Establishment of infectious genotype 4 cell culture-derived hepatitis C virus

To establish infectious genotype 4a (GT4a) cell culture-derived hepatitis C virus (HCVcc), we constructed full-length ED43 and 12 mutants possessing single or double mutations that increase ED43 replicon replication, and performed cell culture after RNA transfection. Sequential long-term culture of full-length ED43 RNA-transfected cells showed increased viral production in two ED43 mutants named ED43 QK/SI and TR/SI among the tested clones. These ED43 mutants possessed a common mutation, R1405G, in the NS3 helicase region and another mutation, D2413G or V2414A, in the NS5a-NS5b cleavage site. Furthermore, serial reinfection of naïve Huh7.5.1 cells accelerated peak HCV production at an earlier time point after every infection. After the fourth infection, we found a common mutation, R1405G, and six additional mutations in both ED43 QK/SI and TR/SI mutants. All seven mutations supported continuous viral production for more than 40 days in both ED43 QS-7M (QK/SI with seven mutations) and ED43 TS-7M (TR/SI with seven mutations). In addition, ED43 TS-7M did not require additional mutations for continuous virus culture up to 124 days. Both ED43 QS-7M and TS-7M were sensitive to the neutralizing E2 antibodies HCV1 and AR3A and the direct-acting antivirals, simeprevir, ledipasvir and sofosbuvir. In conclusion, we established an infectious ED43 strain containing adaptive mutations, which is important for the analysis of HCV genotype-specific pathogenesis, development of pan-genotypic agents and analysis of drug resistance.