西亞試劑：A novel strategy to develop robust infectious hepatitis C v

A novel strategy to develop robust infectious hepatitis C virus cell culture system directly from a clinical isolate

Jie Lu1， Yu Xiang1， Wanyin Tao1， Qingchao Li1， Na Wang1， Yongfeng Gao1， Xiaogang Xiang2， Qing Xie2 and Jin Zhong1

HCV infection is a leading cause of chronic liver diseases. The progress in HCV field was greatly enhanced by constructing infectious cDNA clone of JFH-1. Since then， JFH-1-based intra- and inter-genotypic recombinants were developed and allowed the study of vaccines and antiviral inhibitors for all genotypes. Only until recently， highly efficient HCV culture systems have been established by using consensus sequence-based clones. In this study， we developed a novel strategy to construct infectious HCV cDNA clone by combining functional screening of sequences directly from a genotype 2a clinical isolate (PR63) and cell culture adaptation. Using JFH-1 cDNA as the starting backbone， we sequentially replaced the JFH-1 fragments with a sequence from the pools of PR63 sequences. Through engineering adaptive mutations that improve HCV infectivity， we finally established a full-length cell culture-derived infectious clone of PR63， named PR63cc， which could efficiently produce virus particles in Huh7 derived cells， with peak titers of 1.6×105 ffu/mL. The PR63cc could be neutralized by an anti-E2 antibody and inhibited by antiviral agents， but appeared more resistant to an NS5A inhibitor than JFH-1. In summary： we developed a new approach to construct infectious HCV cDNA clone that can produce viruses efficiently in cell culture. This approach could be applied to other viral isolates， with potential implications for individualized treatments of HCV patients.

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