Characterization of HCV replication and pathogenesis in animal model

[目的]丙型肝炎病毒(丙型肝炎病毒)通过肝内接种合成RNA传播的研究已有报道。然而，到目前为止，从这些黑猩猩或体外培养系统中抢救出可存活的丙型肝炎病毒颗粒还没有成功。在本研究中，我们建立了丙型肝炎病毒的反向遗传系统，以研究其基因组功能。[方法]从慢性肝炎患者血浆中构建完整的丙型肝炎病毒c DNA克隆，并利用双核酶剪裁系统表达丙型肝炎病毒基因组的准确m RNA。将该克隆的RNA转录物导入人原代肝细胞与HepG2融合细胞系Imy-N9。[结果与结论]将合成的丙型肝炎病毒RNA和c DNA导入Imy-N9细胞可感染丙型肝炎病毒，但在HepG2细胞中不能支持丙型肝炎病毒的复制。免疫电子显微镜观察培养上清液中密度为1.1g/ml的颗粒。该颗粒被认为是成熟颗粒。这种培养上清液即使在DNase和RNase处理后仍含有丙型肝炎病毒颗粒，并可能再次感染幼稚的Imy细胞，并可能导致复制。这些结果表明，该克隆挽救了具有感染性的丙型肝炎病毒颗粒，为逆转丙型肝炎病毒的遗传途径开辟了可能。

[Purpose] Previous studies of transmission of hepatitis C virus (HCV) by intrahepatic inoculation with synthetic RNA have been reported. However, rescue of viable HCV particle from these chimpanzees or in vitro culture system had not be successful, so far. In this study, we established reverse genetical system of HCV to characterize genome function. [Methods] We constructed the entire HCV cDNA clone from chronic hepatitis patient plasma and expressed exact mRNA of HCV genome using double ribozyme trimming system. The RNA transcripts from this clone have been transfected into IMY-N9 cells, which was the fused cell line of HepG2 and human primaly hepatocyte. [Results and conclusions] Transfection of the synthetic HCV RNA and cDNA into IMY-N9 cells produced infectios HCV, however in HepG2 cells, they could not support HCV replication. The particle with density of 1.1 g/ml in the culture supernatant was visualized by immunoelectron microscopic study. This particle was considered to be the mature particle. This culture supernatant contained HCV particles even after the DNase and RNase treatment and could re-infect to the naive IMY cells and could cause replication. These results indicated that this cDNA clone rescued the infectious HCV particle and has opened the possibility to the reverse genetical approach of HCV.