Development of plaque assay to isolate cytopathic HCV clones

HCV-JFH 1产生细胞病变斑的亚克隆（Sekine-Osajima Y，et al.，Virology 2008 ; 371：71）。在这里，我们研究了细胞病变突变HCV-JFH 1克隆中的病毒氨基酸取代及其体外和体内特征。具有单独的C2441 S、P2938 S或R2985 P特征替换以及具有所有三种替换的突变体病毒在体外显示出比亲本JFH 1显著更高的细胞内复制效率和更大的细胞病变效应。在感染的早期，突变型HCV感染小鼠的血清HCVRNA和ER应激相关蛋白的表达水平显著升高。在接种后8周，这些特征突变已恢复为野生型序列。HCV诱导的细胞致病性与细胞内病毒复制水平相关，并由HCV-NS 5A和NS 5 B区域的某些氨基酸取代决定。细胞病变的HCV克隆在体内表现出高复制能力，但在感染的早期阶段可能被消除

HCV-JFH1 yields subclones that develop cytopathic plaques(Sekine-Osajima Y, et al., Virology 2008 ; 371 : 71). Here, we investigated viral amino acid substitutions in cytopathic mutant HCV-JFH1 clones and their characteristics in vitro and in vivo. The mutant viruses with individual C2441S, P2938S or R2985P signature substitutions, and with all three substitutions, showed significantly higher intracellular replication efficiencies and greater cytopathic effects than the parental JFH1 in vitro. The mutant HCV-inoculated mice showed significantly higher serum HCV RNA and higher level of expression of ER stress-related proteins in early period of infection. At 8 weeks post inoculation, these signature mutations had reverted to the wild type sequences. HCV-induced cytopathogenicity is associated with the level of intracellular viral replication and is determined by certain amino acid substitutions in HCV-NS5A and NS5B regions. The cytopathic HCV clones exhibit high replication competence in vivo but may be eliminated during the early stages of infection