Development Of A Novel Approach To Establish HCV Replicon And Characterization Of Direct-acting Antiviral Agent Resistance Of Geneotype 3a And 6a HCV Isolates

Hepatitis C virus(HCV)is a positive-stranded RNA virus belonging to the Hepacivirus genus in the Flaviviridae family.HCV exists in forms of chronic infection in about 1% of total population worldwide,which greatly strains social resources.In recent years,advent of highly potent and safe direct antiviral agents(DAA)has greatly improved prognosis of HCV patients,yet clinical data indicates emergence of resistance associated mutations is still a formidable challenge.Most HCV patient sera cannot establish robust infection in hepatoma cell lines,which is still a great mystery.The first robust HCV infectious model was reported by 3 different labs in 2005,which all relied on consensus sequence of JFH1(Japanese fulminant hepatitis 1).Up till now,JFH consensus is the only one that could mount a robust infection in-vitro without any pre-requisite adaptation.Since HCV consensus sequence cannot reflect the de facto mixture of various HCV quasispecies that is circulating in patients,we speculated by screening these natural diversity in forms of recombinant HCV cDNA library,the chance of obtaining viable HCV cDNA sequence may be boosted.To that end,we developed an efficient approach in building library of HCV replicon cDNA clones,the viability of which is further tested in Sec14L2-expressing Huh 7.5.1 cell lines.Our functional screening approach resulted in generation of genotype 1b,3a,6a replicon clones,subsequent subcloning led to establishment of robust cDNA clone.Following sequencing analysis indicated that varying amount of quasispecies can be observed in these replicon clones.Our data indicated quasispecies can assist HCV replication in hepatoma cell lines.Research indicated that sofosbuvir resistance associated mutation(RAS)NS5B S282 T existed at a relative high frequency in base lines of GT6 patients,which suggests that GT6 strains generally may share a low genetic barrier to emergence of NS5 B S282T.To test our hypothesis,we investigated sofosbuvir resistance property of genotype 6a PR58C4 replicon cells.The result indicated that genetic barrier for NS5 B S282T to emerge in PR58C4 is significantly lower than that of Con1.NS5 B S282T confers sofosbuvir resistance in context of PR58C4,and K535 R may boost resistance capacity of S282 T.In conclusion,we established a method in constructing HCV replicon cDNA clones compatible with various genotypes.Establishment of GT1 b PR50,GT3 a PR87,GT6 a PR58 proved the effectiveness of our method.Our work in genotype 3a PR87 provided new insights as to the balance between RAS emergence and fitness maintenance.Our work in genotype 6a PR58 uncovered its low genetic barrier to S282 T emergence and possible ‘auxiliary’ RAS NS5 B K535R,which could facilitate understanding of complex RAS patterns observed in clinical settings.