Full-length sequence analysis of hepatitis C virus genotype 3b strains and development of an in vivo infectious 3b cDNA clone

Worldwide, genotype 3 is the second most prevalent major variant among patients with chronic hepatitis C virus (HCV) infection and the most difficult to treat with direct-acting antivirals (DAAs). Further, subtype 3b, which is highly prevalent in Southeast Asia with increasing transmission in high-risk populations, carries paired NS5A resistance-associated substitutions (RAS), NS5A-A30K+L31M, conferring resistance to DAA therapy and lowering cure rates with pan-genotypic regimens. However, no complete genomic sequence or infectious clone exists for HCV genotype 3b. We determined the entire genome sequences, including 5 ' and 3 ' termini, of HCV geno type 3b isolates from three treatment na & iuml;ve chronic hepatitis C patients, and by clonal analysis of the entire coding sequence demonstrated heterogeneous genome popula tion compositions all carrying RAS A30K+L31M in NS5A. We generated a full-length HCV genotype 3b cDNA clone (pODN) and transfected Huh7.5 and Huh-Lunet/SEC14L2 cells with derived RNA transcripts without detecting HCV antigens by immunofluorescenc e staining. In contrast, intrahepatic transfection with RNA transcripts from pODN, and subsequent virus passages, in human-liver chimeric mice resulted in robust infection with serum HCV RNA titers of up to 7.9 log10 genome equivalents/mL. Consensus HCV sequences of virus recovered from the transfected mouse contained no coding mutations exceeding 5% frequency, and sequences from the passage-infected mice likewise had no consensus changes. Thus, we developed the first HCV genotype 3b full-length cDNA clone which by its infectivity and genetic stability in human-liver chimeric mice proved functionality, and potential utility in future development of infectious cell culture systems needed for this DAA treatment-resistant subtype.