Characterization of HCV replication and pathogenesis in animal model

[目的]报道了丙型肝炎病毒（HCV）通过肝内接种合成RNA传播的研究。然而，到目前为止，从这些黑猩猩或体外培养系统中拯救活的HCV颗粒还没有成功。本研究建立了丙型肝炎病毒基因组功能的反向遗传学系统。[方法]从慢性肝炎患者血浆中构建HCV全长cDNA克隆，并利用双核酶剪切系统表达HCV基因组mRNA。将该克隆的RNA转录物转染到HepG 2与人原代肝细胞融合的IMY-N9细胞中。[结果与结论]合成的HCVRNA和cDNA转染IMY-N9细胞可产生HCV感染，但在HepG 2细胞中不能支持HCV复制。通过免疫电子显微镜研究观察到培养上清液中密度为1.1 g/ml的颗粒。该颗粒被认为是成熟颗粒。该培养物上清液甚至在DNase和RNase处理后也含有HCV颗粒，并且可以重新感染幼稚IMY细胞并可以引起复制。这些结果表明，该cDNA克隆拯救了感染性HCV颗粒，为HCV的反向遗传学研究提供了可能。

[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.