger (50.9 years; SE
1.9) compared to women with other
types (59.9 years; SE 2.8), suggesting
that HPV16-infected women develop
cervical cancer earlier than the others.
In conclusion, we found that only Ki67 and p16 intensity was associated
with the stage of the ICC. The most
prevalent types found were HPV16,
33 and 35 suggesting that further
studies should be considered for
implementation of vaccination against
HPV in Brazil. LIPMED - IOC – Fiocruz;
CAPES; Fogarty International Center/
US – NIH. Pós-Graduação em Biologia
Celular e Molecular – IOC – Fiocruz.
HV1127 - SELECTION PRESSURE
ANALYSIS OF THE HCMV TEGUMENT
PROTEINS
Stangherlin, L.M., Braz, A.S.K., Silva,
M.C.C.
Universidade Federal do ABC,
UFABC, Rua Santa Adélia, 166. Bairro
Bangu. Santo André - SP - Brasil . CEP
09.210-170 E-mail: lmsbotucatu@
gmail.com
Human
citomegalovirus
is
a
betaherpesvirus present in 50 to 80%
of the human population around the
world. HCMV can cause important
diseases
in
immunosuppressed
individuals such AIDS patients and
transplant recipients. The viral particle
is composed of a double strand DNA
coated by an icosahedral capsid made
of proteins, which is surrounded
by a tegument layer comprised of
proteins and RNAs. The capsid and
the tegument are enclosed by a lipid
envelope containing glycoproteins.
The viral tegument contains 20
to 25 different types of proteins,
present in many copies. Studies about
occurrence of selection pressure in
HCMV proteins are scarce, specially in
the tegument proteins. In the present
work we investigated sites of positive
selection pressure in the pp71(UL82),
pp65(UL83) and pp28(UL99) tegument
proteins. We searched for sequences
in databases through the BLAST tool
and sites under selection pressure
were analyzed considering changes in
codons and amino acids. Our results so
far demonstrated that positive selection
occurs more frequently on the UL99
ORF that encodes the pp28 protein,
responsible by the final acquirement of
the envelop. In addition sites of positive
selection in the pp28 are concentrated
at the C terminal region of the protein.
Human Virology: HV
237
The low frequency of positive selection
at the amino terminus is probably due
to the fact that this region of the protein
is important for virus assembly and
therefore must be conserved. These
results suggest that the C terminal
region of pp28 that is under selective
pressure can have an important
function that confers advantage for the
viral life cycle.
HV1129 - ISOLATION, IDENTIFICATION
AND
MOLECULAR
CHARACTERIZATION OF DENGUE
VIRUS FROM THE DEMAND OF
PATIENTS WITH FEVER, SERVED IN
INSTITUTO EVANDRO CHAGAS
Vasconcelos, N.K.N.S.
Mosquitos infected with Dengue virus
(DENV – Flaviviridae, Flavivirus).
Antigenically, four serotypes can be
distinguished within DENV species (
DENV 1-4) and are all known to cause
similar clinical presentations, ranging
from asymptomatic infection, dengue
fever, and also severe cases, such as
dengue hemorrhagie fever and dengue
shock syndrome (DHF/DSS). In order
to report the current DENV serotypes
circulations and their genotypes at the
metropolitan area of Belem (Pará state,
Brazil), we sought to isolate DENV
from human samples (blood and sera)
and genetically characterize them.
Human samples were derived from
patients admitted during 2010-2012
period by the Medical Care Sector from
Instituto Evandro Chagas (Ananindeua,
Pará, Brazil). Therefore, one hundred
samples from symptomatic patients
(up to the five day of ouset) were
included in the study. Ten pacient
of then were selected for nucleotide
sequencing, and phylogenetic analysis
238
Human Virology: HV
the describe the genotype circulating.
Moreover, information from all patients
were also analyzed regarding gender,
age, place of living, employment, and
clinical symptoms. Hence, we obtained
100% of DENV isolation confirmed by
immunofluorescence assay, from which
21% were DENV1, 41% DENV2, and 38%
DENV4. Afterwards, we hate partially
sequenced four DENV1, two DENV2,
and four DENV4 isolates. Phylogenetic
analysis revelead DENV1 strains as
belonging to genotype V (Americas/
Africa), whereas DENV2 strains were
considered as a distinguished lineage
within the American/Asian genotype,
and DENV4 strains were included into
genotype II. Altogether, we identified
the main circulating serotypes at the
metropolitan area of Belém as well as
indicated their genotypes that are more
likely to be infecting this population.
HV1140 - THREE-DIMENSIONAL
MODELS OF NS3 PROTEASE CONTAINING PRIMARY RESISTANCE
MUTATIONS TO PROTEASE INHIBITORS
FROM
CHRONICALLY
INFECTED HCV PATIENTS
Hoffmann, L., Da Silva, M.L., Ramos, J.A.,
Valentin, E.S., Ramos, A.L.A., VillelaNogueira, C.A., Urmenyi, T.P., Bisch,
P.M., Tanuri, A., Rondinelli, E., Silva, R.
1. Depto
Clínica
Médica,
Janeiro, HUCFF-UFRJ, R. Rodolpho
Paulo Rocco, 255, 9o andar, Serv
de Hepatologia, Ilha do Fundão,
RJ
2. Instituto Nacional para Pesquisa
Translacional , INCT-INPeTAm/
CNPq, Av. Carlos Chagas Filho,
373, CCS, bl G, sl G1-050, Ilha do
Fundão, RJ
3. Instituto Biofísica, Universidade
Federal do Rio de Janeiro, IBCCFUFRJ, Av. Carlos Chagas Filho,
373, CCS, bl G, sl G1-050, Ilha do
Fundão, RJ
4. Instituto Federal de Educação,
Ciência e Tecnologia do RJ, IFRJ,
Rua Pereira de Almeida, 88, Pça
da Bandeira, RJ
5. Instituto Biologia, Universidade
Federal do Rio de Janeiro, IBUFRJ, Av. Carlos Chagas Filho, 373,
CCS, bl A, sl 121, Ilha do Fundão,
RJ E-mail: [email protected]
New compounds are being considered
for anti-HCV therapy, mainly NS3/4A
protease inhibitors (PIs). Previously,
we have analyzed the genetic diversity
of HCV NS3 protease from patients
treated with peg-interferon (PEGIFN) and ribavirin (RBV). We have
positively correlated the outcome of the
treatment to some characteristics of
the predominant NS3 virus sequence.
Also we have found reported resistance
mutations to the PIs (telaprevir and
boceprevir) in 3 (4,4%) of the 68
patients, at least one of the mutations
V36L, T54S and V55A in naïve as well
as during treatment with PEG-IFN and
RBV. We aimed to identify possible
resistance mutations to new PIs by
means of three-dimensional (3D)
prediction models. The NS3 protease
region from HCV were amplified
and sequenced from patients serum
obtained before and during treatment
with PEG-IFN and RBV. The sequences
were analyzed using the softwares
Geneious version 4.7.5 and MEGA
version 4.1. The 3D NS3/4A models were
predicted by comparative molecular
modeling using software MODELLER
and analyzed using software PyMol.
The genetic diversity and phylogenetic
analysis were assessed using H77
as the reference sequence and the
comparative modeling was done using
2FM2 as the reference structure. In 3D
models we can observe some amino
acid alterations near Serine-139.
This is one of those amino acids from
protease active site suggesting that it
can interfere at PI and NS3/4A protease
active site ligation and consequently
interfering in treatment outcome. We
plan to investigate docking of these new
protease inhibitors in these predicted
models. This approach may suggest
which patients will not benefit from
treatment with these drugs. Moreover,
we are performing next generation
sequencing to assess viral quasispecies.
HV1142 - EVALUATION OF HAV
RAPID TEST FOR DIAGNOSIS AND
EPIDEMIOLOGICAL PURPOSES
Carneiro, C.S.C., Amado, L.A., Ribeiro,
C.R.A., de Paula, V.S.
Fundação Oswaldo Cruz, Fiocruz, Av.
Brasil, 4365 - Manguinhos, Rio de
Janeiro E-mail: wanessa_cristiny@
hotmail.com
Hepatitis A is an acute infectious
disease transmitted by fecal-oral route.
The diagnostic routine is performed by
detection of anti-HAV IgM antibodies
in serum samples through EIA
tests. To facilitate the diagnosis and
epidemiological studies, it would be
reasonable the use of a rapid test that is
ease of implementation, not requiring
the use of equipment and the result is
generated within minutes using a visual
reading. This feature would be relevant
to bypass many difficulties mainly in
regions with difficult access and in
outbreaks. The aim of this study was to
evaluate the accuracy of a commercial
Human Virology: HV
239
rapid test (SD Bioline HAV IgG/
IgM®) for diagnosis and prevalence of
hepatitis A. In this study 256 samples
were tested, 152 were serum samples
collected from patients (mean age
of 23 y/o) involved in a hepatitis A
outbreaks; and 111 were plasma
samples obtained from whole-blood
donors used to establish the accuracy
of the rapid test for epidemiological
study. Anti-HAV IgM and IgG were
detected in these samples by rapid test
and the results were compared with
EIA results (standard gold). Among
samples from outbreak, anti-HAV IgG
were detected using rapid tests and the
results were also compared with EIA
results. For anti-HAV IgM, the rapid test
showed a sensitivity of 95.5% (95% IC
0.84 to 0.99) and a specificity of 100%
(95% IC 0.54 to 1.0). While for antiHAV IgG it was observed a sensitivity
of 77.8% (95% IC 0.84 to 0.99) and a
specificity of 100% (95% IC 0.54 to
1.0). In the samples from blood donors
it was observed for anti-HAV IgG a
sensitivity of 18.3% (95% IC 0.087 to
0.32) and a specificity of 100% (95%
IC 1.0 to 0.88). The high sensitivity and
specificity achieved in the diagnosis
of acute hepatitis A (IgM anti-HAV)
by rapid test suggest that this assay
could be used as an alternative tool for
diagnosis of this infection and that it is
especially applicable during outbreaks.
However, the low sensitivity and
specificity detected for anti-HAV IgG
indicates that it is not appropriate test
for prevalence studies of HAV.
HV1144 - REPORTED DENGUE CASES
IN THE CITY OF CARUARU-PE, FROM
2001 TO 2010
Albuquerque, A.C.C., Bezerra, J.S.L.,
Silva, M.R., Paiva, M.H.S.
240
Human Virology: HV
1. Faculdade
Associação
Caruaruense do Ensino Superior,
ASCES, Av. Europa, n. 584 Bairro
Universitário Caruaru-PE
2. Centro de Pesquisa Aggeu
Magalhães, Fiocruz., CPqAM,
Universidade
Federal
de
Pernambuco - Recife-PE E-mail:
[email protected]
Background: Dengue is currently the
most important arboviral disease that
affects humans and is a major public
health problem worldwide. Objective:
To evaluate epidemiological data on
dengue in the city of Caruaru-PE, from
2001 to 2010. Methods: Data regarding
reported and confirmed dengue cases
from 2001 to 2010 were obtained
from the Municipal Health Department
of Caruaru-PE. Collected data were:
reported and confirmed dengue fever
cases, according to the month of
onset symptoms, age, sex and deaths
due to dengue. They were stored and
analyzed in Excel 2007 and results were
presented as graphs and tables. Results:
The most problematic years regarding
dengue cases, from the period 2001 to
2010, in the City of Caruaru-PE, were:
2002, 2003, 2007 and 2010. Regarding
these four years ago, the number
of reported cases exceeded 34.4%,
28.7%, 73% and 81.5% of confirmed
cases. Females and aged 30 years or
older were the most committed. In the
study period there were nine deaths
due to dengue, in which five occurred
in 2007. Conclusions: The years 2002,
2003, 2007 and 2010 were years
of epidemic disease in Caruaru-PE,
however in 2002 there was a greater
number of confirmed cases of the
disease. The months from February to
May were when most occurring signs
and symptoms of the disease, probably
due to more rainfall. In Caruaru, despite
several educational campaigns to
eradicate the Aedes aegypti mosquito,
dengue cases are still observed in the
city because it is an endemic disease
in the region. For dengue control,
epidemiological surveillance should
be strengthened, increasing the
predictive ability of risk factors and
early detection of disease outbreaks as
well as improve the quality of actions
against the vector. Financial support:
Secretaria de Saúde de Caruaru-PE
HV1145 - MOLECULAR EPIDEMIOLOGY OF HIV-1 AND HIV-2 EPIDEMICS
IN CAPE VERDE
Araújo, I.I.M.P., Guimarães, M.L., Bello,
G., Vicente, A.C.P., Morgado, M.G.
1. Universidade de Cabo Verde , UniCV, Praça António Lereno - Praia,
Santiago - Cabo Verde CP 379C
2. Instituto Oswaldo Cruz/Fiocruz,
LabAids - IOC/Fiocru, Av. Brasil,
4365 - Pav. Leonidas Deane Sl.
413 Manguinhos, Rio de Janeiro
- RJ
3. Instituto Oswaldo Cruz/Fiocruz,
Lab. Genética Molecu, Av. Brasil,
4365 - Pav. Leonidas Deane Sl.
607 Manguinhos, Rio de Janeiro
- RJ E-mail: isabel.araujo@ioc.
fiocruz.br
Cape Verde is an archipelago of 10
islands located in Western Africa.
Serological studies indicate the cocirculation of both types of HIV in Cape
Verde, with a predominance of HIV-2 to
the mid-90s, to later be overcomed by
HIV-1. The objective of this study was
to characterize the molecular profile
of the epidemics and identify the
predominant HIV-1 and HIV-2 subtypes
circulating in Cape Verde. A total of 89
HIV-positive samples (HIV-1 = 60, HIV2 = 29) from patients living in Cape
Verde collected from 2004 to 2011
were analyzed. Molecular subtyping
was based on the pol (PR/RT) region
and subtype determination was
performed by phylogenetic inferences
and bootscan analysis. Among the 60
HIV-1 patients, 70% were female and
28% were male, the mean age of this
group was 37 years (SD=15,4 years).
Among the 29 HIV-2 patients, 62%
were female and 34% male, the mean
age was 49 years (SD=16,1 years). On
the basis of pol sequence analysis, the
60 HIV-1-positive specimens were
classified as subtypes G (35.0%),
CRF02_AG (33.3%), A (8.3%), F1
(6.7%), URF_CRF02/CRF06 (6.7%), B
(3.3%), C (3.3%), CRF05_DF (1,7%) and
CRF49_cpx (1,7%). All HIV-2-positive
specimens belonged to subtype A.
According to this analysis, the HIV
epidemic in Cape Verde is dominated
by HIV-1 subtypes G, CRF02_AG and
A, and HIV-2 subtype A, similar to the
molecular epidemiologic scenario
observed in some West African
countries.
HV1164 - SEQUENCE ANALYSIS OF
HEPATITIS C VIRUS AND ITS IMPLICATION TO THE MOTHER-TO-CHILD
TRANSMISSION
Dias, T.T., Passini, S.S.S., Zarife, M.A.S.,
Reis, M.G., Silva, L.K.
1. Gonçalo Moniz Research Center
(Fiocruz-BA),
CPqGM/FiocruzBA, Rua Waldemar Falcão, 121,
Candeal - Salvador/BA CEP:
40296-710
2. Professor
Magalhães
José
Maria
de
Netto
Reference
Human Virology: HV
241
Maternity , MRPJMMN, Rua
Marquês de Maricá, s/nº. Pau
Miúdo. Salvador/BA. CEP: 40320350
3. Central Public Health State
Laboratory,
Lacen-BA,
Rua
Waldemar Falcão, 123 – Candeal
– Salvador/Bahia. CEP: 40296710
4. Northeast
Biotechnology
Network, Renorbio, Av. Paranjana,
1.700 - Campus do Itaperi. UECE.
Fortaleza/CE. CEP: 60740-000
E-mail: tamitdias@gmail
Mother-to-child transmission (MTCT)
is the main pathway of hepatitis C
virus (HCV) infection in children. The
aim of this study was to evaluate the
molecular basis of the HCV MTCT. A
cross-sectional study was conducted
in the Prof. Jose Maria Magalhaes
Netto Reference Maternity Hospital,
Salvador-BA and blood from mother
and from the umbilical cord was
screened using an anti-HCV rapid test.
HCV-RNA detection was performed
for diagnostic confirmation. Different
subgenomic regions of the HCV (5UTR,
E1/HVR1, E2) were amplified for
sequence analysis. From a total of 3,254
screened pregnant women, six (0.18%)
tested HCV-RNA detectable in the
serum (Amplicor, Roche). MTCT was
identified in two newborns (33.3%),
showing evidence of HCV intrauterine
transmission. This was confirmed
through phylogenetic analyses. Viral
sequences from mother and newborn
showed high similarity between each
other in all regions, including the
E1/HVR1. It was also shown a high
similarity between the isolates from
the two mothers who transmitted
the HCV to their newborns. This may
242
Human Virology: HV
indicate that there are variations within
the virus genome associated to MTCT.
Identification of common viral epitopes
could lead to the identification of
vaccine-candidate antigens and to the
development of neutralizing antibodies
and therefore prevent transmission.
Financial support: CAPES, PRONEX
PNX0017/2009.
HV1167 - SURVEILLANCE AND MONITORING OF THE CIRCULATION OF
INFLUENZA STRAINS RESISTANT TO
ANTIVIRALS IN BRAZIL FROM 2011
TO 2012
Resende, P.C., Fintelman-Rodrigues, N.,
Mesquita, M., Machado, V., Motta, F.C.,
Oliveira, M.L.A., Pinhão, A.T., Gregianini,
T.S., Cury, A.L.F., Fernandes, S.B., Souza,
T.M., Siqueira, M.M.
1. Instituto Oswaldo Cruz - Fundação
Oswaldo Cruz, IOC - FIOCRUZ, Av.
Brasil, 4365 Manguinhos, Rio de
Janeiro, RJ - Pav. HPP - B105 CEP:
21040-360
Pública do Rio Grande do Sul,
LACEN-RS, Av.Ipiranga, 5.400 –
Bairro Jardim Botânico – Porto
Alegre/RS CEP 90610-000
Pública de Minas Gerais, LACENMG, Rua Conde P. Carneiro, 80,
Gameleira, Belo Horizonte MG FUNED - CEP: 30510-010
Pública de Santa Catarina, LACENSC, Rua Felipe Schmidt, 788,
Centro, Florianópolis, SC. CEP:
88010-002 E-mail: paolabmrj@
gmail.com
Antiviral drugs represent an important
line of defense against influenza. As
virtually all current circulating strains
of influenza are resistant to M2 proton
channel blockers, neuraminidase
inhibitors (NAI) represent the main
class of drug in clinical use. The NAI
oseltamivir (OST) has been widely
used since the 2009 pandemics, and
increasing reports of OST resistance
have been registered thereafter. In
Brazil, we have been studying influenza
virus in a population in which resistance
was likely to emerge, individuals
that underwent hospitalization, that
are immunocompromised or have
comorbidities. In our previous letter,
we have described that OST resistant
strains of pandemic influenza A virus
(H1N1pdm09) were not detected in
2009-2010 – suggesting that if this
variant was circulating in our country,
it would be at very low frequency.
Here, we describe our continuous
surveillance for NAI resistant strains of
H1N1pdm09 southern, southeastern,
northeastern and north regions of
Brazil from 2011 (n = 142) to 2012 (n =
100). And the large majority of samples
were from southern (82,39 % in 2011
and 76,25 % in 2012) region of Brazil.
Neuraminidase gene was sequenced
by Sanger or by pyrosequencing for
residues associated with NAI resistance
or decreased sensitivity. Viruses were
also isolated in cell culture to perform
functional assays of NAI resistance. We
found that the vast majority of samples
were sensitive to OST, with an median
IC50 value of 2,23 ±1,2 nM. However,
in states at southern region of Brazil
in which influenza circulation is more
intense due to their geographical
localization at a temperate region, we
were able to detect H1N1pdm09 virus
with decreased sensitivity or resistant
to OST. In 2011 at the RS state, we found
1 virus carrying the S247N mutation,
and other 25 sequences with the V106I
mutation, specially the former change
has been associated with decreased
sensitivity to OST in H1N1pdm09 virus.
More recently, in 2012 at the SC state,
we detected a sample with the SNP
associated to OST resistance, H275Y.
Due to the confirmation of a resistant
virus, according to the WHO criteria,
we investigated retrospectively, in
samples not tested for OST resistance,
the presence of the H275Y mutation.
Indeed, we detected in 2009 at the
RS state another H275Y mutation.
Altogether, this report expands our
laboratory-based surveillance for NAI
resistance and confirms low frequency
of OST-resistant virus detected in
throughout the world.
HV1176 - COMPARATIVE ANALYSIS
BETWEEN TWO MOLECULAR ASSAYS
FOR THE DIAGNOSIS OF ACUTE
DENGUE IN BRAZIL
de Meneses, M.D.F., de Souza, L.M.,
Veiga, C.S.B., Campos, R.M., Fernandes,
C.A., de Albuquerque, J.P., De Aguiar,
S.F., Ferreira, D.F.
Janeiro, Rio de Janeiro, UFRJ,
Cidade Universitária, Rio de
Janeiro, RJ
Janeiro, Rio de Janeiro/Macaé,
UFRJ/Macaé, Departamento de
Ensino, Universidade Federal do
Rio de Janeiro, Macaé Laboratório
Central de Saúde Pública Noel
Nutels, LACEN, Rua do Resende,
118, Rio de Janeiro E-mail:
[email protected]
Dengue virus (DENV) belongs to the
genus Flavivirus, family Flaviviridae,
and is represented by four distinct
Human Virology: HV
243
serotypes – DENV-1, 2, 3 and 4. The
virus is associated with outbreaks of
Dengue Fever, which is an important
urban disease and became a major
public health issue in many tropical
Countries in the world. Currently, the
World Health Organization estimates
that 50-100 million people are infected
annually with DENV worldwide with
an estimated 5% cases of dengue
hemorrhagic fever (DHF) and 0,5%
deaths occurring. The laboratory
confirmation of dengue infection is
mainly performed by virus isolation,
IgM anti-NS1 antibodies (serology)
and viral genome detection (RTPCR). Virus isolation, despite being
the gold standard methodology, has
a low sensitivity and high cost. The
techniques currently used for the
typing methodology of DENV at the
Public Health Central Laboratory of
Rio de Janeiro (LACEN-RJ) are virus
isolation and conventional RT- PCR for
the envelope glycoprotein. In this study,
we intended to test a new commercial
kit, the SimplexaTM Dengue Kit (Focus
Diagnostics) developed for use in a Realtime RT-PCR platform (3M Integrated
Cycler), and compare the results to the
methods used at the LACEN-RJ. All the
samples tested were from LACEN’s sera
collection. The sera were previously
screened using an ELISA NS1 Test
(Panbio) and all positive samples that
were negative by RT-PCR methodology
were selected. We analyzed 47 samples
and observed that SimplexaTM Dengue
Kit was able to detect 34 samples
(72%), typifying 5 samples as DENV1 and 29 as DENV-4. We conclude that
the Real-time RT-PCR tested in this
study was successful in detecting and
typifying samples that were negative
for the current methodologies used.
The data reinforces the use of Real
244
Human Virology: HV
Time technology for more accurate
detection and typing of DENV in the
Reference Center. Financial support:
CAPES, FAPERJ, CNPq, INBEB
HV1179 - PREVALENCE AND VACCINATION AGAINST HEPATITIS B
AMONG FEMALE SEX WORKERS IN
GOIÂNIA, CENTRAL BRAZIL
Matos, M.A., Caetano, K.A.A., França,
D.D.S., Moraes, L.C. , Souza, D.H.R.,
Pinheiro, R.S. , Araújo, G.C., Silva, A.M.C.,
Carneiro, M.A.S., Martins, R.M.B., Teles,
S.A.
1. Faculdade
de
Enfermagem/
UFG, FEN/UFG, S. Universitário,
Pça
Universitária
Hospital
das Clínicas/UFG, HC/UFG, S.
Universitário, Pça Universitária
Secretaria Municipal de Saúde de
Goiânia-GO, SMS-GO, Secretaria
Municipal de Saúde de Jataí-GO,
SMS- Jataí-GO, Jataí-GO
2. Laboratório de Virologia /IPTSP/
UFG, IPTSP-UFG, S. Universitário,
Pça
Universitária
E-mail:
[email protected]
Female sex workers (FSW) are at
high risk for hepatitis B virus (HBV)
infection, that in turn has been cause of
acute and chronic hepatitis, cirrhosis
and liver cancer. In Brazil, there is few
data on HBV infection among FSW.
Thus the present study estimated the
hepatitis B virus infection prevalence,
compliance with and response to
hepatitis B vaccine among female sex
workers in Goiânia-GO, Central Brazil.
Between May 2009 and June 2010, a
total of 402 women were interviewed
and tested for detection of HBV
markers (HBsAg, anti-HBc total and
anti-HBs) by ELISA (Hepanostika Uniform Organon Téknika B.V., Boxtel,
Holland). Hepatitis B vaccination was
offered to FSW found susceptible for
HBV infection, and the vaccine doses
were administered at their workplace.
An overall HBV infection prevalence
of 16.4% was found: 4 (1.0%) showed
positivity to HBsAg, 65 (16.2%) to
anti-HBc, and 164 (40.8%) to antiHBs. 109 (27.1%) women showed
positivity to anti-HBs only, suggesting
previous hepatitis B vaccination. 227
FSWs (54.5%) were susceptible for
hepatitis B, and 170 (75%) accepted
the first vaccine dose. The second and
third vaccine doses were administered
in only 97 (57%) and 68 (40%) FSW,
respectively. In 60 women, blood
samples were available for quantitative
detection of anti-HBs. Of them, 88.3%
developed protective titers of anti HBs.
The geometric mean titers of antiHBs was 256.4 mIU/mL. These results
reinforce the importance of Public
Health programs including health
education, health promotion. The low
frequency of FSWs immunized and the
low compliance with HBV vaccination
highlight the need for strategies aimed
to reach this population at high risk.
Financial support: CNPQ
HV1181 - IDENTIFICATION OF CIRCULATING DENGUE VIRUS TYPE 4
(DENV-4) IN RIO DE JANEIRO, BRAZIL
AS A RESULT OF THE SURVEILLANCE
SYSTEM
Campos, R.M., Veiga, C.S.B., de Souza,
L.M., de Meneses, M.D.F., da Silva,
K.N., Fernandes C.A., Neto, J.B.A., de
Albuquerque, J.P., Tanuri, A., de Aguiar,
S.F., Marinelli, R.S., Ferreira, D.F.
Janeiro, Rio de Janeiro, UFRJ,
Cidade Universitária, Rio de
Janeiro, RJ
Janeiro, Rio de Janeiro/Macaé,
UFRJ/Macaé, Departamento de
Ensino, Universidade Federal do
Rio de Janeiro, Macaé Laboratório
Central de Saúde Pública Noel
Nutels, LACEN, Rua do Resende,
118, Rio de Janeiro E-mail:
[email protected]
In Brazil, DENV-4 was first detected
during a short period in 1982 in
the Amazon region and again only
in 2008, in three patients without
any traveling history. At the Rio de
Janeiro State, DENV-4 was detected in
2011, during an outbreak of DENV-1
in Niterói City. Since the first reports
about the circulation of DENV-4 in
Rio de Janeiro, surveillance systems
have supported the implementation
of laboratorial methodologies capable
of quickly characterizing the four
dengue serotypes. According to the
surveillance program, 10% of the
samples from patients presenting fever
for 5 days or less are directed to the
State Central Laboratory (LACEN-RJ)
for serological tests for the detection of
NS1 Dengue virus protein, anti-Dengue
IgM, virus isolation in C6/36 cells and
serotype identification by RT-PCR. As a
result of this surveillance, the first case
of DENV-4 was detected in the City of
Rio de Janeiro. The patient went to the
hospital one day after the symptoms
started in December 2011. The second
case, about 26 Km distance from the
first case, was attended with three days
after the symptoms initiated and the
sample was sent to LACEN in January
2012. The two cases were positive
for NS1 ELISA, and both RT-PCR and
RT-PCR Real Time identified DENV4. The nucleotide sequencing of the
structural polyprotein was performed
for both samples. The two sequences
Human Virology: HV
245
had 99,3% homology, and also
presented homology superior to 99%
when compared to samples isolated
from Roraima/2010, São Paulo/2011,
Colombia and Venezuela in 2005, 2006
and 2007. This data suggests that the
DENV-4 virus detected in the City of
Rio de Janeiro is related to the virus
that is circulating in Latin America.
It is also important to note that the
screening methodology based on NS1
detection for sentinel samples followed
by PCR typing allowed an increase
in sensitivity of virus isolation and
serotype identification in the scenario
of DENV-1 co-circulation. Financial
support: CAPES, FAPERJ, CNPq, INBEB
HV1182 - INCIDENCE OF NOROVIRUS,
ASTROVIRUS AND ADENOVIRUS
INFECTION
AMONG
CHILDREN
WITH ACUTE GASTROENTERITIS IN
PORTO VELHO-RONDONIA, BRAZIL
Amaral, M.S.
CEPEM/Fiocruz Rondônia, Fiocruz,
Av.Guaporé 215, Bairro Lagoa, Porto
Velho/RO E-mail: sandra.amarall@
hotmail.com
Acute gastroenteritis is a common
disorder in young children. It is
associated with dehydration, a
leading cause of hospital admissions
in industrialized nations and a major
source of mortality in developing
countries. Enteric viruses have been
identified as the most significant
etiological agents of the disease,
with four categories of viruses being
considered clinically relevant: Group
A rotavirus, norovirus, adenovirus,
and astrovirus. With the exception
of Rotavirus, whose importance has
been well established in the medical
community due to its high prevalence
and worldwide impact, little is known
246
Human Virology: HV
about the epidemiology of the other
three groups of viruses. This study
aimed to determine the incidence of
infection the norovirus, adenovirus
and astrovirus in children 0-6 years of
age admitted with acute gastroenteritis
to a public children's hospital in the
city of Porto Velho, Rondonia. Between
the periods of February 2010 and
February 2012 a total of 591 stool
samples from children were analyzed
for the presence of adenovirus type F
40/41, using the enzyme immunoassay
(ELISA) and confirmed by PCR
methodology. Detection of norovirus
and astrovirus was accomplished by
RT-PCR using primers specific for
each viral type. There were 98 cases
(98/591) of enterovirus infection
detected, with infection rates of 8.9%
(53/591), 5.2% (31/591) and 2.3%
(14/591) for norovirus, astrovirus,
and the adenovirus respectivelly.
9.1% of the children were co-infected
with norovirus and astrovirus. There
was a higher incidence of infection in
children ages 0-24 months. All children
had typical symptoms associated
with enteroviral infection, including
diarrhea, vomiting and fever. Bloody
stool was found in 16.9% (6/31) of the
norovirus infected children, in 19.3%
(6/31) of astrovirus infecion and in
21.4% (3/14) of adenovirus infection.
Viral infection was detected mainly
in the months of February through
May, the period corresponding to the
rainy season in Porto Velho. A higher
incidence of norovirus was detected in
the periods of March and April of 2010
with 20.4% (20/98) of cases, with
lower rates in the subsequent year. The
data presented here may contribute to
a better understanding of the role of
enteroviral infection in the pediatrics
population of Porto Velho, Rondonia
and may be important in the strategic
planning of control of the disease in
this region. Financial support: Instituto
Oswaldo Cruz-Fiocru-RO and CNPq
HV1184 - EXPRESSION OF VARIABLE
AND CONSERVED REGIONS OF
CAPSID PROTEIN OF NOROVIRUS
GII/4
Oliveira, L.M., Nagata, T.
Universidade de Brasília, UnB,
Campus Darcy Ribeiro E-mail:
[email protected]
Norovirus genome consists of a
single-stranded RNA which encodes
six non-structural and two structural
proteins. The capsid protein (VP1)
gene contains variable and conserved
region in its sequence. This study
proposed the production of antibody
of the variable and conserved regions
of VP1 of norovirus GII/4 through the
prokaryotic expression system. Protein
expression in E.coli system of both
regions was confirmed by Western
blotting which showed high expression
level of both after induction of 2 hours
with IPTG. By solubility test the variable
region was shown to be insoluble, hence
denaturing condition was used for
purification. These results confirmed
that the system chosen for expression
and purification was effective and it
was expected for the development
of immunoassay using these partial
capsid proteins. Financial Support:
Cordenação de Aperfeiçoamento de
Pessoal de Nível Superior-CAPES
HV1185 - A RETROSPECTIVE SEROLOGICAL SURVEY OF HANTAVIRUS
INFECTIONS IN THE COUNTY OF
CÁSSIA DOS COQUEIROS, STATE OF
SÃO PAULO, BRAZIL
Badra, S.J., Maia, F.G.M., Moreira,
E.A., Figueiredo, G.G., Junior, G.S.S.,
Figueiredo, L.T.M., Passos, A.D.C.
Universidade de São Paulo, USP,
Centro de Pesquisa em Virologia,
Faculdade de Medicina de Ribeirão
Preto E-mail: [email protected]
In recent years, hantavirus infections
producing severe diseases have
obtained an increased attention from
public health authorities from the
countries of Eurasia to the Americas.
Brazil has reported 1,300 cases
of
hantavirus
cardiopulmonary
syndrome (HCPS) from 1993 to 2010,
with about 80 of them occurring in
the northeast of the State of São Paulo,
with 48% fatality rate. Araraquara
virus was the causative agent of
HCPS in the region. Considering that
hantaviruses causing human disease
in the Americas were unknown until
1993, we have looked for hantavirus
infections in the population of Cássia
dos Coqueiros county, northeast of
the State of São Paulo, Brazil, before
this time. This county has about 2,800
inhabitants and an economy based
on agriculture, including cultivation
of Brachiaria decumbens grass. The
grass seeds are an important rodent
attraction, facilitating transmission of
hantavirus to man. Four HCPS cases
were reported so far in the county. In
this study, 1,876 sera collected from
1987 to 1990 were tested for IgG to
hantavirus by IgG-ELISA, using the N
recombinant protein of Araraquara
virus as antigen. Positive results
were observed in 89 (4.7%) samples,
which were all collected in 1987. The
positivity among urban inhabitants
was 5.3%, compared with 4.3% among
those living in rural areas. Our results
showed that hantavirus infections
Human Virology: HV
247
occurred in Cássia dos Coqueiros,
completely unrecognized, even before
hantaviruses were described in the
Americas.
HV1187 - LABORATORY DIAGNOSIS
BY PCR OF INFECTION CAUSED BY
ORTHOPOXVIRUS IN BRAZILIAN
SAMPLES IN THE PERIOD OF 1999
TO 2011
Gomes, G.M., Lancelloti, S.R., BarretoVieira, D.F., Barth, O.M.
br
The Poxviruses are one of the most
feared virus in the history of virology.
The Poxviridae family comprises
viruses that infect mammals, insects
and brids. Among the genus belonging
to this family four have the capacity
to infect humans. The Orthopoxvirus
wich the Smallpoxvirus, the virus
Vaccinia (used in vaccine production),
Monkeypox, Cowpox, among others,
is the most important genus related
to diseases in humans and animals
(Oliveira, 1994). The Poxviruses are
one of the largest viruses already
described. The viral particles can reach
about 400 nm in length. These viruses
have a complex morphology; the
genome comprising a DNA molecule
of linear single-stranded helix, and
the conserved sequences stored in
the central region (in Orthopoxvirus)
(Schatzmayr & Barth 2005). This
work was carried out with samples
from several Brazilian states received
by de Laboratório de Morfologia e
Morfogênese Viral during a period
of 12 years (1999 – 2011). A total of
104 samples (human and bovine)
were subjected to the technique of
polymerase chain reaction (PCR) for
detection of Orthopoxvirus infection,
248
Human Virology: HV
71% of bovine samples (74 samples)
and 29% of human samples (30
samples). Among the bovine samples
were obtained 46% of positivity (34
samples) while in human samples
while the percentage positive was 77%
(23 samples). According to the data
analyzed during the entire period of
study we concluded that the number
of humans and animals infected with
poxviruses has become constant. Thus
we emphasize the need to use a rapid
and accurate method of diagnosis,
and that allows the identification and
classification of infectious agents
reliably, as well as its use as an
epidemiological monitoring tool of
viral dissipation in our country.
HV1189 - NORWALK VIRUS LIKE
PARTICLES ASSEMBLY IN PLANTS
USING
VIRAL
SUPPRESSOR
PROTEINS OF POSTTRANSCRIPTIONAL GENE SILENCING
Souza, A.C., Bonnet, R.M.V., Inoue
Nagata, A.K., Lacorte, C., Nagata, T.
1. Universidade de Brasília, UNB,
Departamento
de
Biologia
Celular,
Laboratório
de
Microscopia Eletrônica.
UCB, Campus Avançado Asa Norte
SGAN 916 Módulo B B Avenida
W5 - CEP: 70790-160
3. Bras Embrapa Hortaliças, CNPH,
Km 09 BR 060, Rodovia BrasíliaAnápolis, Caixa Postal 218, CEP
70359-970
4. Embrapa Recursos Genéticos
e Biotecnologia, CENARGEN,
Parque Estação Biológica PqEB - Av. W5 Norte (final) , CEP
70770-917 Brasília E-mail: ana.
[email protected]
The virus like particles (VLPs) of
norovirus (NV) is mainly produced
by baculovirus expression system,
however, this system is expensive as
other cell culture systems. Aiming
to use alternative system with high
thoughput quality but reduced cost,
VLPs of NV was expressed using plants
in transient manner. The transient
expression of NV VLP in plant using
binary vector has the disadvantage
of low yields by RNAi machinery. To
improve the production efficiency, the
co-expression of plant virus suppressor
proteins of post-transcriptional gene
silencing (PTGS) was applied. Three
candidates of PTGS suppressors:
126K of Pepper mild mottle virus
(PMMov), Hc-Pro of Brugmansia
suaveolens mottle virus (BsMoV) and
AC2 of Tomato severe rugose virus
(ToSRV), were co-expressed together
with green fluorescent protein (GFP)
using binary vector (pGreenII)Agrobacterium infiltration system and
evaluated their increasing abilities of
GFP accumulation in plant. The 126K
protein of PMMoV showed better
GFP accumulation effect, and it was
selected to NV VLP production. The NV
capsid (CP) was also cloned in binary
vector pBINPLUS and Agro-infiltrated
with the construct of 126K of PMMoV
in Nicotiana benthamiana. Three days
after the infiltration, the leaves were
harvested and VLPs were purified
with sucrose gradient. The NV CP
expression was confirmed by Western
Blotting and the successful formation
of NV VLPs by transmission electron
microscopy.The antigenicity of these
VLP will be evaluated by injecting these
VLPs preparations in animal models in
further study.
HV1191 - LOOP MEDIATED ISOTHERMAL AMPLIFICATION (LAMP)
FOR DETECTION OF MAYARO VIRUS
Souza, V.C., Nascimento, V.A., Silva,
G.A.V., Naveca, F.G.
Instituto Leônidas e Maria Deane/
Fiocruz Amazônia, ILMD/FIOCRUZ,
Rua Teresina, 476, Adrianópolis,
Manaus, AM, Brasil E-mail: victor@
amazonia.fiocruz.br
The molecular diagnostic is dominated
by
polymerase
chain
reaction
(PCR), although new techniques are
emerging, such as Loop-mediated
Isothermal Amplification (LAMP). This
method does not require sophisticated
equipment for thermal cycling or
detection process, since the reaction
occurs on isothermal conditions, usually
carried out in 1 hour. The positive
reaction can be identified directly in a
tube by naked eye, thus reducing the
risk of cross-contamination. Mayaro
virus (MAYV) is an RNA genome
arbovirus which belongs to Alphavirus
genus (Togaviridae family) that can
cause a febrile syndrome in humans,
characterized by rash and severe
arthralgia, which can persist for months,
being more disabling than Dengue. The
virus is endemic to the Amazon region,
where it can be detected in patients,
vectors and animal reservoirs. The
aim of this study was to evaluate the
applicability of a methodology based
on LAMP to detect the MAYV genome.
All GenBank available MAYV complete
sequences were used in an alignment
for LAMP target selection. In order
to standardize the initial reaction
conditions a synthetic control was
constructed carrying the selected
target downstream of a T7 RNA
polymerase promoter site. The limit of
detection for the MAYV LAMP method
Human Virology: HV
249
was determined by testing serially
10-fold diluted copies of the synthetic
control. Each dilution point was
double-checked for accuracy by a MAYV
real-time PCR previously developed
by our group. Under the conditions
tested so far, and with regards to
detection of the amplification products
by color change or electrophoresis on
agarose gels, stained with Gel Red and
observation under UV light, our results
demonstrated a detection sensitivity
about 103 copies per reaction. These
indicate that the LAMP technique can
be a useful tool for molecular diagnosis
of MAYV virus, especially in resourcepoor settings. Financial support: PPSUS
/ FAPEAM
HV1192 - MULTIPLEX REAL-TIME
PCR FOR QUANTIFICATION OF
EPSTEIN-BARR VIRUS, CYTOMEGALOMAVIRUS AND PARVOVIRUS B19
IN HUMANS
Silva, G.A.V., Souza, V.C., Nascimento,
V.A., Passos, L.F.S., Naveca, F.G.
Fiocruz Amazônia , FIOCRUZ/
ILMD - AM, Rua Terezina, 476.
Adrianópolis. Manaus - AM. CEP:
69057-070
2. Hospital Universitário Getúlio
Vargas, HUGV, Av. Apurinã, N
4, Praça 14 de Janeiro, CEP:
69020-170, Manaus - AM E-mail:
[email protected]
Infections
caused
by
Cytomegalomavirus are involved in
complications with opportunistic
infections, as Epstein-Barr virus, which
may began indirect effects on host
immune system in immunosuppressed
patients, resulting an ample range of
pathologies and clinical manifestations.
250
Human Virology: HV
As well as, the Human parvovirus B19
can cause asymptomatic infections. We
develop a multiplexed real-time PCR
with high sensitivity and specificity to
Epstein-Barr, Human parvovirus B19
and Citomegalomavírus viruses in
humans. The sequences of each virus
were selected from GenBank database
and aligned for each target with
purpose to select conserved regions
for primers and probe design. The
selected sequences were then used
for synthese a plasmid containing the
chosen targets as an insert and was
use as positive control encompassing
both virus selected target regions.
Due the assay developed to detection
in humans, we also insert the target
B-Actin as internal control positive.
We calculate the DNA concentration
based on size and amplicon sequence
used for knowledge of the exact copy
numbers of targets. The TaqMan
Fast master mix was used in all qPCR
amplifications with recommended
cycling parameters and a volume of 2µL
of template DNA in 20µL reaction, the
PCR were performed with amplification
efficiency approximately 101.18%
in mean. According to the standard
curve results and if considered Ct
<37 as positive, the assay has a limit
of detection for 2 copies (mean 36.5,
36.9 and 36.3 for EBV, B19V and CMV,
respectively), the internal control
B-Actin present in mean Ct = 36.1. We
report the development of a sensitive
and specific method for detection both
viruses in multiplex real-time PCR in
humans, the assay described here will
help as a tool for viruses detection and
assisting in the control other diseases,
as autoimmune diseases, as well as, in
cases of coinfection. Finacial Support
Fundação de Amparo à Pesquisa
do Estado do Amazonas, Conselho
Nacional de Desenvolvimento Científico
e Tecnológico and Instituto Leônidas e
Maria Deane Fiocruz Amazônia.
HV1193 - ANALYSIS OF THE
EFFICIENCY OF THE TECHNIQUES IMMUNOFLUORESCENCE, “IN HOUSE”
RT-PCR AND REAL TIME RT-PCR FOR
DETECTION OF IN HUMAN PARAINFLUENZA VIRUS
Parmezan, S.N., Camargo, C., Guatura,
S.B., Watanabe, A.S., Silva, E.R.M.
UNIVERSIDADE FEDERAL DE SÃO
PAULO, UNIFESP, R. Pedro de Toledo,
781. 15° andar - CEP 04039-032
E-mail: sheilaparmezan@hotmail.
com
Sensitive detection of respiratory
viruses is important for early diagnosis
of infection. Highly sensitive methods
are needed to detect respiratory virus
infections in patients with few or no
symptoms. Human Parainfluenza
Virus (HPIV) is a common cause
of viral infection in patients with
hematologic hematopoietic stem cell
transplantation (HSCT). The present
study analyzed the diagnostic methods
of immunofluorescence (DFA), “in
house” Reverse Transcription-PCR (RTPCR) and Real Time RT-PCR for HPIV
1, 2, 3 and 4 detection in patients with
acute respiratory symptoms attended
in a Sao Paulo tertiary hospital. Included
patients were those who had a clinical
picture of acute respiratory infection
or possibly asymptomatic patients who
have contact with patients presenting
with infection. These patients were
evaluated by an infectious disease
physician who came into contact with
the laboratory staff when there were
suspected cases. We studied 202
nasal washes from patients (mean
of 45 year of age, variation of 5 to 80
years) attended between March 2008
to December 2009 in a hematology
ward or outpatient HSCT of São Paulo
hospital. Among analyzed samples 4%
and 10% were positive for HPIV by “in
house” RT-PCR and real time RT-PCR,
respectively, all positive samples by “in
house” RT-PCR were positive in real
time RT-PCR. Imunofluorescence did
not resulted positive among samples.
Considering the real time RT-PCR as
gold standard sensitivity, specificity
and accuracy of the “in house” RT-PCR
method were 36% [95% confidence
interval (CI) 19,7 – 50,0], 100% (95% CI
97,9 – 100), 93% (95% CI 88,7 – 93,5),
respectively. This study retrospectively
examined the detection of HPIV for
three different diagnostic methods and
pointed to the need highly sensitive
methods to detect respiratory virus
infections in immunocompromised
patients for better understand the
impact of parainfluenza viruses among
high risk patients.
HV1194 - MOLECULAR CHARACTERIZATION OF BK AND JC
POLYOMAVIRUSES DETECTED IN
URINE SAMPLES OF RENAL TRANSPLANT PATIENTS AND HEALTHY
INDIVIDUALS FROM THE SOUTH OF
BRAZIL
Comerlato, J.
Grande do Sul, UFRGS, Rua
Sarmento Leite 500, Porto Alegre,
CEP 90050-170, Rio Grande do
Sul/ Brasil
2. Instituto de Pesquisa Veterinária
Desidério
Finamor,
IPVDF,
Eldorado do Sul, CEP 92990-000,
Rio Grande do Sul/Brasil
3. Universidade Feevale, Feevale, RS
Human Virology: HV
251
239 - Campus II, Novo Hamburgo,
CEP 93352-000, Rio Grande do
Sul/Brasil E-mail: jucomerlato@
gmail.com
The human polyomaviruses JC (JCV) and
BK (BKV) are widespread in the human
population. Following the primary
infection, viral reactivation may lead
to nephropathy and graft rejection in
renal transplant patients (RTPs). This
study was carried out to access the
presence and the phylogenetic analysis
of BKV and JCV DNA in urine samples
collected from RTPs and healthy
individuals in Porto Alegre, Rio Grande
do Sul. Ninety-two samples from RTPs
and 88 samples from control group
were collected and submitted to a
nested-PCR. Sequencing and molecular
characterization was performed of
selected positive samples. A significant
higher frequency of BKV was found
in RTPs (65.2%) in comparison to
the control group (32.9%). JCV was
equally detected in the RTPs (45.6%)
and in the control group (36.4%).
Phylogenetic analysis of both BKV and
JCV amplicons show that all subtypes
of BKV were found, whereas for JCV,
four different groups are described (1,
2, 3 and 4). BKV is most closely related
with RTPs than healthy individuals,
whereas for JCV this association is not
observed. The molecular detection
proposed by this article could be an
alternative tool to control of viruria in
RTPs preventing the graft lost through
the
immunosuppressive
therapy
modulation. The phylogenetic analysis
demonstrated circulation of different
JCV and BKV variants in the study
population which was described for
the first time in the South of Brazil.
HV1195 - HEPATITIS A OUTBREAK IN
A SEASIDE TOWN IN RIO DE JANEIRO
252
Human Virology: HV
STATE, MAY 2012
Mendes de Oliveira, J., Pinto, M.A.,
Miagostovich, M.P., Lewis, L.L., Melgaço,
J.G., de Paula, V.S., Gaspar, A.M.C.
1. INSTITUTO OSWALDO CRUZ/
FIOCRUZ, IOC/FIOCRUZ, AVENIDA
BRASIL 4365 Manguinhos Pavilhao Helio e Peggy Pereira
2. Universidade
Federal
Fluminense, UFF, Laboratório de
Desenvolvimento
Tecnológico
em Virologia, , Laboratório
de Virologia Comparada e
Ambiental, , Ambulatório de
Hepatites Virais, , E-mail: jackie@
ioc.fiocruz.br
The incidence of hepatitis A virus
(HAV) infection in Brazil has declined
in recent years, with a progressive
shift of the average age of infection
towards late childhood and adulthood.
Even though the global improvement
of sanitation levels in Brazil, HAV still
causes periodic outbreaks. In May
2012, approximately 150 cases of
acute hepatitis A were reported in
Mangaratiba, a seaside town in Rio
de Janeiro state. Contamination of
the public water supply was initially
suspected by local public health
care, which reported an increasing
number of acute hepatitis A cases
covering several localities at the First
District of the town. We carried out
investigations in order to characterize
the etiological agent, identify the
source of infection, and implement
appropriate control measures. Sera
from 63 symptomatic individuals and
86 asymptomatic contactants were
tested for detection of HAV antibodies
and RNA. Water samples, collected by
the local epidemiological surveillance
from 18 spots including public water
supply and alternative sources, had
no HAV detected by RT-PCR, after
concentration by the Katayama´s
method. Phylogenetic analysis of the
VP1/2A region of HAV genome linked
the outbreak to the IA HAV genotype,
which was detected in 22 (88%) out of
25 sera from a subset of anti-HAV IgM
positive patients. The detection of the
IB genotype in seven individuals (three
symptomatic and four asymptomatic,
without
epidemiological
link)
suggests the co-circulation of these
two subgenotypes of HAV during
the outbreak. The primary source of
transmission of the virus could not be
confirmed. However, the identification
of acute cases of HAV asymptomatic
infection (anti-HAV IgM negative,
HAV RNA positive) among household
contacts of patients with recent past
infection (anti-HAV IgM positive)
shows the secondary spread of the
virus during the outbreak. Financial
support: CNPq; IOC/Fiocruz.
HV1212 - DEVELOPMENT OF REAL
TIME PCR FOR QUANTIFICATION OF
HEPATITIS B VIRUS DNA (HBV DNA)
Portilho, M.M., Marques, V.A., do
Espírio-Santo, M.P., Lewis-Ximenes, L.L.,
Lampe, E., Villar, L.M.
Av. Brasil, 4365, Rio de Janeiro -RJ
Nowadays, commercial molecular
methods are available for Hepatitis
B virus DNA (HBV DNA), but these
methods are very expensive to
be introduced in limited resource
laboratories. This study aims to develop
a real time PCR for quantification of
HBV DNA among sera samples using
taqman methodology. A total of 62
sera samples (32 HBsAg reactive and
30 HBsAg not reactive) was employed
for validation of the method. DNA
was extracted using a commercial
kit (High Pure Viral Nucleic Acid kit,
Roche Diagnostics, USA) according
to manufacturer’s instructions. For
HBV DNA quantification, primers and
probes were constructed for surface
region of HBV genome (position 593
to 672 nt) and amplified by taqman
methodology using Icycler equipament
(Biorad). The standard curve was
constructed by cloning a sample from
HBV Quantification Panel (OptiQuant,
Acrometrix) that contains 2 x 107
copies HBV DNA/mL. A ten fold serial
dilution of this curve (viral load varying
from 10-1 to 10-10 copies per reaction)
was employed in five PCR runs in a total
of 40 reactions. After that, standard
curve and sample volume, and melting
temperature (TM) were evaluated.
As results, standard curve presented
linear regression coefficient of 0.997
and the method was able to detect until
20 copies of HBV DNA per reaction. A
good reproducibility and specificity
were observed based on coefficient
of variation and absence of signal in
negative controls, respectively. Best
reaction conditions were observed
when 1µL of standard curve, 5 µL of
DNA and TM of 62ºC were used. Using
optimized protocol, HBV DNA was
detected among 17 of 32 HBsAg reactive
samples giving a median viral load of
1.51 X 107 copies HBV DNA/mL. HBV
DNA was not detected among 30 HBsAg
not reactive samples. Homemade real
time PCR was effective for detection
and/or quantification of HBV DNA
and can be a tool for HBV diagnosis in
limited resource laboratories.
HV1215 - DIFFERENTIAL DIAGNOSIS
OF ACUTE RESPIRATORY DISEASE IN
Human Virology: HV
253
PATIENTS PRESENTING INFLUENZA
LIKE ILLNESS, BY REAL TIME PCR
Paiva, T.M., Paulino, R.S., Benega, M.A.,
Santos, K.O.C., Silva, D.B.B., Pereira, J.C.,
Silva, P.E., Borborema, S.E.T., Curti, S.P.,
Oliveira, M.I., Peret, T., Erdman, D.
1. Instituto Adolfo Lutz, IAL, Av. Dr.
Arnaldo 355, CEP 01246/902,
São Paulo, SP,
2. Brasil Centers for Disease Control
and Prevention, CDC, 1600 Clifton
Road, N. E. Atlanta, GA 30333
INTRODUCTION – The pandemic
influenza occurred due to the influenza
virus of type A (H1N1) pdm09 emerging
has changed the respiratory viruses
investigation scenarios. Taking into
account the impact of acute respiratory
diseases worldwide the development
of laboratory methodologies towards
to detect non influenza respiratory
viruses is urged globally. OBJECTIVE To improve the differential diagnosis
of acute respiratory disease towards
to provide public health answers
facing respiratory disease outbreaks.
MATERIAL AND METHODS - In
the present study we selected 108
respiratory secretions collected from
individuals
presenting
influenza
like syndrome by the three National
Influenza Network Sentinel Units
(two located in São Paulo city and
one in Guarulhos, SP) during JanuaryJune 2010. These samples has
already presented negative results
for influenza viruses type A and B,
adenovirus, respiratory syncytial virus,
parainfluenza viruses 1, 2, 3 by indirect
immunofluorescence (IF) the routine
assay to perform the differential
diagnosis of acute respiratory illness.
These negative samples were submitted
254
Human Virology: HV
to a non- influenza respiratory virus
test kit containing primers and probes
for adenovirus(ADV), rhinovirus (RVs),
respiratory syncytial virus (RSV),
human metapneumovirus (hMPV ),
parainfluenza 1, 2, 3(PV1, PV2, PV3)
by using the Real Time PCR protocol
developed at the Centers for Disease
Control and Prevention. RESULTS - Of the
108 samples collected from individuals
presenting acute respiratory illness
symptoms, presenting negative results,
49 (45,37%) were positive by Real Time
RT- PCR. The following etiologic agents
has been identified: RSV 16 (32.60%);
PV1 3(6.10%); PV2 6(12.30%); PV3
8(16.30%); ADV 10(20.40%); RVs
6(12.30%) . In addition two samples
has shown mix infection: ADV+RVs;
RSV+RVs.This methodology also enable
us to identify the etiology of acute
respiratory disease in individuals that
has been vaccinated with influenza
seasonal vaccine; in these individuals
rhinovirus and adenovirus infections
has been detected. DISCUSSION - This
study demonstrates the high sensibility
of Real Time RT-PCR to attend the
differential diagnosis; in addition to
provide quickly answer to the public
health authorities towards to apply
the control and prevention measures
timely. Financial support: Adolfo Lutz
Institute, Secretariat of Health of São
Paulo State, Ministry of Health of
Brazil, Centers for Disease Control and
Prevention, Atlanta, GA.
HV1219 - EVALUATION OF RAPID
TEST FOR DETECTION OF HEPATITIS
B VIRUS ANTIBODY (ANTI-HBS)
Medina, H.C., Miguel, J.C., Silva, E.F.,
Oliveira, J.C., Scalioni, L.P., de Paula,
V.S., do Ó, K.M., Milagres, F.A., LewisXimenes, L.L., Lampe, E., Villar, L.M.
Viral Hepatitis Laboratory, Oswaldo
Cruz
Institue,
LHV/FIOCRUZ,
Laboratory
of
Technological
Development in Virology, LDTV/
FIOCRUZ, Clementino Fraga Filho
Hospital, UFRJ, Federal University
of
Tocantis,
UFTO,
E-mail:
[email protected]
Being able to test for the presence
of immunity against hepatitis B
virus (HBV) could be important for
prevention of this disease. Rapid test
for antibodies against HBV (anti-HBs)
detection would be very useful for large
scale community studies, since there
is no need of special equipaments and
results can be release in few minutes. The
objective of this study was to evaluate
the performance of commercial rapid
test for anti-HBs detection in serum
samples of individuals from high and
low HBV endemicity. A total of 565
sera samples from individuals referred
to Viral Hepatitis Ambulatories from
Rio de Janeiro and healthy individuals
from North and Southeast region
of Brazil were included. Most of the
individuals were female (53%) and
mean age was 39.9 years (±18.9). AntiHBs was detected using commercial
enzyme immunoassay (EIA) (ETI-ABAK-3, Diasorin, Italy) and rapid test
(Imuno-Rápido anti-HBsAg, Wama,
Brazil). The same sample volume was
employed in both assays (100L)
whereas time of execution was 280
minutes to EIA and 20 minutes to rapid
test. As results, anti-HBs was detected
in 177 and 80 samples by EIA and
rapid test, respectively. On the other
hand, anti-HBs was not detected in
388 and 385 samples using EIA and
rapid test, respectively. These results
gave 82.3% of concordance, 99.2% of
specificity and 45.2% of sensitivity
of rapid test compared to EIA. When
sensitivity was evaluated only among
anti-HBs reactive samples comparing
antibody levels, sensitivity increased to
86% showing more concordant results
among samples presenting anti-HBs
titers higher than 100UI/mL. It is
concluded that rapid tests presented
high specificity in this population,
but the method was mainly sensitive
among samples presenting high antiHBs levels.
HV1221 - EVALUATION OF SEROPROTECTION AND SEROCONVERSION
OF HIV-1 POSITIVE INDIVIDUALS
VACCINATED WITH TRIVALENT
INACTIVATED INFLUENZA
Marttorelli, A., Fintelman-Rodrigues,
N., Sacramento, C. Q., Grinsztejn, B.,
Camacho, L., Santini-Oliveira, M., Souza,
T. M. L.,
Fundação
Oswaldo
Cruz,
FIOCRUZ,
Rua
Leopoldo
Bulhões,1480,Manguinhos.Pavilhão
HPP,sala B109B.Rio de Janeiro.
Individuals infected with human
immunodeficiency virus (HIV) have
an higher risk of being affected by
serious diseases, such as respiratory
virus
infections,
including
the
influenza virus. It has been described
controversial clinical outcomes from
these patients infected with pandemic
influenza virus infection. Although
the immunosuppression of these
individuals may affect their ability
to response to active immunization.
Vaccination against influenza still
represents the primary way of
reducing the impact of this virus.
Due to the circulation of pandemic
influenza virus A/H1N1, A/H3N2 and
B viruses, current vaccine composition
include antigens from these three
Human Virology: HV
255
agents in its formulation. Thus,
analysis of the impact of trivalent
inactivated influenza vaccine (TIV)
in HIV-1-infected individuals merits
further studies. A cohort of 119 HIV1-infected individuals with controlled
viremia received two single doses of
the TIV with 21 days interval. The antihemagglutinant titers of their sera was
evaluated at the baseline, 21 and 42
days after vaccination. About 61%, 70%
and 28% of individuals were already
protected for pandemic influenza A/
H1N1,A/H3N2 and B, respectively.
Relative to the seroconvertion after
vaccination, we found that at day 21 86,1
% of the individuals seroconverted for
A/H1N1 and that at day 42, it reached
93,6 % of seroconversion. In regard to
A/H3N2 rates of seroconversion were
80,3% at day 21 and 81,8% at day
42. Finally, with respect to Influenza
B, 67 % and 70,3% of the individuals
seroconverted 21 and 42 days after
vaccination, respectively. Altogether,
our results indicate that two doses of
the TIV were more effective after for
A/H1N1 virus. Further studies are
being carried out to investigate the
sustainability of the immune response.
HV1223 - GENOTYPING AND
MOLECULAR CHARACTERIZATION
OF STRUCTURAL GENES FROM
SPECIE A ROTAVIRUS CIRCULATING
IN NORTHERN BRAZIL BEFORE AND
AFTER ROTARIX® VACCINE INTRODUCTION
Farias, Y.N., Soares, L.S., Mascarenhas,
J.D.P., Gabbay, Y.B., Linhares, A.C., Leite,
J.P.G.
1. Laboratório
de
Virologia
Comparada e Ambiental, LVCA,
IOC/FIOCRUZ, Av. Brasil, 4365 Manguinhos - Rio de Janeiro.
256
Human Virology: HV
2. Laboratório de Rotavírus, Seção
de Virologia, SEVIR, SVS/MS,
Br 316 Km 07 S/N - Levilândia
- Ananindeua, Pará. E-mail:
[email protected]
Specie A Rotavirus (RVA) is the leading
cause of acute diarrhea in children
under 5 years old, responsible for
453.000 deaths each year. The RVA
genome is composed by 11 segments of
double-stranded RNA (dsRNA), which
encodes six structural (VP1-4, VP6-7)
and six nonstructural proteins (NSP16).Recently, was proposed a new
classification system based on wholegenome sequence analysis, providing
important information about the
genomic diversity of the RVA, such as
reassortment events and interspecies
transmission. This study aimed to
characterize genes that encode for six
VP’s from RVA genotypes G1, G2, G4
and G9 circulating in Northern Brazil,
before and after Rotarix® vaccine
introduction and has been approved by
the Committee in Ethics and Research
from the IEC. A total of 22 fecal
specimens were selected between 1994
and 2010 from children hospitalized
due to acute diarrhea, being 12
samples from pre and 10 post vaccine
period. The viral dsRNA was extracted,
reverse transcribed (RT), amplified
by polymerase chain reaction (PCR)
and the amplicons were sequenced.
The sequences obtained were aligned
using the BioEdit, compared to
sequences available in GenBank and at
phylogenetic analysis was performed
using MEGA software.Based on the
phylogenetic analyses, 45% (10/22)
of analyzed strains were classified
as G1P[8]; 36% (8/22) as G9P[8];
14% (3/22) as G2P4 and one sample
was classified as G4P[6]. The strain
HSE005 showed close relationship
with porcine origin prototype for the
VP3 and VP6 genes, presenting M1-I3
genotypes, respectively. The G1 and G9
strains were related with R1-C1-M1
genotypes, for the VP1, VP2 and VP3
genes, respectively; and G2 strains
were classified as R2-C2-M2. The
current work is one of the few studies
about molecular characterization of
all structural RVA genes in Northern
Brazil. These results help to increase
the knowledge on the genomic diversity
of RVA, aiming detect new variants and
possible antigenic changes, whose
potential effect on vaccine effectiveness
should be studied. Financial support:
CNPq, IEC, IOC-FIOCRUZ.
HV1224 - DEVELOPMENT OF A
SENSITIVITY AND COST-EFFECTIVE REAL TIME PCR: MEASURING A
WIDE RANGE OF HBV DNA CONCENTRATIONS
Santos, A.O., Souza, F.B., Nicolete, L.D.F.,
Salcedo, J.M.V., Vieira, D.S.
1. Instituto
de
Pesquisa
em
Patologia Tropical, IPEPATRO,
Rua da Beira, 7671, Lagoa, Porto
Velho-RO.
2. Centro de Pesquisa em Medicina
Tropical, CEPEM, Av. Guaporé,
215, Lagoa, Porto Velho-RO
3. Fundação
Oswaldo
Cruz
Rondônia, FIOCRUZ-Rondônia,
Rua da Beira, 7671, Lagoa, Porto
Velho-RO. E-mail: alcione.m@
hotmail.com
The quantification of HBV viral load is
indispensable to start the treatment
of patients and the following up of
them, so quantitative assays must
measure a wide range of viral DNA
concentrations. For this reason,
the aim of this study was develop a
sensitivity and low cost in house Real
Time PCR method. A fragment with
109 bp was cloned and serial diluted
to standard curve construction. The
calibration of the HBV - DNA values
was performed against OptiQuant®
HBV-DNA
Quantification
Panel,
AcroMetrix Europe B.V.). Specifically,
serial dilutions of the standard ranging
from 2 to 2x106 were tested. Based
on a linear regression, a conversion
formula was calculated for the in -house
measurements (copies/mL) to the
international standard units (IU/mL).
The correlation between AcroMetrix
kit and in house assay was performed
by Pearson´s test, using GraphPad 5.0
to fit regression lines between IU/
mL and copies/mL. The following
equation was obtained: [Log(IU/ml)=
- 0,5249 + 0,6618Log10(copies/mL)],
consequently 1UI/ml = 6,21 copies/
ml. These findings suggest that the
performance of in-house assay is equally
as well as the commercially available
kit. To test assay´s sensitivity we used
samples 30 negative from Rondonia
blood bank and 26 indeterminate for
HBsAg and 40 positives for HBsAg
and HBeAg to Ambulatorio Specializes
of Rondonia Viral Hepatitis tested
previously by ELISA. They were
performed again using AcroMetrix and
the in house assay. The negative (n=30)
and positive (n=40) samples were
confirmed in both methods (AcroMetrix
and in house) and unresolved cases
(n=26) were identified as positive
samples by AcroMetrix and also by in
house test. These initial data suggest a
100% of sensitivity. The method used
in this study suggests a lower final
cost and it can be used as acid nucleic
test to resolve indeterminate cases.
On the same way it can be a tool for
Human Virology: HV
257
management of chronic HBV patients
in Amazonic region. Consequently, the
validation of this in house assay is the
initial step for implementing on the
blood banks´ trials and clinical routine.
Finnancial support: Sistema Único de
Saúde (SUS); CNPq.
HV1225 - MOLECULAR CHARACTERIZATION OF NOROVIRUS GII.4
VARIANTS IN BRAZIL, 2005-2010
Fioretti, J.M., Ferreira, M.S.R., Victoria,
M.M., Leite, J.P.G., Miagostovich, M.P.
Fiocruz, Instituto Oswaldo Cruz,
Fiocruz, IOC, Av. Brasil, 4365, CEP:
21040-360, Manguinhos, Rio de
Janeiro. Pav. HPP, sl B203 E-mail:
[email protected]
The genus Norovirus belongs to
Caliciviridae family and is divided
into 5 genogroups (G), which GI,
GII and GIV are described infecting
humans. The GI and GII are the most
prevalent, containing the the greatest
diversity of genotypes described,
and the GII.4 responsible for the
majority of outbreaks worldwide. The
norovirus (NoV) are non-enveloped,
with icosaedrichal symmetry virus, its
genome is composed by ssRNA with
approximately 7,5 kb, divided into
3 open reading frames (ORF). ORF1
encodes non-structural proteins, ORF2
encodes structural protein VP1 that
composes the viral capsid and ORF3
encodes structural protein VP2. The
aim of this study was characterize the
different genotypes and variants of
GII.4 of NoV circulanting in different
Brazilian states and regions during
the years 2005-2010. For this purpose
were selected 265 stool samples from
cases of acute gastroenteritis received
in the Laboratory of Comparative
and Environmental Virology, through
258
Human Virology: HV
spontaneous demand, and previously
diagnosed as NoV by polymerase
chain reaction preceded by reverse
transcription (RT-PCR). For the
molecular
characterization
was
performed partial sequencing of two
regions of the NoV genome, region D
for genotyping and P2 subdomain for
characterization of variants GII.4. By
sequencing the region D, 213 samples
were characterized as GII (80,4%)
and four GI (1,55). It was not possible
characterize 48 samples (18,1%). Were
detected the genotypes GI.2, GI.5, GI.8,
GII.4, GII.6, GII.7, GII.12, GII.16, GII.17 e
GII.20, and the most prevalent was the
GII.4 (79%), found in 13 of the 15 states
evaluated, followed by GII.6 (13%). This
is the first description of the circulation
of GI.5, GII.12, GII.16, GII.17 and GII.20
in Brazil. Posteriorly, by sequencing
the region P2 were characterized
five variants GII.4 called 2003, 2004,
2006a, 2006b and 2010, and the most
prevalent was 2006b (54,4%), followed
by 2010 (21,9%) and 2006a (17,5%).
The characterization of a subcluster
consisting of 22 samples within the
2006b variant suggests the emergence
of a new variant. The high genetic
diversity of NoV circulating in Brazil, as
well as the possibility of the emergence
of new variants demonstrates the need
to establish a national network of
surveillance that would make available
information regarding the geographical
and temporal spread of these viruses
as it does other countries. Financial
Support: IOC, CNPq, CGLAB/SVS-MS.
HV1227 - SEVERE METAPNEUMOVIRUS
INFECTIONS
AMONG
IMMUNOCOMPETENT AND IMMUNOCOMPROMISED PATIENTS
Sinohara, J., Watanabe, A., Carraro, E.,
Granato, C., Bellei, N.
UNIFESP, Rua Pedro de Toledo, 781
15° andar E-mail: juliana.sinohara@
gmail.com
Human
coronaviruses
(HCoVs)
cause upper respiratory illness and
occasionally lower tract disease in
susceptible populations. Five HCoVs
are known: OC43, 229E, SARS-CoV and
the recently identified NL63 and HKU1.
Since there is a little knowledge on the
epidemiological features among the
different HCoVs species, we conducted
a comprehensive study by analyzing
the non-SARS HCoVs on 1.137
respiratory samples from subsets
of patients from Sao Paulo, Brazil,
between 2001 and 2010. Subjects were
50 asymptomatic and 1.087 presenting
acute respiratory infections: 465
patients from community (adults and
children), 410 at-risk patients (renal
transplanted patients, children with
heart diseases and patients under
stem cell transplantation program)
and 212 hospitalized patients (adults
and children). To identify the HCoVs
in samples, species-specific real-time
RT-PCR assay were performed. Human
coronaviruses were detected in 88 out
of 1.137 (7.7%) of the samples. The
most frequently detected HCoV species
were NL63 (50.0%) and OC43 (27.3%).
HCoV-NL63 was the species most
frequently associated with children,
both from the community (100%)
and presenting heart diseases (50%),
while there was a high rate of HCoV229E detection among renal transplant
patients (44%). Patients in stem cell
transplantation programs were more
frequently infected with HCoV-OC43
(47%). There were inter-seasonal
differences in the detection frequencies,
with HCoV-229E being predominant in
the year 2004 (61.5%) and HCoV-NL63
(70.8%) in 2008. Besides, HCoV-229E
displayed a marked autumn seasonality
while HCoV-HKU1 and HCoV-OC43
predominated in winter. Dyspnea was
more associated with HCoV-229E
infections (66.6%) and cyanosis was
reported only in HCoV-OC43 infections.
Our data provide an insight into the
epidemiology knowledge of HCoVs
among different subsets of patients,
supporting the notion that HCoVs
have different circulation trends
and play an important role among
patients with comorbidities. Financial
support: FAPESP (nº09/17307-6 and
09/54640-5)
HV1231 - PAIRED ANALYSIS OF
RNA DETECTION OF HEPATITIS C
VIRUS (HCV) IN DRIED BLOOD SPOT
(DBS) AND SERUM SAMPLES FOR
REAL-TIME PCR
Marques, B.L.C., Portilho, M.M.,
Espirito-Santo, M.P., Lewis-Ximenez,
L.L., Lampe, E., Villar, L.M.
de Janeiro E-mail: miss.maques@
gmail.com
Several studies have demonstrated the
importance of DBS samples as a good
alternative specimen once it represents
a non-invasive blood collection with
no need of phlebotomist. Moreover,
samples can be storage and sent to
other laboratories without freezing.
The present study aims to compare
two commercial enzyme immunoassay
(EIA) for anti-HCV detection among
DBS samples. Ten individuals were
selected and gave paired sera and
DBS samples, where 5 were anti-HCV
reactive and 5 were anti-HCV negative
among sera samples by commercial
EIA 1 (HCV Ab, Radim). DBS samples
Human Virology: HV
259
were assayed by EIA 1 increasing
sample volume ten-fold compared to
serum. Using commercial EIA 2 (Murex
Anti-HCV kit, Abbot), manufacturer’s
instructions and sample volume (five
and nine-fold increase compared to
sera) were evaluated. Using EIA1,
100% of concordance for anti-HCV
detection was observed among sera
and DBS. Using EIA2 among DBS
samples according manufacturer’s
protocol gave 80% of concordance
compared to sera results, where two
false negative results were obtained
(mean OD value=1.13). When sample
volume was increased 5 and 9- fold
compared to sera samples, 100% of
concordant results among sera and
DBS samples were observed. Mean OD
values for anti-HCV positive samples
were 3.63 and 3.98 using five and nine
fold increase, whereas mean OD values
for negative samples were 0.070 and
0.226 using five and nine fold increase.
It is concluded that both EIAs can be
used for anti-HCV detection among
DBS samples, but sample volume has
to be increased. Financial Support:
FAPERJ, CNPQ.
HV1237 - HEPATITIS A AND E
VIRUS INFECTIONS IN WESTERN
BRAZILIAN AMAZON
Pereira, R.C.C., Ferreira, M.U., Cardoso,
M.A., Fontoura, P.S., Gaspar, L.P., Freire,
M., Oliveira, J.M., Gaspar, A.M.C., Vitral,
C.L.
1. Universidade
Federal
Fluminense, UFF, Rua Professor
Ernani Melo, 101, Centro, Niterói,
RJ CEP: 24210-130
2. Instituto Oswaldo Cruz, (IOC/
Fiocruz), Av. Brasil, 4365 Manguinhos - Rio de Janeiro
260
Human Virology: HV
Av. Prof. Lineu Prestes, 1374 - Ed.
Biomédicas II - CEP: 05508-000 São Paulo E-mail: rebecapereira@
id.uff.br
To compare the epidemiologic profiles
of hepatitis A virus (HAV) and hepatitis
E virus (HEV) infections in Western
Brazilian Amazon, the prevalences of
HAV and HEV infections were studied
in two Amazonian populations: (a)
toddlers and preschoolers who live in
a small border town (Acrelândia) and
(b) preschool children, schoolchildren
and adults living in an agricultural
settlement (Granada) distant 30-50 km
from Acrelândia, Acre, Brazil. A total
of 1508 serum samples (1103 from
the urban area and 405 from the rural
area) were obtained in a population
based survey conducted in 2007 and
tested for anti-HAV antibodies. For HEV
prevalence investigation, a sample of
364 individuals was randomly selected
and tested for IgG anti-HEV. Anti-HAV
was detected in 47.7% and 81.5%
subjects from Acrelândia and Granada,
respectively, whereas IgG anti-HEV
was detected in only 10 individuals
(2.8%). The anti-HAV prevalence of
children aged five or less from both
urban and rural areas was only 4050%, increasing to 80% among those
aged 10 or more. Seropositive IgG
anti-HEV samples where all from
subjects living at the rural area with
ages ranging from 17 to 90 years
old (mean ± standard deviation,
54.5 ± 2.8 years). HAV infection was
shown to be highly prevalent in both
population groups from the western
Brazilian Amazon, although the antiHAV prevalence observed among
children has been lower than the one
expected for endemic areas. This fact
corroborates with several studies that
have been shown a large proportion
of Brazilian children under the age
of five susceptible to HAV infection,
even from low socioeconomic groups.
On the other hand, in spite of being
transmitted by similar routes, HEV
infection was uncommon. The antiHEV IgG prevalence observed was
consistent with those found in other
Brazilian studies that characterize the
country as a non-endemic area for HEV
infection. Financial support: CNPq,
Finep, FAPERJ
HV1239 - PICORNAVIRUSES IN
HYPERPLASTIC LYMPHOID TISSUES
OF CHRONIC ADENOTONSILLAR
DISEASE
Paula, F.E., Proença-Modena, J.L.,
Valera, F.C.P., Tamashiro, E., Buzatto,
G.P., Saturno, T., Silva, M.L., AnselmoLima, W., Arruda, E.
Otorhinolaryngology and Head and
Neck Surgery, , Av. Bandeirantes,
3900, Ribeirão Preto Virology
Research Center, , Av. Bandeirantes,
3900, Ribeirão Preto University of
São Paulo School of Medicine, FMRP,
Av. Bandeirantes, 3900, Ribeirão
Preto E-mail: [email protected]
Chronic
adenotonsillar
diseases
are frequent otorhinolaryngologic
disorders due to chronic inflammation
of adenoids and palatine tonsils. They
frequently lead to surgery, for removal
of hypertrophic tissues. Although
diverse respiratory viruses can be
detected in adenotonsillar tissues of
diseases, the role of these agents as
triggers of chronic inflammation in
those tissues is poorly understood.
Human rhinoviruses (HRVs) together
with the closely related human
enteroviruses (HEVs), both belonging
to the Picornaviridae family, cause
most of the acute respiratory illnesses
in humans and are frequently detected
in adenoids and palatine tonsils from
diseases. This study was done to
characterize the replicative status
and sites of replication/persistence
of picornaviruses in adenoids and
palatine tonsils from patients with
adenotonsillar diseases. To assess
the picornavirus replication activity
in adenoids and palatine tonsils,
the viral loads of HRV/HEV and
presence of anti-sense RNA species
were determined by qPCR in 121
disease, without symptoms of acute
respiratory infections. The sites of
viral detection were determined
by
immunohistochemistry
(ISH)
and in situ hybridization (ISH). The
5’ UTR PCR fragments obtained
were sequenced, which allowed
for identification of species. Of 121
enrolled patients, respectively 50%
and 55.4% had palatine tonsils and
adenoids positive for picornaviruses.
HRV was more frequently detected in
adenoids while HEV was more common
in palatine tonsils. ISH with antibody
for VP1 capsid protein indicated the
presence of virus capsids within the
lymphoid tissue, in the lymphoid
follicles of palatine tonsils, and also in
epithelial cells from adenoids. The high
frequency of detection of picornavirus
genomes and protein expression in
tissues from patients with chronic
adenotonsillar diseases, suggests that
these agents may play important roles
in the pathogenesis of these frequent
diseases. Financial support: CAPES,
FAPESP,CNPQ
HV1245
-
SAPOVIRUS-LIKE
Human Virology: HV
261
PARTICLES EXPRESSION IN BACULOVIRUS SYSTEM
dos Anjos, K., Souza, A.C., Lima, L.M.P.,
Silva, P.A., Inoue Nagata, A.K., Ribeiro,
B.M., Nagata, T.
1. Universidade de Brasília, UNB,
Departamento
de
Biologia
Celular,
Laboratório
de
Microscopia Eletrônica, CEP 709
Laboratório Central do Distrito
Federal, LACEN-DF, R Sgan Q., 601
Lotes O e P Asa Norte - Brasília DF CEP: 70830-010
UCB, Campus Avançado Asa Norte
SGAN 916 Módulo B B Avenida
W5 - CEP: 70790-160
3. Bras Embrapa Hortaliças, CNPH,
E-mail: dosanjos.karol07@gmail.
com
The family Caliciviridae consists of
viruses belonging to five genera:
Sapovirus,
Norovirus,
Lagovirus,
Vesivirus and Nebovirus. In Brazil
it is known that the most causative
gastroenteritis viruses are rotavirus
and norovirus, whereas the occurrence
of sapovirus is sporadic. The sapovirus
genome is linear, positive-sense,
single-stranded RNA, of approximately
7.5kb that is polyadenylated at the
3’ terminus. Sapporo virus is divided
into, at moment, seven genogroups (GIGVII) and, causes acute gastroenteritis
in human, porcine and mink hosts.
This work aimed to produce viruslike particles (VLPs) of sapovirus
GI isolated form human diarrheic
stool sample in Brasília, DF, Brazil,
for physicochemical study of VLPs
and future use of vaccine. For this
purpose, firstly, the partial capsid
protein (P domain) was expressed by
262
Human Virology: HV
Gateway system (Invitrogen) using
pENTR 2b and subsequently pDEST
17 and two rabbits were immunized to
produce antisera in order to confirm
VLPs expression by Western blotting.
Then, Baculovirus expression system
was used for sapovirus-like particles
production. The expression gene
cassette containing complete capsid
gene and downstream regions were
subcloned into pFastBac1 after PCR.
Subsequent bacmid was transfected
to insect cells (Trichoplusia ni, Tn5)
using Bac-to-bac system (Invitrogen).
The VLPs were purified by CsCl
gradient centrifugation. The possible
sapovirus-like particles were observed
by transmission electron microscopy,
showing the abundant spherical
particles with the expected particle
size of 30 nm in diameter by negative
staining with 2% phosphotungstic acid
solution. This purified preparation now
is analyzing by Western blotting using
antiserum against P domain previously
described.
HV1246 - SEQUENCING AND
MODELING OF THE ENVELOPE
PROTEIN OF A YELLOW FEVER VIRUS
STRAIN (BEAR378600) ISOLATED
IN THE BRAZILIAN AMAZON
Sousa Júnior, E.C., Cardoso, J.F., VianezJúnior, J.L.S.G., Oliveira, R.L.S., Inada,
D.T., Lima, C.P.S., Franco, E.C.S., Silva,
F.R., Silva, J.L., Nunes, K.N.B., Nunes,
M.R.T., Vasconcelos, P.F.C.
1. Instituto
Evandro
Chagas,
s/n - Levilândia - 67030-000 Ananindeua / Pará / Brasil
UFPA, Rua Augusto Corrêa,
01 - Guamá. CEP 66075-
110. Caixa postal 479 E-mail:
[email protected]
The yellow fever virus (YFV) belongs
to the family Flaviviridae , genus
Flavivirus. In Brazil, the YFV is mainly
transmitted by the mosquito species
Haemagogus janthinomys. According
to the World Health Organization,
approximately 200.000 yellow fever
cases and 30,000 deaths are reported
annually worldwide. YFV is endemic in
tropical areas of Africa and Americas
(Central and South) and the number
of cases have increased due to
deforestation, urbanization and climate
changes. The molecular modeling of
proteins is considered a crucial step in
the development of new drugs, since
there are no specific treatments for
this disease and vaccine adverse events
have been reported. The objective of
this study was to elucidate the threedimensional (3D) model of the E of YFV
strains isolated in the Amazon region.
The complete genome of the BeAr
378600 strain, isolated in 1980 from
Haemagogus sp. in Uruaçu state of Goiás
was completely sequenced using the
GSFLX 454 (Roche ). The 3D modeling
of the E protein was performed using
the software MODELLER with seven
reference models (1OAN, 1P58, 1TG8,
1TGE, 3C6D, and 3G7T 3P54), and
validation performed using software
Procheck. The post translational
analyzes using the BeAr 378600 E
gene coding sequence, demonstrated
high similarity with other YFV
proteins previously studied. The
modeling showed a high quality threedimensional stereochemistry and
globally residue-residue geometry with
99.5% of the waste accepted regions.
Multiple sequencing alignment carried
out among BeAr378600 and several
other YFV E gene sequences revealed
high similarity with strains isolated in
the Amazon region. No mutations in key
regions of the protein E were observed,
as well as no amino acid changes were
detected in important regions of the
E protein. Due to the high 3D model
quality
(stereochemistry
quality
exceeding 99%) presented to the
BeAR 378600 YFV strain, this model
can be further used in studies on the
development of potential inhibitors for
the YFV.
HV1247 - DETECTION OF ANTIBODIES WITH POTENTIALLY
NEUTRALIZING
ACTIVITY
IN
NATURAL CHRONIC HCV INFECTIONS
Marins, R.S.Q.S., Moraes, M.T.B., LewisXimenez, L.L., Gomes, S.A.
1. Laboratory of Molecular Virology,
Oswald Cruz Institute, IOC FIOCRUZ, Brasil Avenue, 4365
- Manguinhos, Rio de Janeiro,
Brazil
2. Laboratory of Viral Hepatitis,
Brazil E-mail: rmarins@ioc.
fiocruz.br
Hepatitis C Virus (HCV) affects more
than 170 million people and causes
chronic hepatitis in about 70% of cases,
with progression to cirrhosis, hepatic
failure, and hepatocellular carcinoma.
The envelope glycoproteins E1 and E2
are the natural targets of neutralizing
antibodies responses but are also the
two of the most variable HCV proteins.
Several neutralizing antibodies that
target HCV epitopes have been described
in the literature. One of these regions,
encompassing amino acids (aa) 412
Human Virology: HV
263
to 423 of the E2 protein, is the target
of several neutralizing monoclonal
antibodies described in the literature.
To determine the immunogenic
characteristic of this small well
conserved amino acid sequence (412419) inside this important neutralizing
region and to detect the presence of
antibodies reacting with this small 412423 sequence in natural infection, a in
house enzyme linked immunosorbent
assay (ELISA) was developed and
tested against serum samples from
chronic HCV infected patients. Twentyfive serum samples from chronic HCV
patients and 25 samples from healthy
individuals who tested negative for
anti-HCV antibodies were used to
establish the reactivity of these sera
with the small 412-419 synthetic
peptide. Cutoff value was established
calculating mean absorbance value
of HCV negative sera plus three times
the standard deviation. A serum antipeptide 412-419 previously obtained
in a immunized rabbit was used as
positive control. Two of the 25 sera
derived from chronic HCV patients
reacted specifically with this small
peptide. These results indicate that the
selected epitope was able to induce
humoral immune responses during the
natural infection in a small proportion
(8%) of the studied patients. Further
studies were necessary to associate
the presence of these antibodies
with progretion of liver diseases. The
reasons why human anti-E2 antibodies
raised against HCV natural infection do
not neutralize a HCV infection is not
well understood and may be related
to either genetic variations among
viral population, low titers of anti
E2 antibodies or interference among
neutralizing and non neutralizing
antibodies. Could antibodies detected
264
Human Virology: HV
in the present study protect against
a severe hepatitis? If yes, the small
E2 conserved peptide may be used
as a therapeutic vaccine against HCV
infection. Financial support: CNPq and
Fiocruz
HV1252 - A SINGLE DOSE OF INACTIVATED HEPATITIS A VACCINE
PROMOTES
MEMORY
CELL
RESPONSE
Melgaço, J.G., Morgado, L.N., Santiago,
M.A., Sousa, P.S.F., Velloso, C.E.P., Vieira,
Y.R., Gaspar, L.P., Pinto, M.A., Vitral, C.L.
1. Universidade Federal Fluminense,
UFF, Instituto Biomédico, NiteróiRJ
2. Instituto Oswaldo Cruz, IOC, Av.
Brasil, 4365, Manguinhos-RJ
Biomanguinhos,
Bio-Fiocruz,
Av. Brasil, 4365, Manguinhos-RJ
Although T cells are known to have
important functions in immune
protection against viral diseases,
only a few studies have addressed
the cellular basis of immunity to
hepatitis A virus (HAV) in humans.
In order to further identify the roles
of HAV-specific T cell responses, we
investigated the generation of cellular
immunity after HAV vaccination in
a time elapse study in a small study
population. Ten subjects received
two doses of inactivated hepatitis A
vaccine (0-6 months). For monitoring
the antigen-specific immune response,
blood samples were collected before
the 1st dose (T0), 6 months after the
1st dose (T1) and 24 months after
the 2nd dose (T2). Cell responses
was measured by proliferation
assay (CFSE), memory phenotype
CD4+/CD8+/CD22+/CD45RO+
and
cytokine production (IL-10, IFN-y,
TNF-a) to identify antigen-specific
lymphocytes in vaccinees. Results of
the proliferation assays showed HAV
specific cellular response soon after
the 1st dose application. There was
an increase in the proliferation index
(PI) 2.65 fold in comparison with the
PI obtained with control samples (T0)
(p<0.05), which remained elevated
(2.95, p<0.05) 24 months after the
2nd dose (T2). There was no statistical
difference (p>0.05) between PI rates
induced by lymphocytes obtained
after the 1st (T1) and 2nd doses (T2)
of vaccine. Although no statistical
difference had been observed with
regard to memory cell phenotype
expression (p>0.05), an increase in
CD8+ production could be detected
with a positive correlation between
1st and 2nd dose (p<0.05; r2=0.70).
Moreover, an increased in IL-10, TNF-a
and IFN-g cytokines production could
be detected after 1st and 2nd dose,
with a positive correlation between
IL-10 (T2) and IFN-g (T1) production
(p<0.05;r2=0.60), IL-10 (T1) and TNF-a
(T1) production (p<0.05;r2=0.54),
TNF-a (T1) and TNF-a (T2) production
(p<0.05;
r2=0.70).
Inactivated
hepatitis A vaccines have been licensed
in multiple countries, protecting
against the disease with nearly 100%
efficacy. However, their use have been
limited by cost considerations. One
approach for reducing the cost of its
implementation would be the use of
a single dose schedule like the one
adopted in Argentina. Results of this
study suggest that a single dose of
inactivated hepatitis A vaccine is able
to promote memory cell response.
Financial Support: Capes, Faperj, CNPq
Virologia Humana
HV1253 - HTLV-1 INFECTION IN
ASYMPTOMATIC PERUVIAN PEOPLE
Mayta, E.M.H.
1. Major National University of
San Marcos Biologycal Sciences,
UNMSM, Lima, Peru
2. Universidade de Sao Paulo,
USP-ICB-II, São Paulo, Brasil
Universidade de Sao Paulo Facultade de Medicina Humana,
USP, São Paulo, Brasil E-mail:
[email protected]
Human Lymphotropic Virus Type 1 T
cells (HTLV-1) is a delta-retrovirus that
causes a severe lymphoproliferative
disorder to CD4+ T cells. It causes
leukemias,
neurodegenerative
diseases, inflammation myelopathy
associated, uvieitis with strong unique
support epidemiological and clinical
characteristics that distinguish them
from other similar ones. It is a complex
RNA virus with a single strand of
positive sense; its genome expresses
unique proteins with oncogenic
potential. The aim of this study was to
determine the presence of HTLV-1 in
asymptomatic persons. We evaluated
78 samples of peripheral blood by
venipuncture with vacuum extraction
system (vacutainer) to individuals
attending the Health Campaign at
the San Fernando Health Center
(Augustino, Lima-Peru). Samples
were processed it at the Laboratory
of Clinical Virology and Molecular
FCB UNMSM to obtain the serum by
centrifugation and subsequently used
for the serological diagnosis of HTLV1. We used the ELISA HTLV-I/II BIOKIT
and confirmed results by Western blot
HTLV Innolia I / II Innogenetics. We
obtained 7.7% positive samples to
HTLV-1. The confirmatory test showed
Human Virology: HV
265
intense bands (3 +, 1 +, + / -), the
positive control bands do not show gag
specificity (p19 I / II and p24 I / II) and
two env bands (gp 46 I / II and gp 21 I
/ II). In addition, the env gp 46-I bands
are specific to HTLV-I while env gp 46II bands to HTLV-II. Of those positive
samples for HTLV-1, 1.3% were male
and 6.4% were women. Also, we found
that HTLV-1 was prevalent in infected
individuals from 26 to 60 years old.
In determining the place of birth of
seropositives of HTLV-1, it was found
one person from Junin locality and five
people from Lima. Our results indicate
that HTLV-1 virus is circulating mostly
in healthy individuals. Some studies
reveal that HTLV-1 infected people
remain asymptomatic. Also, HTLV-1 is
present in endemic areas, as well as
Lima, in people of economically active
ages with female gender predominance.
Keywords: HTLV-1, asymptomatic,
HTLV-1 antibodies. Financial support:
CSI-UNMSM
HV1260 - RECOMBINANT OF
HEPATITIS
B
VIRUS
(HBV)
GENOTYPES A AND G DETECTED
AFTER A FOLLOW UP OF A PATIENT
IN THE CHRONIC PHASE OF AN
OCCULT HBV INFECTION
Moraes, M.T.B., Lewis-Ximenez, L.L.,
Peres, L.R., Sousa, P.S.F., Mello, F.C.A.,
Gomes, S.A., Barros, J.J.F.
1. Lab de Virologia Molecular,
Instituto Oswaldo Cruz/Fiocruz,
LVM, IOC/FIOCRUZ, Av. Brasil,
4365, Manguinhos, Rio de Janeiro,
Brasil
2. Lab de Hepatites Virais, Instituto
Oswaldo Cruz/Fiocruz, LAHEP,
IOC/FIOCRUZ, Av. Brasil, 4365,
Manguinhos, Rio de Janeiro, Brasil
266
Human Virology: HV
Chronic hepatitis B virus (HBV)
infection is a serious global health
problem and an important cause of
morbidity and mortality in endemic
areas. Eight HBV genotypes (HBV/A
to HBV/H), have been identified. HBV
genotypes and their subgenotypes
have distinctive geographical and
ethnic distribution around the World.
In Brazil, HBV/ A, HBV/D (worldwide)
and HBV/F (Native from Indians of the
New World) are the most prevalent,
rarely occurring HBV/G. Genotype G
is found in the USA and Europe. Occult
HBV infection is defined by detectable
HBV genome in the absence of surface
antigen (HBsAg, the main serological
marker of active infection). The cause
of an overt HBV infection becoming an
occult one is unknown. In this study, a
recombinant of HBV A/G isolated from
a patient with occult HBV infection is
reported. The patient was a 23-yearold Brazilian male with classical acute
infection (seropositive for HBsAg,
HBeAg and anti-HBc in 10/2008. After
six months, all serological markers
became negative except anti-HBc but
HBV DNA was detectable in low titles
<104 copies/mL. This patient was
monitored for HBV markers for about
two years with a total of 14 serial
serum samples available. Genomic
regions of S, pre-C and C genes were
PCR amplified, followed by nucleotide
sequencing. The first and second last
sample were cloned and 16 and 10
clones of each sample were analyzed,
respectively. PCR products were also
subjected to pyrosequencing in order
to verify the presence of mixture of
genotypes. Possible recombination
was analyzed by SIMPLOT program.
Based on phylogenetic analyses of the
S, pre-C/C genes and pyrosequencing
results of the first six serial samples
and clones from the first sample,
all amplicons clustered on a branch
within subgenotype A2, however,
analysis of the last two samples along
with their clones showed mixtures
of genotypes A and G and suggested
recombination A/G for S region.
The possibility of the development
of occult HBV infection after acute
hepatitis B, leads to a discussion of the
need to expand monitoring to patients
with undetectable HBsAg and without
seroconvertion to anti-HBs, through
molecular tests for detection of HBV
DNA prior to outpatient release. In this
study we identify a recombinant HBV
A/G in the chronic phase whereas only
HBV/A was detectable in the acute
phase. These data should encourage
further epidemiological and virological
investigations on the clinical evolution
of co-infected (HBVA/G) patients.
Financial support: FIOCRUZ
HV1281 - HUMAN PAPILLOMAVIRUS
IN CANCER OF DIVERSE HEAD AND
NECK SUBSITES
Betiol, J.C., Sobrinho, J.S., Costa, M.C.,
Rossi, L., Costa, H.O., Villa, L.L., Sichero,
L.
1. Instituto do Câncer do Estado de
São Paulo, ICESP, Av. Dr. Arnaldo,
251 - Cerqueira César - São Paulo
- SP - CEP: 01246-000
2. Instituto do HPV, INCT-HPV, Rua
General Jardim, 618, 3º andar
sala 32, São Paulo - SP
3. Departamento de Radiologia FMUSP, DR-FMUSP, Av. Dr. Enéas
de Carvalho Aguiar, 255 E-mail:
[email protected]
Head and neck cancer (HNC) is the sixth
most common neoplasia worldwide,
and HPV infection is causally associated
with a subset of these tumours. We
sought to characterize HPV genome
diversity
in
paraffin-embedded
samples from individuals diagnosed
with HNC. Extracted DNA was amplified
using a very sensitive PGMY/GP+
nested PCR protocol. Adequacy of DNA
was assessed by beta-globin gene PCR.
Amplicons were cloned and sequenced
for HPV genotyping. To data, we have
analyzed DNA derived from 62 patients.
These included laryngeal (n=50),
oropharyngeal (n=4), vocal cord (n=6)
and other sites (n=2) specimens. The
overall HPV prevalence was 38.71%
(24/62). HPV positivity was higher
among oropharyngeal samples (75%)
followed by laryngeal (38%) and
vocal cord (16.6%) specimens. Seven
positive samples were genotyped as
yet. The most prevalent type was HPV83 (42.86%; 3/7) followed by HPV18 (28.57%; 2/7), HPV-16 (14.3%;
1/7) and HPV-51 (14.3%; 1/7). Our
preliminary results indicates a different
pattern of HPV type-specific infection
among HNC tumors, once studies
performed worldwide observed HPV16 prevalence of up to 90% in some
head and neck subsites. Our study will
provide epidemiological evidences of
HPV types distribution among HNC
in samples from São Paulo, data that
is actually lacking in our country and
are important for the establishment
of effective governmental policies,
reaffirming the emergent necessity
of vaccine implantation in Brazilian
health programs in order to reduce
the bound of tumors in our population.
Financial support: FAPESP 11/096169, 12/01513-9 and 08/57889-1; CNPq
573799/2008-3; Ludwig Institute
for Cancer Research Área: Virologia
Human Virology: HV
267
Humana
Tema:
Papilomavírus
Humano Arquivo: Betiol et al.,2012
Apresentador: Julio Cesar Betiol
HV1293 - VARIABILITY IN THE S
GENE SEQUENCE OF HEPATITIS B
VIRUS FROM CHRONIC CARRIERS
WITH SIMULTANEOUS POSITIVITY
OF HBSAG AND ANTI-HBS SEROLOGICAL MARKERS
Ferreira, A.C., Pinho, J.R.R., MendesCorrêa, M.C.J., Nastri, A.C.S.S, Soares,
M.C.P., Gomes-Gouvêa, M.
1. Instituto de Medicina Tropical
- USP, IMT/USP, Av. Dr. Éneas
Carvalho de Aguiar n° 500 Prédio
2, 2 ° andar
2. Faculdade de Medicina da
Fundação do ABC, FMABC,
Hospital das Clínicas da Faculdade
de Medicina da USP, HC/FMUSP
E-mail:
ariana86carolina@
hotmail.com
HBV
clearance
is
classically
characterized by the emergence of antiHBs antibodies that may be detected
by serological tests after Hepatitis B
surface antigen (HBsAg) clearance,
although it is produced early in the
course of infection. Detection of HBsAg
and anti-HBs concomitantly comprise
a peculiar serological profile in the
chronic hepatitis B context, which
may be related with the selection
of HBsAg variants that escape from
immune response. This study aimed
to assess the relationship between
the coexistence of HBsAg and antiHBs in chronic hepatitis B carriers
and the presence of HBsAg variants.
Serum samples from chronic HBV
carriers both seropositive for HBsAg
268
Human Virology: HV
and anti-HBs (case group=16) and
only seropositive for HBsAg (control
group=21) were analyzed in this study.
HBV S gene sequences were obtained
by direct sequencing and its amino acid
changes were identified by comparison
with consensus sequences belonging
to the different HBV genotypes. HBV
genotypes A and D were found in
both groups, genotype F was detected
only in one sample from case group.
HBV/A was the most prevalent in both
groups, but HBV/D was found more
frequently in the control group (48%
vs 18%). HBV variants with amino acid
mutations within the HBs sequence
were found more frequently among
control group (76% vs. 63%) and these
mutations were more common in N
and C-terminal regions of the S protein.
Variants with mutations at the major
hydrophilic region of the HBsAg (AA 99
– 160) were also found more frequently
among individuals from control group
(48% vs 19%), however none of these
mutations were located in the second
‘a’ determinant loop (AA 139 -148) or
were previously related with anti-HBs
escape. In conclusion, these results
shows that the simultaneous positivity
of HBsAg and anti-HBs in chronic HBV
carriers studied was not related with
the presence of specific HBsAg variants,
suggesting that in these patients the
presence of anti-HBs did not lead to
a selection of HBV escape mutants.
The higher variability of the S gene
observed among strains isolated from
control group may be related with the
higher frequency of genotype D in this
group, but it was not related to a lower
sensitivity of the kit in detecting these
variants. Financial support: FAPESP
(2010/50081-9 and 2010/51208-2)
HV1294
-
MAYARO
VIRUS
PATIENTS FROM ACRE STATE,
BRAZIL (2010)
Sabino, G.F., Mondini, A., Colombo, T.E.,
Nogueira, M.L.
Universidade Estadual Paulista
“Júlio de Mesquita Filho” , UNESP,
José do Rio Preto, FAMERP, E-mail:
[email protected]
Arboviruses usually have very similar
symptoms in mild or severe cases, such
as fever, malaise, headache, nausea,
vomiting, arthralgia, myalgia among
others. This wide range of symptoms
may lead to inaccurate diagnosis and
an increase in unreported cases of
several diseases in official health data.
This situation usually occurs in regions
where there are outbreaks caused by
enzootic circulation of arboviruses. The
implementation of surveillance systems
and an improvement in diagnostic
expertise has allowed the detection
of new human cases of arboviruses
in regions where their presence was
previously underestimated. Therefore,
arboviruses remain largely unknown
or not reported in the regions where
refined diagnosis is not available. Our
goal was to detect dengue (DENV)
and other arboviruses in patients that
presented febrile illness and searched
health facilities for medical care.
We analyzed medical records of 400
patients from the Hospital de Base of
São José do Rio Preto, São Paulo (Brazil)
whose clinical diagnosis was dengue.
The analysis of these patients from São
José do Rio Preto suggests that 84.75%
of the febrile illnesses might be caused
by other agents than DENV. Actually,
only 14.5% of the patients were
DENV positive and 0,75% presented
unknown ethiology. These results raise
IN concerns on the parameters that have
been used for arbovirus diagnosis.
The great similarity of the symptoms
in diseases caused by arboviruses
requires better analyses, training of
health professionals and tests that
present high sensitivity and specificity.
In order to improve the health policies
needed to strengthen surveillance, the
identification of vectors and reservoirs,
the development of effective diagnostic
tests and strategies to control diseases
are extremely important to uncover
arboviruses that hide under dengue
umbrella. Financial Support: INCTDegue/CNPq;
FAPESP;
CAPES;
PRONEX;
HV1295 - ARBOVIRUS CASES HIDDEN
UNDER DENGUE UMBRELLA IN
NORTHWEST REGION OF SÃO PAULO
STATE
Sabino, G.F., Mondini, A., Colombo, T.E.,
Karam, C.F., Nogueira, M.L.
Faculdade de Medicina de São José
do Rio Preto, FAMERP, Universidade
Estadual Paulista “Júlio de Mesquita
Filho”, UNESP, E-mail: guitosabino@
gmail.com
Arboviruses usually have very similar
symptoms in mild or severe cases, such
as fever, malaise, headache, nausea,
vomiting, arthralgia, myalgia among
others. This wide range of symptoms
may lead to inaccurate diagnosis and
an increase in unreported cases of
several diseases in official health data.
This situation usually occurs in regions
where there are outbreaks caused by
enzootic circulation of arboviruses. The
implementation of surveillance systems
and an improvement in diagnostic
expertise has allowed the detection
of new human cases of arboviruses
in regions where their presence was
previously underestimated. Therefore,
Human Virology: HV
269
arboviruses remain largely unknown
or not reported in the regions where
refined diagnosis is not available. Our
goal was to detect dengue (DENV)
and other arboviruses in patients that
presented febrile illness and searched
health facilities for medical care.
We analyzed medical records of 400
patients from the Hospital de Base of
São José do Rio Preto, São Paulo (Brazil)
whose clinical diagnosis was dengue.
The analysis of these patients from São
José do Rio Preto suggests that 84.75%
of the febrile illnesses might be caused
by other agents than DENV. Actually,
only 14.5% of the patients were
DENV positive and 0,75% presented
unknown ethiology. These results raise
concerns on the parameters that have
been used for arbovirus diagnosis.
The great similarity of the symptoms
in diseases caused by arboviruses
requires better analyses, training of
health professionals and tests that
present high sensitivity and specificity.
In order to improve the health policies
needed to strengthen surveillance, the
identification of vectors and reservoirs,
the development of effective diagnostic
tests and strategies to control diseases
are extremely important to uncover
arboviruses that hide under dengue
umbrella.
HV1302 - SEVERE MYOCARDITIS
AFTER ENTEROVIRUS INFECTION IN
A YOUNG WOMAN
Camargo, C.N., Perosa, A., Granato,
C.F.H., Bellei, N.
UNIFESP, Rua Pedro de Toledo , 781 .
15andar E-mail: claricencamargo@
gmail.com
Although the majority of human EV
infections remain asymptomatic, these
270
Human Virology: HV
viruses are associated with diverse
clinical syndromes, ranging from
minor febrile illness to severe and
potentially fatal pathologies, including
aseptic
meningitis,
encephalitis,
myopericarditis, acute flaccid paralysis,
and severe neonatal sepsis-like
disease. Enteroviral infection of the
heart has been noted in a significant
proportion of cases of myocarditis and
dilated cardiomyopathy. The presence
of enterovirus RNA at stages of disease
after acute infection and correlation
of enterovirus replication with worse
clinical outcome suggests continued
replication of the virus is involved
in the progression of the disease. In
2012, a young woman was admitted to
Sao Paulo Hospital in the 5th months
after her puerperal period. Physical
examination and cardiac laboratory
evaluation revealed the diagnostic
of myocarditis and a low ejection
fraction (<40%). An initial etiology
supposed for the case was puerperal
myocarditis but an infectious diseases
consultation was required to evaluate
the patient. Myocarditis caused by EV
was diagnosed when the virus was
detected in nasal swab and antibodies
to Coxsackie B virus were detected in
serum. Patient was discharged after
10 days of hospitalization with a final
diagnosis of viral myocarditis and
heart failure and is under treatment at
the chronic heart disease ambulatory.
Early recognition of heart failure
and adequate diagnostic testing for
cardiotropic viruses is important to
understand the impact of these viruses
among community. Financial support:
CAPES, FAPESP 2009/17384-0
PAULO CITY
Camargo, C.N., Bellei, N.
UNIFESP, E-mail: claricencamargo@
gmail.com
Enteroviruses (EVs) are among the
most common viruses infecting human
beings worldwide and can induce
non typical respiratory illnesses in
infants or adults, including upper
respiratory tract infections but also
lower respiratory tract infections,
resulting in bronchitis, bronchiolitis,
and pneumonia. The aims of our study
were to investigate the occurrence of
EV in hospitalized and non hospitalized
patients with respiratory symptoms.
The nasal secretion was collected
from 253 hospitalized (96 children
and 61 adults) and 281 community
patients (128 children and 153 adults).
Samples were tested by Real time
RT-PCR to detect EV nucleic acids.
Positive samples for EV were detected
in 3.9% samples (21/ 534), in which,
5,7% (16/281) outpatients and 2%
(5/253) were hospitalized patients. All
hospitalized patients were children,
the majority of inpatients positive
cases occurred in children up to 1 year
old. EV positive infections identified in
21 patients correspond 5.2% (5/96)
of hospitalized children, 4% (5/128)
and 7.2% (11/153) non-hospitalized
children and adults respectively. The
main clinical manifestations related
by patients were fever, coryza, cough,
myalgia and sore throat besides
dyspnea among inpatients. Most EV
positive cases occurred during autumn
and winter. In conclusion, our result
highlighted the importance of EV as a
HV1303 - HUMAN ENTEROVIRUS causative agent of severe respiratory
INFECTION IN COMMUNITY AND infection young children but is rarely
HOSPITALIZED PATIENTS IN SÃO observed among community adults.
Financial support: CAPES, FAPESP
2009/17384-0
HV1309 - CHARACTERIZATION OF
HIV-1 SUBTYPES AND DRUG RESISTANCE
MUTATIONS
AMONG
INDIVIDUALS INFECTED WITH HIV
IN CAMPINAS/BRAZIL
Neto, D.F.L.N., Aoki, F.A., Arns, C.W.
Universidade Estadual de Campinas,
UNICAMP, Instituto de Biologia Rua
Monteiro Lobato - n° 255 E-mail:
[email protected]
In order to describe HIV-1 subtypes
and drug resistance mutations in
Brazil, blood samples from 764
patients infected with HIV-1 collected
from 2001 to 2008 were genotyped. Of
these, 126 samples were from newly
diagnosed, antiretroviral (ARV)-naïve
patients and 27 from ARV-treated
patients. Partial pol and gag region
sequences were used to identify drug
resistance mutations and to conduct
phylogenetic analysis for subtype
determination. The results indicated
that 43% patients harbored subtype C
viruses, 35.2% carried subtype CRF-BC
virus and 18%. Among patients with
no prior exposure to ARVs, mutations
associated with resistance were
detected in five patients: three (2.4%)
patients had reverse transcriptase
(RT) inhibitor mutations and two
other patients had the protease (PI)
inhibitor associated mutation M46I.
PI mutation V77I was found in 42 of
subtype C isolates. Of 27 ARV-treated
patients, 22 (81.5%) harbored at least
one nucleoside reverse transcriptase
inhibitors (NRTI), a non-NRTI (NNRTI)
and/or a PI mutation. The most
common NRTI resistance mutation
Human Virology: HV
271
was M184V/I (74.1%). Frequency
of thymidine analog mutations was
relatively low (25.9%). With regard to
NNRTI mutations, G190S/A was the
most frequent mutation, which might
be a preferred mutations for subtype
C. Brazils HIV epidemic continues
to be dominated by Subtype A FSU.
The prevalence of transmitted drug
resistance is low, but has the potential
to increase with increasing use of ARVs.
HV1311
EPIDEMIOLOGICAL
FEATURES OF HUMAN CORONAVIRUSES SPECIES AMONG BRAZILIAN
PATIENTS
Cabeça, T.K., Watanabe, A.S.A., Granato,
C.F.H., Bellei, N.
1. Clinical Virology Laboratory,
Discipline of Infectious Diseases,
Department of Medicine of The
Sao Paulo Federal University,
Sao Paulo, SP, Brazil. E-mail:
[email protected]
Toledo, 781 15° andar E-mail:
[email protected]
Human
coronaviruses
(HCoVs)
cause upper respiratory illness and
occasionally lower tract disease in
susceptible populations. Five HCoVs
are known: OC43, 229E, SARS-CoV and
the recently identified NL63 and HKU1.
Since there is a little knowledge on the
epidemiological features among the
different HCoVs species, we conducted
a comprehensive study by analyzing
the non-SARS HCoVs on 1.137
respiratory samples from subsets
of patients from Sao Paulo, Brazil,
between 2001 and 2010. Subjects were
50 asymptomatic and 1.087 presenting
272
Human Virology: HV
acute respiratory infections: 465
patients from community (adults and
children), 410 at-risk patients (renal
transplanted patients, children with
heart diseases and patients under
stem cell transplantation program)
and 212 hospitalized patients (adults
and children). To identify the HCoVs
in samples, species-specific real-time
RT-PCR assay were performed. Human
coronaviruses were detected in 88 out
of 1.137 (7.7%) of the samples. The
most frequently detected HCoV species
were NL63 (50.0%) and OC43 (27.3%).
HCoV-NL63 was the species most
frequently associated with children,
both from the community (100%)
and presenting heart diseases (50%),
while there was a high rate of HCoV229E detection among renal transplant
patients (44%). Patients in stem cell
transplantation programs were more
frequently infected with HCoV-OC43
(47%). There were inter-seasonal
differences in the detection frequencies,
with HCoV-229E being predominant in
the year 2004 (61.5%) and HCoV-NL63
(70.8%) in 2008. Besides, HCoV-229E
displayed a marked autumn seasonality
while HCoV-HKU1 and HCoV-OC43
predominated in winter. Dyspnea was
more associated with HCoV-229E
infections (66.6%) and cyanosis was
reported only in HCoV-OC43 infections.
Our data provide an insight into the
epidemiology knowledge of HCoVs
among different subsets of patients,
supporting the notion that HCoVs
have different circulation trends
and play an important role among
patients with comorbidities. Financial
support: FAPESP (nº09/17307-6 and
09/54640-5)
DENGUE 4 (DENV – 4) IDENTIFIED
IN THE STATE OF PERNAMBUCO,
DURING THE PERIOD FROM JANUARY
TO JUNE OF 2012
Alencar, L.X.E., Oliveira, V.F., Santiago,
R.G., Félix, J., Silva, S.G., Ribeiro, M.G.B.
Laboratório Central Dr. Milton
Sobral,
LACEN/SES-PE,
Rua
Fernandes Vieira, s/n, Boa Vista,
Recife-Pe E-mail: licixea@hotmail.
com
Infections caused by dengue virus
(DENV) is one of the most important
diseases transmitted by arthropodborne, about 50 to 100 million people
worldwide are infected each year and
500,000 live in the risk areas.The
increase and spread of dengue cases in
the state of Pernambuco in 2012, may
be due by introduction of DENV-4. In
this study, the serotypes of the dengue
virus were identified, isolated and the
affected counties have been mapped.
Some samples were isolated by viral
isolation in C6/36 cell culture, others
have been screened by the technique
of NS1 ELISA, and then serotyped by
RT-PCR. Of the 254 reagent samples
tested by the NS1, 109 (42.9%) were
positive by RT-PCR where all four
serotypes were identified as 14 (5.5%)
were DENV-1, 2 (0.8%) were DENV-2,
4 (1.6%) were DENV-3 and 89 (35%)
were DENV-4.Of the 504 samples
tested by virus isolation only 37 (7.4%)
were identified as DENV-4. Through
these two methods the serotypes
were mapped across the state of
Pernambuco. However, despite the four
serotypes are circulating in the state,
there was a predominance of DENV4, where its introduction is a recent
event and the population was more
HV1312 - INCIDENCE OF CASES OF susceptible to infection.The knowledge
of these data is important to trace the
involved population's epidemiological
profile and to adopt effective control
measures.
HV1314 - MOLECULAR EPIDEMIOLOGY OF NOROVIRUS IN CHILDREN
Paula, F.L., Sardi, S.I., Pinho, A.C.O.,
Peixoto, I.B., Welby-Borges, M., Brandão,
C.J.F., Bandeira, A., Campos, G.S.
1. Universidade
Federal
do
Recôncavo da Bahia, UFRB,
Av.Carlos Amaral, 1015 - Santo
Antônio de Jesus - BA. CEP:
44.570-000
2. Universidade Federal da Bahia,
UFBA, Av. Reitor Miguel Calmon
s/n - Salvador - BA. CEP 40.110100
3. Hospital Aliança, HA, Av Juracy
Magalhães Jr, 2096 - Salvador
- BA. CEP 41920-900 E-mail:
[email protected]
Viral gastroenteritis is one of the most
common diseases of humans and it
is estimated to occur in more than
700 million cases in children under
5 years old. The common viral agent
that causes gastroenteritis outbreaks
worldwide is Norovirus (NoV). The
viral transmission is mainly by the
fecal-oral route via person to person
or food and water contaminated.
NoV, a member of Caliciviridae family,
is a RNA virus, classified into five
genogroups (GI to G IV) from of which
GII is the most prevalent in humans. The
objective of this study was to identify
and to characterize the NoV during
an acute gastroenteritis outbreak
in children. It was collected stool
samples (n=206) from children from
the Aliança Hospital in Salvador, Bahia,
Human Virology: HV
273
during an outbreak from March-July
2010 from children under 5 years old.
The commercial immunoenzymatic
assay
(ELISA
RIDASCREEN®
Norovirus 3rd Generation R-Biopharm,
Germany) and Reverse TranscriptionPolymerase chain reaction (RT-PCR)
were performed to detect NoV in stool
samples. From the total of two hundred
and six samples, NoV was detected in
26.21% (54/206), where the median
age was 1 year and 6 months, using
an ELISA test. From the total of ELISA
positive stool samples, twenty-three
were submitted and confirmed the
presence of NoV by RT-PCR using the
primers CAL-32/MO3-N and JV-12/
ACAL-36. Then, fifteen of them (15/23)
selected at random were subjected
to sequencing. After the analysis on
NCBI/BLAST, it was found that all
samples exhibited a high similarity
to GII.4 strains (96-99% homology).
Concluding, the acute gastroenteritis
outbreak in children during 2010
confirmed the presence of NoV GII.4.
Financial support: Fundação de
Amparo à Pesquisa do Estado da Bahia
– FAPESB
HV1318 - THE ROLE OF EAST-WEST
BALANCE IN THE SOUTHERN HEMISPHERE INFLUENZA VACCINE
RECOMMENDATION
Born, P.S., Bentancor, G.B., Siqueira,
M.M., Motta F.C.
INSTITUTO
OSWALDO
CRUZ/
FUNDAÇÃO OSWALDO CRUZ, IOC/
FIOCRUZ, AV BRASIL, 4365 RJ 21040360 E-mail: priscilaborn@yahoo.
com.br
Influenza infections are the principal
cause of severe respiratory disease,
affecting individuals worldwide on
a yearly basis. The vaccination is the
274
Human Virology: HV
main strategy for infection control,
but the vaccine needs to be updated
every season to avoid mismatches
between vaccine prototypes strains
and those circulating in the population.
The choice of vaccine prototypes is
driven by a comparison of viruses
circulating throughout the year in the
Northern or Southern Hemisphere
(SH). Historically, the greatest amount
of virus isolates come from Oceania,
particularly Australia, which raises
the question if the samples present in
the SH vaccine recommendations are
closer to the viruses from that region,
therefore, promoting less protection to
virus in South America (SA). Aiming to
evaluate this question, we compared
influenza
A(H3)
hemmaglutinin
sequences
from
Australia
and
Brazil throughout 2004-2011 with
respective vaccine prototypes by
year. The sequences were aligned
using the MEGA v5.1 software and
the comparison between samples and
prototypes were carried-out using JTT
algorithm (1000 bootstrap replicates).
During this period the most important
prevalence of A(H3) in SA was detected
in 2004, 2007 and 2011, just after the
years were recommended vaccine
prototypes showed less identity with
community isolates in Brazil. Our results
demonstrated divergence among the
geographic distinct groups during most
influenza seasons evaluated when
samples were compared with vaccine
prototypes. These differences were
remarkable in 2006 and 2009, when
samples circulating in Australia were
closer to vaccine strains in comparison
to Brazilian ones. In this analysis we
could identify differences between
vaccine and circulating strains along
two distinct regions in SH. In addition,
we demonstrated a simple method
to generate data that, combined to
serological results, allow a rapid
analysis of influenza vaccines annually
administrated in South Hemisphere.
Financial support: DECIT/MS, IOC/
Fiocruz, CAPES
HV1319 - DISTRIBUTION OF
IL28B SINGLE-NUCLEOTIDE POLYMORPHISMS IN PATIENTS WITH
HEPATITIS C VIRUS INFECTION
Pelegrini, A., Passos, A.M., Granato,
C.F.H.
1. Grupo Fleury SA, Fleury, Av.
Gal. Valdomiro de Lima, 508,
Jabaquara, São Paulo
Toledo, 781, 15º andar, Vila
Clementino, São Paulo E-mail:
andreia.pelegrini@grupofleury.
com.br
Hepatitis C virus (HCV) infection is
a global health problem that affects
a significant population worldwide.
HCV causes chronic hepatitis, which
may progress to liver cirrhosis and
hepatocellular carcinoma. Factors
related to the virus, host, environment,
and their interplay have an important
role in determining the disease
progression. Recently, the singlenucleotide polymorphisms in the
interleukin 28B gene (IL-28B SNPs)
have been associated to virological
response to interferon-based therapy,
but it remains unclear whether IL28B
SNPs influence the severity and
progression of liver disease. The aim of
this retrospective study was to assess
the distribution of IL28B genotypes
in HCV patients and investigate a
possible impact of the polymorphisms
on laboratorial findings. A total of 45
patients were enrolled. Genotyping
of the rs12979860 SNP and plasma
HCV viral load were determined using
real-time quantitative RT-PCR assays.
Blood and biochemical tests were
performed using standard methods.
Most patients were males (62.2%),
and the overall mean age was 53.1
years (range 29 - 67 years). The
distribution of IL28B genotypes was
15.6% for the CC genotype, 66.7% for
the CT genotype, and 17.8% for the TT
genotype. Differences in mean serum
value of AST, ALT, total bilirubin, AFP,
platelets, and HCV viral load were
observed when CC group and nonCC group were compared; however,
there were no statistical significant
differences between them. Some
studies have indicated the involvement
of the CC genotype at rs12979860
in both spontaneous and treatmentinduced control of HCV infection. Our
results demonstrated a low frequency
of the CC genotype and there were
no evident association between
the polymorphism and laboratorydetectable abnormalities. As a
preliminary study, the small number
of samples influenced our results and
further information can contribute for
a better understanding of the role of
IL28B SNPs in the progression of the
HCV infection.
HV1320 - DETECTION OF HEPATITIS
B SURFACE ANTIGEN (HBSAG) IN A
NEONATE BORN TO A HBV VACCINATED WOMEN: A CASE REPORT
Pelegrini, A., Justa, M.T.R., Rocha, L.S.A.,
Granato, C.F.H.
1. Grupo Fleury SA, Fleury, Av.
Gal. Valdomiro de Lima, 508,
Jabaquara, São Paulo
Human Virology: HV
275
Toledo, 781, 15º andar, Vila
Clementino, São Paulo E-mail:
andreia.pelegrini@grupofleury.
com.br
Hepatitis B is a vaccine-preventable
disease that has been estimated
to have infected over 2 billion
people worldwide. The likelihood of
progression to chronic infection is
inversely related to age at the time
of infection. Around 90% of infants
infected perinatally become chronic
carriers, unless vaccinated at birth. In
Brazil, the hepatitis B vaccination is
recommended for all infants, regardless
of the HBsAg status of the mother, and
the first dose is administered preferably
within 12 hours of birth. In this case
report, we describe the detection of
hepatitis B surface antigen (HBsAg)
in a neonate born to a HBV vaccinated
women. Furthermore, we discuss the
proper interpretation of the serological
results for the correct diagnosis. A
serum sample of a 3-day-old neonate
was sent to our laboratory for screening
for evidence of HBsAg. The sample was
reactive in the Roche Modular E170
assay (reading/cut-off: 2.39/1.0) and
was confirmed by neutralization in
the HBsAg Confirmatory Test. After
six days, a second sample was sent for
screening for evidence of HBsAg, antiHBs, and anti-HBc markers. HBsAg and
anti-HBc results were negative and antiHBs were positive (reading/cut-off:
447.0/10 UI/L). The woman presented
negative evidence of HBsAg, anti-HBs,
and anti-HBc markers. Analyzing all
the serological markers results, the
presence of HBsAg in the neonate
sample represented a possible crossreactivity with vaccine antigens, since
the mother did not presented a prior
evidence of infection and consequently
276
Human Virology: HV
the mother-to-child transmission
was excluded. The development of
serological assays to detect HBsAg and
other hepatitis B markers has played a
major role in the diagnosis of infection.
This case highlights the importance
of not assessing the HBV status of the
neonate before the completion of the
course of vaccination since serological
profiles can at times be ambiguous and
thus additional evidences are widely
useful for the proper interpretation of
the results.
HV1322 - COMPARISON OF LABORATORY TECHNIQUES FOR HUMAN
RESPIRATORY SYNCYTIAL VIRUS IN
CLINICAL SAMPLES OF OUTPATIENT
CHILDREN AND BONE MARROW
TRANSPLANTED ADULTS WITH
SUSPECT ACUTE RESPIRATORY
INFECTION TREATED AT SAO PAULO
HOSPITAL
Moreira, L.P., Watanabe, A.S., Carraro,
E.M., Silva, E.R.M., Guatura, S.B.,
Granato, C., Bellei, N.
1. UNIVERSIDADE FEDERAL DE SÃO
PAULO, UNIFESP, – Rua Pedro de
Toledo, 781, Vila Clementino – SP,
CEP: 04039-032 – Brazil
2. Universidade Estadual do Centro
Oeste, Unicentro, Guarapuava,
Paraná,
Brasil
E-mail:
[email protected]
Human respiratory syncytial virus
(HRSV), an important agent in the
acute respiratory tract infections
of immunocompromised patients,
accounts for more than 50% of the
deaths of hematopoietic stem cell
transplant patients. Infection control
and clinical management rely on the
prompt diagnosis of suspected cases.
This study was performed to evaluate
an improved diagnostic flow for the
detection of HRSV among patients
of the Haematopoietic Stem Cell
Transplant program (HSCT) using
a direct immunofluorescence assay
(DFA), immunochromatographic pointof-care RSV Bio Easy® (PC) assay and
a polymerase chain reaction assay
used as the gold standard. Laboratory
surveillance guided by viral seasonality
according to community surveys
among children was also evaluated. A
total of 230 nasal wash samples, 102
from HSCT patients and 128 from
children, revealed a viral detection rate
of 14.1% for children and 18.6% for
HSCT patients. An overall concordance
of 84.6% was obtained among the
three methods, and 88.4% for DFA
and PCR. For the samples collected
5 days after the onset of symptoms,
PCR exhibited the highest sensitivity.
We conclude that the low sensitivity
of the tested immunocromatographic
assay do not support its use on routine
practice. For the children group,
DFA was considered sufficient for
epidemiological surveillance. Routine
laboratory surveys based on DFA and
a combination of both DFA and RT-PCR
methods for HSCT high-risk patients
provided the best diagnostic flow for
HRSV diagnosis among these patients.
Financial support: CNPq/FAPESP
HV1324 - MOLECULAR EPIDEMIOLOGY OF G9 ROTAVIRUS GENOTYPE
INFECTION AMONG CHILDREN IN
NORTH REGION, BRAZIL FROM 1999
TO 2011
Guerra, S.F.S., Soares, L.S., Lima, C.S.,
Oliveira, D.S., Oliveira, A.S.L., Gabbay,
Y.B., Linhares, A.C., Mascarenhas, J.D.P.
Rodovia
Br-316-Km07,
s/n,
Levilândia,
Ananindeua,
Pará
Núcleo de Medicina Tropical - UFPa,
NMT-UFPa, E-mail: sylviaguerra@
iec.pa.gov.br
Rotavirus
(RV)
is
the
most
common etiological cause of acute
gastroenteritis in infants and young
children worldwide, representing a
significant cause of morbi-mortality
among children aged less than five
years old. Rotavirus belongs to the
genus Rotavirus, family Reoviridae,
with a genome consisting of 11
segments of double-stranded RNA
(dsRNA) that encodes 12 proteins. Two
proteins, VP7 and VP4, independently
elicit neutralizing responses and
define different genotypes of RV, G and
P, respectively. At least 27 G-types and
35 P-types have been described. The
VP7 gene encodes 326 amino acids and
carries six antigenic regions. The G9
genotype is one of the most frequent
genotype with distinct genetic and
molecular characteristics. Currently, the
phylogenetic analyses of G9 genotypes
confirm the existence of 6 lineages. The
aim of this study was characterize VP7
gene of RV G9 strains detected in north
region, Brazil, between 1999 and 2011
from children with acute. The dsRNA
viral of 35 samples was extracted
from fecal suspensions and submitted
to reverse transcription and then
amplified by PCR. The products were
submitted to a sequencing reaction
and thereafter phylogenetic analysis
was performed. The phylogenetic
analysis demonstrated that all G9
strains grouped into lineage III,
showing great similarity and being
very conserved. The VP7 sequences
had high identities among themselves
ranging from 96,8% to 100%, and
have shown major divergences when
compared to strains from Acre state
Human Virology: HV
277
(2005) and strains from 2010 and 2011
years, which showed modifications
in the amino acid residue located in
the antigenic region A (residue 100
aa). The phylogenetic analysis of G9
genotype that is currently spread on
a global scale, seems very important
mainly if we consider the present postrotavirus vaccine introduction scenario
when possible emergent new strains
may pose a challenge to rotavirus
vaccination. Financial support: CNPq,
Instituto Evandro Chagas
HV1327
ASSESSMENT
OF
ARBOVIRUS
IN
MOSQUITOES
COLLECTED FROM URBAN/FOREST
TRANSITION AREAS OF SÃO JOSÉ DO
RIO PRETO, SÃO PAULO AND SINOP,
MATO GROSSO (BRAZIL)
Ozanic, K., Parra, M., de Carvalho, C.P.T.,
Vedovello, D., Machado, D.C., Nogueira,
M.L., Bronzoni, R., Mondini, A.
São Pedro - 15090-000
UNESP, Rod. Araraquara-Jaú
Km 1 Machados 14800-901 Araraquara, SP
3. Universidade Federal do Mato
Corrêa da Costa, nº 2367 - Bairro
Boa Esperança. Cuiabá - MT 7806 E-mail: katiaozanic@gmail.
com
Arboviruses are zoonoses that depend
on animal specimens to replicate
and spread within the environment.
In terms of public health, the most
important arboviruses are the ones
transmitted by mosquitoes since they
278
Human Virology: HV
are usually found in cities and forests
alike. It is possible to investigate
viral circulation patterns within the
vectors through mosquito collection.
Our goal was to assess the presence
of arboviruses in mosquitos collected
at urban/forest transition areas of São
José do Rio Preto, São Paulo and Sinop,
Mato Grosso (Brazil) from October 2011
to April 2012. The specimens of several
species were grouped in 141 pools
according to date of collection, site,
gender and genus/species. Viral RNA
was extracted using TRIZOL and the
pools were tested with a Hemi-NestedMultiplex-RT-PCR that uses generic
and specific primers for Flavivirus and
Alphavirus. Seventy-seven samples
(54,6%) were analyzed at the moment.
One pool collected in September
2011 was positive for Culex flavivirus,
which was confirmed by sequencing
of NS5 region. This prelimary data is
in accordance with was found in the
city of São José do Rio Preto in 2007
and 2008, when Culex flavivirus was
circulating within the city. It is likely
that this virus have established a
continuous circulation in the region
and the reflex of its circulation may be
the hampering of the transmission of
other flaviviruses. Financial support:
CNPq (480945/2010-1)
HV1328 - MOLECULAR CHARACTERIZATION OF DENGUE VIRUS
SEROTYPE 1 IN SÃO JOSÉ DO RIO
PRETO - SP
Biselli, J.M., Vedovello, D.
do Rio Preto, FAMERP, Av. Brigadeiro
Faria Lima, 5416, São Pedro, São José
do Rio Preto, SP, 15090000 E-mail:
[email protected]
In 2010, Brazil registered more than
one million probable cases of Dengue
in consequence of the recirculation
of DENV1 and Sao Jose do Rio Preto
(SJRP), SP, had the largest Dengue
outbreak, with DENV1 as a the most
important agent after over 10 years
without its detection in 2008. The
introduction of new serotypes/
genotypes is the main risk factor
for Dengue outbreaks; however, it is
not clear if the outbreaks reported
in Brazil have occurred due to clade
replacement, population susceptibility
or secondary infections. Thus, DENV1
samples collected between 2009-2012
in SJRP are under investigation of
Envelope (E) gene sequence to identify
predominant genotypes and eventual
clades of DENV1 that have contributed
to the raise of DENV1 infection in SJRP.
This study included serum samples
sent to the Laboratorio de Pesquisa
em Virologia of FAMERP for Dengue
diagnosis, with 414 positive samples
for DENV and 368 for DENV1. Since
virus isolation in cell lines can lead to
selection of virus strains, we decided to
sequence the complete E gene directly
from serum of infected patients. A
PCR strategy was optimized in order
to amplify a 1855 bp fragment of
DENV1 genome and the samples will
be sequenced. Until now only four
DENV1 samples have been subjected
to sequence analysis of E gene. After
specific RT-PCR, purified PCR amplicons
were sequenced using the BigDye
v3.1 in ABI3130 automatic sequencer
(Applied Biosystems). Derived DENV1
nucleotide sequences were aligned
using Accelrys Gene 2.5 software
(Accelrys) with previously published E
gene sequences from GenBank and the
sequences were confirmed as DENV1 E
gene. Phylogenetic analysis performed
using Mega 5.05 software showed that
these four samples of DENV1 belong
to genotype V and are grouped in a
same clade. We will sequence up to
100 samples from every year with
DENV1 circulation. This study can
be an important tool for monitoring
the introduction and propagation of
viruses and to predict their potential
epidemiological
consequences.
Financial
support:
INCT/CNPqDengue; FAPESP; CAPES; FAMERP/
FUNFARME; Secretaria Municipal de
Saude.
HV1331 - RESISTANCE TO NS5A
ANTIVIRAL AGENTS IN THERAPY-NAÏVE BRAZILIAN PATIENTS WITH
HEPATITIS C VIRUS INFECTION
Peres da Silva, A., de Almeida, A.J.,
Lampe, E.
1. Universidade Federal do Estado
do Rio de Janeiro, UNIRIO, Rua
Mariz e Barros, 775 - Tijuca, Rio
Av. Brasil, 4365 - Manguinhos,
Rio de Janeiro - CEP: 21040-360
br
The current therapy to Hepatitis C
virus (HCV) infection are suboptimal
especially in patients infected with HCV
genotype 1 and are poorly tolerated
because of its side effects. Major
researches efforts focused on new
therapeutic approaches are based on
NS3/4A protease and NS5B polymerase
inhibitors which are currently in most
advanced clinical development. Among
the nontraditional targets, NS5a protein
has shown potentially active across
different HCV genotypes and promising
antiviral efficacy in clinical studies.
However, several resistance variants to
Human Virology: HV
279
NS5a inhibitors have been described
both in in vivo and in vitro studies and
may represent an important factor
that limit the effectiveness of therapy
with direct-antiviral agents (DAAs). In
this context, the aim of this study was
to analyze the genetic variability of
NS5a gene in HCV genotype 1 isolates
circulating in our region. A total of 26
samples from therapy-naïve Brazilian
patients chronically infected with
HCV subtype 1a were studied. Viral
RNA was extracted and the region
encompassing the NS5a gene (6258nt7602nt) was reverse-transcribed and
PCR-amplified and submitted to direct
sequencing. Nucleotide sequences
were analyzed by multiple alignment
using the MEGA 4.0 program and
deduced amino acid sequences were
inferred by the same program. The
analysis revealed the presence of
variants in two Brazilian samples,
BR137 and BR157, at sites Y93 (Y93H)
and H58 (H58P), which confers high
resistance to the inhibitor Daclatasvir
(BMS-790052). These mutants were
not related to occur in European
therapy-naive patients with HCV
subtype 1a infection, corroborating to
the fact that analysis of viral sequences
from different geographical regions
may show significant differences in the
frequencies of resistance to new DAAs
inhibitors. Information on patterns
of resistance to new DAAs may be
determinant for future decisions on
how to combine drugs to achieve
an optimal antiviral effect. Financial
support: Capes-Papes V/ CNPq
HV1334 - HEPATITIS D VIRUS
SCREENING IN PATIENTS WITH
ACUTE OR CHRONIC HEPATITIS
B VIRUS INFECTION IN RIO DE
JANEIRO, BRAZIL
280
Human Virology: HV
Marques, V.A., Miguel, J.C., Silva, E.F.,
de Almeida, A.J., Lewis-Ximenez, L.L.,
Lampe, E.
Av. Leopoldo Bulhões, 1480.
Manguinhos - Rio de Janeiro - RJ
Hepatitis D virus (HDV) is a defective
virus that requires the presence of
hepatitis B virus (HBV) to complete
its replicative cycle. HDV infection
is associated with more severe form
of hepatitis and increased risk of
progression to complications such
as cirrhosis and hepatocellular
carcinoma. In Brazil, the Amazonian
Basin is an endemic area for HDV
infection, however, few date is available
for others regions in the country. This
study evaluated the seroprevalence of
HDV in patients with acute or chronic
infection with HBV followed at the Viral
Hepatitis Ambulatory, Rio de Janeiro,
Brazil, between 2006 and 2011. A
total of 370 samples from patients
presenting hepatitis B surface antigen
(HBsAg) positivity were tested for the
presence of serum anti-HDV antibodies
using the commercial assay ETI-ABDELTAK-2 (Diasorin, Italy), according
to the manufacturer’s instructions.
Reactive or indeterminate results were
retested in duplicate to confirm the
result. Patients’ samples with anti-HDV
positive were tested by PCR to amplify
a fragment of delta antigen (HDAg)
genomic region. HDV RNA positive
samples were submitted to direct
nucleotide sequencing and phylogenetic
analysis using MEGA v. 5.0 software
package to characterize HDV genotype.
Our study population consisted of 243
males and 127 females, with a median
age of 43 years (1 – 82 years), being
138 patients with acute HBV infection
and 232 with chronic. Six patients
were positive for anti-HDV antibodies
(acute HBV, n = 1; chronic HBV, n = 5),
one of the patient with chronic HBV
infection had detectable HDV RNA.
The phylogenetic analysis showed that
the HDV sequence clustered within
genotype 3. In conclusion, despite the
HDV seroprevalence found to be low
in our cohort, this results highlighted
the importance of HDV infection
investigation in non endemic areas.
Financial support: FIOCRUZ
HV1337 - EPIDEMIOLOGIC IMPACT
OF HEPATITIS D (DELTA) IN THE MUNICIPALITY OF GUAJARÁ-MIRIM/RO
Justiniano, R.L., Santos, A.O., Vieira, D.S.
1. Oswaldo Cruz Foundation RO,
Brazil, FIOCRUZ RO, RUA DA
BEIRA, 7671, LAGOA
2. CENTRO DE PESQUISA EM
MEDICINA TROPICAL DE RO,
CEPEM, AV. GUAPORE, 215, LAGOA
Delta virus (HDV) infection is highly
pathogenic and transmitted in the
presence of surface antigen of the
virus causing hepatitis B (HBsAg). The
transmission is given by a co-infection
or superinfection. In areas endemic
for hepatitis B, the HDV infection
represents a serious public health
problem, and the state of chronic HBV
(HBsAg positive) constitutes the main
factor for the epidemiological spread of
HDV, which occurs, for example among
the native populations of the Brazilian
Amazon, Peru and Venezuela and, in
certain areas of Africa. The municipality
of Guajará-Mirim located in the
western Brazilian Amazon, belonging
to the state of Rondônia an estimated
population of 41.933 inhabitants,
considered the eighth most populous
county in the state has characteristics
of mixing of various races with the
native (indigenous culture), resulting in
a population Amazon typically with the
predominance of "shifting cultivators"
and a strong presence of mixing with
immigrants from the border (Bolivia).
This study evaluates the epidemic
incidence of HDV infection in this city
through exploratory and descriptive
research. Data were obtained through
the Program STD/HIV/AIDS and Viral
Hepatitis of the Municipal Health
Guajará-Mirim RO-2010 - 2011, by
which it was estimated 138 cases
of HBV infection and 12 cases of coinfection with HDV. The most affected
age group was the between 20 and 39
years of age, with 66% of the total, and
the male was the most affected by HDV,
registering 55.8% of cases. By these
data we can see the need to program
or improve measures that can reduce
the incidence and prevalence rates of
Hepatitis Delta. Financial support: SUS.
HV1344
COMPARISON
OF
DIRECT FLUORESCENCE ASSAY
AND REAL-TIME PCR FOR THE
DETECTION OF INFLUENZA VIRUS A
AND B IN IMMUNOCOMPROMISED
PATIENTS
Perosa, A., Watanabe, A.S.A., Ricci, E.,
Guatura, S., Bellei, N.
- 15o andar E-mail: anaperosa@
gmail.com
During 2009 H1N1 pandemic,
morbidity was high in Brazil and
hospitalizations
resulting
from
severe respiratory disease due to
suspected influenza infection were of
concern during the subsequent years.
According to health authorities, during
Human Virology: HV
281
2011 influenza A virus caused eight
deaths in São Paulo state, but this year
number of cases and deaths has been
increased. The aim of this study was
evaluate de influenza virus A and B
prevalence among samples collected
from hospitalized patients with severe
acute respiratory infection (SARI) and
from outpatients who presenting with
influenza-like illness (ILI) received
at Virology Laboratory of São Paulo
Hospital. Influenza A and B were
investigated by CDC Real-time PCR
(RT-PCR) with minimal modifications
to include influenza B. During 2011,
we analyzed 169 respiratory samples
(nasal swabs/washes) and influenza
prevalence was 21.3%. In 2012, we
received 103 respiratory samples
and influenza prevalence was 30.1%.
During 2011, influenza was detected
in 4.8% (1/21) of patients with SARI
(only seasonal influenza A) and in
29.5% (33/112) of ILI patients, of
whom 60.6% (12/33) were influenza
B, 36.4% (12/33) were influenza A
H3N2 and 3% (1/33) H1N1pdm/09.
On the other hand, 2012 influenza
detection was 36.1% (13/36) in
hospitalized patients and 42.9% (9/21)
in ILI patients, out of this 89% (8/9)
were influenza A. We documented
that influenza is responsible for mild
and severe respiratory infections and
accounted for a large proportion of
hospitalizations for SARI during 2012
influenza season. Financial support:
CNPQ, FAPESP
HV1345 - THE USE OF SOCIAL NETWORKING FACEBOOK AS A TOOL
FOR DISSEMINATION OF SCIENTIFIC
KNOWLEDGE IN VIROLOGY
Silva, G., Vedovello, D., Ozanic, K.,
Nogueira, M.
282
Human Virology: HV
do Rio Preto , FAMERP, Av. Brigadeiro
Faria Lima, 5416 - Vila São Pedro 15090-000 E-mail: gislaine.cds@
gmail.com
The use of social networks like
Facebook, Orkut, MySpace or Twitter
crossed the personal relationships
and became a great way to Internet
marketing and dissemination of
knowledge. For dissemination of
exclusive scientific knowledge there
are networks like ResearchGate, that
includes more than 35,000 Brazilian
researchers, BiomedExperts, SciLink,
and others. These networks are
restricted to a specific audience and
they aren’t used by the remainder
of the population. The Facebook has
become the larger tool of relationship
in the world and are widely used by
scientific research groups worldwide.
By having the largest number of users
this may contribute to the access and
disclosures of scientific papers, closer
relationship between the Brazilians
laboratories and the others labs in the
world. Using the search engine of the
Facebook we found 42 groups in the
social network (restricted or open)
that have specific content of Virology
and has over 2300 members. Among
them we can highlight our group page
Laboratório de Pesquisa em Virologia
- FAMERP (http: // www. Facebook .
com / groups / 205746489469432).
This group is the largest in number
of members, totaling 405 people,
consisting of undergraduate and
postgraduate students, teachers of
Secondary and Higher Education and
renowned scientific researchers. In the
LPV group there are members of all
regions of Brazil; 260 (64,3%) members
are from the Southeast Region, 9 (2,2%)
are from the Southern Region, 6 (1,4%)
are from the Midwest, 5 (1,2%) from
Northern and 3 (0,7%) from Northeast.
There are also members of the U.S. and
the others countries, 12 (2,9%) and
112 (27,7%) members did not specify
the place of residence/ origin. Created
in June 2011 the our group on Facebook
aims to disseminate information in the
scientific area, promote discussions,
and facilitate the exchange of
manuscripts (within the area of human
virology, plant and animal) published
in Brazil and abroad. From January to
July 2012 more than 114 articles were
shared and discussed among the group
members. The tool also allowed the
dissemination of courses and events
related to this science – Virology.
This shared of information can have a
positive effect making when learning
becomes more enjoyable.
HV1348
PREVALENCE
OF
INFLUENZA VIRUS IN HOSPITALIZED PATIENTS WITH SEVERE ACUTE
RESPIRATORY INFECTION AND OUTPATIENTS WITH INFLUENZA-LIKE
ILLNESS DURING 2011 – 2012 IN
SÃO PAULO HOSPITAL
Perosa, A., Camargo, C., Guatura, S.,
Bellei, N.
- 15o andar E-mail: anaperosa@
gmail.com
During 2009 H1N1 pandemic,
morbidity was high in Brazil and
hospitalizations
resulting
from
severe respiratory disease due to
suspected influenza infection were of
concern during the subsequent years.
According to health authorities, during
2011 influenza A virus caused eight
deaths in São Paulo state, but this year
number of cases and deaths has been
increased. The aim of this study was
evaluate de influenza virus A and B
prevalence among samples collected
from hospitalized patients with severe
acute respiratory infection (SARI) and
from outpatients who presenting with
influenza-like illness (ILI) received
at Virology Laboratory of São Paulo
Hospital. Influenza A and B were
investigated by CDC Real-time PCR
(RT-PCR) with minimal modifications
to include influenza B. During 2011,
we analyzed 169 respiratory samples
(nasal swabs/washes) and influenza
prevalence was 21.3%. In 2012, we
received 103 respiratory samples
and influenza prevalence was 30.1%.
During 2011, influenza was detected
in 4.8% (1/21) of patients with SARI
(only seasonal influenza A) and in
29.5% (33/112) of ILI patients, of
whom 60.6% (12/33) were influenza
B, 36.4% (12/33) were influenza A
H3N2 and 3% (1/33) H1N1pdm/09.
On the other hand, 2012 influenza
detection was 36.1% (13/36) in
hospitalized patients and 42.9% (9/21)
in ILI patients, out of this 89% (8/9)
were influenza A. We documented
that influenza is responsible for mild
and severe respiratory infections and
accounted for a large proportion of
hospitalizations for SARI during 2012
influenza season. Financial support:
CNPQ, FAPESP
HV1358 - DETECTION OF NOROVIRUSES IN FECAL SAMPLES OF
CHILDREN WITH AND WITHOUT
RIO BRANCO, ACRE
Rodrigues, E.L., Silva, L.D., Lucena, M.S.S.,
Menezes, E.M.F., Lima, I.C.G., Medeiros,
T.B., Soares, L.S., Mascarenhas, J.D.P.,
Loureiro, E.C.B., Rodrigues, I.R.C., Silva,
M.C.M., Gabbay, Y.B.
Human Virology: HV
283
Rodovia Br 316, Km 07 E-mail:
[email protected]
Noroviruses (NoVs) are the main cause
of diarrheic outbreaks non-bacterial
origin, transmitted primarily by fecaloral route, through contaminated water
and food or by person to person contact.
The NoVs belong to Caliciviridae family
and the main symptoms caused by
these agents are vomiting and diarrhea.
Populations of developing countries
are more susceptible to diarrhea by
this virus and studies that demonstrate
the etiology of these infections are
important to guide public policies of
prevention and control. The objective
of this investigation was to detect
NoVs in fecal specimens collected from
children under five years old, during
three expeditions carried out in Rio
Branco, Acre, in February, April and
June of 2012. The fecal specimens were
collected from children attended in
the Emergency unit (UPA) of the I and
II district. The samples were initially
tested by enzyme immunoassay (EIA)
and after by reverse transcriptasepolymerase chain reaction (RT-PCR)
using primers Mon 432-434/ 431433 specific for NoVs genogrupos
GI and GII, respectively. Of the 277
samples collected, a positivity of
11.9% (33/277) was observed to
NoVs for at least one technique, among
which, 8.7% (9/103) was collected in
February, 3.8% (4/106) in April and
29.4% (20/68) in June. Therefore, the
viral detection decreased from January
to April, with considerable increase
of the positivity in June. Furthermore,
molecular characterization will be
done for NoVs genotypes identification.
NoVs have been identified as relevant
etiological agent of diarrhea in many
places. In Belém (35.4%- 171/483)
284
Human Virology: HV
and São Paulo (29.2%- 26/89), NoVs
infections were also detected in high
percentage in cases of diarrhea among
hospitalized children less than five
years old. This is the first report about
NoVs detection in Rio Branco, Acre
and demonstrates the epidemiologic
importance of this virus in that region.
Financial Support: IEC/SVS/MS.
HV1360 - STRUCTURAL FEATURES
OF HIV-1 C2-V3-C3 REGIONS OF
GP120: SELECTIVE PRESSURE AND
DIVERSITY BETWEEN CLADES B
AND C FROM BRAZIL
Araújo, L.A.L., Junqueira, D.M.,
Medeiros, R.M., Matte, M.C.C., Graf, T.,
Almeida, S.E.M.
Fundação Estadual de Produção e
Pesquisa em Saúde, FEPPS/RS, Av.
Ipiranga, 5400 - Porto Alegre/RS
- CEP: 90610-00 E-mail: leonardo_
[email protected]
In Brazil, HIV-1 subtypes B and C have
accounted for the majority of infections.
Nearly half of the infections caused by
the subtype B strain are due to viruses
with the unusual GWGR motif in the
V3 loop of the envelope (Env) gene.
Understanding how and why inter- and
intra-subtype differ in Env is necessary
to tackle the genetic diversity of HIV-1
in vaccine design and treatment. For
this purpose, all available gp120 C2V3-C3 (HXB2 6816-7380) for clades
B and C from Brazil were downloaded
from the Los Alamos National
Laboratory HIV Sequence Database
and GenBank. Additional sequences
obtained from samples collected in
Porto Alegre, Brazil, were included in
the data set totalizating 297 subtype
B and 166 subtype C sequences.
Shannon entropy was estimated for
each individual amino acid position
in order to reflect the variability of
that position across all sequences.
Positive selection was assessed using
the Maximum Likelihood approach
implemented in SLAC and FEL in the
DataMonkey package. The sequences
of subtype B are from the North (6.6%),
Northeast (8.2%), Midwest (14.8%),
Southeast (43.8%) and South (26%)
regions of the Brazil. The sequences
of subtype C are mostly from South
region (94.4%). Our analysis among
Brazilian subtypes B and C exhibits
that the structural domain encoded
in the C3 region overlapping sites
under positive selection, suggesting a
convergent evolution of these clades.
Examination of dN/dS ratios in V3
revealed much higher diversifying
selection in subtype B than in subtype
C. It is belived that C clade V3 domain
lacks sites of strong positive selection
due the formation of a cluster of
hydrophobic residues (I307, I309, and
F317). The analysis of subtype B GWGR
amino acid frequencies show that
position I309 tends to not preserve
specific hydrophobic residue (>94%).
The impact on the fitness of virus
might be relevant, since that V3 may
be more exposed in the GWGR viruses
serving as an antibody-mediated
neutralization target. Our data show
structural differences of the Brazilian
subtype B compared to subtype B
analysis worldwide.
HV1362 - THE LARGEST OUTBREAK
OF DENGUE IN THE STATE OF CEARÁ
Teixeira, F.M., Roriz, M.L.F.S., Melo,
M.E.L., Perdigão, A.C.B., Cavalcanti,
L.P.G., Vilar, D.C.L.F., Ramalho, I.L.C.,
Lima, E.G., Silva, L.B., Sá, R.C.A., Araújo,
F.M.C.
CEP:60120-002
600 - Centro Rede Nordeste de
Biotecnolgia - UECE, RENORBIOUECE, Av. Paranjana, 1.700 Campus do Itaperi - 60740-000
Fortaleza/CE E-mail: briciamt@
yahoo.com.br
Dengue virus infections are a major
concern in developing countries,
especially those located in subtropical
and tropical areas, as Brazil. The
disease affects, approximately, 100
million people/ year. In the state of
Ceará in Northeast Brazil, dengue
epidemics have been described since
1986, initially with involvement of
DENV-1, followed by DENV-2 in 1994,
DENV-3 in 2002, and DENV-4 in 2011.
Twenty-six years of endemic dengue
were evaluated by the number of cases
reported and confirmed. Data collection
was based on the Epidemiological
Bulletins of dengue published by the
Health Department of the State of
Ceará. Among the analyzed period,
5 epidemic peaks were reported in
1987, 1994, 2001, 2008 and 2011. Of
these, the highest number of cases was
observed in 2011, with 56,714 cases
with laboratory confirmation, with
457 cases of DCC and 174 cases of DHF.
The total number of DCC and DHF were
behind only the data for year 2008. The
incidence was of 670.98 per 100,000
inhabitants, with predominance of
children and young adults. Secondly,
the year 1994 had 47,789 confirmed
cases with an incidence of 732.31 and
with predominance of serotype DENV2. In the year 2011, was detected
serotype DENV-1 mainly (98.7%), and
Human Virology: HV
285
serotypes DENV-4 and DENV-3 (0.9%
and 0.4% respectively). The highest
number of cases observed in 2011
was probably due to recirculation of
DENV-1 in recent years, having had
similar prevalence in the epidemic of
1987. The DENV-1 circulated widely in
Ceará by the year 2002. The population
born after this period was susceptible
to the virus that was circulating again.
Only one genotype of DENV-1 has been
circulating since the first epidemic
reports in Brazil. However, two different
lineages of DENV-1 genotype 5 have
been found around the country, so as
in Ceará. Financial support: FUNASA.
HV1381 - RT-PCR AMPLIFICATION
OF DENGUE VIRUS RNA USING A
MAGNETIC EXTRACTION METHOD
Barboza, M.M.O., Araújo, F.M.C.,
Perdigão, A.C.B., Cruz, J.N.M., Lima,
D.M., Pires Neto, R.J.
UFC, Rua Alexandre Baraúna, 949
- Rodolfo Teófilo - CEP 60430-160
- Fortaleza - CE
Pública, LACEN-CE, Av. Barão
de Studart, 2405 - Aldeota,
Fortaleza-CE
3. Universidade
de
Fortaleza,
UNIFOR, Av. Washington Soares,
1321, Edson Queiroz E-mail:
[email protected]
Dengue virus is an RNA virus
belonging to the Flaviviridae family
and the etiologic agent of today's
most important arthropod-borne
disease. It is estimated that about half
of the world’s population is at risk of
contracting the disease. It is transmitted
by the bite of the female Aedes aegypti
286
Human Virology: HV
mosquito. The disease can be caused
by any of the four serotypes (DEN1DEN4) and possesses a spectrum of
clinical presentations ranging from
undifferentiated fever to dengue
hemorrhagic fever and dengue shock
syndrome. Laboratory confirmation of
dengue depends on the time-of-sample
collection. Viral isolation, serological
tests and molecular methods are
currently available for diagnostic of
dengue infection. The polymerase
chain reaction preceded by reverse
transcription (RT-PCR) is an important
molecular method for early diagnosis of
dengue infection. However, for a good
yield in the RT-PCR is of paramount
importance that the RNA extraction
method is suitable. Our aim is to
evaluate the performance of magnetic
extraction method followed by Onestep RT-PCR. We used serum samples
from twenty patients known to be
positive for DEN-1 by viral isolation
and immunofluorescence technique.
The magnetic extraction of viral RNA
was performed with Biomerieux
NucliSense miniMAG kit following the
manufacturer's recommendations. For
RNA amplification in one step we used
the QIAGEN OneStep RT-PCR kit. The
primers used were described by RicoHesse (1990) and corresponds to the
E/NS1 junction region. The amplified
fragments were separated on 2%
agarose gel plus 5ul etidium bromide at
a voltage of 103 for 1 hour. The bands
were visualized under ultraviolet light.
Of the 20 samples tested, only 9 (45%)
had the E/NS1 region amplified. We
conclude that the magnetic extraction
method was suitable for RT-PCR of
dengue viruses but a greater number
of samples are necessary to better
evaluate performance.
HV1382 - PREVALENCE OF ANTI-HBC
ALONE IN PATIENTS TREATED AT
THE SEROLOGY LABORATORY OF
THE AMBULATORY SPECIALIZING
IN VIRAL HEPATITIS IN THE CENTER
FOR RESEARCH IN TROPICAL
MEDICINE IN 2010 AND 2011,
RONDÔNIA, BRAZIL
da Silva, M.F., Botelho, L.F.S., Salcedo,
J.M.V., Vieira, D.S., Santos, A.O.
1. Fundação Oswaldo Cruz de
Rondônia, FIOCRUZ Rondônia,
Porto Velho, Rondônia, Brazil
2. Fundação Universidade Federal
de Rondônia, UNIR, Porto Velho,
Rondônia, Brazil
3. Research Center for Tropical
Medicine , CEPEM, Porto Velho,
Rondônia, Brazil
4. Tropical Pathology Research
Institute, IPEPATRO, Porto Velho,
Rondônia, Brazil
The world has two billion people infected
with hepatitis B, and of these about
360 million are chronic carriers. HBV
is a DNA virus belonging to the family
Hepadnaviridae and preferentially
infects hepatocytes. Detection of antiHBc alone in the absence of anti-HBs
HBsAg and corresponds to a serological
profile that is unknown how the
clinical importance, and can mean past
infection without seroconversion or
with decreasing to undetectable levels
of anti-HBs. This profile can also be
observed in the phase of the window in
the cases of acute hepatitis B resolution.
However, this serological profile can
also be suggestive of occult hepatitis
B infection with HBsAg undetectable,
because of a low amount of virus in
the blood or mutations intrinsic to
the virus. Therefore it is important
to characterize the epidemiology
of the prevalence of anti-HBc alone
serological tests in patients attended
in Serology Laboratory Specialized
of Viral Hepatitis Ambulatory in
Rondônia. To that end, we consulted
the results of examinations of patients
seen at the Serology Laboratory of
Viral Hepatitis Ambulatory between
January/2010 and December/2011.
4645 examinations were consulted
where 483 patients as anti-HBc alone
were selected for study. In 2010 found
175 patients, 66% male, with higher
incidence in the age group of 40-60
years and 77% living in the capital.
In 2011 found 308 patients, 53%
male, with higher incidence in the age
group of 40-60 years, 55% living in the
capital and 45% inside the state. These
results corroborate other studies on
the prevalence corresponding to a
high prevalence of anti-HBc alone in
Serology Laboratory of Viral Hepatitis
Ambulatory in Rondonia. A solution to
solve this frame anti-HBc alone is the
test nucleic acid amplification (NAT),
which can detect a possible occult HBV
infection and to elucidate the molecular
mechanisms that cause this serological
profile. Financial support: IPEPATRO,
CEPEM/SESAU, FIOCRUZ RONDÔNIA e
UNIR
HV1392 - DETECTION OF POTENTIALLY NOVEL FLAVIVIRUSES IN
MOSQUITOES OF THE BRAZILIAN
PANTANAL
Pauvolid-Correa, A., Couto-Lima, D.,
Schatzmayr, H.G., Nogueira, R.M.R.,
Komar, N.
1. Fundacao
Oswaldo
Cruz,
FIOCRUZ, Avenida Brasil 4365,
Rio de Janeiro, RJ, 21045-900,
Brasil
Human Virology: HV
287
2. Centers for Disease Control and
Prevention, CDC, 3150 Rampart
Rd, Fort Collins, CO 80521, USA
The Brazilian Pantanal hosts large
concentrations of diverse wildlife
species, and therefore this region is a
hotspot for arbovirus studies in South
America. A recent study reported
serological evidence of various
arboviruses, including West Nile virus
and Ilheus virus (ILHV). To extend
this study, we captured 3111 adult
mosquitoes of 16 species from the
Nhecolandia sub-region of Pantanal
during 2009 and 2010. Mosquito
pool homogenates were assayed for
infectious viruses in C6/36 and Vero
cell monolayers and also tested for
flavivirus RNA by a group-specific
Real-Time RT-PCR using a SYBR-green
detection platform. In addition to a
single isolation of Ilheus virus from
Aedes scapularis, several unidentified
flaviviruses were detected by Real-Time
RT-PCR from Mansonia pseudotitillans
and Culex chidesteri. Amplicons in the
NS5 gene region of the 11 kb flavivirus
genome were cycle-sequenced and
compared to known NS5 sequences
in Genbank. The sequences had less
than 78% identity with other known
flaviviruses. The present data report the
circulation in mosquitoes of potentially
novel flaviviruses in the Nhecolandia
sub-region of Pantanal, Brazil.
HV1393 - SEROEPIDEMIOLOGICAL
STUDY INFECTION OF HEPATITIS C
VIRUS IN A PRISON IN MALE-ARAPIRACA ALAGOAS-BRAZIL
Santos, E.O., Souza, A.R., Silva, E.E.,
Morais, V.M.S.
1. Universidade
Federal
de
288
Human Virology: HV
Moraes Rego, 1235 - Cidade transfusion was performed in 22% of
Universitária, Recife - PE - CEP: those investigated. The absence of HCV
50670-901
infection among inmates shows that
this population does not constitute
2. Universidade
Estadual
de a risk group for this type of infection.
Alagoas, UNEAL, Rua Governador Financial support: State University of
Luiz Cavalcante, S/N - Alto Alagoas.
Cruzeiro - Arapiraca - Alagoas CEP. E-mail: erlon.medtropical@
hotmail.com
HV1394 - HIV-1 DIVERSITY AND
The infection caused by hepatitis C PREVALENCE OF TRANSMITTED
virus (HCV) is a serious public health RESISTANCE MUTATIONS OF ANproblem in Brazil and worldwide. The TIRETROVIRAL DRUGS AMONG
prison population has a high risk for HCV TREATMENT-NAÏVE CHILDREN AND
infection due to the high prevalence of ADOLESCENTS OF RIO DE JANEIROrisk factors related to sexual practices, -BRAZIL
the use of tattoo / piercing and injecting Azevedo, S.S.D., Delatorre, E.O., Ribeiro,
drug use. The present study aimed R.M.M., Couto-Fernandez, J.C.
to evaluate the seroepidemiological
profile of HCV infection among 1. Laboratório de AIDS e Imunologia
Molecular, FIOCRUZ, Avenida
inmates of Arapiraca-AL. A total of
Brasil,4365 - Manguinhos, Rio de
100 peripheral blood samples (10mL),
Janeiro - CEP: 21040-360
from the prison population in the
medium-security male prison Judge 2. Departamento
de
AIDS
e
Luis de Sousa Oliveira, were collected
Hepatites Virais, Ministério da
and sent to the Laboratory of General
Saúde, , E-mail: suwellendias@
Biology, State University of Alagoas for
gmail.com
serum separation and detection antiHCV. To detect the anti-HCV was used Brazil presents a HIV-1 epidemic
a commercial kit from the Wiener® characterized by the co-circulation
lab following the manufacturer's of the subtypes B, F1 and C, as well
instructions. Of the total of 187 inmates as mosaic genomes involving the
of the closed regime, 100 of them recombination of these subtypes.
agreed to participate. Anti-HCV hasn't Currently, about 32 thousand children
been identified in any of the processed and adolescents are infected with this
samples. The ages of the prisoners virus in the country. This study aimed
was between 18 and 60 years, mostly to evaluate pol gene diversity and
mulatto (65%), low educational level the prevalence of transmitted drug
(55%). All from Alagoas. With regard resistance mutations among vertically
to sexual orientation, 3% said being infected children and adolescents
homosexual. The most (58%) reported in Rio de Janeiro State, Brazil. HIV-1
not using condoms. The use of inhaled from plasma samples of 94 patients
drugs was referenced by 64%, since with age <19 years collected between
the use of injecting drug use and 2008 and 2012, at Laboratory of AIDS
tattooing / piercing were reported and Molecular Immunology of the
by 1% and 54% respectively. Blood Oswaldo Cruz Institute-Fiocruz, were
genotyped based on phylogenetic and
bootscanning analyses of the pol (PR/
RT) gene and their drug resistance
profile was analyzed. Among the
samples analyzed, 70 (74.6%) strains
belonged to subtype B, 10 (10.6%)
to subtype F1 and 2 (2.1%) to
subtype C. Two samples (2.1%) were
classified as intersubtype recombinant
CRF28/29. Unique recombination
forms were observed in ten patients
(10.6%). Transmitted drug resistance
mutations were detected in 14% of
the naive-patients analyzed and the
most common mutations to PI, NRTI
and/or NNRTI found were M46I,
M184V and K103N respectively. These
results demonstrate a heterogeneous
distribution of HIV-1 genetic variants
across different age ranges and the
frequency of transmitted resistance
mutations found emphasizes the
importance of genotyping of the HIV-1
before initiating therapy.
HV1399 - ABSENCE OF PRIMARY
MUTATIONS ASSOCIATED WITH
DRUG RESISTANCE IN ANTIVIRAL-NAIVE PATIENTS WITH CHRONIC
HEPATITIS B INFECTION
Santos, E.O., Souza, A.R., Silva, E.E.,
Morais, V.M.S.
1. Centro de Pesquisa Gonçalo
Moniz , CPqGM/Fiocruz-BA, Rua
Waldemar Falcão, 121, Candeal Salvador/BA
2. Hospital Cruzeiro do Sul, Acre,
Brasil,
3. Hospital Universitário Professor
Edgar Santos , HUPES, Rede
Nordeste
de
Biotecnologia,
Renorbio, E-mail: mariaisabel_
[email protected]
Human Virology: HV
289
Hepatitis B virus (HBV) infection is a
public health issue, one of main causes
of death from infectious diseases
worldwide. Brazil public health system
(SUS) has provided antiviral drugs for
chronic hepatitis B treatment for over
10 years, but a system for monitoring
for drug-related resistance mutations
is not available. This study aims
to determine the presence of HBV
primary mutations associated with
nucleoside and nucleotides analogs in
antiviral-naïve patients with chronic
hepatitis B infection. HBV reverse
transcription (rt) gene sequences
from 37 isolates from antiviral-naïve
patients from Cruzeiro do Sul Hospital
were analyzed. These sequence data
were obtained to validate molecular
methods and HBV genotyping from
previous collaborating studies with
HUPES and FIOCRUZ-BA. Briefly, HBVDNA was amplified with a nestedPCR with primers FHBS1-RHBS1 and
FHBS2-RHBS2, and sequenced using
ABI Prism 3100 (Applied Biosystems,
USA). Sequences from forward
(FHBS2) and reverse (RHBS2) primers
were aligned to obtain a contig with
length ranging from 310 to 369 bp,
corresponding to the rt amino acid
position from 51 to 172. Conflicting
sites were edited by comparison with
reference sequence X04615 after visual
inspection. Consensus sequences were
used for interrogating a local HBV
drug resistance database (HBVrt DB,
Stanford University, USA) to retrieve the
prevalence of each mutation according
to genotype and treatment. HBV
genotype A (62.1%) was most prevalent
followed by genotype F (31.0%) and
D (6.9%). Despite the high rate of coinfection with Delta virus (58.6%),
no primary drug-related resistance
mutation was observed in this rt
290
Human Virology: HV
region. Other regions will be evaluated
in the future. After the initiation of
drug therapy it is extremely important
to monitor viral load and identify drugrelated resistance mutations in order
to support clinical decision about
the patient management in addition
to preventing the emergence of
multidrug-resistant viruses. Financial
support: FAPESB/CNPq No 020/2009
PRONEX/CNPq/FAPESB, Application
number: 7201/2009.
private medical health institution.
136 clinical samples were analyzed,
119 of them were fecal material and
17 vomit. The collection period was
from February 2011 to June 2012. The
samples were collected from pediatric
patients (most of them between 0 and
5 years old). Viral RNA extraction was
conducted from the clinical samples,
and cDNA was generated from the RNA
extracted through Retrotranscription
(RT) using random hexamer primers.
Worldwide standardized specific
PCR protocol directed against capsid
gene(s) were conducted for RVA, NV
and HAstV molecular identification
and genotyping. The RT-PCR analysis
of the samples showed the following
results: 39% were positive for RVA
(n=48), 8% for NV (n=12) and 13%
for HAstV (n=18). Thirty-eight DNA
sequence were obtained from RVA RTPCR positive samples (VP7 and/or VP4
genes), and according with the sequence
information the genotypes distribution
were as follow: P[4]G2 (n=9), P[8]G2
(n=4), P[8]G3 (n=1), P[8]G12 (n=1),
G2P[ND*] (n=14), P[4]GND (n=4), P[8]
GND (n=5). From the 13% RT-PCR
positive samples for HAstV, 78% (n=14)
were confirmed by DNA sequencing,
and the genotype distribution were as
follow: 42% Genotype 1 (n=6), 29%
Genotype 2 (n=4), and 29% Genotype
3 (n=4). RT-PCR NV positive samples
are actually being sequenced. These
results represent the first report of
the circulation of RVA, NV and HAstV
in the country outside the Capital city
(Montevideo), and the first report of
the circulation of NV and HAstV in
Uruguay. * (ND = Not-determined)
HV1404 - MOLECULAR CHARACTERIZATION OF ROTAVIRUS, NOROVIRUS
AND ASTROVIRUS FROM PATIENTS
WITH ACUTE GASTROENTERITIS IN
SALTO CITY, URUGUAY
Tort, L.F.L., Victoria, M., García, M.,
Lizasoain, A., Arreseigor, E., Lopez, P.,
Cristina, J., Colina, R.
Laboratorio de Virología Molecular,
Regional Norte, LVMS, RN, UDELAR,
Gral. Rivera 1350, Salto, Uruguay
Hospital Departamental de Salto,
HDS - ASSE, Cervantes esq. 18 de
Julio, Salto, Uruguay Centro Medico
Salto, CAM, Artigas 937, Salto,
Uruguay Laboratorio de Virología
Molecular, CIN, UdelaR, CIN, Mataojo
2055 E-mail: fernandolopeztort@
gmail.com
Group A Rotavirus (RVA), Norovirus
(NV) and Human Astrovirus (HAstV)
are the major cause of acute
gastroenteritis (AG) in children under
five years old worldwide. These
viruses are the leading cause of
hospitalization and death due to AG
among infants of this age group, mostly
in developing countries. In this study,
we analyzed clinical samples of young
children with AG who were treated in HV1405 - HEPATITIS B SUBGENOtwo health institutions of Salto city: TYPES CHARACTERIZATION IN
the public hospital of the city and a CHRONIC HEPATITIS B PATIENTS IN
BRAZIL: A MORE ACCURATE VIEW
ON HBV VARIABILITY
Gomes-Gouvêa, M.S., Mendes-Corrêa,
M.C.J., Ferreira, A.C., Teixeira, R.,
Andrade, J.R., Barros, L.M.F., Ferreira,
A.S., Rezende, R.E.F., Nastri, A.C.S.S.,
Leite, A.G.B., Piccoli, L.Z., Galvan, J.,
Conde, S.R.S.S., Soares, M.C.P., Carrilho,
F.J., Pinho, J.R.R.
1. Instituto de Medicina Tropical FMUSP, IMT- FMUSP, Av. Dr. Enéas
Carvalho de Aguiar, n° 500/
Prédio II 2°andar Faculdade de
Medicina da Universidade de São
Paulo, FMUSP
2. Faculdade de Medicina do ABC,
FMABC
Gerais
4. Universidade
Maranhão
Federal
do
5. Secretaria Municipal de Saúde de
Ribeirão Preto
6. Serviço Municipal de Infectologia
de Caxias do Sul
7. Santa Casa de Misericórdia do
Pará
8. Instituto Evandro Chagas, , E-mail:
[email protected]
Hepatitis B virus (HBV) shows great
variability, at least 10 genotypes
(HBV-A through J) have been identified
and subgenotypes have been classified
within some these genotypes. Most
genotypes and some subgenotypes
show heterogeneity in their global
distribution that may reflect the
different patterns of human migration.
Progression to chronic infection, the
outcome of chronic hepatitis B (CHB)
Human Virology: HV
291
and the response to HBV treatment have
been associated with this variability.
Some studies suggest important
pathogenic differences between HBV
genotypes and subgenotypes. In
Brazil, HBV genotype distribution was
described, but the current available data
is still incomplete, as few of them have
characterized the HBV subgenotypes.
In this study, we identified HBV
subgenotypes isolated from 557
chronic hepatitis B carriers originating
from five different Brazilian states (SP,
MG, RS, PA and MA). A fragment of 1306
bp partially comprising HBsAg and the
DNA polymerase coding regions (S/
POL) was amplified and sequenced.
HBV genotypes/subgenotype were
determined by Bayesian phylogenetic
analyses. HBV genotype A was the most
prevalent (69.1%; 385/557) followed
by genotype D (23.7%; 132/557)
and F (4.8 %; 27/557). Genotypes
B, C, E and G were also found in few
samples. HBV/A was more common in
all regions, except in RS where HBV/D
prevails. In MG, almost all patients were
infected by HBV/A (91.7%). HBV/A1
and A2 were identified among HBV/A
genotypes with higher prevalence of
A1 (97%; 372/385). HBV/D showed
high variability: subgenotypes D1, D2,
D3 and D4 were found. HBV/D2, D3
and D4 were the most prevalent: 18%
(24/131), 51% (66/131) and 28%,
respectively. HBV/D1 only was found
in three cases (2%): two from RS and
one from SP. HBV/D subgenotypes
showed a heterogeneous geographic
distribution, but HBV/D3 was the most
prevalent subgenotype in all regions
excluding MA where D4 prevailed.
HBV/F was found in samples from
all studied regions, with almost all of
them classified as subgenotype F2.
Only two cases from SP were infected
292
Human Virology: HV
by F4. Among genotypes B cases,
subgenotypes B1 and B2 were found
and genotype C cases were classified
as C2. In conclusion, in this study, we
observed that HBV strains circulating
in Brazil shows great diversity and that
genotype identification did not reflect
the accurate variability of the virus,
especially in genotype D cases. Financial
support: FAPESP (2010/50081-9 and
2010/51208-2)
HV1406 - ROTAVIRUS INFECTION
AMONG COMMUNITY CHILDREN IN
GUARAPUAVA, PARANA
Ambrosini, V.A., Semaan, L.M., Campos,
D.A., Pedrosa, F.C., Machado, A., Orsi,
C., Gauer, A.G., Carraro, E.
Universidade Estadual do CentroOeste-PR, Unicentro-PR, Rua Simeão
Camargo Varela de Sá, 03. Vila Carli,
85040-080, Guarapuava, PR. E-mail:
[email protected]
Rotavirus is the main etiological agent
of diarrhea in childhood. Rotavirus
immunization was introduced in
Brazilian 6-month-old children in 2006.
The present study was aimed to evaluate
human Rotavirus occurrence in stool
samples obtained from community
children with gastroenteritis in a
municipal laboratory of health public
service. 83 stool samples collected in
the 2011-2012 period were analyzed
by RT-PCR for human Rotavirus
detection. Rotavirus was detected
in 4.1% of samples in a population
with a coverage upper 80% previous
immunization. These results indicate
low incidence of human Rotavirus
infection in community children with
higher vaccine coverage. Financial
Support: Fundacao Araucaria (Gestao
compartilhada em saude - PPSUS).
HV1407 - STUDY OF TYPE I IFN
LEVELS ON SERUM SAMPLES
OF PATIENTS WITH DIFFERENT
CLINICAL FORMS OF DENGUE
Silva, M.M.C., Carvalho, A.G.O., Oliveira,
R.A.S., Calzavara-Silva, C.E., Marques,
E.T.A., Gil, L.H.V.G.
1. Universidade
Federal
de
Universitária, Recife - PE
2. Centro de Pesquisas Aggeu
Magalhães, CPqAM, Av. Professor
Moraes Rego, s/n - UFPE - Cidade
Universitária | Recife/PE
3. Centro de Pesquisas René
Rachou, CPqRR, Avenida Augusto
Lima, 1715, Barro Preto, Belo
Horizonte - MG
4. Center for Vaccine Research,
University of Pittsburgh, CVR,
Pitt, 9014 Biomedical Science
Tower 3, 3501 Fifth Avenue,
Pittsburgh, Pennsylvania E-mail:
[email protected]
Dengue fever (DF) and dengue
hemorrhagic
fever
(DHF)
are
increasingly important public health
problems in the tropics and subtropics.
Emphasis has been placed on the role
of the adaptive immune system in
dengue pathogenesis. However, there
is increasing evidence about the role
of innate immune system in regulating
dengue infection and possibly
influencing the disease outcome. The
signaling system of type I interferon
(IFN-I/ IFN alpha-beta) is integral to
the innate immune system’s ability to
create an antiviral state. Counteracting
mechanisms developed by viruses often
hinders the antiviral IFN-mediated
response, creating the mechanisms
necessary for the disease installation.
In the present study, IFN alpha and
beta levels on serum samples of 50
dengue virus infected patients (25 DF
and 25 DHF), collected at the acute
phase of the disease (within 3-5 days
of fever), were quantified using Human
IFN-alpha Matched Antibody Pairs
kit (eBioscience) and ELISA Human
IFN beta kit (Invitrogen), respectively,
according to the manufacturers
instructions. We observed that
circulating levels of IFN-I were similar
for both DF and DHF patients with
no statistical significance. Therefore,
we observed no correlation between
mRNA and protein levels of type I IFN.
HV1410 - VIRAL MENINGITES:
INVESTIGATION OF THE HUMAN PARECHOVIRUS AS ETIOLOGIC AGENT
OF LINFOCITARY MENINGITES IN CEREBROSPINAL FLUID SAMPLES
Vidal, L.R.R., Almeida, S.M., Nogueira,
M.B., Raboni, S.M., Cavalli, B., Rossa,
M.C.D., Cavalcanti, E.
1. Universidade Federal do Paraná,
UFPR, Rua Pe. Camargo, 280,
Setor de Ciências da Saúde, Alto
da Glória, Cep; 80060240
2. Laboratório Central do Estado do
Paraná, LACEN, Rua Sebastiana
Santana Fraga 1001 - Guatupê,
Curitiba
Paraná
E-mail:
[email protected]
Meningitis can be caused by several
agents is attributed to enterovirus
(EV) a rate of 90% of cases of viral
meningitis, including the Human
Parechovirus (HPeV) that has been
described as the second most common
pathogen in neurotropic diseases in
humans. Global data related to HPeV
are scarce, in Brazil there is a reporting
Human Virology: HV
293
of cases of HPeV type 8 in an outbreak
of gastroenteritis. The study aims to
define the epidemiological profile and
characterize the molecular detection of
HPeV, using CSF samples collected from
patients with signs and symptoms of
viral meningitis treated at Hospital de
Clinicas, Federal University of Parana.
The methodology used for the detection
of HPeV genome 5'NCR was reverse
transcription followed by real-time
PCR. Positive samples were sequenced
targeting the VP1 gene. Phylogenetic
analysis of the positive samples showed
a 96-100% similarity with the strain of
the Netherlands (AMS721), confirming
the circulation of HPeV in our region.
Patients, with positive results for HPeV,
presented ages ranging from 7 to 12
years, with prevalence of symptoms of
fever, vomiting and headache, 60% of
patients had neck stiffness and 20%
showed signs of Kernig-Brudzinski.
The median age was 8 years (p = 0.03),
lactic acid 2.4 mmol / L (p = 0.03), 4.8
log10 viral load. There was a greater
number of positive samples during the
months with higher temperatures and
coincide with the summer and spring,
however, sporadic cases were observed
in other months. In 2006, a greater
number of positive samples also
occurred when the rainfall was higher.
Most samples were collected in March.
Infection of the central nervous system
(CNS) by HPeV was observed during
the summer months and the use of the
technique of real-time PCR was rapid
and effective to define the etiological
agent of infections of the CNS.
HV1413 - SEROLOGICAL EVIDENCES
OF INFECTION BY FLAVIVIRUS
ROCIO, SAINT LOUIS ENCEPHALITIS
AND WEST NILE IN RIO GRANDE DO
NORTE STATE, BRAZIL
294
Human Virology: HV
Romeiro, M.F., Reis, V.P., Moreira, E.A.,
Tolardo, A.L., Delsin, D.L., Badra, S.J.,
Farias, K.J.S., Rebecchi, I.M.M., Silbiger,
V.N., Machado, L.P.R., Figueiredo, L.T.M.
Ribeirão Preto- USP, FMRP- USP,
Av. Bandeirantes 3900 - Monte
Alegre
Grande do Norte, UFRN, Av.
General Gustavo Cordeiro de
Farias S/N. CEP 59012-570
E-mail:
mafarignoli@hotmail.
com
The Flavivirus genus includes the
most important causatives of arboviral
disease in tropical and subtropical
countries. In Brazil, dengue viruses
are responsible for several outbreaks
every year. Other flaviviruses also
reported in Brazil are yellow fever and
those related to Japanese Encephalitis
Complex (JECV) such as Saint Louis
encephalitis virus (SLEV) and Rocio
virus (ROCV). Although West Nile virus
(WNV) has not been isolated in our
country, serological evidence of WNV
infection in horses has been reported.
We show here results of a serologic
survey including 85 participants from
the County of Espirito Santo, Rio
Grande do Norte State (RN). These
participants, all healthy at the time
of blood collection, were 55 females
and 30 males, 2 to 59 years old (y.o.).
Sera of the participants were tested by
an in-house IgG-ELISA using specific
recombinant peptides of domain III
of E protein (rDIII) from ROCV, SLEV
and WNV as antigen. It was observed
that 8 samples (9.41%) from 5
women and 3 man, 4-45 y.o. presented
monotypic IgG antibodies to WNV; one
serum (1.17%) from a 20 y.o. woman,
presented monotypic IgG antibodies to
ROCV; one serum from a 59 y.o. woman
presented monotypic IgG antibodies to
SLEV. Our results suggest that ROCV,
SLEV and WNV could circulate in RN,
being misdiagnosed with DENV or
even remaining undiagnosed due to the
limited health assistance sources to this
population. Further studies including
neutralization test are necessary in
order to confirm these ROCV, SLEV and
WNV infections. Financial Support:
FAPESP and FAEPA
HV1414 - ANALYSIS OF RESISTANCE MUTATIONS TO ANTIVIRAL
THERAPY USED IN THE TREATMENT
OF HEPATITIS B IN PATIENTS FROM
RONDONIA STATE
Santos, A.O., Gomes-Gouvêa, M.S.,
Silva, M.F., Souza, F.B., Borzacov, L.M.,
Silva, A.S., Pinho, J.R.R., Salcedo, J.M.V.,
Vieira, D.S.
1. Instituto
de
Pesquisa
em
Patologia Tropical, IPEPATRO,
Rua da Beira, 7175, Lagoa.
2. Centro de Pesquisa em Medicina
Tropical, CEPEM, Av. Guaporé,
215, Lagoa.
3. Fundação
Oswaldo
CruzRondônia, FIOCRUZ, Rua da Beira,
7175, Lagoa.
4. Faculdade de Medicina da
Universidade de São Paulo,
USP, Av Dr Enéas de Carvalho
Aguiar, 470 05403-000 E-mail:
[email protected]
Patients with chronic hepatitis B
(CHB) can be successfully treated
using nucleos(t)ide analogs (NA), but
the occurrence of mutations in the
HBV genome that confers resistance
to these drugs is one of the most
important factors in treatment failure.
Drug resistance may emerge during
prolonged
treatment,
therefore
monitoring of HBV variability,
specifically at polymerase gene, is very
important to more efficient treatment.
The aim of this study was identify the
prevalence of HBV with resistance
mutations to NA and characterize HBV
genotypes in samples from patients
that were followed up at the specialized
clinic for Viral Hepatitis at Rondonia
state. Twenty-two patients were
included in this study and among these
18 were under NA therapy. A fragment
of 741 bp comprising partially S and
POL genes of HBV DNA was amplified
by nested PCR and its sequence
determined by direct sequencing.
HBV genotypes were determined by
phylogenetic analysis and the presence
of mutations was determined by
analysis of the amino acid changes
associated with resistance at POL
sequence. HBV DNA was detected
in 22 samples and these 16 shows
sequences with good quality. Among
these samples genotype D was the
most frequent (9/16; 56%) followed
by genotype A (6/16; 38%). Genotype
F was only detected in one sample.
HBV harboring lamivudine resistance
mutations (rtM204V or rtL180M +
rtM204V) was detected in 38% (6/16)
of the patients. One of these samples
also carrier a mutation rtS202G
associated with rtL180M + rtM204V
mutations, a pattern that additionally
confers resistance to Entecavir.
Another mutation (rt238T) that was
potentially associated with Adefovir
resistance was identified in only one
sample. This study shows a higher
prevalence of resistance mutation in
therapy-experienced CHB patients
from Rondonia. Considering that some
mutations confers cross-resistance to
Human Virology: HV
295
different drugs its characterization is
of great value to adequate treatment
management and consequently to
prevent disease progression. Support:
IPEPATRO, CEPEM, FIOCRUZ.
HV1426 - EVALUATION OF TWO
DIFFERENT RT-PCR PROTOCOLS
FOR DETECTION OF DENGUE-1
Barboza, M.M.O., Araújo, F.M.C.,
Perdigão, A.C.B., Cruz, J.N.M., Lima,
D.M., Pires Neto, R.J.
UFC, Rua Alexandre Baraúna, 949
- Rodolfo Teófilo - CEP 60430-160
- Fortaleza - CE
Pública, LACEN-CE, Av. Barão
de Studart, 2405 - Aldeota,
Fortaleza-CE
3. Universidade
de
Fortaleza,
UNIFOR, Av. Washington Soares,
1321, Edson Queiroz E-mail:
[email protected]
Dengue is the most important arboviral
infection that affects humans, causing
epidemics in more than 100 countries in
tropical and subtropical regions of the
world. The dengue virus belongs to the
Flaviviridae family that comprises four
serotypes (DENV1-4) involved in both
dengue fever and dengue haemorrhagic
fever. Early identification of dengue
infection facilitates monitoring and
treatment of patients while minimizing
risks of complications. Laboratory
confirmation is based upon direct or
indirect methods which are applied
according to infection period. During
the acute phase, the virus or viral
components are detectable. The aim
of this study was to test the sensitivity
of two different RT-PCR protocols
296
Human Virology: HV
for molecular diagnosis of DENV-1
infection. Serum samples from 20
patients in the acute phase of dengue
fever were inoculated into C6/36 cells.
DENV-1 strains were identified by
indirect immunofluorescence assay
using monoclonal antibodies. The
protocol I was a semi-nested RT-PCR
amplifying the C/prM region (Lanciotti
et al, 1992). The protocol II was a One
Step RT-PCR amplifying the E/NS1
region (Rico-Hesse, 1990). DENV1 RNA was extracted by magnetic
method. Amplification by protocol I
was possible in only 2 samples (10%).
Amplification by protocol II was
possible in 9 samples (45%). Both
protocols had low sensitivity. The
small number of samples is a limiting
factor in this work. The low sensitivity
observed for DENV-1 in the protocol
I had been previously described.
Protocol II was originally validated for
genotyping. In the state of Ceará DENV1 began to run since 1985, and there
may be inadequate homology between
the sequences of the primers and the
genome of the viral strain currently
circulating in the state. In addition,
other factors, including the RNA
extraction method could explain the
low sensitivity. Using a more significant
number of samples is necessary for
a more accurate assessment of both
protocols.
HV1431 - HTLV-DNA DETECTION IN
PREGNANT WOMEN BY IN HOUSE
NESTED-PCR
Santos, E.O.S., Silva, C.K.C., Silva,
C.W., Silva, R.R., Silva, J.L.A., Morais,
V.M.S., Andrade, M.P., Cahú, G.G.O.M.,
Coêlho, M.R.C.D.
1. Universidade
Federal
de
Pernambuco , UFPE, Av. Prof.
50670-901
2. Universidade Estadual de Alagoas,
UNEAL, Rua Governador Luiz
Cavalcante, S/N - Alto Cruzeiro,
Arapiraca-AL CEP: 57312-000
E-mail:
erlon.medtropical@
hotmail.com
The human T cell lymphotropic
virus (HTLV) is a retrovirus which is
associated with some diseases, like
adult
T-cell
leukemia/lymphoma
and
myelopathy/tropical
spastic
paraparesis. The HTLV-1 is present in
all Brazilian regions, but its prevalence
varies among the states, being highest
in Bahia, Pernambuco and Pará. HTLV
testing is very important in prenatal
care of pregnant women to avoid
the virus transmission during breast
feeding of newborns.The purpose
of this study was to investigate the
presence of HTLV-DNA by in house
nested polymerase chain reaction
(PCR) in pregnant women followed
in the Family Health Program in
Penedo, Alagoas, Brasil. The pregnant
women were screened for HTLV 1/2
antibodies by enzyme immunoassays
kit (Ortho HTLV-I/HTLV-II ab-capture
ELISA test system), according to the
manufacturer’s
instructions.
For
peripheral blood mononuclear cells
(PBMC) isolation, EDTA-anticoagulated
blood samples were treated with
Ficoll-Hypaque (Sigma-Aldrich, USA).
After isolation, the PBMC was stored in
RPMI medium at -80ºC until the DNA
extraction. A commercial kit (QIAamp
DNA Blood Mini Kit) was used for
genetic material extraction, following
the manufacturer’s instructions. An
in house nested-PCR was applied for
detection the sequence env-tax of
HTLV-1. The SK110 and SK44 primers
were used in the first part of the
reaction and SK248 and SK249 in the
second part, generating a fragment
of approximately 400 pairs of bases.
Two hundred and nine pregnant
women were analyzed and 8.2%
(17/209) shown a positive serology
for HTLV 1/2, of which 7.8% (9/17)
were in the 18 to 28 years age group.
However, 11.76% (02/17) refused to
submit to nested-PCR. Thus, the HTLVDNA was detected in 46.7% (7/15)
of the other participants. Therefore,
the HTLV infection was present in
3.4% (7/209) of evaluated pregnant
women. The presented data show
evidences to support the introduction
of HTLV serological test in prenatal
care by public health service to avoid
the possible clinical consequences
in newborns infected during breast
feeding in the study area. Financial
support: Universidade Estadual de
Alagoas (UNEAL)
HV1437 - GENETIC CHARACTERIZATON OF HCV GENOTYPE 2 VARIANTS
IN ISOLATES FROM BRAZIL
Pereira, S.A., Espirito Santo, M.P.,
Martins, R.M.B., Lampe, E.
1. Laboratório de Hepatites Virais,
Instituto Oswaldo Cruz, , IOC, Av.
Brasil 4365, 21045-900 Rio de
Janeiro, RJ, Brasil
Saúde Pública, Universi, UFG,
Hepatitis C is a single-strand, positive
RNA virus classified within the
Hepacivirus genus of the Flaviviridae
family. The sequencing of HCV isolates
is genetically identified into 6 major
genotypes, each of which may have
Human Virology: HV
297
various subtypes. Phylogenetic analysis
of NS5B region has been commonly
used for identification of HCV subtypes
and epidemiological applications. In
Brazil, HCV subtypes 1a, 1b followed
by 3a are the most prevalent strains,
the 2 and 4 are less common strains
and the genotype 5 uncommonly
detected. Few Brazilian studies have
analyzed genotype 2 and the molecular
characterization
is
practically
unknown. Moreover, its origin is not
fully clear. The aim of this study was
to characterize the HCV genotypes 2
circulating in Brazil. Between 2007
and 2012, a total of 8 serum samples
were collected and tested for anti-HCV
and HCV-RNA by RT-nest PCR of the
NS5B region (nt 8279-8619) of HCV
genome. For sequence analysis, the
NS5B region was amplified and the
PCR product obtained was purified
and submitted to direct sequencing.
The DNA alignments of obtained
nucleotide sequences were generated
with Clustal X program and Neighborjoining
and
maximum-likelihood
phylogenetic analysis were performed
by MEGA3 program.A multiple
sequence alignment of the examined
region and the related sequences in
the GenBank/EMBL database was
performed. The phylogenetic tree
showed that six isolates belonged to
genotype 2 subtype b and another one
was classified as subtype 2c. Besides,
one isolate was found out of the group
of isolates subtype 2b of Brazil´s
cluster. This study suggests that the
frequency of subtype 2b is higher than
subtype 2c in Brazil. However, analyses
of other regions are necessary to better
characterize this genotype. Financial
support: CAPES, CNPq and FDTIS/
FIOCRUZ
298
Human Virology: HV
HV1438 - DETECTION OF DENGUE
VIRUS
DIRECT
IN
VECTORS
CAPTURED IN THE CITY OF MANAUS,
AM, BRAZIL
Cardoso, A.J.L., Pereira, S.R., Luz, S.L.B.,
Costa, C.A.
da Amazonia, INPA, Av. André
Araújo, 2936, Aleixo, CEP 69060001, Manaus – AM, Brazil.
2. Instituto Leonidas e Maria Deane
/ Fundação Oswaldo Cruz, ILMD
/ FIOCRUZ, Rua Terezina, 476.
Adrianópolis, CEP: 69.057-070,
Manaus – AM, Brazil. E-mail:
[email protected].
br
Dengue virus (DENV) is an RNA
genome arbovirus, family Flaviviridae,
genus Flavivirus, caused by four
distinct serotypes (DENV 1-4). Dengue
is the most important arbovirus in
Brazil, in number of cases and lethality.
Differences in severity are associated
with serotypes or particular genotypes.
The city of Manaus, with 1.739.000
inhabitants has been infested by
Aedes aegypti since 1996 and the first
case of dengue fever (DF) episode
appeared in 1998 and, in 2008 was
the first case of DENV 4. This study
was part of a dengue virus surveillance
performed in the districts of Manaus.
The mosquitoes were collected in the
year 2011. In the Tropical Virology
Laboratory of the National Institute
of Amazon Researches (INPA), the
specimens captured were identified
and grouped in numbers up to 10
specimens per microtube (pool). A
number of 165 Aedes aegypti were
captured and pooled into 28 lots and
five Aedes albopictus captured were
pooled into four lots. Each pool of
mosquitoes was macerated and diluted
in a 1% solution of bovine albumin
in phosphate buffered saline (PBS).
Mosquito macerates had the RNA
extracted and then were submitted to
three different techniques, an RT-PCR
(Reverse Transcriptase – Polymerase
Chain Reaction) for detection of
flavivirus genus followed by a NestedPCR for identification of DENV species,
both based on size of amplicons, a
Multiplex-Nested-PCR and a Real
Time PCR (qPCR) to detect the DENV
direct in vectors. The three techniques
confirmed the presence of the virus in
different pools, being DENV 2, DENV 3
and DENV 4 serotypes. According to the
molecular studies, which detected the
presence from three of the four DENV
serotypes, it demonstrates that the
circulation and transmission of DENV
is occurring at different locations in
Manaus by Aedes aegypti mosquitoes
during the last year as a part of a
continuous transmission situation.
Financial support: CNPq Pronex /
Fapeam
HV1439
INCIDENCE
OF
CO-INFECTION BY HUMAN IMMUNODEFICIENCY VIRUS (HIV) IN
INDIVUDUALS WITH PULMONARY
TUBERCULOSIS TREATED IN THE
CENTER OF TESTING AND COUNSELING IN STD/AIDS FROM ILHÉUS,
SOUTHERN FROM BAHIA
Almeida, C.S., Santos, S.S., Costa, G.B.,
Oliveira, F.C.S.
1. Centro
de
Testagem
e
Aconselhamento
em
DST/
HIV,
CTA/DST/HIV,
Avenida
Canavieiras, s/n, Centro, Ilhéus,
Bahia
Gerais, UFMG, Avenida Antônio
Horizonte, Minas Gerais E-mail:
[email protected]
The
infection
by
Human
Immunodeficiency
Virus
(HIV)
is one of the most important risk
factors to contract tuberculosis
due to immunological changes that
it determines, creating favorable
conditions for the activation of
tuberculosis infection and disease
development. The aim of this
study was to assess the HIV coinfection in individuals infected with
Mycobacterium tuberculosis who
sought the services of the Counseling
and Testing Center for STD/AIDS from
Ilheus, southern Bahia, during the year
2011. The diagnosis of HIV infection
was performed using the rapid test
and by ELISA, and confirmed by
immunofluorescence and/or western
blot. The study included a total of 134
individuals, 81 men and 53 women,
aged between 7 and 80 years. After
serology, it was found that 10 patients
(7,4%) were HIV positive. It is worth
adding that all HIV-positive patients
had pneumonia and/or liver disease.
Most individuals (88%) belongs to
the city of Ilheus, while the other
12% belong to other municipalities in
southern Bahia, as Canavieiras, Itacaré,
Ubaitaba, Una and Uruçuca. Understand
the psychodynamics involved in the
interaction process between the
patient and the disease is of utmost
importance for surveillance strategies
are planned and enhanced. Still, it must
be emphasized the need to return the
individuals to confirm the diagnosis
and therapeutic management.
HV1440 - EVALUATION OF SUSCEPTIBILITY OF CHILDREN TO HEPATITIS
Human Virology: HV
299
A VIRUS ALONG WITH THE IMPLEMENTATION OF A CHILDHOOD
VACCINATION
PROGRAM
FOR
HEPATITIS A IN THE MUNICIPALITY OF CAMPOS DOS GOYTACAZES,
BRAZIL
Kury, C.M.H., Cruz, O.G., Teixeira, C.L.,
Riguetti, T.P., Conte, P.H., Pereira, R.C.,
Melgaço, J.G., Silva, J.P., Pinto, M.A.,
Vitral, C.L.
1. Departamento De Microbiologia
E Parasitologia Inst.Biomedico,
Uff , Rua Hernani Mello Nº 101, 3º
Andar, São Domingos, Niterói, Rj.
Cep: 24210-350
2. Pós-Graduação Em Microbiologia
E Parasitologia Aplicadas, Uff ,
Rua Hernani Mello Nº 101, 3º
Andar, São Domingos, Niterói, Rj.
Cep: 24210-350
3. Faculdade De Medicina De
Campos-Rj, Fmc, Avenida Alberto
Torres 217 Centro, Campos
Dos Goytacazes-Rj Programa
De
Computação
Científica,
Fiocruz, Avenida Brasil, 4365,
Manguinhos. Rio De Janeiro-Rj
4. Laboratório De Desenvolvimento
Tecnologico Em Virologia, Ioc /
Fiocruz, Avenida Brasil, 4365,
Manguinhos. Rio De Janeiro-Rj
5. Secretaria Municipal De Saude
De Campos Dos GoytacazesRj, Sms/Campos-Rj, Rua Gil De
Góis, 157, Altos, Centro, Campos
Dos
Goytacazes-Rj
E-Mail:
[email protected]
The municipality of Campos dos
Goytacazes is the only city in Brazil that
implemented hepatitis A vaccination
for all toddlers under the age of two
in its public immunization program.
300
Human Virology: HV
In order to evaluate the future impact
that this immunization program will
bring in the epidemiology of hepatitis
A, a seroprevalence study is being
conducted in individuals under the age
of 19 years randomly selected at public
and private schools from all 14 districts
of this county. Sample size calculation
based on 50% HAV estimated
prevalence, 5% precision rate, and 80%
confidence level yielded a total of 1028
subjects. Herein, anti-HAV results from
the first 415 individuals included in the
study are shown. After formal consent,
blood spot samples were obtained for
subsequent anti-HAV testing (Bioelisa
HAV IgG, Symbiosys). Each participant
or legal tutor was submitted to an
interview using a standardized
questionnaire. The overall prevalence
of anti-HAV was 17,8%, being 96,2%
of children under the age of five
susceptible to HAV infection. Risk
factors associated with seropositivity
were: age (>5 years old) (RR=0,84 (95%
CI:0,76-0,92) and skin color negro and
mulatto (RR=0,85 (95% CI:0,78-0,96).
No statistical difference was observed
in gender (RR=0,94 95% CI: 0,86-1,03)
and water source (filtered, mineral and
untreated), with RR= 0,91 (95% CI:
0,78-1,05). An impressive increase in
the anti-HAV prevalence was observed
from the 1-4 year group (3,8%) to the
5-9 year group (15%), which may be
related with the beginning of school
attendance. Results obtained so far
supports the municipality decision
to introduce Hepatitis A vaccination
in children before school admittance,
and corroborate with data from other
Brazilian
seroprevalence
studies
that have been shown that a large
proportion of children under the age
of five are susceptible to HAV infection.
The introduction of a hepatitis A
vaccination program may be an
important strategy for controlling HAV
infection in Brazil.
HV1444 - ISOLATION AND TYPING
OF DENGUE VIRUS FROM VECTORS
AEDES AEGYPTI CAPTURED IN
THE URBAN AREA OF MANAUS,
AMAZONAS
Pereira, S.R., Cardoso, A.J.L., Luz, S.L.B.,
Costa, C.A.
da Amazonia, INPA, Av. André
Araújo, 2936, Aleixo, CEP 69060001, Manaus – AM, Brazil.
2. Instituto Leonidas e Maria Deane
/ Fundação Oswaldo Cruz, ILMD
/ FIOCRUZ, Rua Terezina, 476.
Adrianópolis, CEP: 69.057-070,
Manaus – AM, Brazil. E-mail:
[email protected]
Dengue is the most important
arboviruse in Brazil, in number of cases
and lethality, it is an old zoonosis of the
primates in the Southeast Asia that
adaptated to humans taking as vectors
Aedes spp. mosquitoes and, thus,
eliminating a sylvatic cycle.The city of
Manaus, with 1.739.000 inhabitants
has been infested by Aedes aegypti
since 1996 and the first case of dengue
fever (DF) episode appeared in 1998,
caused by the DENV 1 and DENV 2
serotypes. Were collected mosquitoes
of the genus Aedes aegypti in all areas
of the city of Manaus. This work aims at
the isolation and typing, from vectors
A. aegypti, the four dengue serotypes
(DENV-1, DENV-2, DENV-3 and DENV4). The captured mosquitoes were
separated into pools according to
species and genus. The pools were
soaked in phosphate buffer pH 7.4
with salt, containing bovine serum
albumin. An aliquot of the macerated
inocula were prepared for isolation
attempts and typing of dengue virus.
To isolate viral cell culture were used
mosquito Aedes albopictus, clone
C6/36. The culture medium used was
supplemented MEM medium at 20%
Fetal Bovine Serum and 10.000U/ml
penicillin and streptomycin 1.000μg/
ml. The monolayers were inspected
daily using an inverted microscope for
detection of cytopathic effect (CPE).
On the seventh day after infection, the
tubes were removed and centrifuged
two aliquots, one for the second
pass of infection and another that
was subjected to polymerase chain
reaction reverse transcription RT-PCR
for detection of the dengue virus. The
samples of the second passage was also
subjected to RT-PCR. Isolation attempts
were made in 24 samples of vectors, of
which eight samples were positive by
RT-PCR for dengue virus. The DENV-2
was isolated in 50% of the eight positive
samples, and DENV-4 corresponded to
the other 50%. Of the isolates, some
tubes showed cytopathic effect, so its
no detection shall be conclusive for
negativar a sample for dengue virus.
In some isolates took two passages of
infection for detection of dengue virus
by RT-PCR, that because the viral load
in the first pass may be very low and is
not detected by RT-PCR.
HV1445 - GENOTIPING OF HEPATITIS
B AND D VIRUS IN ISOLATED OF
PATIENTS IN STATE OF AMAZON,
BRAZIL
Oliveira, C.M.C., Braga, W.M., Castilho,
M.C., Galvao, R.S., Vasconcelos, H.L.,
Gimaque, J.B.L., Filho, S.A.
Amazonas, UEA, Av. Pedro Texeira
Human Virology: HV
301
n° 25 Dom Pedro I Fundação de
Medicina Tropical Heitor Vieira
Dourado, FMT-HVD, Av. Pedro
Texeira n°25 Dom Pedro I
2. Universidade
Federal
do
Amazonas,
UFAM,
General
Rodrigo Otávio Jordão Ramos
3000 - Campus Universitário
E-mail: cmaraoliveira@hotmail.
com
Introduction: co-infection between
hepatitis B (HBV) and D (HDV) virus
is an important public health problem
worldwide. Both are responsible for
causing chronic disease with high
evolutionary potential gravity. In the
Amazon, the coinfection HDV and
HBV causes severe forms of hepatitis,
leveraging the rapid progression to
liver cirrhosis and fulminant hepatitis.
In addition to a worse prognosis,
complicates the host immune response.
In view of this situation, the work was
Objectives: To detect and characterize
the genotypes of the virus of hepatitis
B and D in blood samples of patients
treated in ambulatory viral hepatitis of
Foundation of Tropical Medicine Heitor
Vieira Dourado. In the period January
to July 2012. Materials and methods:
the extraction of viral nucleic acid was
extracted from 200 uL serum using a
High Pure Viral Nucleic Acid Kit (Roche
Diagnostics, Germany), according
to the manufacturer’s instructions.
The cDNA was obtained using the
VHD RevertAid First Strand cDNA
Synthesis (Fermentas-Life Sciences).
After DNA amplification of HBV and
HDV cDNA the PCR products were
submitted to cycle sequencing with
the ABI Prism BigDye Terminator cycle
sequencing kit (AppliedBiosystems,
Foster City,CA,USA), according to
the
manufacturer’s
instructions.
302
Human Virology: HV
Sequencing were run on an automated
ABI Prism 3130 DNA Sequencer
(Applied Biosystems). Results and
conclusions: The study included a total
of 29 blood samples from patients coinfected with HBV and HDV of patients.
The characterization of HBV genotype
was identified genotypes A and F
each in 10/29 (34.5%) of the samples
followed by genotype D in 9 (31.0%).
As for the VHD all samples belong to
the genotype 3. Demonstrating that
these results are in agreement with
the literature in relation genotyping
of HDV on HBV virus and Highlighting
the importance of genotyping VHD
genotype 3 is associated with form
most divergent and most aggressive,
often causing fulminant disease
through a cytopathic noninflammatory
process of liver microsteatosis. CNPq
CAPES FAPEAM
HV1451 - EVALUATION OF SINGLE-NUCLEOTIDE
POLYMORPHISMS
(SNPS) IN THE GENE OF THE INTERLEUKIN-10 -1082A/G (IL-10)
AMONG BRAZILIAN WOMEN WITH
HUMAN PAPILLOMAVIRUS RELATED
CERVICAL LESIONS
Chagas, B.S., Cordeiro, M.N., Gurgel,
A.P.A.D., Cruz, H.L.A., Silva Neto,
J.C., Serra, I.G.S.S., Muniz, M.T.C.,
Albuquerque, E.M.B., Freitas, A.C.
1. Universidade
Federal
de
50670-901
2. Centro de Medicina Integrada
de Sergipe, CEMISE, Hospital
Universitário Oswaldo Cruz, ,
Universidade de Pernambuco,
UPE, E-mail: babisimas@gmail.
com
Human papillomavirus (HPV) is the
main etiologic agent of malignant lesions
such as cervical cancer. The mechanism
of HPV infection is a process dependent
on basal keratinocytes differentiation.
Thus, HPV infection promotes little or
no alert to the human immune system,
since the viral release does not promote
cell lysis. Interleukin-10 (IL-10) has
an important role in the immune
response against viral infection, acting
through the determination of a pattern
of immune response and inhibition
of viral replication. Polymorphisms
in IL-10 have been related to the
susceptibility of the development of
cancers. In our study, we analyzed
the single nucleotide polymorphisms
-1082A/G present in the promoter of
the gene IL-10 in a group of 77 patients
infected with HPV and cervical lesions
of low degree and high degree in order to
evaluate their influence on the onset of
infection and the cancer development.
As a control group, we studied 77
healthy individuals not presenting
HPV infection. The biological material
was obtained from peripheral blood
and cytobrush. The detection of HPV
was performed by PCR (MY09/11).
The SNP located in the promoter of
the IL-10 were genotyped by real-time
PCR using High Resolution Melt. The
results allowed the identification of
three possible genotypes AA, AG and
GG. All populations analyzed were
found in Hardy-Weinberg equilibrium.
In relation to the allele frequencies
no evidence of any association of IL10 SNP (-1082A/G) between patients
with HPV/cervical lesion and the
control group was found (p = 1). For
the genotypic frequencies, as observed
before, no differences were identified
between the groups (p = 0.72). Although
interleukin is an important molecule in
the immune system, our results did not
show any direct association between
the SNPs presence in the IL-10 gene and
an increased susceptibility to cancer
development when associated with
HPV. However, this is a preliminary
study and the results need to be
confirmed in a larger cohort. Financial
support: FACEPE
HV1452 - DENGUE VIRUS: ANALYSIS
OF EPIDEMIOLOGICAL PROFILE IN
PARÁ STATE, BRAZIL, IN THE FIRST
HALF OF THE 2012
Carvalho, V.L., Silva, E.V.P., Lima, C.S.,
Gonçalves, E.J., Vasconcelos, H.B.,
Lima, M.F., Lima, C.P.S., Azevedo, R.S.S.,
Rodrigues, S.G., Vasconcelos, P.F.C.,
Arbovirology & Hemorrhagic F.
Dept.-Evandro Chagas Institute,
IEC, BR 316, KM 7, s/n E-mail:
[email protected]
Introduction. Dengue fever is a
mosquito-borne virus infection that
in recent decades has become a major
international public health concern.
Dengue is found in tropical and subtropical regions around the world,
predominantly in urban and semiurban areas. The incidence of dengue
has grown dramatically around
the world in the recent decades. At
least 2.5 billion people are now at
risk for dengue. The World Health
Organization currently estimates an
annual occurrence of 50 million dengue
infections worldwide, mainly in the
Americas and Southeast Asia. The
spread of dengue is attributed to the
expanding of geographic distribution
of the four dengue virus serotypes
and their mosquito vectors. On the
other hand, in the last decades, only
the serotypes 1 (DENV-1), 2 (DENV-
Human Virology: HV
303
2) and 3 (DENV-3) circulated in Brazil.
However, in July 2010, the serotype 4
(DENV-4) reemerged in Boa Vista, the
capital of Roraima State, in Northern
Brazil after an absence of 28 years in
Brazil. The study aimed to perform
a passive virologic surveillance of
dengue in the Pará, Northern Region,
Brazil, in the first half of the 2012 from
samples received by the Department
of Arbovirology and Hemorrhagic
Fevers - Evandro Chagas Institute.
Material and Methods. A total of 1009
samples of suspect patients were
inoculated into C6/36 cells culture for
attempt of virus isolation. The indirect
fluorescence assay using monoclonal
antibodies was used to confirm viral
infection. Results. A total of 280 (28%)
Dengue Virus strains were isolated as
follows: 12 (4.3 %) DENV-1; 24 (8.6
%) DENV-2; and 244 (87.1 %) DENV4. Conclusion. These results showed
an intense circulation of the DENV-4 in
Pará State, Brazil and low circulation of
DENV-1 and DENV-2 with an apparent
absence of DENV-3. Financial support:
Evandro Chagas Institute, CNPq.
HV1453 - INCIDENCE OF DENGUE
VIRUS INFECTION IN A COHORT
OF CHILDREN, GOIÂNIA, GOIÁS,
CENTRAL BRAZIL, 2010-2012
Oliveira, A.C.M., Martelli, C.M.T., Argolo,
A.F.L.T., Silveira, L.A., Féres, V.C.R.
Faculdade de Farmácia Universidade
Federal de Goiás, CEP: 74605-220,
Brasil
acmessiasoliveira@gmail.
com, [email protected]
Recent studies in Brazil reported a
change in the prevalence of dengue
cases in children with increasing
cases hospitalized for DHF. In children
below one year the potential risk to
development of severe cases from
304
Human Virology: HV
decay of maternal antibodies has been
reported in the literature. This study
aims to determine the incidence of
infection by dengue virus in a cohort
of children born in public hospital in
the city of Goiania-GO (~1.2 million
inhabitants), Central Brazil, 2010-2012.
The baseline prevalence of dengue in
Goiás refers to a study of prevalence
(53.9%) of dengue virus infection in
pregnant women conducted in 2010.
The IgM antibodies anti-dengue
(PanBio) were tested in samples from
neonates to evaluate acute dengue
infection. Monitoring the reporting of
dengue cases was done by the national
dengue surveillance system and active
investigation of symptomatic dengue
cases in children was conducted by
telephone interviews with the mothers
to identify clinical care for suspected
dengue in the follow-up period of 2
years. Data collected during the study
were analyzed in SPSS.17. The incidence
of dengue virus infection in neonates
was 8/235 (3.4%) (95%CI 1.36.0%). The kappa index evaluated the
concordance of IgM positivite between
mothers and neonates (87.4%).
Monitoring of reported cases of dengue
in the cohort was 3/235 (1.3%) (95%CI
0.0-3.0%). There was significant loss
to follow-up of mothers to contact by
phone. Of the 68 children studied 0,8%
were reported for suspected dengue
clinical care. These results reflect the
accumulated incidence of the disease
among children (5,5%) in Goiania-GO,
an endemic area for viral circulation
the four serotypes of dengue. Children
in the cohort had dengue fever after
one year of age, suggesting protection
by maternal antibodies against severe
infection. Epidemiological studies
that assess the incidence of dengue
in children may contribute to age-
appropriate vaccinations and are in
line with recommendations of the
WHO. Financial support: Pronex Rededengue/FAPEG/CNPq/2009
HV1455
AUTOIMMUNE
HEMOLYTIC ANEMIA IN THERAPY-NAÏVE PATIENTS WITH CHRONIC
HEPATITIS C VIRUS INFECTION: EVALUATION OF CLINICAL, BIOLOGICAL
AND VIROLOGICAL PARAMETERS
de Almeida, A.J., Muniz, L.P., Campos
de Magalhães, M., Brandão-Mello, C.E.,
Lampe, E.
1. Laboratório de Hepatites Virais
(LAHEP)/IOC, FIOCRUZ, Av Brasil,
nº4365, Manguinhos, Rio de
Janeiro, RJ, Brasil
2. Setor de Hematologia-CMB/H.U.
Gaffrée e Guinle (HUGG), EMCUNIRIO, Rua Maria e Barros, nº
775, Tijuca, Rio de Janeiro, RJ,
Brasil
3. Setor de Hepatologia-CMA/H.U.
Gaffrée e Guinle (HUGG), EMCUNIRIO, Rua Maria e Barros,
nº 775, Tijuca, Rio de Janeiro,
RJ, Brasil E-mail: adilsonjoal@
ig.com.br
Chronic hepatitis C virus (HCV) infection
is widely reported in association with
an array of extrahepatic manifestations,
including autoimmune disorders
such as thrombocytopenia, thyroid
autoimmunity, and hemolytic anemia.
Uncommon reports of Coombs’positive
autoimmune
hemolytic
anemia
(AIHA) related to chronic HCV
infection have been published in
the literature, however, its clinical,
biological, and virological features
are not well known. The aims of this
study were to evaluate the prevalence
and immunohematological profile
of HCV-related AIHA, as well as its
association with other immunological
abnormalities and virological aspects
in a cohort of antiviral therapy-naïve
patients with chronic HCV infection.
HBsAg and anti-HIV positive patients
were excluded from the study. Between
January 2011 and June 2012 a total of
190 patients, 84 (44.2%) males and
106 (65.8%) females with a mean
age of 49.9 ± 11.0 (21-70) years were
studied. Five (2.6%) patients were
found to have AIHA, 3/5 females,
with age ranging from 32–66 years.
Regarding
immunohematological
features, all cases were due to
warm-reacting autoantibodies (IgG
isotype), with distinct specifities
(anti C, anti-D, anti-E, and anti-k)
and variable intensity (1+ to 2+/4+).
Other immunological abnormalities
were detected in association with
AIHA
cases:
antithyroperoxidase
(n=1), antithyroglobulin (n=2), and
anticardiolipin IgM antibodies (n=1).
Infecting HCV genotypes were 1a
(n=2), 1b (n=2), and 3a (n=1), with
only one patient presenting high
plasma viral load (>600,000 IU/mL).
In conclusion, the low prevalence of
AIHA suggests that this alteration is an
infrequent extrahepatic manifestation
of chronic HCV infection. HCV-related
AIHA cases were found to be caused
by warm-active antibodies of IgG
isotype with distinct specificities. HCVrelated AIHA was also diagnosed in
association with other immunological
abnormalities and appears not to be an
extrahepatic manifestation dependent
on a particular HCV genotype. Financial
support: PROEP/CNPq
HV1456 - EVALUATION OF IL28 POLYMORPHISM IN PATIENTS INFECTED
Human Virology: HV
305
WITH ACUTE HEPATITIS C
Delvaux, N., Costa, V.D., Costa, M.M.,
Sousa, P.S.F., Lewis-Ximenez, L.L.,
Lampe, E.
de Janeiro - CEP: 21040-360 E-mail:
[email protected]
Hepatitis C virus (HCV) infects
approximately 170 million people
worldwide. Only 20-30% of individuals
spontaneously clear acute HCV
infection, most of infected will develop
chronic hepatitis. Patients with acute
HCV infection are difficult to identify
due to the lack of symptoms and signs
of acute phase. The therapy available
based on interferon and ribavirin is
effective in about half of the patients.
Recently, several studies have shown
that host genetic factors exert influence
on the response to antiviral therapy
and the spontaneous resolution
of infection. A single nucleotide
polymorphism (SNP) upstream of the
IL28B gene (rs12979860) has been
associated with chronic hepatitis C
and also with spontaneous clearance
of HCV. The homozygous CC genotype
is rather associated with spontaneous
resolution
of
infection
than
heterozygous CT or the homozygous
TT genotype. Aim: To evaluate the
SNP rs12979860 genotype and its
relation to the outcome of the disease
towards spontaneous viral clearance
or chronicity. Methods: 14 well
characterized patients acutely infected
with HCV (clearance, n=6 vs chronic,
n=8) were analyzed by restriction
using BstUI and real-time PCR with
a commercial kit (Roche). Results:
The rs12979860-CC and CT variants
were detected, respectively, in 83%
306
Human Virology: HV
(5/6) and 17% (1/6) of patients
which spontaneous clearance of HCV.
Concerning patients with chronic HCV,
50% (4/8) showed CC genotype, 25%
(2/8) genotype CT and 25% (2/8) TT
genotype. A complete concordance
between the two methods was
observed and both are suitable for
routine testing. However, screening by
real-time PCR detection had a reduced
execution time. Conclusion: These
preliminary results demonstrated that
the protective C allele of rs12979860
was more common in patients with
spontaneous clearance (83% vs 50%)
suggesting that IL28B polymorphism
has a marked impact on natural
clearance of acute HCV infection in the
cohort studied.
HV1457 - RECOMBINANT VESICULAR
STOMATITIS VIRUS-BASED DENGUE
VACCINES
Lauretti, F., Chattopadhyay, A., Rose, J.,
Fonseca, B.A.L.
Av Bandeirantes 3900
2.
recombinants (rVSV) were constructed
to express envelope (E), membrane and
its precursor (prM) proteins of dengue
1 and 2 to be tested as live vaccine
candidates. rVSVs have successfully
been used as vaccine vectors for several
viruses to induce strong humoral and
cellular immune responses. The DENV1 recombinant was constructed using
codon optimization for mammalian
cell expression of prM-E sequence
of a Brazilian strain (DENV-1/BR/
BID-V2392/2005) cloned downstream
a VSV-G signal peptide sequence. The
DENV-2 construct was prepared by
RT-PCR cloning of E protein domain
III (EDIII) of the Thailand/16681/84
strain. Each construct was inserted
in the in the full length VSV plasmid
DNA for recombinant VSV-DENV
recovery. Infectious recombinants
were recovered and plaque purified in
BHK-21 cells. The preliminary results
shown that recombinants grew at high
titers (3.5 x 107) and (2 x 108) for
VSV-DENV-1 and -2, respectively. VSV
proteins could be detected four hours
post-infection by immunofluorescence
(IF) and western blot (WB) analysis. A
22 kDa EDIII of DENV-2 was detected
in WB analysis of VSV-DENV-2 but
not by IF. Confirmation of prM and E
proteins expression by VSV-DENV-1
recombinants are under way to test
these viruses in mice in order to
determine if they are able to elicit
neutralizing antibodies and protect
mice against lethal challenge with
wild type DENVs. If this strategy
proves successful, manufacturing
VSV recombinants could be an useful
strategy for production of tetravalent
vaccine studies.
Yale University School of
Medicine, Yale, New Haven,
Connecticut
06510
E-mail:
[email protected]
Dengue is a viral disease with
widespread distribution throughout
tropical and subtropical areas of
the world. It is transmitted by a
mosquito bite and most infections
are asymptomatic or present with
mild symptoms, but some patients
progress to a severe disease (dengue
hemorrhagic fever/dengue shock
syndrome). Although experimental
vaccines are undergoing clinical trials,
there is no licensed vaccine. In this HV1461 - BLOOD CELL COUNT
study, vesicular stomatitis viruses CHANGES IN DENGUE DIAGNOSIS: A
STUDY OF 1269 CASES IN UBERABA,
MINAS GERAIS
Oliveira, A.C.S., Dulgheroff, A.C.B.,
Ferreira, D.A., Terra, A.P.S., Sarmemto,
R.R., Teixeira, D.N.S., Abreu, M.T.C.L.,
Martins, P.R.J.
1. Universidade
Federal
do
Triângulo Mineiro, UFTM, R Frei
Paulino, 30 - Nossa Senhora da
Abadia -Uberaba-MG
2. Universidade
de
Uberaba,
UNIUBE, Avenida Nenê Sabino,
1801- Uberaba-MG
3. Unversidade Federal da Paraíba,
UFPB, Cidade Universitária, S/N
- Castelo Branco-João Pessoa-PB
Sabin laboratório clínico, sabin,
E-mail:
anasantanoli@yahoo.
com
Dengue is an important infectious
disease present in a large number of
tropical countries around the world.
It is an acute febrile disease caused by
an arbovirus, a member of the family
Flaviviridae. Dengue virus (DV) has
four antigenically related serotypes
known as DV-1, DV-2, DV-3 and DV-4
which has the vector mosquitoes of the
genus Aedes (aegypti and albopictus).
The diagnosis of dengue is based on
clinical and laboratory findings. Among
the laboratory tests blood cell count
shows a good ally in the diagnosis and
monitoring of disease progression.
Therefore, the objective of this study
was to evaluate changes in the blood cell
count of patients affected by dengue in
Uberaba (Minas Gerais) comparing the
2005-2006 and 2009-2010 biennia.
The choice of the biennia was based on
the fact that occurred a large number of
dengue cases in these periods and also
because different serotypes of the virus
circulated in each biennium: serotype 2
Human Virology: HV
307
and 3 in 2005-2006 and predominance
of serotype 1 in the biennium 20092010. We analysed blood cell counts of
patients with serology (IgM) positive
for dengue, attended in a private
clinical laboratory and in a public
emergency unit of the city of Uberaba.
Blood cell counts were performed
using the automated method and
differential counts in lamina and the
detection of IgM antibodies of dengue
virus was performed by capture elisa.
The analysis was made using X2 (chisquare). In biennia 2005-2006 and
2009-2010 were selected, respectively,
1061 and 208 blood cell count for
inclusion in the study. The most
frequent findings were: leukopenia in
2005-2006 (66%) significantly higher
than in the subsequent biennium
(32%), lymphopenia in 2005-2006
(45.12%) that were also significantly
higher than the 14.9% observed in
2009-2010 and thrombocytopenia
40% in 2009-2010, significantly
higher than in 2005-2006 (16.12%).
In the other parameters of white and
red series there were no significant
changes. Thus, it was possible to better
understand the profile of changes that
infection with this flavivirus causes in
blood cell count of individuals from
the region of Uberaba. Haematological
abnormalities were similar in both
periods studied, so the clinical
symptoms combined with classical
results found in the blood cell count,
can anticipate the treatment of patients
especially during epidemics, when the
demand of serology extrapolates the
stocks of public and private laboratories
and seropositivity delay.
HV1465 - ANALYSIS OF HIV VIRAL
LOAD AND GENOTYPIC RESISTANCE
PROFILE IN VAGINAL SECRETION OF
308
Human Virology: HV
PREGNANT WOMAN SUBMITTED TO
CHEMOPROPHYLAXIS FOR MOTHER
TO CHILDREN VERTICAL TRANSMISSION
Rodrigues-Pedro, A., Grinsztejn, B.,
Pilotto, J.H., Morgado, M.G.
1. Instituto Oswaldo Cruz-Fiocruz,
IOC-Fiocruz, Av. Brasil, 4.365,
Manguinhos, Rio de Janeiro Brasil
2. Instituto de Pesquisa Clínica
Evandro Chagas-Fiocruz, IPEcFiocruz, Av. Brasil, 4.365,
Manguinhos, Rio de Janeiro Brasil
The methodology bDNA ® was used
to determine viral load in plasma
and EasyQ NucliSens NASBA ® was
used for the quantification of genital
samples. The HIV-1 RNA in plasma was
compared to that of vaginal secretions.
The HIV-1 RNA was detected in 40
(43.5%) of 92 genital secretion samples
and in 83 (91.3%) of 92 plasma
samples. The comparative analysis of
HIV-1 viral loads between these two
compartment was performed for 74
samples. It was shown that the viral
load in about 50% (36/74) of the
plasma and genital secretion samples
were found to be in the same range. Of
these, 13 (36%) were in the detection
range below 1.000 copies/ml, 9 (25%)
in the intermediate range (1.000 to
10.000 copies/ml) and 14 (39%) were
above 10.000 copies/ml. Thirty-eight
samples (51%) were discordant in viral
load analysis in both compartments,
with a greater discrepancy observed
in the intermediate range (1.000 to
10.000 copies/ml), confirming the
differences of plasma and genital
secretions viral loads before the
introduction highly active antiretroviral
therapy (HAART) (p=0.013). After
HAART discontinuation, 43 women
had samples of plasma and genital
secretion available for HIV-1 viral load
analyses. These results suggest that
HIV-1 viral load detection in plasma
is an important predictor of HIV-1
viral load in the genital secretions.
However, there may be excretion of
HIV-1 in genital secretions, even in the
absence of detectable HIV-1 viral load
in plasma, suggesting that the genital
compartment may act as a reservoir
for HIV-1 replication.
3. Hospital Geral de Nova Iguaçu Rio de Janeiro, HGNI-RJ, Avenida
Henrique Duque Estrada Mayer,
953 - Posse- Nova Iguaçu- E-mail:
[email protected]
The HIV-1 in the female genital tract
has an important role in mother-tochild transmission (MTCT) of virus.
Quantitation of HIV-1 levels in mucosal
secretions is essential to study
compartmentalization of HIV-1. The aim
of this study was to analyze the profile
of HIV-1 viral load in genital secretions
in HIV-infected pregnant women
submitted to chemoprophylaxis for the
prevention of MTCT before treatment
and after its discontinuation at delivery.
The quantification of HIV-1 viral load
was undertaken in the women’s genital
tract to evaluate a possible correlation
with plasma HIV viral load. A cohort of
274 HIV-1 infected pregnant women,
antiretroviral-naïve, was followed up.
Of that group, a total of 92 pregnant
women agreed to participate in this
study, all women collection of plasma
samples and genital secretions at the
inclusion in the study, before starting
antiretrovirals. HIV-1 RNA has been HV1467 - FIRST DETECTION OF NEW
quantified in these two compartments. ASTROVIRUS MLB1 IN BRAZILIAN
CHILDREN INPATIENT
Xavier, M.P.T.P., Carvalho, C.F.A.,
Miagostovich, M.P., Kreischer, F.B.D.,
Ferreira, M.S.R., Andrade, J.S.R., Leite,
J.P.G.
Oswaldo Cruz, IOC - FIOCRUZ, E-mail:
[email protected]
The family Astroviridae comprises
non-enveloped, positive-sense, singlestranded RNA viruses. The classical
human astroviruses are genetically
closely related and can be classified
into eight serotypes (HAstV1–8). In
addition, several genetically distinct
human astroviruses (e.g. MLB1, MLB2,
VA1, VA2 and VA3) have been recently
identified in stool samples from
patients suffering from gastroenteritis.
Although astrovirus is one of the major
causative agents for gastroenteritis,
there is relatively little information
on the prevalence and circling these
viruses. The recent discoveries of
genetically diverse astroviruses in
human highlight the genetic diversity
of astroviruses in nature and suggest
that there might be many more novel
astroviruses circulating in children
and adults human. The currency
study, described a case of AstV-MLB1
infection in a pediatric patient with
diarrhea in Sao Luis, Northeastern
Brazil. 200 stool samples from acute
gastroenteritis in children under
two years old were selected from
a Laboratory of Comparative and
Environmental Virology collection. A
two-fhases screening strategy to detect
and identify astrovirus was used. The
first phase, the OneStep RT-PCR Kit was
used to screen 5 µL of extracted material
from sample using consensus primers
that target highly conserved regions
in the ORF 1b (RNA polymerase) of
Human Virology: HV
309
astrovirus. The second phase was used
primers specific for classic human
astrovirus [Mon 269/270], AstVMLB1 [SF0053/SF061] and AstV-VA1
[SF0178/SF0179], using OneStep RTPCR. Amplicons were sequenced by
the Genomic Plataform of DNA PDTIS/
Fiocruz. The results confirm the MLB1 for two different regions ORF1b and
ORF2. This is the first description of
AstV MLB-1 in Brazilian sample and
corroborate with the possibility that
Astv-MLB-1 is associated whit acute
diarrhea disease in children. Finantial
support: Financial support: CNPq; IOCFiocruz.
HV1468 - MOLECULAR CHARACTERIZATION OF HUMAN ASTROVIRUS
IN CASES OF ACUTE GASTROENTERITIS IN THE STATE OF RIO GRANDE
DO SUL
Kreischer, F.B.D., Xavier, M.P.T.P.,
Carvalho-Costa, F.A., Andrade, J.S.R.,
Ferreira, M.S.R., Miagostovich, M.P.,
Volotão, E.M., Leite, J.P.G.
Fundaçao Oswaldo Cruz, IOC
FIOCRUZ, Av Brasil 4365 Manguinhos
Rio de Janeiro E-mail: fernanda.
[email protected]
The acute gastroenteritis is a public
health aggravator because of the
high rate of morbidity and mortality,
particularly in children under five
years old. Among the etiologic agents,
associated with acute gastroenteritis,
Humans Astrovírus (HAstV) are
described as the third in clinical
importance after the Group A rotavirus
(RV) and norovirus (NoV). The
family Astroviridae comprises nonenveloped, positive-sense, singlestranded RNA viruses. The classical
human astroviruses are genetically
closely related and can be classified into
310
Human Virology: HV
eight genotypes (HAstV1–8). In order
to investigate the circling of different
genotypes of HAstV in Rio Grande do
Sul, Brazil, we analyzed 65 positive stool
samples from acute gastroenteritis
in children under five years old from
a Laboratory of Comparative and
Environmental Virology collection,
during
January
2005
through
December 2010. All samples had
prior negative results for other enteric
viruses (Rotavirus and Norovirus). The
OneStep RT-PCR was used to screen 5
µL of extracted material from sample
using primers specific for classic
human astrovirus [Mon 269/270].
For molecular characterization, the
products generated by One Step RTPCR were sequenced in both directions.
The sequences were aligned and
edited using the program BioEdit and
compared to eight prototypes of HAstV
available in GenBank. The analysis
of a 449 bp ORF2 fragment revealed
that HAstV-1 was the predominant
genotype detected (65%), followed
by HAstV-2 (12%), HAstV-4 (3%) and
HAstV-8 (3%). This study is the first
of HAstV in the State of Rio Grande do
Sul, Brazil. Our findings pointed out the
importance of epidemiology molecular
of HAstV in cases of infantile acute
gastroenteritis showing the circulation
of different strain contributing for
further designing to prevention and
control strategies of gastroenteritis
transmitted by this important etiologic
agent. Finantial support: Financial
support: CNPq; IOC-Fiocruz.
HV1471 - MOLECULAR EPIDEMIOLOGY OF NOROVIRUS FROM
OUTBREAKS OF ACUTE GASTROENTERITIS OCURRED IN THE STATE OF
RIO GRANDE DO SUL 2004- 2010
Andrade, J.S.R., Ferreira, S.R.M., Xavier,
M.P.T.P., Fioretti, J.M., Leite, J.P.G.,
Miagostovich, M.P.
Laboratório de Virologia Comparada
e Ambiental-Fiocruz, LVCA-FIOCRUZIOC, Avenida Brasil, 4365 E-mail:
[email protected]
Norovirus (NoV) is recognized as the
leading etiological agent in sporadic
cases and outbreaks of nonbacterial
gastroenteritis
worldwide
in
individuals of all ages. They are
divided into five genogroups (G): GIGV, within which 35 genotypes were
identified, classified according to the
molecular analysis of the amino acid
sequence of the viral capsid protein
VP1. This study of NoV in the state of
Rio Grande do Sul aims to perform
a molecular characterization of NoV
circulating in the state, contributing
to the establishment of molecular
surveillance of NoV from outbreaks of
acute gastroenteritis occurred between
2004 and 2010. These samples
were received at the Laboratory of
Comparative
and
Environmental
Virology (LVCA) for elucidation of
acute gastroenteritis cases attended
at the Central Laboratory (LACEN)
of Rio Grande do Sul. One hundred
and twenty two samples previously
positive for Nov by RT-PCR (region B)
were tested by RT-PCR for region D
to determine the GI (Cap A, B1 e B2)
and GII (Cap C, D1 e D2) genotypes. A
total of 88 samples were amplified for
GII and 2 to GI. It was not possible to
characterize 32 samples. Analysis of
partial nucleotide sequences showed
that genotype GII.4 was prevalent and
was found in all years studied. The
second prevalent was the genotype
GII.6 and, the remaining samples were
characterized as GII.2, GII.3, GII.12,
GII.13, GII.14, GII.17, GII.21, and GI.1
GI.3. These results show the great
diversity of NoV circulating in the state
of Rio Grande do Sul, also observed in
other Brazilian states. The relevance
of the molecular characterization of
these viruses show their impact on
health of the population and provide
important information for developing
an effective vaccine against these
viruses. Financial support: CNPq/
FIOCRUZ-IOC/ Plataforma Genomica
de Sequenciamento PDTIS-FIOCRUZ
Acknowledgments to staff of LACEN of
Rio Grande do Sul.
HV1480 - ENTEROVIRUS DETECTION
IN CASES OF ASEPTIC MENINGITIS
IN MOZAMBIQUE
Pinto, G.C., Costa, E.V., Gudo, E.S., Jani,
I.V., da Silva, E.E.
1. Instituto Nacional de Saude Moçambique, INS - MISAU, 264
Av. Eduardo Mondlane/Salvador
Allende MAPUTO – Republica de
Moçambique
2. Fundação
Oswaldo
Cruz
(Laboratório de Enterovirus),
FIOCRUZ, Av. Brasil. 4365. Pav.
Helio e Peggy Pereira. Sala B217
E-mail: gabbydocarmo@gmail.
com
Aseptic meningitis refers to a clinical
syndrome of meningeal inflammation in
which common bacterial agents cannot
be identified in the cerebrospinal
fluid (CFS). Rapid diagnosis of viral
meningitis using PCR testing of CSF can
help shorten hospitalization, and avoid
the unnecessary use of antibiotics.
Non-polio human enterovirus is the
leading recognizable cause of aseptic
meningitis, accounting for 80–92%
of all the cases in which a pathogen
Human Virology: HV
311
is identified. Most of the cases occur
during the summer and autumn
although sporadic cases can occur
throughout the year. In our study
163 CSF samples were collected in
three Hospitals in Southern (Maputo),
Northern (Nampula) and Center
(Beira) of Mozambique, from July 2011
to February 2012. All 163 CSF samples
from suspected aseptic meningitis were
negative when submitted to culture
for bacterial and fungi and Indian ink
stain negative for fungi. Seventy CSF
samples have been so far tested using
two methods; TaqMan real-time RTPCR (rRT-PCR) and virus culture using
RD cells. Twenty-six specimens were
enterovirus positive by rRT-PCR. From
these, 13 were positive regarding
enterovirus isolation. This is the first
set of data associated to a broader
study concerning the etiology of aseptic
meningitis in Mozambique. Financial
support: CNPQ, FIOCRUZ, INS
Posters Immunobiological Virology - IV
IV688 - SELECTION OF PEPTIDES
BY PHAGE DISPLAY AS NOVEL EPITOPE-BASED DIAGNOSTIC PROBES
FOR SEROLOGICAL DETECTION OF
HUMAN T-CELL LYMPHOTROPIC
VIRUS 1 (HTLV-1)
Machado, L.F.A., Santos, F.A.A., Lima,
M.I.S., Goulart, V.A., Tafuri, S.M., Ishak,
M.O.G., Azevedo, V.N., Vallinoto, A.C.R.,
Ishak, R., Ueira-Vieira, C., Goulart, L.R.
1. Universidade Federal do Pará
- ICB - Lab. Virologia, UFPA,
Universidade Federal do Pará Rua Augusto Corrêa 01 - Guamá
- Belém-Pará
2. Universidade
Federal
de
Uberlândia - Inst. Genética e
Bioqu, UFU, Av. Pará 1720 Campus Umuarama - Bloco 2E Sala 246 - Uberlândia - MG E-mail:
[email protected]
Human T-cell leukemia vírus 1 (HTLV-1)
is endemic worldwide and the infection
results in a variety of hematological and
neurological diseases, including Adult
T-cell Leukemia (ATL) and myelopathy/
tropical spastic paraparesis (HAM/
TSP). Currently, the molecular
methods for diagnosis are indirect
immunofluorescence or western blot,
which are either not available or too
expensive to adapt for routine use. In
this study, IgG from patients with HTLV1 and negative controls were covalently
coupled to protein G-magnetic beads.
After coupling, beads were washed
and incubated with a 12-mer random
peptide phage display library (PhD12, NEB) for peptide selection. Our
aim was to identify and characterize
specific ligands to IgG that mimic
HTLV-1 epitopes. After selection, 38
clones were tested in ELISA assays
against sera from HTLV-1-infected
313
patients and negative controls, and
the most reactive clones had their
DNA sequenced and translated.
After validation with additional
immunoassays, the selected clones that
showed significant differences between
patients and controls were further
analyzed by bioinformatics. Linear
and structural alignments showed
similarity to relevant viral antigenic
proteins, such as envelope glycoprotein
(ENV_HTL1C, ENV_HTL1A) and GagPro-Pol polyprotein (POL_HTL1A,
GAG_HTL1M). The selected peptides
present great potential for diagnostic
improvement of HTLV-1, and the novel
mimetic phage-fused peptides may
be readily used in immunosensor
platforms. Financial Support: CNPq,
CAPES, FAPEMIG, UFU and UFPA.
IV731
CONSTRUCTION
OF
PLASMIDS FOR EXPRESSION IN
MAMMALIAN CELLS OF CHIMERIC
ANTIGENS CONTAINING SEGMENTS
OF HEPATITIS C VIRUS ENVELOPE
PROTEINS E1 AND E2
Freitas, G.R.O., Oliveira, T.F.M., Chávez,
J.H., Yokosawa, J.
Laboratório
de
Virologia,
UFU, Av. Amazonas, 2210 - Bloco
4C SL 216 Campus Umuarama.
Uberlândia, MG E-mail: guibio_ufu@
yahoo.com.br
Hepatitis C is a liver infectious disease
caused by the hepatitis C virus (HCV),
a member of the family Flaviviridae.
Development of an effective vaccine
has been difficult due to high virus
variability. HCV envelope proteins E1
and E2 induce neutralizing antibodies
and are considered major targets for
prophylactic HCV vaccine development.
In this study, we constructed three
October 2012 Volume 17 (2) – Supplement 1 / Foz do Iguaçu, Paraná, Brazil - Posters - Immunobiological Virology: IV
314
plasmids containing one, two or three
segments of HCV E1 and E2 genes for
expression in mammalian cells, since
these segments induce the production
of neutralizing antibodies. To each
coding sequence, the Kozak sequence
and a sequence that encodes for 8xHistag were added to the 5’ and 3’ end,
respectively, and inserted into pcDNA3
vector. The plasmids were transfected
into CHO-K1 cells by using a liposomebased reagent, and selection was done
with G418. At the end of eight weeks,
presence of E1E2 nucleotide sequences
was detected by testing the DNA from
resistant cells by PCR. In addition,
transfected cells showed to be reactive
in indirect immunofluorescence assay
by using a monoclonal antibody against
6xHis-tag, indicating expression of the
E1E2 proteins. These three plasmids
will be used to immunize mice in order
to evaluate their immunogenicity, as
well as the capability of the antibodies
to neutralize HCV in vitro. Financial
support: CNPq, FAPEMIG, UFU, PROPPUFU
IV758 - CLONING AND EXPRESSION IN BACTERIA OF A CHIMERIC
ANTIGEN CONTAINING SEGMENTS
OF THE ENVELOPE PROTEINS E1
AND E2 OF HEPATITIS C VIRUS
Tostes, R.O., Froelich, L., Chávez, J.H.,
Yokosawa, J.
UFU, Av. João Naves de Ávila,
2121, Uberlândia, MG E-mail:
[email protected]
It is estimated that about 170 million
people in the world are suffering
from chronic hepatitis C and that
every year there are 3 to 4 million
new cases. Acute infections caused
by hepatitis C virus (HCV) are usually
asymptomatic and chronic infections
in advanced stage, often progress to
hepatic cirrhosis and to hepatocellular
carcinoma. Transmission of the
virus occurs by contact with infected
blood, primarily through sharing of
needles by intravenous drug users and
accidents with sharp objects by health
care professionals. It can also occur by
vertical and sexual transmission. So far
there is no vaccine available to prevent
hepatitis C. The main objective of this
work is to clone an artificial gene that
encodes for various regions of the E1 and
E2 glycoproteins of the HCV envelope
for the expression of a chimeric antigen
for vaccine development purposes.
The nucleotide sequence of E1E2 was
synthesized and cloned into vector
pGS-21a, for expression in Escherichia
coli of a protein fused with glutathione
S-transferase (GST) and 6XHis-tag. The
plasmid DNA (of the construct and the
vector) was used in transformation of
bacteria BL21 cells. In parallel, pGEX4T-2 vector DNA was also used in the
transformation. Various clones of
each transformation were inoculated
into culture medium and protein
expression was induced by adding IPTG
(isopropyl β-D-tiogalactopiranosid).
Bacteria cell lysates were submitted to
electrophoresis in SDS-polyacrylamide
gel. However, the GST protein (29
kDa) was observed only in clones
transformed with pGEX-4T-2. EnzymeLinked
Immunoabsorbent
Assay
(ELISA) using monoclonal anti-6X-Histag was also performed and no increase
in reactivity was observed in lysates
of induced cells in comparison with
those of non-induced cells. The results
showed that the pGEX-4T-2 may be a
more appropriate vector for expression
of the HCV chimeric protein.
IV787 - ENZYME IMMUNOASSAY
BASED ON ENVELOPE RECOMBINANT PROTEIN AS ANTIGEN FOR
DIAGNOSIS OF EQUINE INFECTIOUS
ANEMIA: AN ALTERNATIVE FOR
CONTROL AND PREVENTION OF THE
DISEASE
Franco-Luiz, A.P.M., Souza, A.M.R.,
Santos, J.R., Bonjardim, C.A., Trindade,
G.S., Abrahão, J.S., Ferreira, P.C.P.F.,
Kronn, E.G.
Universidade Federal Minas Gerais,
UFMG, Av. Antonio Carlos, 6627
- Pampulha, Belo Horizonte - MG
Equine Infectious Anemia (EIA), also
known as “swamp fever”, is a viral
disease of equids, including horses,
mules and donkeys. EIA has a great
economic impact, compromising the
fitness of infected animals. This disease
has as etiologic agent the Equine
infectious anemia virus (EIAV), which
belongs to the Retroviridae family. The
clinical course of the disease may be
acute, chronic or asymptomatic. The
clinical signs which predominate are
recurrent fever, weight loss, hemolytic
anemia and ventral edema. There is no
available treatment or vaccine to EIA
and disease control is performed by
segregation or sacrifice of seropositive
animals. The official diagnostic test
to detect the disease is the agar gel
immunodiffusion (AGID), however
this test has limitations such as low
sensitivity and misinterpretation
of results. More sensitive tests have
been proposed mainly based on
enzyme linked immunosorbent assay
(ELISA). The aim of this study was to
standardize an ELISA for detection of
IgG antibodies in sera of horses using
a recombinant protein (rgp90) as
antigen. The ELISA showed a sensitivity
315
of 92.0% and specificity of 96.0%
when it was compared with AGID.
Serum samples of 1198 horses were
submitted to ELISA. Prevalence rates in
equines were 15,6% (187), 1,1% (13)
were indeterminate and 83,3% (998)
were negative by IgG ELISA-rgp90.
Furthermore, the IgG ELISA using the
recombinant rgp90 provided a high
level of reproducibility on different
days, suggesting robustness of the assay.
The coefficient of variation of 12%
indicated adequate repeatability at the
current stage of assay development.
The obtained results indicate that the
IgG ELISA rgp 90 is a good alternative
methodology to be employed in the
routine screening of EIA.
IV840 - DEVELOPMENT OF AN
ENZYME IMMUNOASSAY FOR ANTI-HEPATITIS C VIRUS ANTIBODY
DETECTION
Pedrazza, EL, Moreira, L, Renner, R,
Woltmann, R, Scarton, G, Silva, M G,
Bender, A L, Kreutz, F , Schmitt, V M,
1. FK Biotecnologia S.A., FK Biotec,
Rua da Várzea, 22, Porto Alegre RS Pontifícia
2. Universidade Católica do Rio
Grande do Sul , PUCRS, Av.
Ipiranga, 6681 - Partenon, Porto
Alegre - RS, 91530-000, BRA
E-mail:
imunodiagnostico@
pucrs.br
Hepatitis C virus (HCV) is a public
health problem worldwide, with about
3% of world population being infected.
Hepatitis C laboratory diagnosis is
based on serologic testing for the
detection of antibodies against HCV
and molecular detection of HCV RNA.
In Brazil, technical standards for
hemotherapy services are currently
316
regulated by the RDC No. 153 of
June 4, 2004, which highlightes
the detection of antibodies against
HCV as a crucial test for candidate
donation. In 2009, the Laboratory
of Imunodiagnostics Research (LIR)
was created in connection with
the “triple helix” concept, joining
expertise of FK Biotechnology, Faculty
of Pharmacy (PUCRS) and Lifemed
with governmental financial support
(FINEP), aiming the development of
immunoassays with fully national
technology in all steps of production.
Here we report the development of an
enzyme immunoassay for detection
of antibodies to HCV, based on ELISA
technique, for future use in blood
screening in clinical laboratories or
blood banks. The first development
step was standardization of all reagents
concentrations and incubation times.
The established conditions were:
antigen concentration of 2.5 µg/
mL; sample dilution 1:20; secondary
antibody dilution 1:1000; 30 minutes
of incubation time for sample and
secondary antibody. The second step
was validation, with tested 250 negative
and 209 positive samples tested. The
resulting test sensitivity was 99.5%
and specificity 98.4%. In addition,
possible interference was evaluated in
100 samples positive for anti-HIV, 100
for anti-HBs, 100 for anti-HBc and 63
samples positives to HBsAg. The test
showed no interference of any of the
tested parameters. The performance
reported here is comparable to systems
available at the market and currently
in use for blood screening in clinical
laboratories and blood banks, with the
adavantage of being produced with
national technology. Financial support:
FINEP
IV915 - DEVELOPMENT OF TWO
MOLECULAR METHODS BASED ON
REAL TIME PCR BY SYBR GREEN AND
TAQMAN TO DETECTION AND QUANTIFICATION OF YELLOW FEVER 17D
VIRUS
Oswaldo Cruz Foundation, Fiocruz,
Av. Brazil 4365
For the development of live attenuated
flavivirus vaccines, it is important to
estabilished the viral fitness. In this
context, the real time PCR technique
allows the quantification of nucleic
acids directly from the supernatant of
infected cell cultures. Furthermore, it
can be used to determine the viral load
in serum of yellow fever vaccinees. The
aim of this work was to establish this
methodology based on Sybr Green and
Taqman approaches to quantify yellow
fever 17D virus. Two distinct regions of
NS5 gene were chosen to design Sybr
Green primers and Taqman primers
and probe. The standard curve to
TaqMan assay was constructed with the
target region cloned in plasmid which
was used to quantify samples ranged
on 109 to 102 copies/reaction. The
first results for TaqMan demonstrated
reproducibility intra and inter assay.
The sensitivity was 100 copies/
reaction and detection limit was 25
copies/reaction.
To
demonstrate
specificity to 17D virus, this method
was used to detect dengue and
Japanese encephalitis virus in clinical
samples and the results were negative.
About Sybr Green, the technique was
performed through a serial dilution of
viral RNA and the copy numbers was
previously determined by TaqMan.
The results about the melting curve
revealed a peak at 81˚C for all dilutions
and 76˚C for the negative control. The
reproducibility and sensitivity was
similar to that observed on Taqman
assay. However, dengue positive
samples showed a peak amplification
corresponding to yellow fever in the
melting curve. The search on BLAST
revealed that primers design was not
successful. So, these data impaired this
test since it proved to be specific for
another flavivirus, including dengue.
Other sets of primers are being tested
for NS5, NS3 and 3’-UTR regions and
NS5 showed high specificity for 17D
virus. It was concluded that the TaqMan
showed better results than the Sybr
Green but the validation process is still
ongoing to implement this method in
the monitoring of viral load.
IV918 - AN IGG-ELISA BASED ON
RECOMBINANT ANTIGENS FOR
DETECTION OF ANTI-DENGUE ANTIBODIES IN DRIED BLOOD SAMPLES
DEMONSTRATES
COMPARABLE
RESULTS TO NEUTRALIZATION TEST
Rocha, E.S.O., Gaspar, C.H.P., Vilela,
A.P.P., Ferreira, T.A.R., Cursino, A.E.,
Marinho, P.E.S., Figueiredo, L.B.,
Oliveira, J.G., Ferreira, P.C.P., Kroon, E.K.
1. Laboratório de Vírus, Depto de
Microbiologia, UFMG, , Av. Antônio
Carlos, 6627, Belo Horizonte, MG,
Brazil.
2. Laboratório de Imun. Cel. e
Molecular, Fundação Oswaldo
Cruz, , Av. Augusto de Lima 1715.
Belo Horizonte, MG, Brazil.
E-mail:
eliseubiologia@gmail.
com
Several serological methods have
been developed to assess laboratory
confirmation of dengue infection which
includes a large number of ELISA tests.
The present study attempted to evaluate
the effectiveness of recombinant
317
proteins for detection of Dengue virus
(DENV) specific IgG antibodies in dried
blood samples absorbed onto filter
paper compared to sera samples results
by plaque reduction neutralization test
(PRNT). Recombinant proteins used as
antigens in a standardized IgG-ELISA
were expressed in Escherichia coli
system and purified by nickel affinity
chromatography. A panel of 48 blood
samples was obtained from dengue
fever suspected individuals residents in
Caratinga (Minas Gerais State, Brazil).
Half of blood sample was absorbed
onto filter paper and the other half
was centrifuged to obtain sera. Blood
samples were tested by the IgG-ELISA
based on recombinant DENV antigens
and serum samples for neutralizing
antibodies detection. Thirty-eight
(79.2%) samples were concordant
between the two sampling preparations,
19 positives and 19 negatives. On the
other hand, 9 samples were ELISA+ and
PRNT- probably associated to higher
sensitiveness of ELISA on detection of
specific antibodies and only 1 sample
was ELISA- and PRNT+ hypothetically
associated to IgM neutralizing
antibodies detection. Overall, IgG-ELISA
sensitivity was 95.0% and specificity
was 67.9% when using dried blood as
sample. This study presents the IgGELISA as an inexpensive and suitable
kit for use in clinical laboratory and
the use of dried blood samples as an
alternative for sample transportation
and storage. In conclusion, IgG-ELISA
based on recombinant DENV antigens
is able to detect anti-DENV IgG in
dried blood samples and should be
potentially as useful as sera samples
to neutralizing antibodies detection of
dengue infections. Financial support:
CNPq, CAPES, DECIT/MS, FAPEMIG,
INCT-Dengue and Pronex-Dengue
318
IV946 - CLONING AND EXPRESSION
OF DOMAIN III OF THE ENVELOPE
GLYCOPROTEIN OF DENGUE VIRUS 3
Moreira-Vieira, F.F., Yokosawa, J.
Universidade
Federal
de
Uberlândia, UFU, Av. Pará, 1420,
Campus Umuarama Bl. 4C. 38401136 Uberlândia, MG, Brasil E-mail:
[email protected]
The Dengue Virus (DENV) belongs to
the Flaviviridae family and Flavivirus
genus, and there are four serotypes.
The transmitting vector is mostly
mosquitoes Aedes aegypti. Dengue
is considered the most important
arboviral disease according to the
World Health Organization, causing
high rates of morbidity and mortality.
There is no specific treatment or a
preventive vaccine. A reinfection with
heterologous serotype may cause severe
dengue and research for development
of vaccine using full virus may cause
unwanted reactions. The domain III
(DIII) of the envelope glycoprotein
is the determinant of neutralization
and has low cross-reactivity, avoiding
antibody-dependent
enhancement.
Therefore, domain III-based vaccine
may be a better option for development
of a recombinant dengue vaccine. The
objective of this study was to express
the DIII of DENV-3 for investigation of
virus neutralizing activity by elicited
antibodies, for the development of a
vaccine candidate with reduced side
effects. DIII sequence was cloned
into vector pET-14-b for expression
in E. coli protein fused with 6XHistag. The plasmid and pET-14-b
DNA’s were used in transformation
of bacteria BL21. Three colonies
transformed the construct and one
colony transformed with the vector
was used for protein expression by
induction with IPTG (isopropyl β-D-1thiogalactopyranoside). These bacteria
cells were lysed and, after centrifugation,
the supernatant was submitted to
electrophoresis in SDS-polyacrylamide
gel. The expression was observed
only with the supernatant from the
clone transformed with pET-14-b.
Enzyme-Linked
Immunoabsorbent
Assay (ELISA) using monoclonal
anti-6X-His-tag was carried out and
reactivity to lysates of induced cells in
comparison with those of non-induced
cells was not increased. The presence
of the recombinant DIII in the inclusion
bodies remains to be investigated and
expression using other vector will be
evaluated. Key words: Dengue, DENV,
rDIII, vaccine Financial support:
FAPEMIG, CNPq, PROPP-UFU.
IV954 - BRAZILIAN PRODUCTION
OF REFERENCE REAGENTS TO
IDENTIFY DIFFERENT SUBTYPES OF
AVIAN INFLUENZA VIRUS
Orsi, M.A., Camillo, S.C.A., Domingues,
R.D., Fortunato, E.C., Ribeiro, S.A.M.,
Ramazzoti, A., Almeida, F.S., Sobreira,
K.L., Jenson, T.C. , Pedersen, J.C.
1. Laboratorio
Nacional
Agropecuário, Lanagro-SP, Rua
Raul Ferrari Sn, Campinas- Sp,
Brasil
2. National Veterinary Services
Laboratories, Nvsl, Ames, Ia, Usa,
Usa. E-Mail: Giulia96@Yahoo.
Com
Avian Influenza (AI) is considered
one of the most relevant diseases of
the poultry industry. The Brazilian
Reference Laboratory for Avian
Influenza (Lanagro-SP), established
cooperation
with
the
World
Organization for Animal Health (OIE)
to assure the production and control
of quality diagnostic reagents and the
capability to diagnose Avian Influenza
in accordance with OIE standards. This
research reports the production of the
master seed virus, inactivated antigens
and antiserum for all 16 subtypes of AI
and quality analysis of the produced
reference reagents, allowing to apply
as an OIE resource for avian influenza
reagents. Antiserum was produced in
specific-pathogen free (SPF) chickens
with live virus, and the chicken
antisera were evaluated using the HI
and NI methods. The source of seed
stock virus came from NVSL and was
produced by inoculating SPF chicken
embryos and collecting allantoic
amniotic fluid. Part of the fluid was
conserved as master seed and part
was inactivated using ß-propiolactone.
To ensure virus inactivation, the
antigen was passed in embryonated
eggs twice. Quality assurance was
conducted on all products including
master seed virus, antigens and
antisera by evaluation for sterility
and titer by using hemagglutinationinhibition or hemagglutination tests.
Specificity tests were also conducted
on all products using a panel of avian
influenza antigens or antisera (H1H16). Lanagro produced 16 lots of
Avian influenza master seed with titers
of (HA types of ranges 1:16 to 1:1,024),
12 lots of antiserum with (HI titers of
ranges 1:256 to 1:2,048), and 12 lots
of inactivated antigens (HA= 1:16 to
1:512). The results obtained showed
Lanagro-SP has met the conditions
to produce influenza reference
reagents Financial Support: Ministry
Agriculture, Livestock and Food Supply,
OIE and NVSL
IV959 - BRAZILIAN PRODUCTION OF
319
REAGENTS FOR EQUINE INFLUENZA
DIAGNOSIS
Orsi, M.A., Camillo, S.C.A., Domingues,
C.S., Reischak, D., Fortunato, E.C.,
Jenson, T.A., Pedersen, J.C.
1. Laboratorio
Nacional
Agropecuário, Lanagro-SP, Rua
Raul Ferrari Sn, CampinasSp, Brasil National Veterinary
Services L
2. aboratories, NVSL, Ames, IA, USA,
USA. E-mail: giulia96@yahoo.
com
Equine influenza (EI) is an acute
respiratory and highly contagious
disease caused by influenza virus A. It is
an endemic disease in most part of the
world and causes high economic impact
and because of its high infectivity can
stop the activity of one large numbers
of horses simultaneously. The diagnosis
is made by rapid detection of virus
in nasal swabs by ELISA, RT-PCR and
virus isolation. National Agricultural
Laboratory (Lanagro-SP) the Brazilian
Reference Laboratory for Avian
Influenza, established cooperation
with the World Organization for
Animal Health (OIE) to assure the
production and control of quality
diagnostic reagents for Avian influenza
in accordance with OIE standards.
The aim of this study was to produce
reagents for Equine influenza and
quality control of the produced
reference reagents, allowing Lanagro
to apply as an Brazilian resource for
Equine Influenza reagents. Antiserum
was produced in specific-pathogen
free (SPF) chickens with live virus, and
the chicken antisera were evaluated
using the hemagglutination-inhibition
(HI) and Neuraminidase inhibition
methods. The source of seed stock virus
320
came from NVSL and was produced by
inoculating SPF chicken embryos and
collecting allantoic amniotic fluid. Part
of the fluid was conserved as master
seed and part was inactivated using
ß-propiolactone to produce antigen. To
ensure virus inactivation, the antigen
was passed in embryonated eggs twice.
Quality assurance was conducted on all
products including master seed virus,
antigens and antisera by evaluation for
sterility and titer using HI or HA tests.
Specificity tests were also conducted
on all products by using a panel of
avian influenza antigens or antisera
(H1-H16). Lanagro produced 07 lots
of master seed (HA= 1:16 to 1:512),
03 lots of antiserum (HI =1:256 to
1:4,096), and 05 lots of inactivated
antigens (HA= 1:16 to 1: 128). The
results obtained showed that LanagroSP can produce these reference
reagents. Financial Support: Ministry
Agriculture, Livestock and Food Supply,
OIE and NVSL
IV1027 - PRE-CLINICAL TRIAL OF
AN INACTIVATED YELLOW FEVER
VACCINE IN C57BL/6 MICE
Pereira, R.C., Silva, A.N.M.R., Souza,
M.C.O., Gaspar, L.P., Caride, E., Galler, R.
Oswaldo Cruz Foundation, FIOCRUZ,
Av. Brasil, 4365 E-mail: renata.
[email protected]
The Yellow fever is an acute infectious
disease caused by a virus that belongs
to the Flaviviridae family, Flavivirus
genus. It is endemic in Africa and
South America and is transmitted by
mosquitoes of the genera Aedes and
Haemagogus. The available yellow fever
vaccines using the attenuated strains
17DD and 17D show safety and efficacy,
but rare occurrence of serious adverse
events such as the vaccine-associated
viscerotropic and neurotropic disease
(YEL-AVD and YEL-AND respectively)
has been reported. Therefore, it
became necessary the development of
a safer alternative vaccine that could
be potentially used in those with
contraindications to the live 17DD and
17D vaccine. The aim of this work was
to develop an inactivated vaccine for
yellow fever virus. This vaccine has been
developed by Bio-Manguinhos using
17DD strain, produced in bioreactors
in serum-free medium and purified
by chromatography. The analysis
of the immunogenicity and level of
protection was done using a murine
model, C57BL/6. The animals were
inoculated with the inactivated vaccine
in the absence or presence of the alum
adjuvant, in different numbers of doses
and challenged with the yellow fever
virus 17DD. The humoral immune
response was analyzed by PRNT
(Plaque Reduction Neutralization Test).
The results showed that only animals
which received three doses of the
inactivated vaccine with alum adjuvant
had neutralizing antibody titers greater
than the cut off considered positive
for yellow fever. The animal’s survival
rate were 100% after intracerebral
challenge with yellow fever 17DD
virus in animals that received 3 doses
of vaccine with adjuvant. Thus, it was
demonstrated that the inactivated
yellow fever vaccine with alum
adjuvant has a protective role when
administered in three doses in mice.
Financial Support: CNPq
IV1047 - PADRONIZATION OF THE
INDIRECT ELISA USING RECOMBINANT GLYCOPROTEIN D AS ANTIGEN
FOR THE DIAGNOSIS OF BOVINE
HERPESVIRUS 5
Araujo, I.L., Dummer, L.A., da Rosa, M.C.,
Oliveira, P.D., Leite, F.P.L., Campos, F.S.
UNIVERSIDADE
FEDERAL
DE
PELOTAS, UFPEL, CDTec - PPGB,
prédio 19, caixa Postal 354, CEP:
96010-900, Pelotas, RS, Brazil.
Considered one of the main pathogens
of cattle, Bovine Herpesvirus type 5
(BoHV5) is responsible for several
economic losses. It is characterized
by its active replication in neural cells
and spread through the host’s Central
Nervous System, being responsible
for outbreaks of Meningoencephalitis.
The glycoprotein D present in the
envelope of BoHV5 is essential for
the virion penetration in the host
permissive cells. Also, the gD stimulate
strong immune humoral responses
in the host which allows its use for
serological based diagnostic. The yeast
Pichia pastoris has been used for the
production of recombinant antigens
and combines advantages in handling
and protein expression by mechanisms
of
eukaryotic
post-translational
modifications. Serological diagnosis of
BoHV5 infection is mainly performed
by ELISA and serum neutralization.
The aim of this study was to develop
and standardize an Indirect ELISA for
detection of antibodies against BoHV5
using the recombinant glycoprotein
D expressed in Pichia pastoris as
antigen. The parameters evaluated
were: different concentrations of
antigens, blocking solution, serum
dilutions and plate brands. The
dilution of 1:400; blocking solution
with 5% skim milk plus 3% casein;
antigen concentration of 100 mg per
plate from NUNC brand provided the
more consistent values and greater
differentiation
between
positive
sera and negative. Consequently, the
321
parameters above mentioned provided
the best padronization criteria for
the development of an Indirect ELISA
against BoHV5. Financial Support:
Capes, CNPq.
IV1063 - DESIGN OF HPV-16 E5 GENE-BASED EXPRESSION VECTORS FOR
GENETIC IMMUNIZATION AGAINST
CERVICAL CANCER
Cordeiro, M.N., Coimbra, E.C., Jesus,
A.L.S., Freitas, A.C.
Universidade
Federal
de
Pernambuco, UFPE, Av. Prof. Moraes
Rego, 1235 - Cidade Universitária,
Recife - PE - CEP: 50670-901 E-mail:
[email protected]
According to World Health Organization,
cervical cancer is the second most
common cancer in women worldwide.
More than 85% of cervical cancer
deaths are in developing countries.
For malignant progression in cervix
lesions, human papillomavirus (HPV)
persistant infection is a common
prior condition. HPV-16 is the most
frequent papillomavirus found in
cervical cancers. Vaccination is one of
the most desirable approaches for anticancer proposal. However, therapeutic
strategies against HPV infection appear
to better attend efficiency and low
costs issues. DNA vaccines have been
described as promising approach to
enhance cytotoxic immune response
against viral proteins, which means
that it could act as gene therapy
against
human
papillomaviruses
and, consequently, preventive tool
against cervical cancer. E5, E6 and E7
oncoproteins are the major therapeutic
targets in order to create anti-HPV
vaccines. Especially, E5 gene product
is highly expressed in infected cells at
premalignant stage and, recently, its
322
role in carcinogenic progress has been
described. These evidences support
the choice of an E5 gene-based strategy,
by construction of an expression
plasmid for an anti-HPV immunization
purpose, which is the main objective of
this work. So we have design a codonoptimized HPV-16 E5-based gene for
expression in mammalian cells. In
order to enhance immune response
and avoiding oncogenic activity, a
second E5 gene-based sequence has
been design, including only previously
described immunogenic epitopes. Each
gene has been successfully inserted into
pCI-neo mammalian expression vector,
as confirmed by restriction assay and
sequencing analysis. Next steps of
this work include in vitro evaluation
of constructs transforming activity,
capability to promote proliferative
response and lymphocytic activation.
Further results may be achieved by
mouse inoculation and comparison of
the immune response generated by
each DNA vaccine. Financial support:
CAPES and FACEPE.
IV1098 - A CHIMERICAL DENGUE
PROTEIN CONTAINING CAPSIDE,
ENVELOPE AND NS1 EPITOPES IS
RECOGNIZED BY SERA OF DENGUE
VIRUS INFECTED INDIVIDUALS
Batista, I.C.A., Pereira, P.V.B., Gonçalves,
C.S., Rocha, E.S.O., Kroon, E.G., CorreaOliveira, R., Oliveira, J.G., CalzavaraSilva, C.E.
1. Departamento de Microbiologia
- ICB – UFMG, UFMG, Av. Antônio
Horizonte - MG, CEP 31270-901
2. Centro de Pesquisas René Rachou
- Fiocruz/MG, CPqRR/FIOCRUZ,
Av. Augusto de Lima, 1715, Barro
Preto, Belo Horizonte - MG - Brasil
E-mail: izabellaandrade@cpqrr.
fiocuz.br
Dengue is caused by Dengue viruses
(DENV), members of the Flaviviridae
family, and is composed by 4 genetically
distinct serotypes referred as DENV
1–4. No vaccines or antiviral drugs
for dengue prevention and treatment
have been approved yet. Moreover,
a safe vaccine demands a balanced
tetravalent immune response. In this
study, we hypothesized that chimerical
proteins
expressing
potential
immunogenic epitopes from DENV 1-4,
could be recognized, of a tetravalent
and balanced way, by sera of DENVinfected patients and, consequently,
useful to develop safe dengue vaccine
compositions. By using bioinformatics
tools, the most potential immunogenic
regions derived from envelope, capsid
and NS1 proteins, focusing in the
DENV 1–4 conserved regions, were
previously selected to design a pilot
1400 bp minigene, named here as
qDV, coding for a 50 KDa chimerical
protein. The qDV minigene was cloned
into the expression vector pMAL-c5x,
which was able to express a 94 kDa
recombinant protein in fusion with the
MBP protein, named qDV-MPB in E.
coli BL21. The aim of this study was to
purify a chimerical protein and test its
recognition by sera of DENV-infected
individuals. The chimerical protein
qDV-MBP and the MBP alone were
purified by affinity chromatography
and used in ELISA assays to test
its reactivity with sera of DENVinfected patients and DENV-negative
sera (identified by plaque reduction
neutralization test) as negative control.
Our results showed recognition of the
protein qDV-MBP by sera of DENVinfected patients, but also reacted
weakly by negative sera of non-infected
individuals, meanwhile MBP protein
alone was also weakly recognized by
both groups. The qDV-MBP protein
will be further tested in ELISA and
Western blot assays to determine if
that recognition is tetravalent and
if qDV-MBP can also be recognized
by anti-YFV antibodies. Our results
suggest that artificial proteins can be
specifically designed to be recognized
by sera of dengue infected individuals.
Financial support: FAPEMIG/CNPq/
Fiocruz
IV1149 - CLONING OF SYNTHETIC
GENES PRM/E OF DENGUE-3 VIRUS
IN A SYSTEM OF EXPRESSION IN
YEAST
Oliveira, M.D., Monteiro, J.M.C.,
Cardoso, S.A., Silva, A.C.A., de Paula,
S.O.
Universidade Federal de Viçosa,
UFV, Avenida PH Rolfs, s/n, Campus
Universitário - CEP 36570000
The dengue viruses are arbovirus
belonging to Flaviviridae family,
which comprises other viruses that
constitute a threat to public health, as
yellow fever virus, West Nile virus and
Japanese encephalitis virus. There are
four serotypes antigenically related for
Dengue viruses (dengue 1-4), which can
cause from mild febrile to more severe
diseases, such as hemorrhagic dengue
fever and dengue shock syndrome, that
can be fatal. The dengue viruses are
enveloped and their genome has 11
Kb. It encodes three structural proteins
and seven non-structural proteins in
the same ORF. Among the structural
proteins, the E protein represents the
main antigenic determinant for these
viruses, being the responsible for their
323
principal biological properties. The
prM protein is important for correct
folding of E protein. Since there is only
treatment to the symptoms but not for
the disease itself, the creation of an
effective vaccine and as well as a fast
and accessible diagnostic method is of
paramount importance. In this study,
the sequences of the genes prM and E of
the dengue-3 virus were optimized in
order to increase the levels of proteins
expression in yeast in an attempt to
provide antigens in large scale. The
synthetic genes obtained were amplified
using specific primers and cloned into
cloning vector pTZ57R/T. Then, the
construction obtained was subjected
to double digestion with the enzymes
EcoRI and NotI and the fragment of
interest was cloned into expression
vector pPICZαA previously digested
with the same enzymes. Afterwards,
Pichia pastoris KM71H yeast was
transformed with the recombinant
plasmid constructed, as confirmed by
PCR reaction. The results indicate that
the cloning of genes of interest into
expression vector was effective as well
as yeast transformation, showing us a
promising attempt for the obtainment
of dengue virus antigens for the use
as immunogens in diagnosis kits.
Financial support: CAPES/FAPEMIG/
CNPq
IV1217
PRODUCTION
AND
CHARACTERIZATION OF IMMUNOGLOBULIN Y ANTI-HSV-1
Silva, A.S., Vasconcelos, G.A., Lanzarini,
N.M., Lima, L.R.P., Guimarães, J.R.,
Pinto, M.A., de Paula, V.S.
Instituto Oswaldo Cruz, FIOCRUZ IOC, Av. Brasil, 4365; Manguinhos
- Rio de Janeiro - RJ - Brasil CEP:
21040-360 E-mail: alexsantos@ioc.
324
fiocruz.br
The Herpes Simplex virus 1 (HSV-1)
has a worldwide prevalence of 70%80% in adults. The principal method of
disease transmission occurs through
direct exposition of mucosal secretions
or skin lesions and viral infection
vary from asymptomatic infections to
vesicular lesions, gingivostomatitis,
keratoconjunctivitis and encephalitis.
The treatment of HSV-1 is based on
antiviral drugs utilization which are
toxic, can induce several metabolic
side effects and resistance of patient to
drug administration. One alternative to
antivirals is the use of immunoglobulins
encountered in birds and reptiles,
the immunoglobulin Y (IgY). The
IgY has several advantages when
compared to immunoglobulin G (IgG):
their antibodies show high response
against mammals antigen; IgY is a noninvasive method because it was found
and harvested in the eggs; IgY is more
affordable than IgG. In this work we
try to develop an IgY anti-HSV-1 who
could be a alternative to treatment
with antiherpetic antivirals. Methods:
Two groups of hens was inoculated
(group 1: inoculated with HSV-1 and
CpG-ODN; group 2: inoculated with
CpG-ODN and incomplete adjuvant of
Freund (IFA), the eggs were harvested
and the yolk was purified by PEG 6000
method. The determination of yolk
protein concentration was realized
through bicinchoninic acid (BCA) assay
and the presence of IgY anti-HSV-1 in
yolk was assessed by electrophoresis
and Western blotting. Results: Protein
concentration of yolk demonstrated
a mean yield of 27,72 mg/ml. The
electrophoresis pattern of gel filtration
was in accordance with the standard
IgY and the presence of anti-HSV-1 IgY
in egg yolks was confirmed by Western
blotting. Conclusion: The method
utilized for IgY anti-HSV-1 production in
hens was efficient. This antibodies will
be evaluated as antiviral to determine
if is possible to use as an alternative
treatment of HSV-1. Financial support:
Cnpq; Fiocruz-IOC
IV1236 - PURIFICATION OF IMMUNOGLOBULIN Y FROM EGG YOLK OF
HENS IMMUNIZED WITH GROUP A
ROTAVIRUS
Lanzarini, N.M., Vasconcelos, G.A.,
Silva, A.S., Guimarães, J.R., Vargas, A.S.,
Volotão, E.M., de Paula, V.S., Pinto, M.A.
Oswaldo Cruz Institute,Rio de
Janeiro, RJ /Brazil, IOC/FIOCRUZ, Av.
Brasil, 4.365 Pav. HPP. LVC Oswaldo
Cruz Institute, Rio de Janeiro, RJ /
Brazil, IOC/FIOCRUZ, Av. Brasil,
4.365 Pav. HPP. LADTV Veterinary
Medicine School, Teresópolis, RJ /
Brazil, UNIFESO, E-mail: natalial@
ioc.fiocruz.br
The rotavirus (RV) belongs to the family
Reoviridae, consisting of 11 segments
of double stranded ribonucleic acid
(RNA) virus. Seven groups from A
to G have been identified, where
the group A is the most prevalent in
the population. The infection with
rotavirus is the major cause of acute
gastroenteritis in children under
five years old, being responsible
for 600,000 deaths annually and
approximately 40% of hospitalizations
due to diarrhea at this age. The RV
infection causes vomiting, dehydration,
and diarrhea lasting 5-6 days, with
high morbidity and mortality mostly
associated with malnutrition. The use
of immunoglobulin Y (IgY), antibody
produced in chickens and purified from
egg yolk, has increased in recent years
because of the advantageous features
as compared with the immunoglobulin
G (IgG), as the obtaining antibodies by
noninvasively technology, production
in large quantities and low cost. In
this study, hens were divided in three
groups: the group one has received
three immunizations at intervals of
one month with group A rotavirus
plus Incomplete Freund`s Adjuvant
(IFA) plus an adjuvant consisting of
oligodeoxynucleotides
containing
C-guanosine
phosphate
(CPGODN). The group two received one
immunization with IFA plus CPG-ODN in
the first month and two immunizations
at intervals of one month of group A
rotavirus plus IFA plus CPG-ODN. The
group three, the control group, was
immunized during three months with
IFA plus CPG-ODN. After twenty days
the eggs were collected and the antirotavirus IgY was purified from egg
yolk by polyethylene glycol 6,000. The
immunoglobulin Y was characterized by
sodium dodecyl-sulfate polyacrilamide
gel electrophoresis (SDS-PAGE) 12%
and specificity determined by Western
blot analysis. The development of a
methodology to purify IgY from egg
yolk was successful, obtaining an
antibody with low interference and
high concentration, which is going to
contribute to use the IgY as an input
in immunoassays. Financial support:
CNPQ, IOC/FIOCRUZ.
IV1242 - COMPARISON BETWEEN
TWO PURIFICATION PROCEDURES APPLIED TO ANTI- HAV IGY
EXTRACTED FROM HENS
Lima,
L.R.P.,
Lanzarini,
N.M.,
Vasconcelos, G.A., Silva, A.S., de Paula,
V.S., Pinto, M.A.
Av. Brasil, 4.365 Centro Universitário
325
Serra dos Órgãos , UNIFESO, E-mail:
[email protected]
Introduction: Hepatitis A is a public
health problem in Brazil. Hepatitis
A infection is easily spread, either
by personal contact or by ingestion
of contaminated food and water.
Recent years have seen an increase in
outbreaks in susceptible persons due
to the improvement of sanitation and
water supply. Antibody production in
immunized hens can be an alternative
technology to diagnosis testes,
which has many advantages as easily
accessible, large-scale production and
appropriate methodology regarding
the bioethical appearance. The aim of
this study was compare two purification
procedures applied to anti-HAV IgY
for possible application this antibody
in diagnostic tests. Methodology:
Two groups of hens were immunized:
the group one was immunized with
hepatitis A vaccine plus adjuvant CPG
oligodeoxynucleotides
(CPG-ODN)
and the group two only with CpGODN. The eggs from both groups were
collected and the IgY was purified
from egg yolk by only polyethylene
glycol 6000 precipitation (PEG) and
by PEG precipitation followed by
thiophilic adsorption. After the IgY was
characterized by the electrophoresis
and Western Blotting methods.
Result and Conclusion: The result of
purification IgY of PEG precipitation
followed by thiophilic adsorption
proved to be more effective in
removing impurities contaminants in
egg yolk than purification using only
PEG precipitation. The methodology
using in this study to purify IgY from
egg yolk was successful, obtaining
an antibody without the presence of
interfering proteins, which contributes
to the possible application from the
326
antibody in diagnostic methods for
detecting hepatitis A virus. Financial
support: IOC/FIOCRUZ, Rio de Janeiro,
RJ, Brazil.
IV1259 - PRODUCTION OF MONOCLONAL ANTIBODIES AGAINST
HEXON PROTEIN OF HUMAN ADENOVIRUS
Paulini, I.J., Silva, S., Marinheiro, J.C.,
Harsi, C.M., Bellei, N., Granato, C.F.H.
UNIVERSIDADE FEDERAL DE SAO
PAULO, UNIFESP, RUA PEDRO
DE
TOLEDO,781.
15
ANDAR
UNIVERSIDADE DE SAO PAULO, USP,
AV. PROF LINEU PRESTES ,1374. ICB
II SALA 229 E-mail: inareip@yahoo.
com.br
Human adenovirus (HAdV) present 52
serotypes which are causative agents
of infections in the gastrointestinal,
uro-genital, and neurological systems,
both in children and adults. Although
many of these infections are severe
requiring hospitalization, HAdV are
rarely diagnosed due to the lack
of sensitive, practical and cheap
diagnostic tests. The aim of this study
is to produce monoclonal antibodies
against HAdV-2 hexon protein which
will be applied in rapid diagnostic test.
First step of this project was to purify
HAdV-2 hexon protein. For this, 48
cell culture bottles of 300cm2 were
seeded with HEK-293 cells and the
cell monolayer infected with 0.1 M.O.I
of HAdV-2. When the cytopathic effect
was evident (between 2-5 days), the
cells were harvested and centrifuged
at 220 g for 10 min. The supernatants
were collected and the HAdV-2
precipitated by ultracentrifugation.
The cell pellets were pooled in 10 mM
HEPES buffer at pH 7.4.Viruses were
released after three cycles of freezing
and thawing. Lysed cells suspensions
were clarified with equal volume
of Vertrel XF, followed by vigorous
vortexing. Cells debris were removed
by centrifugation at 2.619 g for 25min
at 4°C. Viral particles and soluble
proteins in the supernatant were
purified in CsCl gradients prepared in a
10 mM HEPES buffer at pH 7.4. Purified
HAdV-2 complete particles were stored
at -20°C. Soluble proteins fraction of
the gradient were analyses by SDSPAGE, Electronic Microscopy and
hexon protein purified by ion exchange
columns/ultrafiltration. BALB/c male
mice were primed intraperitoneally
with mixtures of purified hexon protein
(50µg/mouse) in Freund’s complete
adjuvant and were then boosted twice
intraperitoneally with these mixtures
in Freund’s incomplete adjuvant with
2-week intervals. The mice were
sacrificed on 45 days. Spleen cells
were harvested and fused with SP2/0
myeloma cells. Then production and
selection of monoclonal antibodies
against hexon protein were performed
as previously described protocol.
Financial support : Fundação de
Amparo à Pesquisa do Estado de São
Paulo – FAPESP (number: 2011/501006).
IV1243 - PRODUCTION OF SPECIFIC
ANTIBODIES IN RABBITS AGAINST
CONSERVED AND POTENTIALLY IMMUNOGENIC PEPTIDES OF THE HCV
ENVELOPE GLYCOPROTEIN E2
Marins, R.S.Q.S., Almeida, E.C.C., Freitas,
L.J., Moraes, M.T.B., Gomes, S.A.
1. Laboratory of Molecular Virology,
Brazil
2. Biotery Helio e Peggy Pereira,
Brazil E-mail: rmarins@ioc.
fiocruz.br
Hepatitis C virus (HCV) is a chronic
infection that affects more than 2% of
the global population and is a leading
cause of hepatocellular carcinoma, and
end-stage liver diseases. HCV displays
a high variability and is classified into
seven genetically distinct genotypes
which differ by approximately 30%
at the nucleotide level. Envelope (E1
/E2) proteins of HCV may generate
neutralizing antibodies. At the end
N-terminus of the E2 protein there is a
region of 27 amino acids hypervariable
region 1 called (HVR1), very important
in neutralizing HCV. Despite the high
degree of variability of E2 protein
some amino acid positions are
conserved. This study was designed
to assess the immunogenic properties
of conserved peptides derived from
E2 and HVR1 regions. Nucleotide
sequences of all available HCV E2
region in the databases were aligned
and four amino acid sequences with
immunogenic potential were selected.
These selected regions were used to
generate synthetic peptides: peptide 1
(aa412-149), peptide 2 (aa517-531),
peptide 3 (aa523-535) and peptide 4
(HVR1, aa373-416) were synthesized
conjugated with KLH and ressuspended
in PBS (2ug/uL). Each peptide (0,5
mg/ml, 250UL) was used to inoculate
one rabbit after emulsification of l
volume of Freund’s complete adjuvant.
Subcutaneous injections in three
different sites of each rabbit were
performed. Two subsequent doses, on
day 14 and on day 28, were performed
327
with emulsified Incomplete Freund’s
adjuvant. One control animal was
injected with 0,5 ml saline solution at
the 14 days intervals of immunization
protocol. One month after the last
immunization, the animals were
scarified. An in house Elisa was
developed to titrate antibodies and test
their specificities by cross reactivity of
each peptides and raised antibodies.
ELISA plates were covered with the
same peptides but conjugated with BSA
(0,5µg/well (100µL). Standardization
determined there is an appropriated
dilution for each antibody that allows
specific detection of these anti-E2
antibodies generated in rabbits with
exception of anti-HVR1 antibody which
shows beyond the specific reaction,
a nonspecific reaction with BSA. In
this case, the standardization was
performed by recovering the plate with
the pure peptide (without conjugation).
In conclusion, all the designed peptides
were able to generate anti-E2 specific
antibodies in rabbits at relatively high
titles. Financial support: CNPq and
Fiocruz
IV1340 - MAPPING OF STRUCTURAL
AND NON-STRUCTURAL PROTEINS
AND CHARACTERIZATION OF THE
MAJOR NEURALIZING SITES ON THE
MAYARO VIRUS
Pêgo, P.N., Teixeira-Pinto, L.A.L., Simone
2. Universidade Federal Fluminense,
UFF, Campus Valonguinho-Inst.
de Biologia- Outeiro de S. João
Batista s/n Niterói -RJ E-mail:
[email protected]
Mayaro virus (MAYV) is an arbovirus
328
of the genus Alphavirus, family
Togaviridae, enzootic in South
America, being held in the sylvatic cycle
involving mosquitoes and vertebrates
Haemagogus. The cases described
outside of the Amazon forest are rare,
however in recent years there has been
an increased incidence in travelers and
people with activities within or near
the forest. As this arbovirus has many
symptoms similar to dengue and other
febrile diseases including hemorrhages,
we have found important to perform
the complete mapping of all immune
proteins of MAYV, to identify epitopes
relevant to protective and diagnostic
purposes. In this study, all the nonstructural (nsP1-4) and structural (E13, C and 6K) polyproteins were mapped
using a pool of patients immune sera
collect in Manaus-Amazon in different
phases of the disease and the parallel
Spot-synthesis peptide technique.
Two libraries of 733 peptides with
a length of 14 amino acids and nine
overlapping residues were synthesized
and evaluated for IgM and IgG
reactivity. Either IgM and IgG B-linear
epitopes were identified including the
shifty of immune response. Forty-six
major epitopes IgG with a length of
9-15 residues were identified in the
nonstructural polyprotein, whereas
only eight epitopes (9-10 amino acids)
were identified in the structural protein.
Bioinformatics tool and molecular
modeling approaches determined that
all linear B epitopes were situated at
the surface of the molecules. In addition
we show that the Cellu-spot arrays
are applicable to diagnosis of Mayaro
infections. Cellu-spot array screen
would provide a new insight in the
identified of relevant epitope reactivity
detected by Spot method and will be
used as point-of-care EIA-test. Thus
now our goal is use the information
obtained to develop a biochip-peptide
method useful in large scale point-ofcare serodiagnostics of MAYV and other
similar febrile syndromes. Financial
Support: MCT-CNPq, FAPERJ, CAPES,
FIOCRUZ (PDTIS/IOC) Mestranda
da Pós-Graduação em Biologia das
Interações-UFF
IV1346 - PURIFICATION OF CHIMERIC
PROTEIN NAMED HBSAGE2RECHIS
PRODUCED FROM THE TRANSIENT
EXPRESSION IN MAMMALIAN CELLS
HEK-293-T USING THE METAL ION
CHROMATOGRAPHY (IMAC)
de Freitas, J.H.R., Junior, M.S.S., Vieira,
M.C.R., de Barros, J.J.F., Bottecchia
Gomes, S.A., Moraes, M.T.B.
L a b . V i ro l . M o l e c u l a r - I n s t i t u to
Oswaldo Cruz- Fiocruz, LVM-IOCFiocruz, Avenida Brasil, 4365,
Manguinhos, Rio de Janeiro-RJPrédio HPP E-mail: jessica.freitas@
ioc.fiocruz.br
About 2 billion people have already
come into contact with the hepatitis
B virus (HBV) and 350 to 400 million
are chronic carriers. Vaccination
against HBV is available, but several
factors keep the HBV circulating in the
population. One hundred and seventy
million people of the world population
are chronic carriers of hepatitis C
virus (HCV). The progression of HCV
infection to hepatocellular carcinoma
is a worldwide public health problem
that grows each year due to the
absence of a vaccine. In this study a
purification process of the envelope
protein of Hepatitis B virus (HBsAg),
containing the epitope of the HCV E2
protein and containing a histidine
tail (HBsAgE2Rec chimeric protein),
derived from the transient transfection
of human embryonic kidney cells with
the SV40 T antigen gene inserted
(HEK-293T) was established. The yield
of the purification protocol was 75%.
The purified protein was assessed
for immunogenicity by Western blot
and ELISA. We also inoculated mice
in order to evaluate antigen and
monoclonal antibody production. The
data obtained mainly serve to validate
the system chimeric HBsAg expression
of heterologous antigens to be used
as vaccines and diagnostics. Financial
support: IOC-Fiocruz, CNPq
IV1424 - QUILLAJA BRASILIENSIS
SAPONINS ARE A POTENT ADJUVANT
OF SPECIFIC CELLULAR AND
HUMORAL IMMUNE RESPONSES
AGAINST INACTIVATED POLIOVIRUS
ANTIGEN
De Costa, F., Yendo, A.C.A., Cibulski, S.,
Colling, L.C., Fleck, J.D., Roehe, P.M.,
Spilki, F.R., Fett-Neto, A.G., Gosmann, G.
Universidade Federal do Rio Grande
do Sul, UFRGS
The use of inactivated polio vaccine has
an important role at the final stages of
poliomyelitis eradication programs.
An affordable option to enhance the
vaccine immunogenicity and reduce
costs is using an effective adjuvant.
The saponins of Q. brasiliensis, a native
species from Southern Brazil, are similar
to those of Q. saponaria, a commercial
adjuvant used in vaccine formulations.
It has been shown that aqueous extract
(AE) and the purified fraction of
saponins from Q. brasiliensis, QB-90,
had adjuvant activity in experimental
vaccines against bovine herpesvirus
type 1 and type 5 in mice. Herein, we
analyze the potential of AE and QB90 adjuvant activity using poliovirus
antigen as model. Five groups of
329
seven mice each (CF-1 breed) were
vaccinated subcutaneously on days
0 and 28. Animals were immunized
with saline, viral antigen plus saline,
or with either Quil-A® (50 µg), AE
(400 µg) or QB-90 (50 µg). Sera from
inoculated mice were collected on days
0, 28, 42 and 56 post-inoculation of the
first dose of vaccine. Specific antibody
titres were evaluated by indirect
ELISA. Delayed type hypersensitivity
responses (DTH) were evaluated in
three mice from each group on day 56.
Serum levels of specific IgG, IgG1 and
IgG2a were significantly enhanced by
AE, QB-90 and Quil-A® compared to
control groups until day 56 (α< 0.05).
DTH response indicated that, similar to
Quil-A®, AE and QB-90 were capable
of stimulating the generation of Th1
cells against the administered antigen
(α < 0.05). Both AE and QB-90 from
Q. brasiliensis are potent adjuvants of
specific cellular and humoral immune
responses and can be considered as
an alternative to Quil-A®. Financial
support: CNPq, CAPES.
IV1473 - EVALUATION OF RABIES
VIRUS GLYCOPROTEIN EXPRESSION
IN MAMMALIAN CELLS USING A RECOMBINANT SEMLIKI FOREST VIRUS
CARRYING THE RESPECTIVE MRNA
Rezende, A.G., Puglia, A.L.P., Astray,
R.M., Bernardino, T.C., Pereira, C.A.,
Lemos, M.A.N., Núñes, E.G.F., Jorge,
S.A.C.
Instituto Butantan, IBu, Av. Vital
Brasil - 1500 Escola Politécnica USP, Poli-USP, E-mail: alerezende@
usp.br
Introduction: The present study aims
to use the Semliki Forest Virus - SFV,
as expression system in order to assess
gene expression in mammalian cells.
330
The rabies virus glycoprotein (RVGP),
recognized as an antigen capable of
conferring immune response against
rabies, was chosen as a target gene in
this approach. Objectives: To establish
protocol for transfection of viral RNA to
produce recombinant SFV (SFV-RVGP);
to analyze the RVGP expression in cells
infected by SFV-RVGP by using the
ELISA; to determine the best conditions
for cell culture and viral infection for the
expression of the heterologous protein.
Methods: Two different plasmids were
used: an expression plasmid containing
SFV genes coding for nonstructural
proteins and the RVGP gene, and a
helper plasmid containing SFV genes
coding for structural proteins. In vitro
transcription was performed and
RNAs were co-transfected in BHK-21
cells, for generation of SFV-RVGP. They
were then activated and used to infect
BHK-21 and Huh 7.0 cells, and induce
the heterologous protein (RVGP).
Expression evaluation was done by
ELISA. Results and Discussion: Using
the SFV-RVGP method of expression,
we evaluated the time of SFV-RVGP
generation, and the RVGP production
after infection. The experiments were
performed in duplicate in 6 wells
plate in CO2 incubator at 37 ° C. The
cell inoculum was of 7x1E5 cells/
well with a working volume of 2 mL.
Based on these results, the method of
transfection electroporation was better
than the method with a commercial kit
Transmessenger, and time 24 hours
after transfection, the best collection of
viruses generated. Another important
fact is that protein

Journal of the Brazilian Society for Virology Editors Editorial Board

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