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 genotype 3b isolates from three treatment naïve chronic hepatitis C patients, and by clonal analysis of the entire coding sequence demonstrated heterogeneous genome population 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 immunofluorescence 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 log ₁₀ 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. IMPORTANCE HCV genotype 3b is a difficult-to-treat subtype, associated with accelerated progression of liver disease and resistance to antivirals. Moreover, its prevalence has significantly increased among persons who inject drugs posing a serious risk of transmission in the general population. Thus, more genetic information and antiviral testing systems are required to develop novel therapeutic options for this genotype 3 subtype. We determined the complete genomic sequence and complexity of three genotype 3b isolates, which will be beneficial to study its biology and evolution. Furthermore, we developed a full-length in vivo infectious cDNA clone of genotype 3b and showed its robustness and genetic stability in human-liver chimeric mice. This is, to our knowledge the first reported infectious cDNA clone of HCV genotype 3b and will provide a valuable tool to evaluate antivirals and neutralizing antibodies in vivo, as well as in the development of infectious cell culture systems required for further research.