Characterization of HCV replication and pathogenesis in animal model

【目的】已有文献报道了人工合成RNA肝内接种丙型肝炎病毒（HCV）传播的研究。然而，迄今为止，从这些黑猩猩或体外培养系统中拯救活的HCV颗粒尚未成功。在这项研究中，我们建立了HCV的反向遗传系统来表征基因组的功能。[方法]从慢性肝炎患者血浆中构建HCV cDNA全克隆，利用双核酶切边系统精确表达HCV基因组mRNA。将该克隆的RNA转录本转染到HepG2与人原代肝细胞融合细胞系IMY-N9细胞中。【结果与结论】将合成的HCV RNA和cDNA转染到IMY-N9细胞中可产生感染性HCV，但在HepG2细胞中不能支持HCV复制。免疫电镜观察培养上清中密度为1.1 g/ml的颗粒。该颗粒被认为是成熟颗粒。即使在DNase和RNase处理后，该培养上清仍含有HCV颗粒，并且可以再次感染初始IMY细胞并引起复制。这些结果表明，该cDNA克隆挽救了感染性HCV颗粒，并为HCV的反向遗传方法开辟了可能性。

[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.