MOLECULAR BIOLOGY OF HEPATITIS C VIRUS

丙型肝炎病毒(HCV)是社区获得性病毒性肝炎的主要原因。包括本实验室发现的一些不同类型的丙型肝炎病毒的原型毒株正在黑猩猩体内进行生物扩增，包装和分发作为挑战接种，用于被动和主动免疫预防等研究。已构建了丙型肝炎病毒的全长cDNA克隆(1a、1b和2a)，并通过体内肝转移将RNA转录用于将丙型肝炎病毒传播给黑猩猩。用丙型肝炎病毒感染性克隆的黑猩猩进行跟踪研究，以确定感染的自然病史。已经从感染了单克隆性丙型肝炎病毒的黑猩猩身上制备了感染池(通过体内转染法获得感染性cDNARNA转录本)；这些感染池已经在其他黑猩猩中进行了感染性滴定。此外，丙型肝炎病毒感染性克隆的出现首次使基因组区域的突变分析成为可能。例如，3‘NCR的个别部分已从全长克隆中删除，并通过肝内转基因将由此产生的缺失突变克隆接种到黑猩猩体内。NCR的某些区域已被确定为在体内复制丙型肝炎病毒的关键。我们构建了与丙型肝炎病毒关系最近的猴病毒GB病毒B(GBV-B)的感染性cDNA克隆。此外，我们已经准备了GBV-B的攻击池，并测定了这些攻击池在罗望子素中的传染性滴度。我们计划使用GBV-B Tamarin系统来研究它与丙型肝炎病毒共享的病毒的特征，这必须在黑猩猩身上进行研究。在其他研究中，我们用丙型肝炎病毒和牛病毒性腹泻病毒的感染性克隆构建了嵌合基因组。这些基因组可以在转基因细胞中复制，但在没有辅助病毒的情况下，产生的病毒产物不能组装成具有感染性的病毒。我们已经确定了从输血后感染的患者身上恢复的丙型肝炎病毒分离株的遗传异质性。对从重型肝炎患者、急性肝炎恢复期患者和慢性丙型肝炎患者获得的多个克隆的包膜蛋白1和2的高变区及其相邻部分的序列进行了测定，观察到在感染的最初几周内克隆序列的动态变化的独特模式。重型肝炎和消退型肝炎患者克隆序列变化不大，而慢性肝炎患者克隆序列变化较大。因此，丙型肝炎病毒感染的结局可以在感染的最初几周内预测出来。

Hepatitis C virus (HCV) is a major cause of community-acquired viral hepatitis. Prototype strains of the various genotypes of HCV, including some of those discovered in this laboratory, are being biologically amplified in chimpanzees, packaged and distributed for use as challenge inocula in studies of passive and active immunoprophylaxis, etc. Full-length cDNA clones of HCV (genotypes 1a, 1b and 2a) have been constructed and transcribed RNA used to transmit hepatitis C to chimpanzees by in vivo hepatic transfection. Chimpanzees, transfected with infectious cDNA clones of HCV, are being followed to determine the natural history of infection. Infectivity pools have been prepared from chimpanzees infected with monoclonal HCV (derived by in vivo transfection with RNA transcripts of infectious cDNA); these have been titered for infectivity in other chimpanzees. In addition, the availability of infectious cDNA clones of HCV has permitted for the first time a mutational analysis of genomic regions. For example, individual portions of the 3' NCR have been deleted from the full-length clone and the resultant deletion mutant clones inoculated into chimpanzees by intrahapatic transfection. Certain regions of the NCR have been identified as critical for in vivo replication of HCV. We have constructed an infectious cDNA clone of GB virus-B (GBV-B), a monkey virus that is the closest relative to HCV. In addition, we have prepared challenge pools of GBV-B and have determined the infectivity titer of these in tamarins. We plan to use the GBV-B tamarin system to study characteristics of the virus that it shares with HCV, which must be studied in chimpanzees. In other studies, we have constructed chimeric genomes from infectious cDNA clones of HCV and bovine viral diarrhea virus. These genomes can replicate in transfected cells but the resultant viral products cannot assemble into infectious virus in the absence of helper virus.We have determined the genetic heterogeneity of HCV isolates that were recovered from patients who were infected following transfusion. The sequence of the hypervariable region and adjacent portions of envelope proteins 1 and 2 were determined for multiple clones obtained from patients who had fulminant hepatitis, from patients who convalesced following acute hepatitis and from patients who progressed to chronic hepatitis C. Distinctive patterns of dynamic change in sequence of clones during the first several weeks of infection were observed. Patients with fulminant or resolving hepatitis had few changes in the sequences of clones, whereas there were many changes in the sequences of clones from patients who progressed to chronic hepatitis. Thus, the outcome of an HCV infection could be predicted in the first few weeks of the infection