Cell Culture Replication of a Genotype 1b Hepatitis C Virus Isolate Cloned from a Patient Who Underwent Liver Transplantation 英文参考文献.docVIP

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Cell Culture Replication of a Genotype 1b Hepatitis C Virus Isolate Cloned from a Patient Who Underwent Liver Transplantation 英文参考文献.doc

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Cell Culture Replication of a Genotype 1b Hepatitis C Virus Isolate Cloned from a Patient Who Underwent Liver Transplantation 英文参考文献

CellCultureReplicationofaGenotype1bHepatitisC VirusIsolateClonedfromaPatientWhoUnderwentLiver Transplantation GeorgeKoutsoudakis*,SofiaPerez-del-Pulgar,MaireneCoto-Llerena,PatriciaGonzalez,JakubDragun, LauraMensa,GonzaloCrespo,MiguelNavasa,XavierForns Liver Unit, Institut D’Investigacions Biome`dics August Pi i Sunyer, Centro de Investigacio′n Biome′dica en Red: Enfermedades Hepa′ticas y Digestivas, Hospital Cl?′nic, Barcelona,Spain Abstract Theintroductionofthegenotype2aisolateJFH1wasamajorbreakthroughinthefieldofhepatitisCvirus(HCV),allowing researchers to study the complete life cycle of the virus in cell culture. However, fully competent culture systems encompassing the most therapeutically relevant HCV genotypes are still lacking, especially for the highly drug-resistant genotype 1b. For most isolated HCV clones, efficient replication in cultured hepatoma cells requires the introduction of replication-enhancingmutations.However,suchmutationsmayinterferewithviralassembly,asoccursinthecaseofthe genotype1bisolateCon1.Inthisstudy,weshowthataclinicalserumcarryingagenotype1bviruswithanexceptionally highviralloadwasabletoinfectHuh7.5cells.Similartopreviousreports,inoculationofHuh7.5cellsbynaturalvirusisvery inefficientcomparedtoinfectionbycellcultureHCV.Aconsensussequenceofanewgenotype1bHCVisolatewascloned fromtheclinicalserum(designatedBarcelonaHCV1),andthensubjectedtoreplicationstudies.Thisvirusreplicatedpoorly inatransientfashioninHuh7.5cellsafterelectroporationwithinvitrotranscribedRNA.Nonetheless,approximately3weeks postelectroporationandthereafter,coreprotein-positivecellsweredetectedbyimmunofluorescence.Surprisingly,small amounts of core protein were also measurable in the supernatant of electroporated cells, suggesting that HCV particles mightbeassembledandreleased.OurfindingsnotonlyenhancethecurrentmethodofcloninginvitroHCVreplication- competentisolates,butalsooffervaluableinsightsfortherealizationoffullycompetentculturesystemsforHCV. Citation: Koutsoudakis