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ISSN 0036-4665ISSN 1678-9946online

Established: 1959.The year 2010 is the 51st anniversaryof continuous publication

UNIVERSIDADE DE SÃO PAULO - BRAZILFACULDADE DE MEDICINA

InstitutodeMedicinaTropicaldeSãoPauloDirector: Prof. Dr. Claudio Sergio Pannuti

EDITOR‑IN‑CHIEF EMERITUSEDITORS Prof.Dr.ThalesF.deBrito Prof.Dr.LuisRey(FoundingEditor)AssociateEditors:Prof.Dr.MarcelloFabianodeFranco Prof.Dr.CarlosdaSilvaLacaz Prof.Dr.PedroPauloChieffi

EDITORIALBOARDAlanL.deMelo(BeloHorizonte,MG)AlbertoDuarte(S.Paulo,SP)AngelaRestrepoM.(Medellin,Colombia)AnnaSaraS.Levin(S.Paulo,SP)AntonioA.Barone(S.Paulo,SP)AntonioCarlosNicodemo(S.Paulo,SP)AntonioSesso(S.Paulo,SP)AntonioW.Ferreira(S.Paulo,SP)BarnettL.Cline(NewOrleans,USA)CarlosF.S.Amaral(BeloHorizonte,MG)CelsoGranato(S.Paulo,SP)CesarA.CubaCuba(Brasília,DF)CésarNaquiraV.(Lima,Peru)ClarisseM.Machado(S.Paulo,SP)ClaudioS.Pannuti(S.Paulo,SP)CláudioSantosFerreira(S.Paulo,SP)DaltonL.F.Alves(BeloHorizonte,MG)EridanCoutinho(Recife,PE)ErnestoHofer(RiodeJaneiro,RJ)EuclidesA.Castilho(S.Paulo,SP)EufrosinaS.Umezawa(S.Paulo,SP)FanHuiWen(S.Paulo,SP)FernandoA.Corrêa(S.Paulo,SP)FernandoMontero‑Gei(SanJosé,CostaRica)

FlairJ.Carrilho(S.Paulo,SP)GilBenard(S.Paulo,SP)GiocondaSan‑Blas(Caracas,Venezuela)GovindaVisvesvara(Atlanta,USA)HeitorF.AndradeJr.(S.Paulo,SP)HenriqueL.Lenzi(RiodeJaneiro,RJ)HiroGoto(S.Paulo,SP)IsesA.Abrahamsohn(S.Paulo,SP)JoãoCarlosPintoDias(BeloHorizonte,MG)JoãoRenatoRebelloPinho(SaoPaulo,SP)JoséEduardoLevi(S.Paulo,SP)JoséM.R.Zeitune(Campinas,SP)JuliaMariaCosta‑Cruz(Uberlândia,MG)JulioLitvoc(S.Paulo,SP)LuizCaetanodaSilva(S.Paulo,SP)LuizCarlosSevero(P.Alegre,RS)LuizJacinthodaSilva(Campinas,SP)LuizT.M.Figueiredo(Rib.Preto,SP)LygiaB.Iversson(S.Paulo,SP)MarcosA.Rossi(RibeirãoPreto,SP)MarcosBoulos(S.Paulo,SP)M.A.Shikanai‑Yasuda(S.Paulo,SP)MariaI.S.Duarte(S.Paulo,SP)MariaL.Higuchi(S.Paulo,SP)

MarioMariano(S.Paulo,SP)MirianN.Sotto(S.Paulo,SP)MoisésGoldbaum(S.Paulo,SP)MoysésMincis(S.Paulo,SP)MoysésSadigursky(Salvador,BA)MyrthesT.Barros(S.Paulo,SP)NilmaCintraLeal(Recife,PE)PauloC.Cotrim(SãoPaulo,SP)PauloM.Z.Coelho(BeloHorizonte,MG)PedroMorera(SanJosé,CostaRica)ReginaAbdulkader(S.Paulo,SP)RicardoNegroni(B.Aires,Argentina)RobertH.Gilman(Baltimore,USA)RobertoMartinez(Rib.Preto,SP)SemíramisGuimarãesF.Viana(Botucatu,SP)SilvinoA.Carvalho(S.Paulo,SP)SilvioAlencarMarques(Botucatu,SP)SumieHoshino‑Shimizu(S.Paulo,SP)ThelmaS.Okay(S.Paulo,SP)TsutomuTakeuchi(Tokyo,Japan)VenâncioA.F.Alves(S.Paulo,SP)VicenteAmatoNeto(S.Paulo,SP)ZiltonA.Andrade(Salvador,BA)

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Thepurposeofthe“RevistadoInstitutodeMedicinaTropicaldeSãoPaulo”(JournaloftheSãoPauloInstituteofTropicalMedicine)istopublishtheresultsofresearchwhichcontributesignificantlytoknowledgeofalltransmissiblediseases.

REVISTADOINSTITUTODEMEDICINATROPICALDESÃOPAULO(JOURNALOFTHES.PAULOINSTITUTEOFTROPICALMEDICINE).

SãoPaulo,SP‑Brasil,1959‑v.ilust.28cm

1959‑2009,1‑511973‑2002(supl.1‑12)2003(supl.13‑on‑lineonly)2005‑2010(supl.14‑16)

ISSN0036‑4665ISSN1678‑9946online

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ADDRESSINSTITUTO DE MEDICINA TROPICAL DE SÃO PAULO

Av. Dr. Enéas de Carvalho Aguiar, 47005403-000 São Paulo, SP - Brazil

Phone/Fax: 55.11.3062.2174; 55.11.3061-7005e-mail: [email protected]

SUBSCRIPTIONSFOREIGN COUNTRIESOne year (six issues) ........ U$ 200.00Single issue ...................... U$ 50.00

International Theoretical CourseVIRAL HEPATITIS AND THE HUMAN HOST

February 22nd to 25th, 2010Rebouças Convention Center

São Paulo- SP, Brazil

Department of GastroenterologyUniversity of São Paulo - School of Medicine

São Paulo - SP – Brazil2010

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SPONSORSHIP AND INSTITUTIONAL SUPPORT


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COURSE ORGANIZERS


Dr. João Renato Rebello PinhoDepartamento de GastroenterologiaFaculdade de MedicinaUniversidade de São Paulo

Dr. Flair José CarrilhoDepartamento de GastroenterologiaFaculdade de MedicinaUniversidade de São Paulo

Dra. Suzane Kioko Ono-NitaDepartamento de GastroenterologiaFaculdade de MedicinaUniversidade de São Paulo

Dr. Esper Georges KallásDepartamento de Clinica MedicaFaculdade de MedicinaUniversidade de São Paulo

Dr. Mário PessoaDepartamento de GastroenterologiaFaculdade de MedicinaUniversidade de São Paulo

Mónica Viviana Alvarado MoraEstudante de DoutoradoDepartamento de GastroenterologiaFaculdade de MedicinaUniversidade de São Paulo

Dra. Maria Cássia CorreaDepartamento de Doenças InfecciosasFaculdade de MedicinaUniversidade de São Paulo

Dr. Diogo MeyerDepartamento de Biologia e Biologia EvolutivaInstituto de BiociênciasUniversidade de São Paulo

Dr. Eduardo Luís Rachid CançadoDepartamento de GastroenterologiaFaculdade de MedicinaUniversidade de São Paulo

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Dr. Stephen LocarniniHead, Research & Molecular Development Victorian Infectious Diseases Reference LaboratoryAustralia

Dr. Richard M. SingleDepartment of Mathematics and Statistics,University of Vermont, Hills Science BuildingUnited States of America

Dr. Jason D. BarbourHIV/AIDS Division, San Francisco General Hospital.Department of Medicine, University of California San FranciscoUnited States of America

Dr. Marcos MirettiDepartamento de Genetica, FCEQyN, Universidad Nacional de MisionesArgentina

Dr. Mary CarringtonCancer and Inflammation Program,Laboratory of Experimental ImmunologyUnited States of America

Dr. Scott MuerhoffAbbott Diagnostics, Molecular Biology ResearchUnited States of America

Dr. Anna KramvisHepatitis Virus Diversity Research Programme Department of Internal Medicine University of the WitwatersrandSouth Africa

Dr. Christopher M. WalkerCenter for Vaccines and Immunity The Research Institute at Nationwide Children’s HospitalUnited States of America

SENIORS INVITED COURSE STAFF - International

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Dr. Flair José CarrilhoDepartamento de GastroenterologiaFaculdade de MedicinaUniversidade de São Paulo – SP- Brasil

Dra. Maria Cássia CorreaDepartamento de Doenças InfecciosasFaculdade de MedicinaUniversidade de São Paulo – SP- Brasil

Dr. Diogo MeyerDepartamento de Biologia e Biologia EvolutivaInstituto de BiociênciasUniversidade de São Paulo – SP- Brasil

Dr. Paolo Marinho de Andrade ZanottoInstituto de Ciências BiomédicasUniversidade de São Paulo – SP- Brasil

Dr. Nelson Jurandi Rosa FagundesInstituto de Biociências Universidade Federal do Rio Grande do SulPorto Alegre, RS – Brasil

Dr. João Renato Rebello PinhoDepartamento de GastroenterologiaFaculdade de MedicinaUniversidade de São Paulo – SP- Brasil

Dra. Ana Rita Coimbra Motta CastroCentro de Ciências Biológicas e da Saúde Universidade Federal de Mato Grosso do SulCampo Grande, MS – Brasil

Dra. Suzane Kioko Ono-NitaDepartamento de GastroenterologiaFaculdade de MedicinaUniversidade de São Paulo – SP- Brasil

Dra. Gilberta BensabathInstituto Evandro ChagasBelém, PA – Brasil

SENIORS INVITED COURSE STAFF - National

Dear Colleagues,

It is indeed a great honor to present to you the program of the INTERNATIONAL THEORETICAL COURSE ON VIRAL HEPATITIS AND THE HUMAN HOST. This course will be held on February 22 -25, 2010 at the Rebouças Convention Center, in Sao Paulo, Brazil.

The course will discuss advances in viral hepatitis diagnosis, epidemiology and treatment, followed by lectures on the monitoring of HBV and HCV drug resistance and genotyping.

The human genetic diversity will also be discussed, especially genetic markers related to viral response, including MHC genes and other genes involved in innate and acquired immune response. These lectures will also discuss the presence of these markers in different human populations. The analysis of the presence of these viruses in different human populations and phylogeographical studies helping to elucidate their spreading pathways in the human population will also be addressed.

For this course, we have invited lecturers from Brazil, Argentina, United States of America, Australia and South Africa. This will be a fine opportunity to establish contacts among researchers and students that will be joining the meeting from Brazil and other countries all over the world.

We are looking forward to welcoming you to São Paulo, Brazil.

João Renato Rebello Pinho Mónica Viviana Alvarado Mora Flair José Carrilho Suzane Kioko Ono-Nita Esper Georges Kallás Maria Cássia Jacinto Mendes Correa Diogo Meyer Eduardo Luís Rachid Cançado Mário Pessoa


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SCIENTIFIC PROGRAM

Monday, February 22, 2010

08:30 – 09:00 Welcome and Introduction – Dr. João Renato Rebello Pinho

CHAIRS: Dr. Suzane Kioko Ono-Nita and Dr. Maria Cássia Jacintho Mendes Correa09:00 – 09:45 Advances in the virology and diagnosis of human hepatitis Dr. A. Scott Muerhoff09:45 – 10:00 Discussion10:00 – 10:45 Epidemiology of hepatitis B and C in Brazil Dr. Flair José Carrilho10:45 – 11:00 Discussion11:00 – 11:20 Coffee break11:20 – 12:00 Update on the treatment of hepatitis B and C viruses Dr. Suzane Kioko Ono-Nita12:00 – 12:15 Discussion12:15 – 14:30 Lunch14:30 – 15:30 Poster presentations

CHAIRS: Dr. João Renato Rebello Pinho and Dr. Ana Rita Coimbra Motta Castro15:30 – 16: 15 Hepatitis B and C viruses coinfection in HIV patients in Brazil Dr. Maria Cássia Jacintho Mendes Correa16:15 – 16:30 Discussion16:30 – 16:45 Coffee break16:45 – 17:30 Hepatitis B, C and D viruses epidemiology in the Amazon Basin Dr. Gilberta Bensabath17:30 – 17:45 Discussion17:45 Close

Tuesday, February 23, 2010

CHAIRS: Dr. Mary Carrington and Dr. Anna Kramvis09:00 – 09:45 Bioinformatics methods to evaluate hepatitis B and C viruses resistance to treatment Dr. Stephen Locarnini09:45 – 10:00 Discussion10:00 – 10:45 Hepatitis B and C viruses genotypes and drug resistance related mutations in Brazil Dr. João Renato Rebello Pinho10:45 – 11:00 Discussion11:00 – 11:20 Coffee break11:20 – 12:00 The Viral Genetics Diversity Network: HCV Dr. Paolo Marinho de Andrade Zanotto12:00 – 12:15 Discussion12:15 – 14:30 Lunch14:30 – 15:30 Poster presentations

CHAIRS: Dr. Diogo Meyer and Dr. Jason Barbour15:30 – 16:15 Adaptive immune responses in acute and chronic hepatitis C vírus infection Dr. Christopher Walker16:15 – 16:30 Discussion16:30 – 16:45 Coffee break

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16:45 – 17:30 Methods applied in association studies among immunogenetics markers and viral diseases Dr. Jason Barbour17:30 – 17:45 Discussion17:45 Close

Wednesday, February 24, 2010

CHAIRS: Dr. Christopher Walker and Dr. Marcos Miretti09:00 – 09:45 Evolutionary genetics of genes that control the immune response Dr. Diogo Meyer09:45 – 10:00 Discussion10:00 – 10:45 Origins of GB virus C determined by complete genome analysis Dr. A. Scott Muerhoff10:45 – 11:00 Discussion11:00 – 11:20 Coffee break11:20 – 12:00 Human migrations and HBV origins in South America Dr. Nelson Jurandi Rosa Fagundes12:00 – 12:15 Discussion12:15 – 14:30 Lunch14:30 – 15:30 Poster presentations

CHAIRS: Dr. Stephen Locarnini and Dr. Flair José Carrilho15:30 – 16: 15 The phylogeography of hepatitis B virus in Africa Dr. Anna Kramvis16:15 – 16:30 Discussion16:30 – 16:45 Coffee break16:45 – 17:30 Molecular epidemiology of hepatitis B virus in Afro-Brazilian population Dr. Ana Rita Coimbra Motta de Castro17:30 – 17:45 Discussion17:45 Close

Thursday, February 25, 2010

CHAIRS: Dr. Eduardo Cançado and Dr. Mario Pessoa09:00 – 09:45 Linkage-Disequilibrium Map of the Human Major Histocompatibility Complex and First Generation of Tag Single-Nucleotide Polymorphis Dr. Marcos Miretti09:45 – 10:00 Discussion10:00 – 10:45 Studies on the allelic frequencies and haplotypes for immunogenetic markers Dr. Richard Single10:45 – 11:00 Discussion11:00 – 11:20 Coffee break11:20 – 12:00 Innate and acquired immune response markers and viral infections Dr. Mary Carrington12:00 – 12:15 Discussion12:15 – 12:30 Concluding Remarks Dr. João Renato Rebello Pinho and Monica Viviana Alvarado Mora

INTERNATIONALTHEORETICALCOURSE.VIRALHEPATITISANDTHEHUMANHOST. February22nd‑25th,2010.SãoPaulo,SP,Brazil.Rev.Inst.Med.Trop.SãoPaulo,52(supplement16),2010.

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SUMMARY OF THE LECTURES

L1 - ADVANCES IN THE VIROLOGY AND DIAGNOSIS OF HUMAN HEPATITIS

Dr.A.ScottMuerhoff

Humanhepatitisisamajorglobalhealthconcern.Anestimated500million people are infected with one of the human hepatitis viruses.Amongtheviralagentsresponsibleforhepatitisinhumans,hepatitisBandCviruses,andinsomecasesD,oftenresultinchronicinfectionwhileinfectionwithAandEvirusesisself‑limiting.ChronicinfectionwithBorCvirusescanleadtofibrosis,cirrhosis,hepatocellularcarcinomaanddeath.Accurateandrapiddiagnosisofhepatitisisrequiredtoprescribethepropercourseoftreatment,preventfurthertransmissionandunderstandtheepidemiologyofthedisease.Currentmethodsrelyondetectionofantibodiestotheantigensencodedbytheseagents.TheseserologicalassayscanbeautomatedforscreeningofthebloodsupplyforBandCviruseswhichareparenterallytransmitted.Recentintroductionofnucleicacidtests(NAT)inNorthAmericaandtheEuropeanCommunityforuseinblooddonorscreeninghasreducedtheresidualriskofpost‑transfusionHCV substantially. Use of highly sensitive assays for detection ofhepatitisBsurfaceantigen(HBsAg)continuestobeusedforblooddonorscreeningasistheanti‑HBcoreassay.TheuseofnucleicacidtestsfordetectionofHBVhasnotbeenimplementedintheUSA.QuantitativeNATforHCVRNAisutilizedfordeterminationofresponsetoantiviraltreatmentandcurrentlylicensedassaysarecapableofdetectingallmajorgenotypes.Withinthelastfewyears,theHCVcoreantigenassayhasbeenintroducedasamethodformonitoringresponsetotherapyasthecoreantigentiterandRNAtitercoincide.BecausethegenotypeofHCVcaninfluencetreatmentoutcomes,genotypingisperformedpriortothestartoftreatment;theseassaycanbestripblotassaysor,morerecently,PCR‑based.Therapidmutationratesofsomeoftheseviruses,aswellasthecontinueddiscoveryofnewvariantsorcirculatingrecombinantforms,requirescontinuoussurveillanceofglobalhumanpopulations.

L2 - EPIDEMIOLOGY OF HEPATITIS B AND C IN BRAZIL

Dr.FlairJoséCarrilho

Hepatitis B (HBV) and C virus (HCV) infections constitute asignificant health problem inLatinAmerica.Approximately400,000newcasesofhepatitisBperyearand10millionpeopleinfectedwithhepatitisCare estimated tooccur. InBrazil, theprevalenceofHBVserummarkersalsovariesdependingonthegeographicalregionbeinganalyzed.TheMinistryofHealthestimatesthatinBrazil,atleast15%of thepopulationhadcame incontactwithHBVand that1%of thepopulationpresentchronicdisease.HBVprevalenceishigherinstatesin the North and Northeast of the country when compared to SouthandSoutheaststates.Theseroprevalencemayreach2.8%to10.3%oftheentirepopulationintheseareas.InEspíritoSantoandParaná,theprevalenceofHBVserummarkershasbeenreportedtovarybetween3.2% and 8.3%, respectively.Anti‑HCV prevalence in blood donorsvariesfrom1.7%to3.4%inNortheastofBrazilandfrom0.8%to2.8%

inSoutheast.OnestudycarriedoutinSaoPaulocityshowedthat5.94%and1.42%ofthepopulationshowedserologicalmarkersforcurrentorpreviousHBVandHCVinfections,respectively.AnotherrecentstudyevaluatedtheprevalenceofHBVandriskfactorsinthecapitalcitiesoftheNortheast,Central‑West,andFederalDistricts(2004‑2005)andfoundthatpositivityforHBsAgwaslessthan1%amongnon‑vaccinatedpersonsandgenotypesA,D,andFco‑circulated.Riskfactorsforhepatitisinfections in Brazil are the same found throughout the world. HBVinfection ismore frequent inpersonshaving initiatedsexualactivity,andhealthcare jobsandpriorhospitalizationwere risk factors.Otherhigher risk populations include hemodialysis patients, hemophiliacs,patients with hepatosplenic schistosomiasis, patients with leprosy,malehomosexuals,prostitutes,drugaddictsandhouseholdcontactofHBVcarriers.ForHCV,riskfactorsforinfectionsarepreviousbloodtransfusion(especiallyupto1993),hospitalizations,intravenousdruguse, sexual promiscuity, tattooing practices, exposure to blood andhemodialysis. For both hepatitis, it is noteworthy that in Brazil, foraboutatleast30%ofcasestherouteoftransmissionisnotknown.SinceBrazilisalargecountrywithmanydifferentpopulationbackgrounds,awidevariationinthefrequenciesofHBVandHCVgenotypeswouldbeexpectedtobefoundthroughoutitsterritory.GenotypesA,DandFofHBVarethemostprevalentamongHBVcarriers.InanAfro‑Brazilian,slave‑descendant community, the genotypeA1 is the most prevalentindicatingtheAfricaninfluxduringthecolonialslaveryperiod.Also,intheAmerindianpeoplethegenotypeF2isthemostfrequentbutothersFsubgenotypesarefound,suchasF1b,showingprobablytheexistenceoftwoormorefounderviralpopulationsofgenotypeFinBrazil.Ontheotherhand,severalstudieshavebeenconducted todetermine thedistributionofHCVgenotypesamongdifferentgroupsofindividualsinBrazil,indicateahigherprevalenceinthecountryofgenotypeHCV‑1,followedbygenotypesHCV‑3andHCV‑2.

L3 - UPDATE ON THE TREATMENT OF HEPATITIS B AND C VIRUSES

Dr.SuzaneKiokoOno‑Nita

OverthepastyearsseveralnewantiviralsforthetreatmentofchronichepatitisBbecameavailable.Despitethepotentactionofthesedrugsthe development of new antivirals and strategies to treat hepatitis Barestill themajorgoal.Lamivudine isanucleosideanaloguereversetranscriptase inhibitor widely used in Brazil, but because its highresistanceratedevelopment,itisgraduallybeingsubstitutedbythenewantiviralsasthefirstlineindicationfornaïvetreatmentpatients.Theneworal antivirals include entecavir, tenofovir, adefovir and telbivudine.Interferonbasedtherapyisalsoanoption.IndicationoftherapycanvaryaccordingtoHBeAgstatus,presenceornotofcirrhosisandpresenceofnotofmutations.WewillreviewthelatestoptionsfortherapyofchronichepatitisB,includingcombinationstrategiesthatcouldbeanapproachto improving the response rate of treatment. During the past decadethetreatmentofchronichepatitisCwasbasedonpegylatedinterferonassociatedwithribavirinwithmodestsustainedvirologicalresponseonclinicalpracticedailybasis.Wewilldiscussthemostcommonadverteventsandsupporttherapy.Inaddition,newapproachessuchusrapid


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andearlyvirologicalresponseandvariationofdurationoftreatmentwillalsobediscussed.WiththedescriptionofthetertiarystructureofHCVproteinssuchuspolymerase,proteaseandhelicase,newstructurebasedtherapyisbeingdeveloped.Manyantiviralsarecurrentlyinclinicaltrials.Wewillreviewthemostpromisingantivirals,itsclinicaltrialsresultsandperspective.

L4 - HEPATITIS B AND C VIRUSES COINFECTION IN HIV PATIENTS IN BRAZIL

Dr.MariaCassiaJacinthoMendesCorrea

HEPATITISCPatientsinfectedwithhumanimmunodeficiencyvirus(HIV)have

prevalenceratesforinfectionwithhepatitisCvirus(HCV)higherthanthoseinthegeneralpopulation.InBrazil,thisprevalencerangesfrom4.1%to83%accordingtodifferentstudies.InBrazil,theprevalenceofHCVintheHIV‑positivepopulationdiffersgeographicallybecauseofthedistributionofriskfactorsthatdeterminesitstransmission.Patientswith parenteral transmission risk factors, such as intravenous drugusers(IDUs),showahigherprevalenceofco‑infectionwithHIVandHCVrangingfrom70%to83%.AfewstudiesinBrazilhaveanalyzedspecificriskfactorsfortransmissionofHCVintheco‑infectedgroupofpatients.Accordingtothesestudies,HCVinfectionwasassociatedwithinhaledillicitdrugs,anage>30yearsold,andahistoryofanalintercourse.ThesestudiesalsoconfirmedthatintravenousdrugusewasthemostimportantriskfactorassociatedwithHCVinfection,asalsoreportedbyothers.AdecreaseinthenumberofnewcasesofinfectionwithHIVamongIDUshasbeenobservedinBrazilandotherpartsoftheworld.RegardinghepatitisCgenotypesamongHIVinfectedpatients;differentstudieshaveshownthathepatitisCgenotype1seemstobethemoreprevalent(68.7%to72%),followedbygenotype3(26.3%to30%).Genotypes2and4arediagnosedinaminorityofpatients,rangingfrom1%to4%.Anassociationbetweengenotype3andintravenousdrugusehasalsobeenobservedamongBrazilianHIV‑HCVco‑infectedpatients.ChronichepatitisC(HCV)infectioniscurrentlyoneofthemostclinicallyrelevantco‑morbiditiesintheHIVpopulation.Progressiontoend‑stageliver disease occurs faster in coinfected patients and decompensatedcirrhosisisoneofthemaincausesofhospitalizationanddeathinthispopulationThe Brazilian literature on HIV/HCV has scarce data onlongitudinalevaluationofco‑infectedpatients.Mostpublishedstudieshavebeenlimitedtoaseriesofcasesanalyzedfromacross‑sectionalperspective or over short time periods. Brazil’sAIDS treatmentprogramguaranteesfreeaccesstohighlyactiveantiretroviral therapy(HAART)forallpeople livingwithHIV/AIDSinneedof treatment.ThelongersurvivalofHIVinfectedpatientsasaresultoftheincreasedeffectivenessofanti‑retroviraltreatmentsindicatesthattheprevalenceofpatientsco‑infectedwithHIVandHCVwillcontinuetobehighinthenearfuture,withmanyofthesepatientsbeingpotentialcandidatesforspecificHCVtreatment.HIVsignificantlyworsensliverdiseaseinHCV‑positivepatientsandappearstoaccelerateprogressiontocirrhosis.Therefore,inHIV‑co‑infectedpatients,treatmentofhepatitisCshouldbeapriority.Reportsfromdifferentpartsoftheworldhavedemonstratedthatonly30%ofco‑infectedHIV‑HCVpatientsareconsideredeligibleforinterferontherapy.ItseemsthatinreallifeinBrazil,morethanhalfofHIV‑HCVco‑infectedpatientshavebeenconsiderednotcandidatesto received anti‑HCV treatment.The most frequent reasons for non‑

treatmentobserved in these studieswere:non‑adherence,drugabuseandpsychiatricdiseaseaccordingtotwodifferentstudies.ThegoalofHCVtreatmentistoeradicatethevirusortodecreasefibrosisprogressioninindividualsinwhomviraleradicationisnotpossible.CombinationtherapywithPEG‑IFN‑andRBV iscurrently the standard treatmentforpersistentHCVinfection.Howeversustainedvirologicalresponses(SVR)areachievedinonly25to49%ofcasesinHCV‑HIVco‑infectedindividualsaccordingtorandomizedtrials.

HEPATITISBDifferentstudiesinBrazilhaveshownthatbetween3.4%and8.5%

ofHIV‑infectedpatientsareco‑infectedwiththehepatitisBvirus(HBV).InformationontheHBVgenotypedistributioninHIV‑HBVco‑infectedpatientsisscarceinBrazil.Inoneseriesof13co‑infectedpatients,HBV‑genotypeAandGwereidentifiedin11and2patientsrespectively.InSãoPaulo,inanotherseriesof34co‑infectedpatients,hepatitisBgenotypedistributionwasasfollows:A(n=22;%),D(n=5;%),G(n=5;%)andF(n=2;%).TheBrazilianliteraturehaslittleinformationonlongitudinalevaluationofHIV/HBVco‑infectedpatients.Inaseriesof86HIV‑HBV(HBsAg+)co‑infectedpatientsunderHAARTinSãoPaulo,highHBVDNA was associated to HBeAg reactivity, abnormalALT levels andthepresenceofadvancedliverdisease.ThepresenceofhepatitisdeltaamongHIV‑HBVpatientsinBrazilhasbeendescribedinthenorthernpartofBrazilandrarelyinthesoutheastpartofBrazil.Lamivudineisanucleosideanaloguethathasbothanti‑HIVandanti‑HBVactivity.Giventhewidespreaduseoflamivudine(LAM)foranti‑HIVtherapysincethemid‑1990s,manyHIV‑HBVco‑infectedpatientshavereceivedprolongedLAMtherapyinBrazil.Tenofovir(TDF),anucleotideanalogue,isalsoactiveagainstHBVandHIVandhasbeen introducedmore recentlyforHIVtreatmentinBrazil.Amatterofconcernamongthesepatientsis theemergenceof lamivudinemutationsin thehepatitisBgenome.InformationonthefrequencyofLAMorTDFresistancemutationsinHIV‑HBVco‑infectedpatientsisscarceinBrazil.InSaoPauloandRiodeJaneiro(SouthernpartofBrazil)afewstudieshaveshownthatprolongedLAMusehasbeenassociatedwiththeemergenceofvariousmutationsintheHBVgenome,includingmutationsthatmayelicitavaccineescapephenotype.Inthissameseriesofpatients,tenofovircontainingHAARTwassuccessfulinachievingHBVvirologicalsuppression.Inco‑infectedpatients,combinationtherapyshouldideallybeusedtoavoidordelaythedevelopmentofantiviralresistance.Regularmonitoringofthesepatientsisimperativetorecognizereactivationandtoidentifydrugresistanceandviralbreakthroughearly.

L5 - HEPATITIS B, C AND D VIRUSES EPIDEMIOLOGY IN THE AMAZON BASIN

Dr.GilbertaBensabath

The largevariation in theprevalenceofhepatitisBVirus (HBV)markers invariousgeographicareasandbetweenpopulationsgroupswithin specific areas is well known as stated by Smuzness in 1978.WithinthehugeBrazilianAmazonregionwouldn’tdedifferent.SoonafterthefirstsurveyinPurusriver(1970)lookingforAustraliaAntigenusinglow sensitivitytestasimmunodiffusion,epidemiologicresearcheswerestartedandshownthatinBelem,Pará,healthyadultstheHBsAgprevalencewas<1%,Itaituba,Pará;4,7%,SenaMadureira,Acre,9,2%.Thesestudieswereconfirmedlatelyusingmoresensitivetestsandby


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othersresearchers.GenerallyinwesternAmazontheHBsAgprevalencehadvariationfrom1,9%to13,5%andanti‑HBsbetween10,4%,90,3%,higherthanthosefromEasternAmazon:HBsAg0%to2,7%andforanti‑HBs,6,7% to13,8%.Surveillancedata fromMinisterofHealthabout confirmed cases of hepatitis B incidence show this differencein the1999through2007:6978cases inWesternAmazonand3.233inEasternAmazonthelaterwithmorepeoplethantheformer.Withinthesegeographic areas there arevariations in endemicity. InwesternAmazon,intheleftsideareaoftheAmazonRiver,theendemicityislowwithexceptionof indigenousgroups suchasYanomamis. In therightside,intheJurua,PurusandMadeiraValley’stheseroprevalenceofHBVmarkersbeforethemassivevaccinationandinclusionofHBvaccineintheNaturalImmunizationProgram,theseroprevalenceofHBVmarkerswerehighandafterthosemeasuresarestillmoderate:HBsAgcarrier3,3%inRioBranco,Acre.Thetransmissionwaspredominantlyhorizontal,byinter‑familiarcontactsbelow15yearsofageandsexualafterthisage.Verticaltransmissionwasof10%ofcases.Inthesevalleysthe incidence of infections by hepatitis delta virus (HDV) with theepidemiologicaspectsofco‑infectionandsuper‑infectionaremajorityofetiologiccauseoffulminanthepatitisknownasLabreahepatitis.ThegenotypesprevalenceofHBVwasidentifiedinTembe,Yanomami,KarajaIndiansasgenotypeF,NotwithstandinginfulminanthepatitisoccurredinBocadoAcre,municipalityAmazonasState,HBVgenotypesF,AandDwerefoundin50,0,28,6and21,4%ofcases,respectively.WithreferencetoinfectionsbyhepatitisCvirus,inthewesternAmazonArea,1636caseswerereportedand710inEasternduringtheperiod1999‑2006.Anti‑HCVserum‑prevalenceinblooddonorsinBelemwas0,37%,inManaus19980,99%andRioBranco1,63%.Seroprevalencewerehigherinhaemodializedpatients.Itwasobserved38,6seroconvertionfrom62patientsunitinahaemodyalisisduringperiod<1year.EpidemiologicresearchinHepatitisCVirusshowbeencouraged.

L6 - BIOINFORMATICS METHODS TO EVALUATE HEPATITIS B AND C VIRUSES RESISTANCE TO

TREATMENT

Dr.StephenLocarnini

INTRODUCTIONAntiviralresistanceisemergingasthesinglemostimportantfactor

intreatmentfailurewhenusingsmallmoleculeinhibitorsforhepatitisBandhepatitisC.Sinceantiviral resistancemutations (bothprimaryandsecondary)selectedunderoneagentmayaffecteitherpositivelyornegativelytheefficacyofsubsequentagents,databaseapproachesallowtheassimilation,correlationandintegrationofviralsequencedata,aswellasin vitroantiviraldrugsensitivityandrelevantclinicalinformationcollected over time [1]. Similar databases/search methods have beendevelopedinHIV[2,3,4].

HEPATITISBSeqHepBiscomposedofahepatitisBvirus(HBV)genomesequence

analysisprogramandarelationaldatabasewhichcanbeusedtocorrelatelarge numbers of patient clinical, routine pathology diagnostic data,viralmutationalsequenceinformation,andin vitroantiviralsensitivityand cross‑resistance phenotypic data in an integrated and structuredway for subsequent patient monitoring [1].The SeqHepB database

currentlycontainsroutinepathologyandspecialisedvirologydatafor3,420patients.Associatedwiththesepatients,thereareover400clinicalhistories, 3,000 treatment histories, 300 biopsy results, and 31,767specimenrecords.Intermsofroutinepathologytestsperformedonthesamples, there are 34,149 records in the database, and these includeHBV, hepatitis C virus (HCV), and hepatitis D virus (HDV) relatedpathologytestresults,aswellasroutineliverfunctionandhaematologytest results. Samples within the database are also associated with5,273HBVgenomicsequenceinformationcorrespondingto180,712nucleotide or amino acid variation data points.The mutation data iscorrelatedtoanextensivedatasetofin‑houseaswellaspublishedin vitrophenotypicdataonHBVantiviraldrugsensitivityandresistance.ThemaindiagnosticobjectiveoftheSeqHepBdatabaseistoprovideadrug‑resistancetestingserviceforpatientsonantiviraltherapy.Thecorrelation of clinical, pathological and viral molecular biologicaldatausingdifferentartificialintelligencetechniquesisfacilitatingtheanalysisofthepathogenesisandnaturalhistoryofchronichepatitisBintheeraofantiviraldrugresistance.Forexample,wehavebeenabletodefineapatternoffour“resistancepathways”duringtheemergenceofantiviraldrug‑resistance:(i)L‑nucleosidepathway(rtM204V/I)forlamivudineandtelbivudine;(ii)Acyclicphosphonatepathway(rtN236T)foradefovir;(iii)“Shared”pathwayfor(i)and(ii)viartA181T/V;and(iv)theD‑cyclopentanepathway(entecavir)(rtL180M+rtM204V/IplusoneofrtT184orrtS202orrtM250substitutions).Likewise,becausetheHBVenvelopeoverlapstheviralpolymeraseina+1frameshift[5],thedata inSeqHepBalso includesanequivalentHBsAgcomponent.Wearenow inaposition to follow theeffectsofdrug‑therapy includingemergenceofresistanceonthesequenceofHBsAg.Studieshaveshownthat lamivudine resistant HBV (rtV173L+rtL180M+rtM204V) has asignificantlyreducedanti‑HBsbindingduetoimportantchangesintheoverlappingHBsAg(sE164D+sI195M)[5].Thesestudieshavesignificantpublichealthimplications.

HEPATITISCHepatitisCvirus(HCV)hasbeenclassifiedintosixmajorgenotypes

but genotype does not appear to influence disease presentation orseverityofdisease.Therehavebeenreportsthatgenotype3ainfectionisassociatedwithsteatosisandgenotype1blinkedwithmorerapidandsevere viral recurrence post‑liver transplantation. However, genotypehasbeenidentifiedasamajorpredictorofresponsetointerferon‑basedribavirin(Rv)containingantiviraltherapies.AntiviralregimeshavebeenoptimizedforinfectionswithHCVgenotypes1‑4,althoughtreatmentstrategies forgenotypes5 and6haveyet tobedeveloped.Thus, forgenotype2and3,24weeksofinterferon‑Rvtherapyissufficient,whilst48 weeks is typically required for non‑genotype 2/3 infections.ThemolecularbasisforthedifferencesinresponseofHCVgenotypeshasyettobedetermined.

Thenextgenerationoftherapiesincludethe“specificallytargetedantiviraltherapyforHCV”orSTAT‑CcompoundsforhepatitisChasstarted,andinitialclinicalstudieshavedemonstratedrapidemergenceofresistancetothesemoleculeswhenusedasmonotherapy.LikewithHBV,particularsignaturesubstitutionsareemergingatspecificcodonssuchasR155,A156,D168,V36andT54oftheHCVNS3/4AproteaseandP495LandS282TfortheHCVNS‑5BRdRppolymerase.Thesechangesdoaffectviralreplicationcompetence/fitnessanditshouldprovepossibletolinkbio‑informaticallygenotypicresistanceresultsandphenotypicmeasurements to allow relevant predictions for patient outcomes,including planning of combination therapies.Thus, a SeqHepC‑type


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modelwillproviderelevantpatientselectionandon‑goingmonitoringduringSTAT‑Ctreatment.

FUTUREDIRECTIONSSeqHepBandSeqHepCwillenablevirologistsandphysicians to

individualisepatientmanagement,copewiththecurrentexplosionofantiviraldrugassociatedviralmutationsbyprovidinganeducationalmodule, and to conduct cross‑sectional retrospective or prospectivestudiesonvirus‑infectedindividualsundergoingantiviraltherapy[6,7],includingthedevelopmentofcombinationtherapies[6].

REFERENCES 1.Yuen,LKW,AAyres,MLittlejohn,DColledge,AEdgely,WJMaskill,SALocarnini,

andA Bartholomeusz. SEQHEPB:A sequence analysis program and relationaldatabasesystemforchronichepatitisB.2007.AntiviralResearch;75:64‑74.

2.Beerenwinkel,N.,Schmidt,B.,Walter,H.,Kaiser,R.,Lengauer,T.,Hoffmann,D.,Korn,K.,Selbig,J.DiversityandcomplexityofHIV‑1drugresistance:abioinformaticsapproachtopredictingphenotypefromgenotype.2002.ProceedingsoftheNationalAcademyofSciencesoftheUSA;99:8271‑8276.

3.Rhee,S.Y.,Taylor,J.,Wadhera,G.,Ben‑Hur,A.,Brutlag,D.L.,Shafer,R.W.Genotypicpredictors of human immunodeficiency virus type 1 drug resistance. 2006.ProceedingsoftheNationalAcademyofSciencesoftheUSA;103:17355‑17360.

4.Saigo,H.,Uno,T.,Tsuda,K.MiningcomplexgenotypicfeaturesforpredictingHIV‑1drugresistance.2007.Bioinformatics;15:2455‑2462.

5.TorresiJ,Earnest‑SilveiraL,DeliyannisG,EdgttonKZhuangH,LocarniniS,FyfeJ,SozziT,JacksonDC.ReducedantigenicityofthehepatitisBvirusHBsAgproteinarisingasaconsequenceofsequencechangesintheoverlappingpolymerasegenethatareselectedbylamivudinetherapy.2002.Virology;293:305‑13.

6.ShawT.,Bartholomeusz,A.,Locarnini,S.HBVdrugresistance:mechanisms,detectionandinterpretation.2006.JournalofHepatology;44:593‑606.

7. Locarnini. S. and Mason.W. S. Cellular and virological mechanisms of HBV drugresistance.2006.JournalofHepatology;44:422‑431.

L7 - HEPATITIS B AND C VIRUSES GENOTYPES AND DRUG RESISTANCE RELATED MUTATIONS

IN BRAZIL

Dr.JoãoRenatoRebelloPinho

HepatitisBandCviruses(HBV/HCV)arethetwomajorvirusesinvolved inchronichepatitiscasesworldwide.These twovirusesarerelatedtoagreatmorbidityandmortality,astheyarefoundinalmostallregionsoftheworldandareassociatednotonlytochronichepatitisbut also to severe liver diseases such as cirrhosis and hepatocellularcarcinoma.TreatmentofHBV/HCV infectionsconsists in theuseofimmunomodulatory and antiviral compounds. Interferon α (IFNα) isconsidered the first option for treatment of both infections and theefficacyof this treatmenthas largely improvedafter the introductionofpegylatedinterferons(PEG‑IFN),leadingtomorestableplasmaticinterferon levels after weekly injections. HCV treatment is currentlycarriedoutusingconjointlyPEG‑IFNandRibavirin,butforHBVtherearecurrentlyseveralnucleos(t)ideanalogs(NA)withspecificantiviraleffectthatareagoodoptionfortreatmentinsomecases.HBVgenotypesmaybeusedtoindicatewhichkindoftreatmentshouldbeusedineachpatient:genotypesAandBareconsideredindicationstostarttreatmentwithIFNαwhilepatientsinfectedwithgenotypesCandDaretreatedwithNAbysomeauthors.Furthermore,methodologiesthatcandeterminethepresenceofmutationsrelatedtoantiviralresistancearenowavailable.Thesemethodologieshavebeenusedtoidentifythepresenceofdrugrelatedresistancemutationsandmaybeusedasaroutinemethodology

during(orevenpreviouslytostart)treatmentassuggestedforHIVasthefindingofsomemutationsmayindicatewhichdrugwillbethebestoptionforeachpatient.ForHCV,genotypes2and3infectedcasescanbetreatedforshorterperiodsthanthoseinfectedwithgenotypes1and4.ThedevelopmentofspecifictargetedantiviraltherapiesforHCV(STAT‑C)willaddnewoptionsforthetreatmentofthisdiseasebutitisalreadyknownthatNAresistancemutationswillbeveryfrequentinHCVandthatsomeresistantvirusmightnaturallyoccurastheirsitemightoverlapwithHLArecognitionones.InBrazil,HBVgenotypesA,DandFandHCVgenotypes1and3arethemostfrequentones.Thegeographicaldifferences in the distribution of the genotypes of these two virusesinBrazilwillbediscussed,aswellastheuseofmolecularbiologicalmethodstodetectHBVdrugresistantmutationsinalargehospitalatSãoPaulocity,wherelamivudineresistanceisfoundinmorethan30%oftheanalyzedpatientsandsomepatientsalreadyshownresistancetoentecavir,adefovirandpartialresistancetotenofovir.

L8 - SOCIAL NETWORKS AND THE EPIDEMIOLOGY OF HEPATITIS C VIRUS IN

BRAZIL

Dr.PaoloMarinhodeAndradeZanotto

Hepatitis C virus (HCV) infects 170 million people worldwide,themajoritychronically.ChronicallyinfectedindividualsareboththesourceoftransmissiontoothersandareatriskforHCV‑relateddiseases,suchaslivercirrhosisandlivercancer.Beforetheadoptionofanti‑HCVcontrolmeasuresinbloodbanks,thisviruswasmainlytransmittedviablood transfusion.Today, however, needle sharing among injectingdrugusersisthemostcommonformofviraltransmission.OfparticularimportanceisthatHCVprevalenceisgrowinginnon‑riskgroups;thatis, in people who have never used injecting drugs or received bloodproducts.SincethereiscurrentlynovaccineagainstHCV,itisessentialtodeterminethefactorsthatcontrolviraltransmissionsoastodevelopmoreefficientcontrolmeasures.Here,wesequencedpartialNS5bgenesequencessampledfrombloodsamplescollectedfrom591patientsinSãoPaulostate(Brazil).CombiningthesewithepidemiologicaldatafromthesamepatientsrevealthatdifferentsubtypesofHCVhavedifferenttransmissiondynamics.Inparticular,subtypes1aand3aenteredSãoPaulomorerecentlyandaregrowingfasterthansubtype1b.WealsofoundacorrelationbetweenthedateofthevirusentryintoSãoPaulo,theageoftheinfectedpatient,andthesocialnetworkofthepatients,sothatsocialcomponentsplayakeyroleindeterminingtherateandpatternofHCVspread.

L9 - ADAPTATIVE IMMUNE RESPONSES IN ACUTE AND CHRONIC HEPATITIS C VIRUS

INFECTION

Dr.ChristopherWalker

ThehepatitisCvirusisamongthemostsuccessfulofallpersistenthumanviruses.WithacompactRNAgenomethoughttoencodeonly11proteins,HCVpersistsinupto70%ofthoseinfectedbysuccessfullyundermining virus‑specific immunity while leaving host defences to


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otherinfectiousagentsintact.Anestimated170millionindividualsareinfected worldwide, and approximately 38,000 new infections occurannuallyintheUnitedStatesalone.TheoutcomeofHCVinfectionisdeterminedwithinsixmonthsofexposuretothevirus.Acuteinfectionisoftenunrecognizedbecausesymptomsareusuallymildorabsent.InitialviewsofimmunitytoHCVwerethereforelargelyshapedbystudiesofchronicallyinfectedindividuals.RecentprospectivestudiesofhumansathighriskofHCVexposureandofexperimentallyinfectedchimpanzeeshaveprovidedamorecompletepictureofadaptiveimmunitytothevirus.In particular, control of acute primary viral replication is associatedwith expansion of antiviral CD4+ (helper) and CD8+ (cytotoxic)Tcells. Moreover, immunological memory conferred by spontaneousresolutionofacutehepatitisCdoesnotprotectagainstreinfection,butdoessubstantiallyreducetheriskofpersistenceuponre‑exposure.HereweoutlinefeaturesofsuccessfuladaptiveimmuneresponsestoHCVand review current concepts of evasion strategies that might explaindefects in humoral and cellular immunity in those individuals whodeveloppersistent infections.ThehepatitisCvirus(HCV)persists inthemajorityofinfectedindividualsandisasignificantcauseofhumanillnessanddeathglobally.RecentstudieshaveyieldedimportantinsightsintoimmunitytoHCV,inparticularrevealingthecentralroleofTcellsinviralcontrolandclearance.Otherkeyfeaturesofadaptiveimmuneresponsesremainobscure,includingmechanismsbywhichTcellscontrolHCVreplication,theroleofantibodiesinconferringprotectionandhowcellularandhumoralimmunityaresubvertedinpersistentinfection.

L10 - METHODS APPLIED IN ASSOCIATION STUDIES AMONG IMMUNOGENETICS

MARKERS AND VIRAL DISEASES

Dr.JasonBarbour

Wewillreviewthestepsinassemblingandperforminggenome‑wideassociationstudiesof infectiousdiseasesusceptibility (encompassingHepatitisvirusesandotherviruses)anddiseaseseverity,withexamplesdrawnfromrecentlypublishedstudies.Earlystepsincludeidentificationofastrongandclearlyoutlinedcase‑ordiseasestate(suchasHCVinfection state, or a measure of disease severity) ‑ definition andquantification,andtheidentificationofappropriatecase(orstudy)andcontrol populations. Based on the case definition, and knowledge ofvariationinstudymeasuressuchaslaboratorydiagnosticsandthegenetic/genomictechnologytobeemployed,statisticalpowercalculationsareperformed to project the required cohort size for a study capable ofrenderingananswertotheresearchquestion.Methodsforprojectingstatistical power will be discussed, including commonly employedcomputationalsolutions.Thechoiceofgenetic/genomictechnologyisdrivenbymultipleconsiderationsincludingbalancingcostversusthesizeofthecohort,thepreviousidentificationofcandidategeneregions,andthenatureofthesamplesavailable.InthislecturewewillfocusontheuseoftargettedcandidategeneandgenomewideapproachesbasedonSNP(singlenucleotidepolymorphism)arraysbasedontheHapMapproject, and appropriate for high throughput study.We will discusstheadvantagesofSNPbasedstudies,howtheywillcompetewiththeemergenceof‘454’orpyro‑sequencingofDNA,thecostandfeasibilityofcompetegenomere‑sequencingandhowthefieldmayevolveoverthenextdecade.Wewilldiscussthecommontoolsandapproachesin

thecomputationalprocessingofgeneticmarkerdata,anditsanalysistoincludedataquality,theapplicationofstandardtestsofdistribution,andHardy‑Weinbergequilibirum.Wewilldiscussthevalueandneedtoaddresspopulationstratification(admixture)ingenomewidestudies,andtoaccountforgeneticvariationintheoriginatingpathogen,suchasHCVorHBV.Finallywewilldiscussrobustdiscoverymethodsencompassingtraditional pairwise comparison methods, approaches to the multiplecomparisons problem, the application of FDR (false discovery rate),alternative discovery methods such as recursive partitioning, itsextensions,andstatisticalvalidationofresults.

L11 - EVOLUTIONARY GENETICS OF GENES THAT CONTROL THE IMMUNE RESPONSE

Dr.DiogoMeyer

Genes that control adaptive and innate immunity are constantlyunder natural selection, since the pathogens that they respond toare themselves evolving strategies to evade the immune response.In this presentation, I will review the methods that detect selectionat these immune function genes, emphasizing that different typesof data (e.g., polymorphisms or sequence divergence amongspecies)canbeusedtostudyselection,andthattheinferencesmaderefertodifferenttimedepths(recentorancientselection).Byanalyzinggenesinvolvedinadaptiveimmunity,Iwillarguethatselectionisfrequentandintense, and periodically introduces profound changes in the geneticconstitutionofthepopulations,bothattheimmunelociandinregionsphysicallylinkedtothem.

L12 - ORIGINS OF GB VIRUS C DETERMINED BY COMPLETE GENOME ANALYSIS

Dr.A.ScottMuerhoff

GB virus C (GBV‑C), a positive‑strand RNA virus, currentlyinfects approximately one‑sixth of the world’s population. Thisvirus exists as a collection of genotypes whose global distributioncorrelateswithgeographicalorigin.GenotypingofGBV‑Cisolatesbyphylogeneticanalysishasreliedupontheuseof5’‑untranslatedregion(5’‑UTR)sequences,however,completegenomesequencesareusedtodemonstratedefinitively their existence andgeographical correlation.InitialidentificationofthefifthgenotypefromSouthAfricawasbaseduponphylogeneticanalysisofthe5’‑UTR.Itwassoughttoconfirmthisclassificationbyanalysisof full‑lengthE2genes fromSouthAfricanisolatesandbyanalysisofacompletegenotype5genome.Analysisoffull‑lengthE2genesfrom28GBV‑C‑infectedSouthAfricanindividualsrevealedtheexistenceofauniquegroupof18isolates,distinctfromtheotherfourgenotypes.Bootstrapanalysisprovidedstrongsupport(95%)forthisfifthgroup.Theremainingisolateswereeithergenotype1(n=8)or2(n=2).AnalysisofhumanE2genesequences,withtheE2genefromthechimpanzeevariantGBV‑Ctroincludedastheoutgroup,producedatreerootedonthegenotype1branch.Thecompletegenomenucleotidesequence of SouthAfrican genotype 5 isolate D50 was determined.Phylogeneticanalysisofthe5’‑UTRandopenreadingframeproducedcongruenttreesthatgroupedthesequencesintofivemajorgenotypes.


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InclusionofthecorrespondingregionofthechimpanzeeisolateGBV‑Ctrointheanalysisproducedtreesrootedonthebranchleadingtothegenotype5isolateD50,suggestinganancientAfricanoriginofGBV‑C.

L13 - HUMAN MIGRATIONS AND HBV ORIGINS IN SOUTH AMERICA

Dr.NelsonJurandiRosaFagundes

Evolutionarystudiesoftenusegeneticdatacollectedinonespeciestoinferpasteventsandprocessesaffectinggeneticvariationinthissamespecies.However,theco‑evolutionofhostsandparasitesrepresentaninterestingcasewherethepasthistoriceventsofonespecieswillalsoaffectgeneticvariationin theotherspecies.HepatitisBvirus(HBV)presentsaremarkablegeographicstructureinhumanpopulations,andunderstandingthegeneticvariationofcurrentviralstrainsmaybeusefultoshedlightinpastdynamicsofhumanpopulations.Inthispresentation,Iwill reviewthegeographicstructureofHBVanddiscusshowsuchdiversity correlates with human demographic history, especially intheAmericas,rangingfromtheearlysettlingoftheNewWorldinthePleistoceneuntilthemuchmorerecenteventsofadmixtureamongNativeAmericans,EuropeansandAfricanssince theColonialperiod.WhilethegeographicstructureshownbytheseveralHBVgenotypesmayberelatedtoveryoldprocessesinhumanevolution,thefastevolutionaryrateinferredforHBVsuggeststhattheHBVspreadinhumanpopulationsisrecent.IwilldiscusshowevolutionaryratesestimatedforpedigreesmayunderestimatethetimeofthemostbasalsplitsinHBVphylogenies.RecentstudiesonthetempoandmodeofevolutionforhumanmtDNAmaybeparticularlyusefultounderstandthisapparentcontradiction,andabetterunderstandingofhownaturalselectionaffectsHBVsequenceevolutionwillbefundamentaltosolvethisissue.

L14 - THE PHYLOGEOGRAPHY OF HEPATITIS B VIRUS IN AFRICA

Dr.AnnaKramvis

An estimated 65 of the world’s 360 million chronic carriers ofhepatitisBvirus(HBV)resideinAfrica.GenotypesA,DandEofHBVcirculate inAfrica and show a distinct geographical distribution thatmaytrackhumanmigrationsandinterventions.ThemajorityofAfricangenotypeA isolates characterizedbelong to subgenotypeA1andarefoundmainlyinthesouthernandeasternregionsofAfrica,includingSouthAfrica,Zimbabwe,Malawi,Tanzania,Uganda, theCongoandSomalia.All the isolates except those fromSomalia cluster together.Somalian isolatesclusterwithAsian isolates, leading to theproposalthatthissubgenotypewasintroducedintoAsiaasaconsequenceoftradeandtravelalongtheeasterncoastofAfricabyAsians.SubgenotypeA1hasalsobeenisolatedinBraziliansofAfricandescent,areflectionofthe17th‑19thcenturytransantlanticslavetrade.SubgenotypeA2orthe“European”subgenotypeofAhasbeenisolatedfromSouthAfricans.IthasbeensuggestedthatthissubgenotypeoriginatedinsouthernAfricaandwasintroducedtoEuropebyPortuguesesailorswhotravelledtoAfricainthe15thcentury.SubgenotypeA3hasbeenisolatedfromtheCameron, Mali, Gabon, Nigeria and the Gambia on the west coast.

AfricangenotypeAisolatesaremorediversethanthosefoundintherestoftheworldsuggestingalongendemicityofthisgenotypeinAfrica.Genotype D isolates from Egyptians and SouthAfricans belong tosubgenotypeD1andD3,respectively.SubgenotypeD1prevailsintheMiddleEastwhereassubgenotypeD3inEuropeandUSA.GenotypeEisthemostprevalentgenotypeofwesternandcentralAfrica,coveringa large expanse from Mali to Namibia. Genotype E is anAfricangenotype.MostgenotypeEstrains,sporadicallyisolatedoutsideAfrica,originatefromcarriersofAfricandescent,regardlessoftheircountryofresidence.TheshallowgeneticdiversityofthisgenotypeanditsrarityintheAmericasissuggestiveofarecentintroductionintotheAfricanpopulation,withan iatrogenicmeansof transmissionpostulated.TheidentificationofthisgenotypeinancientandisolatedpopulationgroupsinAfrica,andinnon‑humanprimates,implicatesthemaspossiblenaturalreservoirsoftheancestorofthisgenotype.FurtherandmoreextensivestudiesofHBVisolatesfromAfricaarethereforenecessaryandmaybeimportant inrevealingtheoriginofHBVandclarifyingthemanyquestionsaboutitsevolution.

L15 - MOLECULAR EPIDEMIOLOGY OF HEPATITIS B VIRUS IN AFRO-BRAZILIAN

POPULATIONS

Dr.AnaRitaCoimbraMottadeCastro

HepatitisBvirus(HBV)infectionisanimportantproblemofpublichealth throughout theworld.In thisstudy,aseroepidemiologicalandmolecularinvestigationofHBVinfectionwascarriedoutin12Afro‑descendantscommunitiesinCentralBrazil,whichhavebeenmaintainedassmallisolatedcommunitiessinceslaveryperiod.Serumsamplesfrom1058African‑descendantswerescreenedforthepresenceofhepatitisBserologicalmarkers.TheoverallprevalenceofHBVinfectionwas19.8%(95%CI:17.5‑22.3),withratesvaryingfrom5.5%(BoaSorte/BS community) to 42.4% (Furnas dos Dionísios/FD community).Multivariateanalysisofriskfactorsshowedthatincreasingage,familyhistory of hepatitis, and sexual activity were associated significantlywiththisinfection.OccultHBVinfectionwasinvestigatedinFDandBS communities, with high and low endemicities, respectively.AhigherrateofoccultHBVinfection(17.9%),associatedtolowHBVloads(mean2.2x104copies/mL),wasobservedinFDcommunitywhencomparedwithBS(2.4%).HBVisolatesofFDcommunitywerefurthercharacterized.RFLPandphylogeneticanalysisofpreS/SregionofHBVisolatesshowedthatthemajorityofthemwereidentifiedasgenotypeA, subtypeAa (HBV/Aa‑FD), suggestinganAfricanorigin for theseisolates.PairwisegeneticdistancesanalysiswasperformedincludingothergenBanksequences.Asexpected,the28HBV/Aa‑FDisolateshadsequencevariation significantly lowerwhencompared to thegeneticdistanceswithin18HBV/AaisolatesfromotherBrazilianregions(1.7± 0.3% vs. 2.5 ± 0.4%). However, the sequence variation withinAaisolatesfromFDcommunitywassimilartothatfoundwithinAesubgroup(1.5±0.4%).ThesefindingssuggestthatHBVwasintroducedinthecommunitybydifferentsourcesofAaisolates,mostofthemprobablyduringtheslavetradefromAfricatoBrazil.Inaddition,demonstrationof close genomic relatedness of HBV/Aa‑FD isolates was the mostconvincingevidenceforhorizontal(intraandinterfamilial)transmissionamong theseAfro‑descendants. Several substitutions (as C100Y in


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smallSproteinandW501Rinpolymerase)wereobservedamongHBVisolatesbyanalyzingtheaminoacidsequencesofsurfaceandpartialpolymerase regions. Sequencing of precore/core promoter region ofHBsAg‑positive isolatesshowed thatseveralmutationscouldexplaintheanti‑HBephenotype,whichwaspredominantamongthem.Finally,inordertoevaluatethecompliancewithandtheresponsetohepatitisBvaccineintheseAfro‑descendants,708subjectsfromeightcommunitieswerevaccinated.Although567(80%)individualscomplainedwiththefirstdose,only198(28%)receivedthefullscheme.Of148subjectswhoagreedtotesttoanti‑HBs,123(83.1%)respondedtothevaccinewithageometricmeantiterof512mUI/mL.Malesexandolderagewereindependently associated with non‑response.These results reinforcethat additional health education programs and alternative hepatitis Bvaccineschedulesareneeded to improve thevaccinationcoverage inAfro‑descendantscommunitiesinCentralBrazil.

L16 - INNATE AND ACQUIRED IMMUNE RESPONSE MARKERS AND VIRAL INFECTIONS

Dr.MaryCarrington

Human leukocyte antigen (HLA) class I loci are essential toan effective immune response against a wide variety of pathogenicmicroorganisms, and they represent the prototypes for geneticpolymorphism that are sustained through balancing selection.ThefunctionalsignificanceofHLAclassIvariationisbetterexemplifiedbystudiesinvolvingHIVtype1(HIV‑1)thananyotherinfectiousorganism.HLAclassImoleculesareessentialtotheacquiredimmuneresponse,buttheyarealsoimportantininnateimmunityasligandsforthekillercellimmunoglobulin‑likereceptors(KIR),whichmodulatenaturalkillercellactivity.HereweconcentrateontheinteractionbetweentheHLA‑BandKIR3DL1/KIR3DS1genes,describetheeffectsoftheselocionHIVdisease,anddiscussquestionsthatremainunresolved

L17 - STUDIES ON THE ALLELIC FREQUENCIES AND HAPLOTYPES FOR IMMUNOGENETIC

MARKERS

Dr.RichardSingle

Theclassicalgenesofthehumanmajorhistocompatibilitycomplex(MHC)andthegenesofthekillerimmunoglobulin‑likereceptor(KIR)systemplaycriticalrolesintheadaptiveandinnateimmuneresponses.There are several well established disease associations of KIR andhuman leukocyte antigen (HLA)alleles andhaplotypeswith specificdiseases.InthistalkIwillprovideanoverviewofgeneandhaplotypefrequency estimation for immunogenetic data, pointing out crucialdifferencesbetweenapplicationsforHLAandKIRgenes,summarizefrequencypatternsforHLAandKIRgenesinglobalandSouthAmericanpopulations,anddiscusscorrelationsamongfrequenciesforthesetwogenetic systems and analysis methods based upon them.Allele andhaplotypefrequencyresultswillbepresentedwithparticularemphasisonHLAandKIRgenesandhaplotypesthatareknowntoimpactdiseaseprogressionandclearanceforhepatitis(e.g.,HLA‑A,‑C,‑DRB1,‑DQB1,KIR2DL2,andKIR2DL3).Resultswillbesummarizedatseverallevels.

Global frequencies, based on a recent meta‑analysis of HLA data in497populations,willbefollowedbyresultsforNativeAmericanandadmixedMestizopopulationsfromSouthAmericaalongwithresultsfromBrazilianbonemarrowdonorregistries.ParticularchallengesforfrequencyestimationinthesedifferentclassesofdatasetswillbediscussedandexampleswillbepresentedusingthePyPop(PythonforPopulationGenomics ‑www.pypop.org) software framework alongwith sampledata files and analysis scripts. In many instances for immunogeneticdatathereisahierarchyofgeneticeffects,rangingfrompredisposingthroughneutraltoprotective,withconsistentpatternsacrossregional/ethnicgroupsseeninassociationstudies.However,casesinwhichthisconsistency of results across ethnic groups is not present have beenreportedforHLAassociationswithHCVinfection.Thus,differencesinthefrequencydistributionsofimmune‑relatedgenesbasedongeographicorethnicbackgroundareimportanttoconsidersinceassociationsmaybe impactedby thesedifferences.Additionally,evenwhenconsistenteffectsareseenacrossgroups,theseregional/ethnicdifferencesinallelefrequencyspectraareimportanttoconsiderinthedesignandanalysisof studieswhenpatientsorcontrolsarecombinedacrossgroupsduetotheimpactofpopulationstratificationonstudyresults.InthistalkIwillpresentmethodsthatallowarankingoftherelativecontributionsofdifferentalleles/haplotypestodiseaseassociation(e.g.,analysisofrelativepredispositionaleffects(RPE)).TheRPEmethodisasequentialprocedurefortestingheterogeneitybetweenallele/haplotypespectraforpatientsandcontrols.ThereissubstantialvariationacrossethnicgroupsinfrequenciesforHLAandKIRallelesandhaplotypes.Variouslinesofevidenceindicatethatthesegenesexperiencebalancingselection(atboththealleleandaminoacidlevel),resultinginmoreevenfrequencydistributions than expected under neutral evolution.The ability ofindividualswhoareheterozygousforcertainimmune‑relatedgenestosuccessfullyrespondtoabroaderrangeofpathogensisbelievedtoplayan important role in shaping these frequency distributions.The highlevelofpolymorphismforHLAgenesisdueprimarilytovariationatfunctionally important amino acid sites. Different HLA alleles sharecombinationsofvariableaminoacidsitesandthesesharedepitopesmayprovidemoreinformationabouttheunderlyinggeneticcausesofdisease.InthistalkIwillpresentrecentcollaborativeworkaddressingthisissueforassociationtestsbasedongenesandproteinsthatarestratifiedintobiologicallyrelevantsequencefeatures(SFs).ThevariantsfortheseSFsdefinestructural(e.g.,beta‑strand1)andfunctional(e.g.,peptidebindingsite)featuresofproteins.ThegoalofthesenewSFvarianttypeanalysesistoenhancethesynthesisofinformationacrossdifferentdiseaseandpopulationstudiesatthelevelofalleles,haplotypes,andaminoacids

L18 - LINKAGE-DESEQUILIBRIUM MAP OF THE HUMAN MAJOR HISTOCOMPATIBILITY COMPLEX AND FIRST GENERATION OF TAG

SINGLE-NUCLEOTIDE POLYMORPHIMS

Dr.MarcosMiretti

Autoimmune,inflammatory,andinfectiousdiseasespresentamajorburdentohumanhealthandarefrequentlyassociatedwithlociinthehuman major histocompatibility complex (MHC). Here, we report ahigh‑resolution (1.9 kb) linkage‑disequilibrium (LD) map of a 4.46‑Mb fragment containing the MHC in U.S. pedigrees with northern


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and western European ancestry collected by the Centre d’Etude duPolymorphismeHumain(CEPH)andthefirstgenerationofhaplotypetag single‑nucleotide polymorphisms (tagSNPs) that provide up to afivefold increase in genotyping efficiency for all future MHC‑linkeddisease‑association studies.The data confirm previously identifiedrecombinationhotspotsintheclassIIregionandallowthepredictionofnumerousnovelhotspotsintheclassIandclassIIIregions.TheregionoflongestLDmapsoutsidetheclassicMHCtotheextendedclassIregionspanningtheMHC‑linkedolfactory‑receptorgenecluster.Theextended

haplotypehomozygosityanalysisforrecentpositiveselectionshowsthatall14outlyinghaplotypevariantsmaptoasingleextendedhaplotype,whichmostcommonlybearsHLA‑DRB1*1501.TheSNPdata,haplotypeblocks, and tagSNPsanalysis reportedherehavebeenentered intoamultidimensionalWeb‑baseddatabase(GLOVAR),wheretheycanbeaccessedandviewedinthecontextofrelevantgenomeannotation.ThisLDmapallowedustogivecoordinatesfortheextremelyvariableLDstructureunderlyingtheMHC.

P1 - CORRELATIONS BETWEEN HBV GENOTYPES AND BASAL CORE PROMOTER

AND PRECORE/CORE MUTATIONS AND THEIR CLINICAL IMPLICATIONS

FláviaMiryanMartinsAlmeida‑Mello1,AlineSatieObaKuniyoshi2,AndréFanhani

Lopes3,MicheleSoaresGomes‑Gouvêa4,DennisArmandoBertolini5.

1MestreemCiênciasdaSaúde.LaboratóriodeRetrovirologia.DisciplinadeInfectologia‑DepartamentodeMedicina.UniversidadeFederaldeSãoPaulo,SãoPauloSPBrasil.2MédicaGastroenterologistadoConsórcioIntermunicipaldeSaúdedoSetentriãoParanaense,MaringáPR,Brasil.3AcadêmicodoCursodeMedicina,UniversidadeEstadualdeMaringá,MaringáPR,Brasil.4LaboratóriodeGastroenterologiaTropical,InstitutodeMedicinaTropical,FaculdadedeMedicina,UniversidadedeSãoPaulo,SãoPauloSPBrasil.5Laboratório de Imunologia Clínica, Departamento deAnálises Clínicas, UniversidadeEstadualdeMaringá,MaringáPRBrasil.

StudieshavedemonstratedacorrelationbetweenhepatitisBvirusgenotypesandmutationsinthebasalcorepromoter(BCP)andprecore/core region.Thus, the objective of this cross‑sectional study was toinvestigate the molecular characteristics of HBV in chronic carriers,identifyinggenotypesprevalentintheregionandthepresenceofBCPandprecore/coremutations,todeterminetheprevalenceofHBeAgandtoevaluatetherelationshipofthesefactorswithclinicalandvirologicalaspects.TheamplificationproductsoftheS‑polymerasegene,BCPandprecore/core region from54 chronicallyHBV infectedpatientsweresequenced and analyzed. Phylogenetic analysis of the S‑polymerasegenesequencesshowedthat66.7%(36/54)ofthepatientswereinfectedwithgenotypeD(D1,D2,D3),25.9%(14/54)withgenotypeA(A1,A2),5.6%(3/54)withsubgenotypeC2,and1.8%(1/54)withE.Wasfoundaprevalenceof14.8%ofpatientsHBeAgpositive.Comparisonof virological characteristics showed significant differences betweengenotypesA,CandD,intermsofthepresenceofmutationsG1896A,C1858TandG1862T(P<0.001).ComparisonbetweenHBeAgstatusand the G1896A stop codon mutation in patients with genotype Drevealedthatmostofthemwereanti‑HBepositive(P=0.001),afindingdemonstratingtheexistenceofarelationshipofHBVG1896AprecoremutantsandgenotypeDwith theprocessofHBeAgseroconversion.Analysisofthemutationsshowedawell‑defineddistributionaccording

POSTER PRESENTATIONS

togenotype.Asignificantdifference(P=0.030)wasalsoobservedwhencomparingHBeAgstatusandALT,withelevatedALTlevelsbeingmorefrequentinHBeAg‑positivepatients.ThedetectionofHBeAgpositivepatients, genotyping and identification of mutant BCP and precore/coreareimportanttoolsandessentialfortheprognosisandtreatmentofpatientswithchronicHBV,sincetheyarefoundinassociationwithincreasedriskforthedevelopmentofhepatocellularcarcinoma.Keywords: HBV, Genotypes, Subgenotypes, Basal Core PromoterMutations,Precore/CoreMutations.

P2 - MOLECULAR CHARACTERIZATION OF THE HEPATITIS B VIRUS: GENOTYPING,

SUBGENOTYPING AND DRUG RESISTANCE ASSESSMENT IN A SINGLE SEQUENCE

REACTION

RobertaSitnik,RúbiaAnitaFerrazSantana,OziresSantosRamos,LetíciaOyakawa,

GregórioTadeuFernandoDastoli,RobertaCardosoPetroni,VanessaFuscoDuartede

Castro,CristóvãoLuisPitangueiraMangueira&JoãoRenatoRebelloPinho.

ClinicalPathologyDepartment,HospitalAlbertEinstein,SãoPaulo,SP,[email protected]

HepatitisBvirus(HBV)isamajorcauseofchronicliverdiseaseworldwide.This virus has been classified into eight genotypes (A‑H) that show distinct geographic distribution worldwide. MolecularcharacterizationofHBVisauseful tool in themanagementofHBVinfected patients and there are few data about genotypes and drugresistance mutations present in our country. To access these data,we have developed a method allowing identifying not only HBVsubgenotypes but also drug resistance mutations to major drugs inonlyonesequencereaction.PrimerswereselectedinordertoamplifyaregionofHBVthatcandistinguishallHBVgenotypesandsubtypesandthatcontainsallimportantmutationsfordrugresistanceassessmentinthepolymerasegeneandresistancetoHBIg,anti‑HBsmonoclonalantibody,orvaccinationintheHBsAgcodingregion.HBVDNAwasextractedwithQIAampDNAMiniKit(QIAGEN,Hilden,Germany)andsubjectedtoanestedPCR.PCRproductswerefurthersequenced


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usingtheABIPrismBigDyeterminatorcyclesequencingkit(AppliedBiosystems,FosterCity,CA,USA).Afterpurification,samplesweredenaturedandloadedinanautomatedABI3130DNAsequencer(AppliedBiosystems).SequenceanalyseswereperformedwithSeqScapeSoftwarev2.5(AppliedBiosystems),bycomparisonwithadatabasecontainingsequencesfromdifferentHBVgenotypesandsubgenotypespreviouslydescribed.Furthermore,specificaminoacidmutationsrelevantfordrugresistanceandHBsAgimmunerecognitionwerereported.TheuseofthisnewsystemallowsimprovingtheinformationobtainedinonlyonePCRproduct.Wehaveanalyzed312patientsfromMarch2006toNovember2009.Allknowngenotypeswerefound,withexceptionofgenotypeH.GenotypeAwasthemostfrequent(43%;A1–87%,A2–13%),followedbygenotypesD(29%;D1–2%,D2–17%,D3–70%,D4–11%);F(14%;F1–7%,F2–70%,F3–10%,F4–13%);C(14%‑allC2);G(2%);B(1%‑allB2)andE(1%).Thenumberofpatientswithoutanyknowndrugresistancemutationschangedfrom70.3%in2006to67.5%in2007,to50%in2008,andin2009to56.5%.ThemostfrequentlyfoundmutationswererelatedtoLamivudine(LMV)resistance,whichwaspresent in18.9%of thepatients in2006and in36.6% in2009.Concluding,wehavefoundsubgenotypesA1,D3andF2asthemorefrequentonesinthispopulation,asdescribedbeforebyotherauthors.WealsoshowedthatinalargecountrysuchasBrazilevenraregenotypesinSouthAmericacanbefound,suchasgenotypesE(3patients)andG(6patients).Ourresultsalsoshowedthatthereareanincreasingnumberofpatientsresistanttomanydrugs,especiallytoLMVthathasalreadybeenusedformanyyears.ThesedatareinforcestheimportanceofthemolecularcharacterizationofHBVinthetreatmentofinfectedpatients.

P3 - MOLECULAR CHARACTERIZATION, DISTRIBUTION AND DYNAMICS OF HEPATITIS C VIRUS (HCV) GENOTYPES IN BLOOD DONORS

FROM COLOMBIA

MónicaVivianaAlvaradoMora,1*CamilaMaltaRomano2,MicheleSoaresGomes‑

Gouvêa,1MariaFernandaGutiérrez,3FlairJoséCarrilho,1JoãoRenatoRebelloPinho.1

1.LaboratoryofGastroenterologyandHepatology,SãoPauloInstituteofTropicalMedicineandDepartmentofGastroenterology,SchoolofMedicine,UniversityofSãoPaulo,Brazil.2.LaboratoryofVirology,SãoPauloInstituteofTropicalMedicine,DepartmentofInfectiousandParasiticDiseases,SchoolofMedicine,UniversityofSãoPaulo,Brazil.3.LaboratoryofVirology,DepartmentofMicrobiology,PontificiaJaverianaUniversityBogotá,Colombia.

HepatitisCvirus(HCV)isafrequentcauseofacuteandchronichepatitisandaleadingcauseforlivercirrhosisandhepatocarcinoma.HCVisclassifiedinsixmajorgenotypesandmorethan70subtypes.InColombianBloodBanks,serumsamplesweretestedforanti‑HCVantibodiesusing thirdgenerationELISA tests.Theaimof this studywastocharacterizetheviralsequencesinplasmaof184volunteerblooddonorswhopresentedthemselvestothe“Banco Nacional de Sangre de la Cruz Roja Colombiana”,Bogotá,Colombia.ThreedifferentHCVgenomicregionswereamplifiedbyNestedPCR:1)asegmentof180bpofthe5´UTRregiontoconfirmthepreviousdiagnosticwithELISAtest;2)Fromthosewhichwerepositivein5UTRregion,asegmentof380bpofNS5Bregionandasegmentof391bpofE1regionwereamplifiedforgenotypingandsubtypingbyphylogeneticanalysis.ThedistributionofHCVsubtypesfoundwas:1b(82.8%),1a(5.7%),2a(5.7%),2b(2.8%)

and3a (2.8%).By applyingMonteCarloMarkov simulation, itwasestimatedthatHCV1bwasintroducedinBogotáaround1950.Also,thissubtypespreadatanexponentialratebetween~1970and~1990,afterthattransmissionwasapparentlycontrolledbytheELISAAnti‑HCVtestinthispopulation.Inconclusion,intheColombianblooddonorsgroup,genotype1bisthemostfrequentespeciallyinlargeurbanconglomeratessuchasBogotá,asinmanyotherSouthAmericancountries.

Financialsupport:FAPESP07/53457‑7and08/50461‑6

P4 - VARIABILITY OF HBV REVERSE TRANSCRIPTASE SEQUENCE IN VIRAL

ISOLATES FROM TREATMENT NAIVE CHRONIC HEPATITIS B PATIENTS FROM BRAZIL

Gomes‑Gouvêa,MS1;Mendes‑Corrêa,MCJ2,3;Pádua,AP1;Silva,MH4;Seixas,ACS1,5;UipDE2;PinhoJRR1

1Departamento de Gastroenterologia, Laboratório de Gastroenterologia e HepatologiaTropical,InstitutodeMedicinaTropical‑FMUSP,SãoPaulo;2UnidadedeReferênciaemDoençasInfecciosasePreveníveisdaFaculdadedeMedicinadaFundaçãodoABC–SantoAndré,SP;3HospitaldasClínicasdaFaculdadedeMedicinadaUSP,SãoPaulo;4ClinicadeespecialidadesdeSãoBernardodoCampo,SãoPaulo;5CentrodeReferênciaeTreinamentoDST/AIDSdaSecretariadeEstadodaSaúdedeSãoPaulo.

Background‑ThehighdegreeofhepatitisBquasispeciesdiversityin chronically infected individuals may suggest that viral variantsfavoring resistance to specificnucleos(t)ideanalogues (NA)maypreexistpriortoantiviraltreatments.Theaimofthepresentstudywastodeterminethenaturallyoccurringpreexistingamino‑acidsubstitutionsrelated to resistance to NA in treatment naive patients with chronichepatitisB(CHB).Methods‑Serumsampleswereobtainedfrom93treatmentnaivepatientswithCHBfromSãoPaulostate,Brazil.HBVgenotypeandmutationstatusanalysiswerecarriedoutbyamplifyinganddirectlyandbidirectionallysequencingofthecompleteHBVRT‑domain.Results‑AsexpectedforthisregionofBrazil,genotypesA(51–54.8%),D(30–32.2%)andF(8‑8.6%)arethemostprevalent,butwealsofindgenotypesB(B1–1.0%),C(C2‑1.0%),G(1.0%),andE(1.0%),whicharegenotypesrarelyfoundinBrazil.SubgenotypeA1wasobservedinallpatientsinfectedwithgenotypeA,4subgenotypesofgenotypeDwereobserved(D1‑D4),howeversubgenotypeD3(73.3%)was the more frequent. For genotype F samples, subgenotypes F2a(87.5%)andF4 (12.5%)wereobserved.Amongpatients infectedbygenotypesA,DandF,13.7%,20.0%and12.5%wereHBeAgpositive,respectively. Mutations that have been potentially associated withresistancetoAdefovirwereobservedin6.4%ofthesamplesstudied,includingrtI233V(3.2%),rtN238D(2.0%),rtV214A(1.0%)andrtQ215S(1.0%).MutationswithestablishedandmarkedeffectsinNAtreatmentforHBVhavenotbeenfoundatall.Conclusions‑Inconclusion,thisstudyshowsthatHBVmutationsthathavebeenpreviouslyassociatedwithdrugresistancemaybepresentinuntreatedpatients.Althoughforsomeofthesemutationsin vitroresultshaveshownsomeeffecttotheviruses,theirpresenceinuntreatedpatientsreinforcestheconceptthattheyarenotreallyassociatedtodrugresistance,especiallyiftheyaredrivenforAdefovirresistance,adrugwithalesspronouncedeffectonHBVreplication.


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P5 - STRUCTURAL STUDIES OF NS3 VARIANTS FROM PATIENTS INFECTED WITH HCV

GENOTYPE 3

PaolaJ.S.Provazzi1,HelenAArcuri2,JoãoRenatoR.Pinho3,MárioS.Palma4,Paula

Rahal1

1–SãoPauloStateUniversity‑UNESP,DepartmentofBiology.SãoJosédoRioPreto/SP,Brazil.2–FacultyofMedicine,UniversityofSãoPaulo–USP,DepartmentofMedicalClinic.SãoPaulo/SP,CEP:01246‑903,Brazil.3–FacultyofMedicine,UniversityofSãoPaulo‑USP,DepartmentofGastroenterology.SãoPaulo/SP,Brazil.4–SãoPauloStateUniversity‑UNESP,CenterofStudyofSocialInsects/DepartmentofBiology.RioClaro/SP,Brazil.

TheHepatitisCvirus(HCV)infects130millionpeopleworldwide.Neverthelessmosteffectivetreatmentoptionsarenotyetavailable.Oneofthemostpromisingtargetsforanti‑viraltherapyisthenon‑structuralprotein3(NS3).Inordertoidentifypossiblechangesinthestructureof theNS3protein,amodelwasconstructedforeachproteincodingsequenceof16patientswithHCV.Throughthestudyofstructuralmodelsby using comparative molecular modeling and docking simulation,changesinthehelicasedomainwereidentified.Theanalysisrevealedthesubstitutionofaminoacidlysinetoglutamicacidatposition210(involvedinhydrolyseoftheATP)oftheNS3inpatientsresistantoftreatment.ThemotifsI,I,II,III,IV,VandVIofHelicase,locatedindomain 1 (region which occurs the hydrolysis ofATP) and 2 of theHelicasearetheenginethatconvertsthechemicalenergyderivedfromthehydrolysisofATP.Here,wehighlight the changeof apositivelycharged residue to a negatively charged which can cause changes inthedistributionofchargedandtheinteractionbetweenaminoacidsandthus interferewith thehydrolysisofATP.This leads toan imbalancein thedistributionof charges leading to lossof connectionsbetweentheHelicaseandATPthatmaycauseaslightchangeinthemolecules’orientation.Wefoundtoothattheaminoacidtryptophanwasreplacedbyarginineatposition501ofNS3.Inthiscase,ocurredthesubstitutionofahydrophobicforahydrophilicresidueleadstothelossofhydrophobicityintheregionwherethechangeoccurredandthusalterstheinteractionoftheproteinwiththeenvironment.Thiscanleadtoaslightchangeinconformationofthemolecule.Thedockingsimulationsshowedthatthisreplacementdoesnotpreventthebindingofribavirin.

Financialsupport:Fapesp,CapesandCNPq.

P6 - THE INFLUENCE OF THE GENETIC DIVERSITY OF THE HOST AND HCV IN PATIENTS WITH CHRONIC HEPATITIS C

RESPONSIVE AND NON RESPONSIVE TO TREATMENT

Ramos,J.A.1,2;Vargas,P.S.2;Hoffmann,L.1;Ramos,A.L.3;Villela‑Nogueira,C.A.3;

Ürményi,T.P.1;Silva,R1;Rondinelli,E.1,3.

1LaboratóriodeMetabolismoMacromolecularFirminoTorresdeCastro,InstitutodeBiofísicaCarlosChagasFilhodaUFRJ;2InstituoFederaldeEducaçãoCiênciaeTecnologiadoRiode Janeiro, IFRJ;3DepartamentodeClínicaMédicadaFaculdadedeMedicinadaUFRJ.E‑mail:[email protected]

Hepatitis C is a health problem in Brazil with 3 million peopleinfected.Theevolutionoftheinfectionandtheresponsetotreatment

variesaccordingtoHCVquasispeciesand/orhostgeneticfactors.Theaimofthisworkistoassesstheviralquasispeciesandthepolymorphismsin thecytokinesgenes inpatientswithHCV1a/b in theextremesoftherapeutical response.The RT‑PCR products for the viral genomeregionsE2,HVR1,NS5AandNS5B,weredirectlysequencedtoassessthepredominantquasispeciesandclonedtoobtain the individualizedquasispecies.Theresultsobservedinthepatientsanalyzedhaveshownthat it ispossible to identifyvariantsbythepresenceofoneormorepoint mutations among the clones. In the region E2 we have foundabout15%ofnonsynonymssubstitutionswhichmight influence theresponsetothetreatment.Toassessthepolymorphismsofthecytokinegenes we analyzed genomic DNA of 186 patients with HCV.TheIL‑10 –1082 polymorphism 70,8% patients were genotyped asAA,26,5%asAGand2,7%asGG.TheIL‑4(+33),19,4%hadgenotypeCC,48,4%hadgenotypeCTand32,3%hadgenotypeTT.The+874IFN,13,1%weregenotypeasTT,45,9%asTAand41%asAA.The‑308TNF62,7%weregenotypedasGG,21,5%asGAand15,8%asAA.Thepolymorphismsinthecodon25intheTGFwefoundmostlygenotypeGG(81,5%)inrelationtogenotypeCC(4,1%).Theallelicand genotypes frequencies were compared with a group of healthyindividualsandwe foundsignificantdifferencesbetween thegroups.Ourresultsdemonstratedthathostfactorscaninfluencetheprogressionoftheinfectionandthatdifferencesobservedinthepredominantviralpopulationandindividualizedquasispecieswillbeusedinthisstudyasapredictivefactoroftheresponsetotreatment.

Financialsupport:CNPq/MS;FAPERJ

P7 - CLONING AND EXPRESSION OF A RECOMBINANT MULTIEPITOPE PROTEIN FOR

HEPATITIS C DIAGNOSTIC

Santos,J.C,Galdino,A.S,Queiroz,M.S,Felipe,M.S,Torres,F.A.

LaboratóriodeBiologiaMolecular‑InstitutodeCiênciasBiológicas‑[email protected]:HepatitisC,Multiepitopeprotein,Diagnostic

HepatitisCvirus(HCV)isanimportanthumanpathogenaffecting3%ofthehumanpopulation.Chronicinfectionarefrequentandamajorcauseof livercirrhosisandhepatocellularcarcinoma.Seroprevalencestudies suggest that at least 170 million individuals have infectedworldwide.InBrazil,approximately2millionacutecasesofhepatitisC have been report till data. Several EIA based diagnostic kits areavailableinthemarketforthedetectionofantibodiestoHCVinplasma.However, due to the distinct geographical distribution of all HCVgenotypesworldwide, thesekits donot alwaysperformequallywellindifferentpartsofworld.Nowadays,thesekitsusepeptideantigens(Thirdgenerationkits)orrecombinantantigens(fouthgenerationkits)frombothstructuralandnon‑structuralregionsoftheviralprotein.Therequirementofmultiplepeptidesand/ormultiplerecombinantproteinsforreliablediagnosisofHCVinfectionaddstothecostoftheseEIAkits.Inthisway,wehavedesignedasinglerecombinantmultiepitopeprotein,consistingofseveralepitopesfromstructuralandnon‑structuralregionsoftheviralprotein.Thissyntheticgenewithc. 936‑pbencondingarecombinantHCVmultiepitope(hereafterrMEHCV)wasclonedinthepET21aandsequenced.TheresultingvectorwasnamedpETMEHCV.Escherichia coli BL21(αDE3)pLyEwastransformedwiththeresulting


11

plasmid.TheE. coli cells wasinducedwith1mMIPTGandthetimecourseofinductionduring8hshowedanproteinwithapparentmolecularmassof~37kDaasestimatedbySDS‑PAGEanalysis.

Soluble recombinant enzyme was purified from the culture tohomogeneitybyasinglestepNi‑NTAchromatography.Thesuccessfulexpression of MEHCV shows that this protein may be used fordevelopment of a genuine Brazilian Hepatitic C kit for diagnosticpurposes.

Financialsupport:CAPES,CNPqeFAPDF

P8 - MOLECULAR EPIDEMIOLOGY AND CHARACTERIZATION OF HEPATITIS B VIRUS

(HBV) GENOTYPES IN HBEAG POSITIVE PATIENTS FROM RONDÔNIA, BRAZIL

AlcioneOliveiraSantos1,DeusileneSouzaVieira1,MónicaVivianaAlvaradoMora2,Lívia

Botelho2,JoãoRenatoRebelloPinho2,FlairJoséCarrilho,2CarinaGodoyPicelli1,Juan

MiguelSalcedo1,EduardoRezendeHonda.1

1.ResearchCenterforTropicalMedicine–CEPEM/TropicalPathologyResearchInstitute–IPEPATRO.PortoVelho,RO,Brazil.2.LaboratoryofGastroenterologyandHepatology,SãoPauloInstituteofTropicalMedicineand Department of Gastroenterology, School of Medicine, University of São Paulo, SãoPauloSP,Brazil

HepatitisBremainsasignificantworldwidehealthproblem.Globally,itisestimatedthat2billionpeoplehavebeeninfectedandmorethan350millionpeoplearechroniccarriers.InBrazil,theendemicityofHBVisveryheterogeneous,beingthemostprevalentinthenorth.HBVcanbeclassifiedintoeightgenotypes(AtoH),definedbysequencedivergenceof more than 8% based on the complete genome.There is growingevidence that differences in clinical data, response to treatment andprognosiscanbedependentonthegenotypeoftheinfection.InRondôniastate,fewstudieshavebeencarriedoutwithHBV,particularlyconcerningviralmolecularcharacterization.ThisstudyisbeingconductedtoidentifygenotypedistributionofHBVinRondôniastatelocatedintheWesternAmazon,northernBrazil and to evaluate if there are anyassociationbetweenthegenotypeswithinfectedpatientsageandviralreplicationactivity.Objectives:TostandardizeaRealTimePCRforqualitativedetectionofHBVDNAandtocharacterizeHBVgenotypedistributionofHBVinthestateofRondônia,Brazil.Methods:Weselected40samplesfrompatientswithHBVinfection,aged18to60years,allHBsAgandHBeAgpositive.HBVDNAwasdetectedbyrealtimeusingaprobeFAM/MGB(AppliedBiosystems)designedforthisstudyamplifyingafragmentwith109pb.For19ofthesesamples,a416bpsegmentoftheSgeneHBVwasamplifiedtocharacterizethegenotypesbysequencing.Amplified DNA was purified using ChargeSwitch ® PCR Clean‑UpKitandsequencingwasperformedinanABIPrism®377AutomaticSequencer(AppliedBiosystems,FosterCity,CA,USA).QualityofeachelectropherogramwasevaluatedusingthePhred‑Phrapsoftware(Ewinget al.1998; Ewing and Green, 1998) and consensus sequences wereobtainedbyalignmentofbothsequencedstrands(senseandantisense)usingCAP3softwareonawebpageforelectropherogramqualityanalysis(http://asparagin.cenargen.embrapa.br/phph/).SequencesobtainedinthisstudywerealignedwithreferencesequencesextractedfromGenBankusingClustalXsoftware(Thompsonetal,1997),andmanuallyeditedintheSE‑ALsoftware.Maximumlikelihood(ML)phylogenetictrees

were inferredusing theGarli 0.9program.Results:The40 selectedsampleswereHBVDNApositivebyusingaqualitativereal‑timePCRsincewas.Weinitiallysequenced19samplesandgenotypesfrequencywere:42.1%genotypeD,36.8%genotypeAand21%ofgenotypeF.Conclusion:GenotypesfoundinthissamplemightreflectthosepresentinAmerindians(F)aswellasthosebroughtbynon‑Amerindians(AandD).Indeed,genotypeDandAfrequenciesinthispopulationaregreaterthanthisfoundtheAmerindiangenotypeF.Amongtheperspectivesofthisworkaretheamplificationofalargerfragmentofthevirusgenome,whichhasahigherphylogeneticsignaltocharacterizethesubgenotypescirculatinginthisregiontobetterinfertheirpossibleorigin,andalsothequantitativedetectionofHBVDNAbyrealtimePCR.

Acknowledgments:IPEPATRO,CEPEM,FAPESP07/53457‑7and08/50461‑6.

P9 - GENOTYPING AND DETECTION OF BASAL CORE PROMOTER AND PRECORE MUTATIONS

IN CHRONIC HBEAG NEGATIVE HEPATITIS B PATIENTS FROM PORTO VELHO, RO – BRAZIL

CarinaGodoyPicelli,1MónicaVivianaAlvaradoMora,2LíviaBotelho,2JoãoRenato

RebelloPinho,2FlairJoséCarrilho,2DeusileneSouzaVieira1,AlcioneOliveiraSantos1,

JuanMiguelSalcedo,1EduardoRezendeHonda,1.

1.ResearchCenterforTropicalMedicine–CEPEM/TropicalPathologyResearchInstitute–IPEPATRO.PortoVelho,RO,Brazil.2.LaboratoryofGastroenterologyandHepatology,SãoPauloInstituteofTropicalMedicineand Department of Gastroenterology, School of Medicine, University of São Paulo, SãoPauloSP,Brazil.

HepatitisBremainsasignificantworldwidehealthproblem.Globally,itisestimatedthat2billionpeoplehavebeeninfected,andmorethan350millionpeoplearehepatitisBvirus(HBV)chroniccarriers.InBraziltherearesomeareaswithhighprevalenceofHBV.InRondôniastate,fewstudieshavebeencarriedoutwithHBV,particularlyconcerningviralmolecularcharacterization,makingdifficulttofollowupthecourseofthediseaseinthepatients.Ontheotherhand,knowledgeofHBVgenotypeisveryimportantforclinicaltreatmentassomestudieshavesuggestedpossiblepathogenicandtherapeuticdifferencesamongHBVgenotypes.TheaimofthisstudywastodetermineHBVsubgenotypesin33HBeAgnegativechronicHBVinfectedpatientsfromPortoVelho,ROandtocorrelatetheseresultswiththepresenceofbasalcorepromoter(BCP)andprecore(PC)mutationsandclinicaldata.HBVDNAextractionwascarriedoutfrom100μLofeachserumsampleusingtheacidguanidiniumthiocyanate/phenol/chloroformmethoddescribedbyChomczynskiandSacchi(1987).GenotypingwasperformedbynestedPCRdescribedbySitniket al.2004coveringa734bpregioncorrespondingtotheHBsAgencodinggeneandapartialfragmentofthepolymerasegene(S/Pol).Detectionofbasalcorepromoter(BCP)andprecore(PC)mutationswereperformedbyNestedPCRdescribedbyTakahashi(1995)coveringa270bp.AmplifiedDNAwaspurifiedusingChargeSwitch®PCRClean‑UpKit.SequencingwasperformedinanABIPrism®377AutomaticSequencer(AppliedBiosystems,FosterCity,CA,USA).QualityofeachelectropherogramwasevaluatedusingthePhred‑Phrapsoftware(Ewinget al.1998; Ewing and Green, 1998) and consensus sequences wereobtainedbyalignmentofbothsequencedstrands(senseandantisense)


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usingCAP3softwaresonthewebpageelectropherogramqualityanalysis,(http://asparagin.cenargen.embrapa.br/phph/).SequencesobtainedinthisstudywerealignedwithreferencesequencesextractedfromGenBankusingClustalXsoftware(Thompsonet al,1997),andmanuallyeditedintheSE‑ALsoftware.Maximumlikelihood(ML)phylogenetictreeswere inferred using the GARLi program.The distribution of HBVgenotypeswas:A1(38.4%),D3(34.6%),F2A(15.3%),D4(7.6%)andD2(3.8%).Infoursamples,precore(PC)mutationwasfoundandtheBCPmutationwasfoundintwosamples.TheseresultsarepreliminarybutapparentlythefrequenciesofBCPandPCmutationsarelowandtheyarenotcorrelatedwithaspecificgenotypeintheseHBV‑infectedchronicpatients.The perspective of this study is to expand these diagnosticprocedurestootherstatesinthenorthofthecountrywhichdonothavethesepracticesasroutine.

Acknowledgments:IPEPATRO,CEPEM,FAPESP07/53457‑7and08/50461‑6.

P10 - QUASISPECIES COMPOSITION FROM HEPATITIS C VIRUS NS5A PROTEIN IN NON-

RESPONDER AND END-OF TREATMENT RESPONDER PATIENTS

Bittar,C.1;Jardim,A.C.G.1;Yamasaki,L.H.T.1;Queiroz,A.T.L.2;Carareto,C.M.A.1;

Pinho,J.R.R.3;Rahal,P.1

1‑UNESP–SãoPauloStateUniversity–InstituteofBioscience,LyricsandExactScience–IBILCE–DepartmentofBiology;2‑USP–SãoPauloUniversity;3‑USP–SãoPauloUniversity–FacultyofMedicine–DepartmentofGastroenterologyLaboratoryofHepatologyandGastroenterologyfromInstituteofTropicalMedicine‑AlbertEinsteinIsraeliHospital‑DepartmentofClinicalPathology.

HepatitisCisaglobalhealthissue,withaprevalenceof130millionseropositive persons worldwide. Because of the high mutation rateof theRNApolymerase, thisviruspresentsavastgeneticvariabilitythat is presented in a variety of levels, the genotypes, subtypes andthequasispecies.Thevariabilityofoneoftheviralproteins,NS5A,isbelievedtoberelatedtotheresponsetotheIFNtherapy,theadoptedtreatmentfortheinfection.Thisstudyaimedtoanalyzethequasispeciescomposition of the NS5A from HCV in patients infected with thegenotype3aduringandaftertreatmentinpatientsNon‑responder(NR)andEnd‑of‑treatmentresponder(ETR).Samplesfrom8patients(4NRand4ETR)werecollected.ViralRNAwasextracted,cDNAsynthesized,theNS5Aregionwasamplifiedandclonedfrom2ETRpatients(Pointofcollectionaftertreatment:RF109‑2m,3m,4m,5mandRF119‑3m,4m,5m).Untilnow110sequenceswereobtainedandpartiallyanalyzed.ResultsshowthatinpatientRF109,oneaminoacidquasispeciesemergedat2months(10%frequency),beingalsofoundat3months(8,33%),by4monthsitsfrequencyrisesto18,18%andonthe5thmonthitisfoundinafrequencyof60%.ResultsfrompatientRF119showsanidenticalnucleotidesequence in the4thand5thmonthafter treatment.As fortheaminoacidsequences,onequasispeciesemergesonthe3rdmonth(7,14% frequency), being found at 4 months with a 25% frequencyandby5monthsitsfrequencyrisesto36,36%.Theseresultsprovideinformationonhowtheselectivepressureofthetreatmentactsontheviralgeneticcomposition.

P11 - ASSOCIATION OF HUMAN HOST POLYMORPHISM AND HEPATITIS C

INFECTION IN A SAMPLE OF RIO DE JANEIRO POPULATION

Fabricio‑SilvaGM1,AlmeidaBS2,MarquesPS2,CardosoJF1,FigueiredoF2,PerezRM2

andPortoLC1.

1HistocompatibilityandCryopreservationLaboratory–StateofRiodeJaneiroUniversity.2Gastronterology Service; Pedro Ernesto University Hospital – State of Rio de JaneiroUniversity.

HepatitisCvirus infection isoneof themostcommonblood‑borneinfectionsworldwide.Approximately20%ofinfectedpatientssuccessfullyeliminatethevirus,whereasthemajorityofpatientsdevelopchronicinfectionwithawidespectrumofliverlesions,rangingfromaminimalinflammationtocirrhosis.Killerimmunoglobulin‑likereceptors(KIRs)expressedonnaturalkillerandnaturalkillerTcellsareinvolvedinactivationofthesecellsandcaninfluenceantiviralimmunityintheliver.TheaimofthisstudywastoexplorethepossibilityoftheinheritanceofKIRgenotypesasacandidateforsusceptibilitytopersistentHCVinfectionorHCVclearance.Thereversesequencespecificoligonucleotidepolymerasechainreaction(rSSO‑PCR)wasemployedtoidentifytheKIRgenesandpseudogenesin86chronichepatitisC(CHC)patients,44spontaneouslyrecovered(SR)controls,and181healthycontrols.Also,theCHCgroupwasdividedinno/lightfibrosis(F0)andmoderate/severefibrosis(F1),accordingIshakclassification.ThefrequencyofinhibitoryKIR2DL2isincreasedinSRgroup(72.0%)thanCHCgroup(49.0%;p=0.02).Even,KIR2DL5isdecreasedinCHCgroup(44.0%)thanControl(63.0%;p=0.008).Whenthestimulatorygeneswereanalyzed,theKIR3DS1genewasdecreasedinCHCgroup(25.0%)thanSRgroup (48.0%;p=0.01)andControl (39.0%;p=0.04).Furthermore,KIR2DS1andKIR2DS5wasincreasedinF1group(42.9%and48.6%,respectively)thanF0group(18.4%;p=0.04and21.1%;p=0.01,respectively).WeconfirmedthatKIR2DL2andKIR3DS1areinvolvedinresolutionofHepatitisCvirusinfection.Also,wesuggestthatKIR2DL5isassociatedwithchronichepatitis.Also,KIR2DS1andKIR2DS5mayinfluencetheseverityoffibrosis in the liver.This is thefirstworkofKIRgenesandHepatitisCvirusinfectionassociationinRiodeJaneiroState.Theresultsof this investigationmayprovideuseful informationforbothpopulationgeneticanddiseasesstudies.

P12 - PREDICTION OF STRUCTURAL AND FUNCTIONAL FEATURES OF NS5A PROTEIN

GENOTYPES 1 AND 3 FROM PATIENTS THAT SHOWED DIFFERENT RESPONSES TO

THERAPY

LílianHiromiTomonariYamasaki1;HelenAndradeArcuri3;AnaCarolinaGomes

Jardim1;CíntiaBittar1;JoãoRenatoRebelloPinho2;PaulaRahal1

1.UNESP/IBILCE–CampusdeSãoJosédoRioPreto;2.USP/FaculdadedeMedicina;3.USP/FaculdadedeMedicina,DepartamentodeClínicaMédicae‑mail:lí[email protected]

HepatitisCvirus (HCV) infects almost3%ofpeopleworldwideand it is considered the main cause of liver chronic diseases. Until


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today,thereisnoeffectivevaccineandthemostusedtherapy,basedonPeginterferon,issuccessfulonlyin50%ofpatientsinfectedbygenotype1.Theoutcomesofthistreatmentresistanceareunclear,itissuggestedhostandvirusfactorsmayparticipateinthismechanism.Non‑structural5A(NS5A)proteinisinvolvedinseveralcellularandvirusprocessesanditisacriticalcomponentofHCV.However,itsstructureandfunctionarestilluncertain.Regardingthesefacts,thepresentstudyattachmentsweretoelaborateamodeloftheNS5Aproteinandtoinvestigatestructuralandfunctionalfeatures,usingin silicotools.Itwasanalyzed345sequencesofHCVNS5Aproteinfrom23patientsinfectedbygenotypes1or3that showed different responses to therapy and reference sequencesfrom Genbank. Residues and secondary structure composition of allsequencesdemonstratedthattherearedifferencesbetweengenotypes.Itmayindicatethattherearedifferencesininteractionsbetweengenotypes,whichcouldberelatedwiththedistinctaverageoftreatmentresistance.FunctionalanalysiswasperformedwithProtFun.ItsuggestedthatNS5Ais involved with metabolism, translation, growth, transport, ligationandhormonefunctions in thecell.These functionsvarybetween thedomains,strengtheningthehypothesisthatNS5Aisamultifunctionalprotein.Prositemotifsearchindicatedthattherearemanyglicosilation,fosforilationandmyristoilationsites,whicharehighlyconservedandmayplayanimportantroleinstructuralstabilizationandfunction.Inaddition,itwasidentifiedahighlyconservedsiteofintegrinligation.Bythreaderandhomologymolecularmodelingmethods,itwaselaboratedthe3DstructureofcompleteNS5A.Nextstepsincludemoleculardynamicsof3DstructureandevaluationofalterationsinNS5Astructure/functioninmutants.

P13 - SEQUENCE ANALYSIS OF NS3 SERINE PROTEASE DOMAIN OF HEPATITIS C VIRUS IN

BRAZILIAN ISOLATES

PeresdaSilvaA,deAlmeidaAJandLampeE.

ViralHepatitisLaboratory,OswaldoCruzFoundation,FIOCRUZ,RiodeJaneiro‑Brazil

TheaminoterminalportionofNS3ofhepatitisCvirus(HCV)isachymotripsin‑likeserine‑proteasethatpossessesanimportantroleinviralreplicationsinceisresponsibleforcleavageofthenon‑structuralproteinsofHCV.Inthissense,theNS3regionhasbeenoneofthemoleculartargetstonewtherapeuticapproachesthatblocksspecificsstepsinviralreplication.ThegenomesequencevariabilityofHCVBrazilianisolatesispoorlydocumentedincreasingtheneedforobtainingmoredetaileddataonthemagnitudeofitsgeneticdiversity.Inthiscontext,theaimofthisstudywastoanalyzethegeneticvariabilityof5’‑terminalregionofNS3geneinthemostprevalentHCVsubtypescirculatinginourregion.Atotalof114samplesfromtherapy‑naïveBrazilianpatientswithchronichepatitisC,infectedwithsubtype1a(n=48),1b(n=53),or3a(n=13)werestudied.ViralRNAwasextractedand the regionencompassingthe NS3 serine protease domain was reverse‑transcribed and PCR‑amplifiedandsubmittedtodirectsequencing.TheobtainedsequenceswerealignedwithcorrespondingnucleotidesequencesofHCVstrainsfromotherscountries,byusingClustalXprogram.Phylogeneticthreeswereconstructedusingtocomputenucleotidedistancesthemaximumlikelihood and Neighbor‑Joining methods of Mega 4.0 program.

Phylogenetic analyses revealed that isolates belonging to subtype 1bshowedahigherdegreeofheterogeneitythansubtype1aand3a,andalsoformedvarioussubclustersinthephylogenetictreeconstructs.Themeanpairwisenucleotidegeneticdistancebetweenthe1bisolateswas0.098,whereasfor1aand3asequenceswere0.060and0.062,respectively.Thesubtypes1aand3aisolatesformedseparatebranchesdistinctfromreferencesequencesfromothergeographicalregions.Inconclusion,aremarkableheterogeneityofBraziliansequenceswasobservedwhichshouldbetakenintoaccountforfuturetherapeuticapproachwithdrugsdesignedtoinhibittheHCVNS3serineprotease.

P14 - HEPATITIS B VIRUS INFECTION IN RURAL COMMUNITIES OF THE PANTANAL WETLANDS,

MATO GROSSO DO SUL STATE, CENTRAL BRAZIL

GláuciaBigaton1,ReginaBringelMartins5,DelsodoNascimento2,ÍrisBuckerFroes2,

AlcioneFaroStief1,GinaJonassonMousquer1,PaulaGuerraMurat1,FernandaRodas

Pires1,JoséRodrigoRégisLopes1,MariaElizabethCavalheirosDorval3,SheilaAraújo

Teles5,SelmaAndradeGomes4,AnaRitaCoimbraMotta‑Castro1

1DepartamentodeFarmáciaeBioquímica,UniversidadeFederaldeMatoGrossodoSul,CampoGrande,MS,Brasil2CentrodeEspecialidadesemDoençasInfecciosaseParasitárias,SecretariadeEstadodeSaúde,CampoGrande,MS,Brasil 3DepartamentodePatologia,UniversidadeFederaldeMatoGrossodoSul,CampoGrande,Ms,Brasil 4DepartamentodeVirologia,LaboratóriodeVirologiaMolecular,InstitutoOswaldoCruz‑Fiocruz,RiodeJaneiro,Brazil 5InstitutodePatologiaTropicaleSaúde‑Pública,UniversidadeFederaldeGoiás,Goiânia,Goiás,Brazil;

The objective of this paper is to investigate the prevalence of theserologicalmarkersforhepatitisBintheriversidepopulationlivinginPantanalsul‑mato‑grossense.Thestudyinvolved321individualslivinginfourremotecommunities in theAltoParaguaiBasin.ThesamplesweretestedfordetectionofHBsAg,totalanti‑HBcandanti‑HBsmarkersusingimmuneenzymaticassay.Outof321riversidedwellerswithagesrangingfrom1to89years,52%weremales,54.2%werecaucasiansand43.3%reportedtohaveasteadyrelationship.Mostindividuals(93.4%)presented low socioeconomic and educational levels, poor hygieneandhousingconditionsand theirhousesdidnothaveproper sewagesystem.TheglobalprevalenceofHBVinfectionwas36.5%,rangingfrom 15.1% (Passo do Lontra community) to 61% (Paraguai Mirimcommunity).Positivityfortotalanti‑HBcassociatedwithHBsAgwas1.6%andtheassociationofanti‑HBcwithanti‑HBs,indicatingformerinfectionandimmuneresponse,wasfoundin32.1%.Isolatedanti‑HBcwas seen in 2.8%.The presence of anti‑HBs as an isolated marker,indicatingantecedentofvaccineresponsewasdetectedin32.4%oftheindividuals.FromthetwoHBsAgpositivesamples,fourwereanti‑HBereagent.HBVDNAwasdetectedbyPCRin40%oftheHBsAgreagentsamplesandin80%ofthepositiveanti‑HBe.TheHBVisolateswereidentified as genotypes D and F.The findings reinforce the need foradditionalprogramsofhealtheducationandalternativeschemesagainsthepatitisBwithaviewtoincreasingthevaccinecoverageinisolatedcommunitiesofCentralBrazil.


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P15 - ADDRESSING THE POTENTIAL ROLE OF OPN PROMOTER ALLELES IN INTERFERON-

BASED TREATMENT OUTCOME AMONG BRAZILIAN HEPATITIS C CHRONIC PATIENTS

PauloH.C.França,ÂngeloA.Mattos,CristianeTovo,CarlosE.Brandão,RaquelF.

L.Garcia,LysandroNader,andLeslieE.Ferreira

Hepatitis C virus (HCV) is well recognized as a major cause ofchronicliverdiseasesworldwide.Despitetheavailabilityofinterferon‑based(IFN)therapies,onlyafractionofpatientsachievetheintended“sustainedvirologicalresponse”(SVR)ordevelopsignificantsideeffectsrequiringtreatmentarrest.Osteopontin(OPN)isanextracellularmatrixprotein,withpleiotropiceffects,expressedinavarietyoftissues.OPNhasbeenimplicatedintheinitiationofTh1immuneresponse.Recently,foursinglenucleotidepolymorphisms(SNPs) in thepromoter regionof the OPN gene (‑155, ‑443, ‑616, and ‑1748nt) were suggested asmarkersreflectinghepatitisactivityinpatientswithHCV.Inthissense,weintendtoassesstherelationof‑443and‑616ntSNPstoanti‑HCVtreatmentresponse.Peripheralbloodhasbeencollectedandkeptdried(FTA Elute Cards®) from, at least, 100 Brazilian patients with wellcharacterizedpegylated‑IFN‑basedtreatmentoutcomedirectedtoHCVgenotype1.GenomicDNAisextractedundermanufacturer´sinstructionsandSNPshavebeenanalyzedbydirectsequencingofDNAfragmentsencompassingthetargetedlocus,amplifiedbypolymerasechainreaction(PCR),andsequence‑specificprimersPCR(SSP‑PCR).TheproportionsofSVRandnon‑SVRpatientswillbecomparedwithdifferentOPNallelesbyFischer´sexacttest.Indeed,wehaveestablishedthebasisfortheinvestigationofthehostgeneticdiversitycontributiontothetherapyoutcomeinHCVchronicinfection.

Support:CNPq/EditalUniversal2008;FAP/UNIVILLE

P16 - STUDY OF THE GENETIC VARIABILITY AND ACTIVITY OF NS3 PROTEASE FROM

HEPATITIS C VIRUS

Hoffmann,L.1;Villela‑Nogueira,C.A.2;Ürményi,T.P.1;Tanuri,A.3;Rondinelli,E.1,2;and

Silva,R.1

1ProgramadeBiologiaMoleculareEstrutural,InstitutodeBiofísicaCarlosChagasFilho,UFRJ,RiodeJaneiro;2DepartamentodeClínicaMédica,UFRJ,RiodeJaneiro;3InstitutodeBiologia,UFRJ,RiodeJaneiro,Brazil.E‑mail:[email protected]

The Hepatitis C is a health problem in Brazil with 3 millionpeople infected.The evolution of the infection and the response totreatmentvariesaccording toHepatitisCVirus (HCV)genotypeandthe heterogeneity of the viral population (quasispecies) among otherfactors.Only50%ofthetreatedpatients,withtheconventionaltherapy(interferonandribavirin),achievesuccess.Nowadays,severalgroups,specially the viral protease inhibitors, are studying other therapeutictargets.TheviralproteaseisresponsibleforcleavageofthepolyproteininthetargetsNS3/4A,NS4A/4B,NS4B/5AandNS5A/5Bandparticipatesinthevirusevasionthroughtheinhibitionofinterferonproductionbythehost.TheaimofthisworkistoassessthegeneticvariabilityofHCVNS3proteaseandtoproduceaphenotypicassayforanalysisofanti‑HCVtherapy.65patientswithHCV1a/bwithchronicHepatitisCare

beingstudied.ToaddressthegeneticvariabilityofNS3proteaseregion,weperformedRT‑PCRofHCVRNA,isolatedfromallthe65patients,with randomprimers followedbynestedPCRwith specificprimers.ThePCRproductsweredirectlysequencedtoassessthepredominantquasispecie.ThesequenceswereanalyzedusingthesoftwareGeneious4.6.4.The sequence analysis identified genetic diversity among thedifferent predominant quasispecies in the patients analyzed thus far,usingtheHCV‑J1basthereferencesequence.WefoundvariationintheNS3proteaseDNAsequences,obtainedfrompatientswithdifferenttherapeuticresponses.ThisfindingmaybeusedtoestimateanassociationbetweenNS3geneticvariabilitywithresponsetodrugtreatment.InordertodevelopthephenotypicassayforNS3/4Aregion,wehavedevelopedanexpressionvectorinpET17bwhichisreadytoreceivethecorrespondentNS3proteasefromvirusisolatedfrompatients tobeanalyzedfor itsproteaseactivityagainstdifferentcandidateanti‑HCVdrugs.Financialsupport:CAPES/FAPERJ/CNPq

P17 - PLAN PILOTO PARA EL TRATAMIENTO Y CONTROL DE VHC EN CHILE

MuñozG,TorresC,VenegasM,RiveraM.

LaboratorioBiologíaMolecular,Gastroenterología,HospitalClínicoUniversidaddeChile.*Div.PrevenciónyControl.DeEnfermedades,MinisteriodeSaluddeChile.

LainfecciónporVirusHepatitisC(VHC)presentaunadistribuciónmundial.EnChile,seestimaunaprevalenciadeinfecciónentre0,13a0,5%,conun75%depacientesvirémicos,dandocuentadel70%deloscasosdehepatocarcinomaydel23%delostransplanteshepáticos.Apartirdelaño2006,seiniciaenChileunPlanPilotoparaeltratamiento(TTO)delVHC(pegIFN+rivabirina),conunincrementoprogresivoanualdelnºdepacientestratados.Objetivo:MostrarlosresultadosdelaboratorioobtenidoseneltratamientodeVHCenpacienteschilenos.Material:Seanalizantodoslosresultadosdelaboratoriodelospacientestratados durante el período 2007 – 2009 y que hayan completado elseguimiento,portérminoosuspensióndelaterapia.Laevaluacióndelaboratorioconsideraunalgoritmoqueincluyeuncontrolbasal(cargaviralygenotipo),yunmonitoreconmuestrasalas24sem.intraTTO(carga viral), al término TTO (PCR) y 24 SEM post TTO (PCR).Resultados:Sehanestudiado478casosantiVHC(+),329deloscualesiniciaronTTO(PCRpositivo).Un71%correspondieronagenotipo1;26%y3%agenotipos3y2,respectivamente.Ochentayunpacientescompletaronsuesquema,alafecha,segúnelprotocoloestablecido.Losresultadosson:

Genotipo Respuestaviralsostenida(RVS)

Fracaso Recaída

1 31% 58% 11%2‑3 73% 5% 22%Total 44.4% 42% 13,6%

Conclusiones:Elgenotipo1eselmásprevalenteenlospacienteschilenosinfectadosporVHC.Estegenotipoeseldemenorrespuestaa la terapia antiviral, lo cual explicaría el 44% de RVS en el grupoanalizado.LaRVSobtenidaenlosgenotipos2y3estádeacuerdoalodescritoenlaliteratura,adiferenciadelgenotipo1cuyosresultadosson


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levementeinferiores.

P18 - GENETIC VARIABILITY OF HEPATITIS C VIRUS IN A POPULATION OF INFECTED

HEMOPHILIACS IN URUGUAY

López,Lilia,Moratorio,Gonzalo,LópezFernando,Recarey,RicardoandCristina,Juan.

Laboratoriodevirologíamolecular,CentrodeInvestigacionesNucleares

Background: Hepatitis C virus (HCV), has been the subject ofintenseresearchasitsmajorroleinhumandiseasehasemerged.Previousstudies have suggested a diversification of type 1 HCV in the SouthAmericanregion.ThedegreeofgeneticvariationamongHCVstrainscirculating in Uruguay is currently unknown. Hemophiliacs treatedwith cryoprecipitate are a group of patients especially vulnerable toinfectionbyHCV.InUruguay,hemophiliacshavehighprevalenceofHCVinfection.Objective:Study thedegreeofgeneticvariabilityofHCV strains circulating in a population of infected hemophiliacs inUruguay.MaterialsandMethod:5´noncodingregion(5´NCR)wasamplifiedandsequenced.Alignmentofthesequenceswasperformedwith the CLUSTAL W program. Phylogenetic tree analysis wasperformedusingtheneighbor‑joiningmethodunderamatrixofgeneticdistancesestablishedundertheKimura‑twoparametermodel.Results:PhylogenetictreeanalysisofHCVstrainsisolatedrevealedthepresenceofanimportantnumberofgenotypesandsubgenotypesco‑circulatingin apopulationof infectedhemophiliacs inUruguay.Furthermore, adistinctgeneticlineageinsidegenotype1wasidentified.Comparisonsof these resultswith theones foundforEuropeorNorthAmericaaswellaswithothersfoundinSouthAmerica,revealedthatthislineageischaracteristicoftheSouthAmericanregion.Conclusion:Phylogeneticanalysisrevealedthepresenceofawidedegreeofgeneticvariabilityaswellasadistincttype1HCVsubpopulationcirculatinginagroupofinfectedhemophiliacpatientsinUruguay.

P19 - DISTRIBUTION OF HEPATITIS C VIRUS (HCV) GENOTYPES IN SAMPLES SENT TO

LACEN-BA 2007 AND 2008

ElineCPOliveira1,AlziratheCosta1TA,HaydéeCLNascimento1,MaryAliceSZarife1

1.CentralLaboratoryofPublicHealthProfessorGonçaloMoniz(LACEN‑BA).

Introduction:ThehepatitisCvirus(HCV)isamajorpublichealthproblem in Brazil and the world. Epidemiological data suggest thatthedistributionofHCVgenotypesvariesbetweendifferentregions.InBrazil,inchronicallyinfectedpatients,thepredominantgenotypeisthegenotupe1,followedbygenotypes3and2.Genotype4wasdescribedinSãoPauloandBahia,andgenotype5onlyinSãoPaulo.Objective:To investigate the distribution of HCV genotypes in samples sent totheCentralLaboratory‑BAintheyears2007and2008.Methods:Thedetection of HCV‑RNA was performed by RT‑PCR by the COBASAMPLICOR(Roche)andgenotypingbyLIPA(Siemens).Results:Ofatotalof1092sampleswithdetectableHCV‑RNAwerefound76.6%(n=837)of1genotype1,3.5%(n=38)ofgenotype2,19.6%(n=214)of

genotype3,0.1%(n=1)ofgenotype4and0.2%(n=2)ofgenotype5.Conclusion:Therewasanagreementwiththeliteraturedatawithrespecttogenotypes1,2,3and4.Theinsertionofgenotype5inBahiashowsachangeintheepidemiologicalprofileofHCVinourstate.

P20 - CAN SALIVA BE USED TO DETECT HBV MARKERS?

VillarLM1,MedinaHC1,Villela‑NogueiraCA2,NabucoLC2,doÓKMR2,daSilvaEF1,

Lewis‑XimenezLL1,YoshidaCFT1,LampeE1.

1ViralHepatitisLaboratory,IOC/Fiocruz,RiodeJaneiro,RJ,Brazil;2HospitalClementinoFragaFilho,UFRJ,RiodeJaneiro,RJ,Brazil.E‑mail:[email protected]

Background and aims: NumerousimmunologicalapproachesexisttodiagnosehepatitisBvirus(HBV)infection.TheaimsofthisstudyweretoadaptacommercialenzymeimmunoassaytodetectHBsAgonsalivarysamplesandtoevaluatetwocollectionsystemstodetectthisantigen.Study Design: Apanelof115pairedwholesaliva,oralfluidandserumsampleswereobtainedfrominfectedandnoninfectedindividuals.Transportbufferfororalfluid;samplevolumeonassay,incubation period of sample and conjugate and cut‑off values wereevaluated to optimize the assay. Results: No significant differenceson salivary HBsAg were observed using different transport buffersandsamplevolume.Timeofsalivasampleandconjugateincubationandcut‑offsamplecalculationclearlyinfluencedassay,soincubationperiodwasextendedto16hoursinsteadof1hourandusingtheareaundertheROCcurve(AUROC),qualityparameters,suchasaccuracyandpositiveandnegativepredictivevaluesweresensitivelyimproved,providingvaluessuperior to89%forbothspecimens.HBsAgweredetectedin40oralfluidand44wholesalivasamplesoutof47pairedpositiveserumspecimensandnotdetectedin64oralfluidand63wholesalivasamplesoutof68matchednegativeserasamplesbyEIA.Theagreement between results obtained in serum and saliva specimenswasexcellentaccordingtotheKappaindex(k:0.80fororalfluidandk.0.87forwholesaliva).Conclusion: DetectionofHBsAgonwholesalivaandoralfluidyieldedoptimalresultsfordiagnosisofHBVusingmodifiedcommercialenzymeimmunoassay.Keywords:HepatitisB,wholesaliva,gengivocrevicularfluid,enzymeimmunoassay.

P21 - USE OF DIFFERENT SALIVA COLLECTION SYSTEMS FOR HEPATITIS C VIRUS DIAGNOSIS

Villar,L.M.1;Medina,H.C.1;Villela‑Nogueira,C.A.2;doÓ,K.M.R.2;Lewis‑Ximenez,

L.L.1;Yoshida,C.F.T1;Lampe,E1.

1ViralHepatitisLaboratory,IOC/Fiocruz,RiodeJaneiro,RJ,Brazil;2HospitalClementinoFragaFilho,UFRJ,RiodeJaneiro,RJ,Brazil.E‑mail:[email protected]

Backgroundandaims:Interestingdevelopmentshaveoccurredinthefieldoflaboratorydiagnosisofinfectiousdiseasesonsalivaspecimens.Blood collection is often difficult in children, and the collection ofsalivaryspecimensforantibodytestingintheseindividualswouldbeadvantageous. However, the accuracy of detection of antibodies insaliva specimens may depend on the collecting system.The presentstudywasdesignedtoevaluatetheuseofdifferentcollectionsystems


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todetectHCVantibodies in saliva samples: a commercial collectiondevice(Salivette)and“dribbledform”inwhichthesubjectallowssalivatofallfromthemouthintoavial.Methods:Matchedplasmaandsalivasamplesof97BrazilianindividualswithorwithoutpasthistoryofHCVinfectionwerecollected.Serasamplesweretestedaccordingsupplier’sinstructionsandinputvolumewas10mL,butforsalivatesting,theinputvolumewasincreased(+190mL).Results:Thesamplesetincluded39anti‑HCVpositiveserumsamplesandacontrolgroupof58anti‑HCVnegativeserumsamples.Meanageofpopulationstudiedwas45.5years(SD: 14.4, range: 20‑77 years) and 51% were male. Using Salivettecollection,sensitivityandspecificityofsalivaryanti‑HCVassaywere64%and100%,respectively.Fordribbledformcollection,sensitivityand specificity of anti‑HCV test were 71% and 100%, respectively.Conclusions:TheseresultsshowedthatbothsalivaspecimenscanbeusedtodetectantibodiesagainstHCVbutdribbledformcollectiongivesmorepreciseresults.HoweverfurtherstudiesshouldbedonetoimprovetheefficiencyofHCVtestonsalivasamples.

Keywords:HepatitisC,collectionsystem,enzymeimmunoassay.

P22 - HEPATITIS DELTA VIRUS (HDV) INFECTION IN HIV-HBV CO-INFECTED PATIENTS IN SÃO

PAULO, BRAZIL

Mendes‑Correa,MCJ1,3;Gomes‑Gouvêa,MS2;daSilva,AC3;Pádua,AP2;LázariC3;

CavalcantiNCS3;UipDE1;Pinho,JRR2.

1UnidadedeReferênciaemDoençasInfecciosasePreveníveisdaFaculdadedeMedicinadaFundaçãodoABC–SantoAndré,SP;2DepartamentodeGastroenterologia,LaboratóriodeGastroenterologiaeHepatologiaTropical,InstitutodeMedicinaTropical‑FMUSP,SãoPaulo;3HospitaldasClínicasdaFaculdadedeMedicinadaUSP,SãoPaulo

Background‑LimitedinformationisavailableabouttheseroprevalenceofhepatitisdeltavirusamongHBV‑HIVco‑infectedpatientsinBrazil.TheobjectiveofthisstudywastoevaluatetheprevalenceofhepatitisdeltainagroupofHIV‑HBVco‑infectedpatients,followedatasingleinstitutionin2007,inSãoPaulo.Methods‑2,412HIVpositivepatientsweretestedforHBVinfection.VirologicalmarkersforHBVincludedHBsAg, HBeAg and total anti‑HBc by ELISA. One hundred‑twentyHBsAg positive patients (4.9%) were identified. Plasma specimenswereobtainedfrom70patients,comprisingthestudypopulationthatunderwentfurtherhepatitisdeltadetectionbyELISAandcharacterizationby PCR. Results ‑ Only one (1.3%) anti‑HDV positive patient wasidentified. Current HDV infection was confirmed by PCR and thegenotypeofthiscasewasdeterminedbyphylogeneticanalysisofthesequenceasHDV‑1.Thepatientwasa37years‑oldmalepatient,activehomosexual, originating from Ceará (Northeast of Brazil) and livinginSãoPauloforthelast20years.Conclusions‑HepatitisdeltaisnotfrequentamongHIV‑HBVco‑infectedpatientsintheSoutheastregionofBrazil.AsHDVgenotype1 isnot common inBrazil, thispatientwaspossiblyinfectedthroughcontactwithinfectedpeoplefromothercountrieswherethisgenotypeisprevalent.Toourknowledge,thisisthefirstdescriptionoftheHDVgenotypeoutsidetheAmazonregionwherethisinfectionismorefrequentinBrazil.

P23 - IMPACT OF HIV CO-INFECTION ON MUTATION PATTERNS OF HEPATITIS B VIRUS

GENOME IN PATIENTS WITH LAMIVUDINE-RESISTANT CHRONIC HEPATITIS B IN BRAZIL

Mendes‑Corrêa,MC1,4;Martinelli,ALC2;Pinho,JRR3;Gomes‑GouvêaMS3;daSilva

AC4;Figueiredo,JF2;Pádua,AP3;UipDE1.

1UnidadedeReferênciaemDoençasInfecciosasePreveníveisdaFaculdadedeMedicinadaFundaçãodoABC–SantoAndré,SP;2DepartamentodeGastroenterologia,FMUSP‑RibeirãoPreto;3DepartamentodeGastroenterologia,LaboratóriodeGastroenterologiaeHepatologiaTropical,InstitutodeMedicinaTropical‑FMUSP,SãoPaulo;4HospitaldasClínicasdaFac‑uldadedeMedicinadaUSP,SãoPaulo

Background ‑ Prolonged lamivudine (LAM) therapy has beenassociated with different mutations in the hepatitis B virus (HBV)genome.Theobjectiveofthisstudywastocomparelamivudine‑resistancemutation patterns after prolonged LAM use between patients withchronichepatitisBinfectionwithorwithouthumanimmunodeficiencyvirus(HIV)co‑infection.Methods‑WeincludedpatientswithchronichepatitisBtreatedwithLAMwithHBVdetectableviremia(HBV‑DNA>50IU/mL)afteratleastsixmonthsofLAMuse.HBVgenotypeandmutationstatusanalysiswerecarriedoutbyamplifyinganddirectlyandbidirectionally sequencing the complete HBV RT‑domain. Results ‑TwentypatientsinfectedwithHBVand26HIV‑HBVco‑infectedpatientswereincluded.ThetimeofexposuretoLAMvariedfrom7to36monthsamongHBVinfectedpatientsandfrom6to96monthsamongco‑infectedpatients.HBVgenotypicdistributionamongco‑infectedpatientswas:A(15–57.6%),D(6–23.0%),G(3‑11.5%)andF(2‑7.6%).AmongonlyHBVinfectedpatients,itwas:genotypeA(7–36.8%)andgenotypeD(12–63%).MutationsthathavebeenassociatedwithresistancetoLAMwereobservedin7(35.0%)ofHBVinfectedpatientsandin15(57.6%)ofHIV‑HBVco‑infectedpatients.Inthislattergroup,thefrequencyofthertV173L+rtL180M+rtM204Vtriplemutationwas19.0%versus5.0%observedamongpatientsinfectedonlywithHBV.AllpatientswiththetriplemutationalpatternshowedsE164D+sI195Mchangesintheenvelopegene.Conclusions‑HIV‑HBVco‑infectedpatientsshowedmultiplemutationsmorefrequently,includingthetriplemutationthatmayelicitavaccineescapephenotype.Amongpatientsco‑infectedwithHIV/HBV,carefulHBVDNAmonitoringisessentialtodetecttreatmentfailuresandtopreventtheemergenceofavaccineescapephenotype.HBVdrugandvaccineresistancestrainmightrepresentamajorpublichealthduetoitspotentialriskoftransmission.

P24 - ANALYSIS OF QUASISPECIES PROFILE OF HEPATITIS C VIRUS NS5A GENOMIC REGION

GENOTYPE 1

MATOS,R.A.M1.;JARDIM,A.C.G1.;BITTAR,C.1;YAMASAKI,L.H.T1.;PINHO,J.

R.R.2;RAHAL,P1.

1.UNESP/IBILCE–CampusdeSãoJosédoRioPreto;2.USP/FaculdadedeMedicina.

HCV genotype 1 is the most prevalent around the world and isassociatedtoalowertreatmentresponse.TheNS5AproteinhasbeenimplicatedintheresistanceofHCVtointerferontherapy.Theobjective


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ofthisstudywastoanalyzethegeneticvariabilityofquasispeciesonthecodingregionofHCVNS5AintwopatientswhodevelopedinfectionwithHCVgenotype1:anon‑responderpatient(EAO)andaresponderattheendoftreatment(CSM).ThecollectionpointsforEAOpatientwere:pre‑treatment,14weeksoftreatment,14days,2and6monthsaftertreatment’send.ForCSMpatient:pre‑treatment,thereboundpoint2,3,4,5and6months.SamplesweresubmittedtoRT‑PCR,clonedandsequenced.TensequenceswereusedintheanalysisofNS5Aforeachcollectionpoint.ItrevealedthatthegeneticdistancesofCSMpatientdecreasedgraduallyuntilthe3rdmonthsandincreaseon4th,5thand6thmonthsgradually.TheanalysisoftheEAOpatientshoweddecreasedvaluesinsamplescollectedat12weeksoftreatment,asubsequentgradualincreaseupto2monthsandafurtherdecreasein6month.ThedN/dSratiovaluesinCSMpatientincreasedprogressivelyfromthereboundpointuntilthe3rdmonthanddecreaseon4th,5thand6thmonthsgradually.SamplesfromtheEAOpatientshowedanincreaseinthedN/dSratiovalueswith12weeksoftreatment,adecreaseintheEAO14days,andanincreasein2ndmonthremainingconstantuntil6thmonth.RegardingtheNS5Aquasispeciesdiversityastrainstartedtobepredominanton2monthsinCSMpatientandfortheEAOpatientadominantvariantcouldbeidentifiedon12weeks,thisvariantremainedpredominantinthenextpointandin6thmonthanothervariantwasidentifiedaspredominant.

P25 - DESENVOLVIMENTO DE UM REPLICON DE HCV COM CAPACIDADE DE SUBSTITUIÇÃO

DO GENE DA RNA POLIMERASE RNA-DEPENDENTE (NS5B) POR FRAGMENTOS

HOMÓLOGOS DE PACIENTES FALHANDO A TERAPIA COM INTERFERON α E RIBAVIRINA

TanuriA.,AlonsoNetoJ.B.,PereiraHS,HorbachI.S.

UFRJ‑UniversidadeFederaldoRiodeJaneiroLaboratóriodeVirologiaMolecular‑InstitutodeBiologia‑DepartamentodeGenética

Estima‑se que 180 milhões de pessoas estejam infectadas nomundopelovírusdaHepatiteC(oquecorrespondea3%dapopulaçãomundial),sendo130milhõesemriscodedesenvolvercirrosehepáticae/oucâncerdefígado.Atualmente,oregimedetratamentoparaHCVpelaFDA,consistedeinterferon‑αpeguilado(PEG‑IFN)administradosubcutaneamenteeribavirinaadministradaoralmente.ANS5B(RNApolimeraseRNAdependente)possuiatividadeúnicadosvírusdeRNA,nãoseencontrandoessaatividadenascélulashospedeiras.DessaformaaRNApolymeraseRNAdependenteéumalvomuitoespecíficoparaoHCV.Opresente trabalho temoobjetivodedesenvolver repliconssubgenômicosrecombinantesdeHCVcomcapacidadedesubstituiçãodogenedaRNApolimeraseRNAdependenteporfragmentoshomólogosdepacientesfalhandoaessaterapiaclássica.Estesrepliconsestãosendoutilizadosparatestagemdenovasdrogas(inibidoresnucleosídicosenãonucleosídicos)contraHCV,determinaçãodoIC50edescobertadenovasmutaçõesderesistênciaàestescompostos.Paratal,umsistemautilizandorepórterdeluciferasepresentenaconstruçãodorepliconétransfectadoemcélulasdeumalinhagemhepatica(Huh7).AoutraalternativaparaoestudodovírusdahepatiteCéautilizaçãodeclonesinfecciosos.Osclones infecciosos possuem o genoma viral completo, com algumasalterações que os fazem mais atenuados in vivo. Nosso laboratório

pretendeestudarocomportamentodeumcloneinfecciosonapresençadenovasdrogascontraHCV.Alémdisso,estamosdesenvolvendoumnovomodeloanimalmurinoquepoderáserutilizadoparaavaliaçãoin vivo doscompostosantivirais.

Financiadores:CNPq,FAPERJ.

P26 - CLONING AND EXPRESSION OF A RECOMBINANT MULTIEPITOPE PROTEIN FOR

HEPATITIS B DIAGNOTICS

Souza,M.Q.;Galdino,A.S.;Santos,J.C.;Torres,F.A.G.;Felipe,M.S.S.

LaboratóriodeBiologiaMolecular‑InstitutodeCiênciasBiológicas‑UniversidadedeBrasí[email protected]

HepatitisBisaliverinflammationcausedbytheHBVvirus.Thefirstantibodydetectedafterexposuretothisvirusistheanti‑HBcIgM.Thepresenceofthisantibodyenablestheidentificationoftheclinicalstageof thedisease andbecamesundetectable after the acutephase.Anti‑HBcIgGappearsquicklyafterIgM,reachinghightitersinchronichepatitisandremainsevenaftercure.Sinceanti‑HBcisthefirstantibodyidentifiedandsometimestheonlymarkerdetectedduringthecourseofinfection,itcanbeusedbothtoindicateHBVinfectionandtoidentifyindividualswhohavecomeincontactwiththevirus.ThegoalofthisworkwastoproducearecombinantHBcmultiepitope(rMEHB)proteinthatcouldbeusedfordiagnosisofhepatitisB.Forthispurpose,agenesequencenamed rMEHBwasdesigned taking into account themostcommon and conserved HBV epitopes.The synthetic gene (609pb)wasclonedintovectorpET21aasaNdeI/XhoIfragmentandbearinga6xHistagattheC‑terminal.TheresultingplasmidwastransformedintoEscherichia coliBL21(DE3)pLysS.Aselectedclonewasgrownfor induction of the recombinant protein in the presence of IPTG.SDS‑PAGEanalysisshowedaninductionbandof~21kDa,whichwasconsistentwiththepredictedsizeofthemultiepitopeprotein.Westernblotusinganti‑polyHisantibodywasfurtherperformedtoconfirmtheidentityofinductionband.ThepurificationtheproteinwasperformedbyasinglestepwithNi‑NTAaffinitychromatography.Inconclusion,ourresultsshownthatthecarefulchoiceofepitopes,theuseofE. colisystemforexpressionassociatedwiththesimplepurificationprotocol,offersthepotentialdevelopmentofaninexpensiveserologicaldiagnostictestwithhighdegreeofsensitivityandspecificity.Keywords:HepatitisB,Multiepitopeprotein,HBVdiagnostic

P27 - SEROPREVALENCE OF HBSAG AND ANTI-HCV DURING THE VII BAHIANA AWARENESS WEEK FOR HEPATITIS C IN

SALVADOR-BA, BRAZIL

PEREIRA,F.M;BERTOLLO,L.A;SANTOS,E.D;SANTOS,N.S;ZARIFE,M.A.S

PublicHealthCentralLaboratoryfortheStateofBahia(LACEN/BA)Salvador‑Ba

BACKGROUND:ViralhepatitisisconsideredtobeamajorpublichealthprobleminBrazilandtheworld.InBrazil,preliminarydatafromapopulation‑basednationalsurveyofthemajorBraziliancitiesrevealeda


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prevalenceof0.9to1.9%forantibodiesagainsthepatitisCvirus(HCV)andalowendemicity(0.11to0.74%)ofchronichepatitisBcarriers‑HBV(Brazil,2008).OBJECTIVE:Toevaluate theepidemiologicalprofile of the prevalence of HBsAg (surface antigen of hepatitis B)andanti‑HCV(antibody tohepatitisCvirus)during theVIIBahianaAwarenessWeekforHepatitisC.

MATERIALAND METHODS:A total of 3075 samples werecollectedfromthe18thto23rdofMay2009insevenhealthcentresandwere analyzed in LACEN / BA.The reagents used were theAbbottARCHITECTHBsAg,AbbottARCHITECTanti‑HCVtestandathirdgenerationrecombinantimmunoblotassay(RIBA).RESULTS:Ofthe3075samplescollected,3044wereanalyzedforanti‑HCVandofthese15(0.5%)werepositiveand11(0.4%)wereindeterminate.TheHBsAgtestwasperformedfor2982samplesofwhich13(0.4%)werepositive.Therewasnoevidenceforco‑

infection in the analyzed samples.Among the samples reactiveforanti‑HCV,nine(60%)werefemaleandsix(40%)weremale.Thepredominantagerangeinbothsexeswas45to59years.Ofthe11anti‑HCVindeterminatesamples,eightwereanalyzedusingtheRIBA,sevensamplestestednegativeandonewasindeterminate.Ofthe13HBsAgpositivesamples,nine(69.2%)werefemaleand4(30.8%)weremale.Thepredominantagegroupwas29to45years.CONCLUSION:Theprevalenceofanti‑HCVwashigherthanthatforHBsAginthesamplesanalyzed. Both markers were predominantly positive in adults, withfemalesbeingthemostprevalent.

P28 - INTERACTION BETWEEN THE ESTABLISHMENT OF HEPATITIS C VIRUS

INFECTION AND THE MUTATION AT AMINO ACID CYS508 OF HUMAN MAVS GENE

ValériaChamasMiura1,PaolaJ.S.Provazzi1,MaurícioL.Nogueira2,JoãoRenatoR.

Pinho3,PaulaRahal1

1.SãoPauloStateUniversity‑UNESP,DepartmentofBiology.SãoJosédoRioPreto/SP,Brazil.2.FacultyofMedicineofSãoJosédoRioPreto‑FAMERP,DepartmentofInfectiousandParasitaryDiseases.SãoJosédoRioPreto/SP,Brazil.3.SchoolofMedicine,UniversityofSãoPaulo‑USP,DepartmentofGastroenterology.SãoPaulo/SP,Brazil.

TheHepatitisCvirus(HCV)isconsideredthemajorcauseofliverdisease, is responsible for acute and chronic hepatitis, cirrhosis andhepatocellularcarcinoma.Itisestimatedthatabout200millionpeopleareinfectedworldwideandinBrazil,thisprevalenceis1,6%.TheHCVgenomehasasinglestrandRNAandencodesapolyproteinthat,whenprocessedbyviralandcellularproteases,generates10proteins(Core,E1,E2,p7,NS2,NS3,NS4A,NS4B,NS5AandNS5B).Recentstudieshaveshowntheexistenceofacatalyticinteractionbetweentheaminoacid serine139 (Ser139)of theviralproteaseNS3/4Aand theaminoacidcysteine508(Cys508)ofthehumanproteincalledMitochondrialAntiviralSignaling (MAVS), resulting in thecleavageof thishumanprotein,whichplaysanessentialroleininductionofInterferons.TheobjectiveofthisstudyistoinvestigatetheoccurrenceoftheaminoacidCys508substitutionatMAVSproteinin100patientspositiveforserumHCV: 50 patients with chronic hepatitis C that underwent standard

treatmentand50withpositiveresultsforviralRNA,buthadcuredthediseasespontaneouslyandtherefore,didnotundergothetreatment.Therewereperformed:DNAextraction,PCRamplification,purificationandsequencingofMAVS generegionofinterestinthechronicpatientsgroup.Thesequencesgeneratedwerealignedandthetripletofbases“TGC”atposition508wasobservedin100%ofthesequences,indicatingthatnopatienthasitscysteine508replaced.ThenextstepistoevaluatetheoccurrenceofthesubstitutionofCys508residueinthegroupofpatientswithspontaneoushealingtoseeifthenon‑establishmentofviralinfectionwasduetoamutationinMAVS gene,whichwouldguaranteetheintegrityoftheMAVSproteinandthereforeoftheimmuneresponse.

Financialsupport:PIBIC/CNPqandFapesp.

P29 - DISTRIBUIÇÃO DE HCV EM PLAQUETAS DURANTE TRATAMENTO ANTIVIRAL COM

INTERFERON E RIBAVIRINA

MPEspírito‑Santo1,AJDeAlmeida2,CFTYoschida1,CEBrandãoMello2,ELampe1

1InstitutoOswaldoCruz–Fiocruz,2UNIRIO

INTRODUÇÃO:A compartimentalizacão do HCV é definidacomo a distribuição não‑randômica de quasispecies virais em sítiosextra‑hepáticos, que podem servir como reservatórios do HCV, bemcomo influenciar a resposta ao tratamento antiviral. OBJETIVO:AvaliaradinâmicadequasispeciesdoHCVantes,duranteepós‑terapiaantiviralemsoroeplaquetassobrearespostaaotratamentoantiviral.MATERIALEMÉTODOS:Amostrasdesoroeplaquetascoletadasnopré‑tratamento,12asemana,e24asemanapós‑tratamentoantiviraldeumpacientedosexofeminino,45anos,infeccãocrônicapeloHCV‑1b,quenãoobteverespostavirológicacominterferonpeguiladoeribavirinaforamavaliadas.AdetecçãodeHCV‑RNAfoifeitaporRT‑nestedPCR,eprimersparaHCV‑1, regiãoNS5a.OsprodutosforaminseridosnovetorpCR4‑TOPOeclonadosemcélulasE.coli.Sequenciamosde19a44clonesdecadaamostra/período.NãodetectamosHCV‑RNAnofinaldotratamento,ena24asemanapós‑tratamentodetectamosapenasnosoro.OprogramaMega4.0foiutilizadoparaanálisedadistribuiçãodasquasispecies.RESULTADOS:Aanálisefilogenéticadasseqüênciasdas amostras de soro e de plaquetas no pré‑tratamento revelou queestes dois sítios formaram dois grupos distintos de quasispecies nãorelacionados entre si. Na 12a semana de tratamento a maioria dasquasispeciesdosdoissítiosformaramumúnicoclusterdequasispecies relacionadasentresi.Na24asemanapós‑tratamento,tambémobservamosaformaçãodecluster,entretantoalgumasquasispecies distribuíram‑senosclustersdosoroprée12asemanadetratamento.CONCLUSÃO:A compartimentalizacão do HCV em plaquetas foi evidenciada pelaformaçãodeclustersdequasispecies divergentesdasencontradasnosoropré‑tratamento,contudo,otratamentoantiviralpareceinfluenciara dinâmica da distribuição das quasispecies.A grande proximidadegenética das quasispecies pós‑tratamento com as do pré‑tratamentopareceindicarumaevoluçãodequasispeciesoriginariasdosoro,asquaissãoresistentesaotratamentoantiviral.


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AUTHOR INDEX

Almeida‑Mello,F.M. P1Almeida,B.S. P11AlonsoNeto,J.B. P25AlvaradoMora,M.V. P3,P8,P9Arcuri,H.A. P5,P12Barbour,J. L10Bensabath,G. L5Bertolini,D.A. P1Bertollo,L.A. P27Bigaton,G. P14Bittar,C. P10,P12,P24Botelho,L. P8,P9Brandao,C.E. P15,P29Carareto,C.M.A. P10Cardoso,J.F. P11Carrilho,F.J. L2,P3,P8,P9Carrington,M. L16Castro,V.D. P2Cavalcanti,N.C.S. P22Costa,A. P19Cristina,J. P18daSilva,A.C. P23daSilva,E.F. P20,P22Dastoli,G.T. P2DeAlmeida,A.J. P13,P29doO,K.R.M. P20,P21Dorval,M.E. P14Espirito‑Santo,M.P. P29Fabricio‑Silva,G.M. P11Fagundes,N.J.R. L13Felipe,M.S. P7,P26Ferreira,L.E. P15Figueiredo,F. P11Figueiredo,J.F. P23Franca,P.H.C. P15Froes,I.B. P14Galdino,A.S. P7,P26Garcia,R.F. P15Gomes‑Gouvea,M.S. P1,P3,P4,P22,P23Gomes,S.A. P14Gutierrez,M.F. P3Hoffmann,L. P6,P16Honda,E.R. P8,P9Horbach,I.S. P25Jardim,A.C.G. P10,P12,P24Kramvis,A. L14Kuniyoshi,A.S. P1Lampe,E. P13,P20,P21,P29Lazari,C. P22Lewis‑Ximenes,L.L. P20,P21Locarnini,S. L6Lopes,A.F. P1Lopes,J.R. P14

Lopez,F. P18López,L. P18Mangueira,C.L. P2Marques,P.S. P11Martinelli,A.L.C. P23Martins,R.B. P14Matos,R.A.M. P24Mattos,A. P15Medina,H.C. P20,P21Mendes‑Correa,M.C. L4,P4,P22,P23Meyer,D. L11Miretti,M. L18Miura,V.C. P28Moratorio,G. P18MottadeCastro,A.R. L15,P14Mousquer,G.J. P14Muerhoff,S. L1,L12Munos,G,P17Murat,P.G. P14Nabuco,L.C. P20Nader,L. P15Nascimento,D. P14Nascimento,H.C.L. P19Nogueira,M.L. P28Oliveira,E.C.P. P19Ono‑Nita,S.K. L3Oyakawa,L. P2Pádua,A.P. P4,P22,P23Palma,M.S. P5Pereira,F.M. P27Pereira,H.S. P25PeresdaSilva,A. P13Perez,R.M. P11Petroni,R.C. P2Picelli,C.G. P8,P9Pinho,J.R.R. L7,P2,P3,P4,P5,P8,P9,P10,

P12,P22,P23,P24,P28Pires,F.R. P14Porto,L.C. P11Provazzi,P.J.S. P5,P28Queiroz,A.T.L. P10Queiroz,M.S. P7Rahal,P. P5,P10,P12,P24,P28Ramos,A.L. P6Ramos,J.A. P6Ramos,O.S. P2Recarey,R. P18Rivera,M. P17Romano,C.M. P3Rondinelli,E. P6,P16Salcedo,J.M. P8,P9Santana,R.A. P2Santos,A.O. P8,P9


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Santos,E.D. P27Santos,J.C. P7,P26Santos,N.S. P27Seixas,A.C.S. P4Silva,M.H. P4Silva,R. P6,P16Single,R. L17Sitnik,R. P2Souza,M.Q. P26Stief,A.F. P14Tanuri,A. P16,P25Teles,S.A. P14Torres,C. P17Torres,F.A. P7,P26

Tovo,C. P15Uip,D.E. P4,P22,P23Urmenyi,T.P. P6,P16Vargas,P.S. P6Venegas,M. P17Vieira,D.S. P8,P9Villar,L.M. P20,P21Villela‑Nogueira,C.A. P6,P16,P20,P21Walker,C. L9Yamasaki,L.H.T. P10,P12,P24Yoshida,C.F.T. P20,P21,P29Zanotto,P.M. L8Zarife,M.A. P19,P27,

issn 0036-4665 issn 1678-9946 on line · 2021. 2. 10. · iii issn 0036-4665 issn 1678-9946 on line...

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