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病因。各种HCV基因型的原型菌株，包括本实验室发现的一些，正在黑猩猩中进行生物扩增，包装和分发，用于被动和主动免疫预防研究中的挑战疫苗等。HCV全长cDNA克隆（基因型1a、1b和2a）已被构建，并转录RNA，通过体内肝转染将丙型肝炎传播给黑猩猩。研究人员对感染了丙型肝炎病毒的感染性cDNA克隆的黑猩猩进行了跟踪，以确定感染的自然历史。从感染单克隆HCV的黑猩猩中制备了传染性池（通过体内转染感染性cDNA的RNA转录物获得）；这些病毒已经在其他黑猩猩中进行了感染滴度检测。此外，HCV传染性cDNA克隆的可用性首次允许对基因组区域进行突变分析。例如，3' NCR的个别部分已从全长克隆中删除，由此产生的删除突变克隆通过肝内转染接种到黑猩猩中。NCR的某些区域已被确定为HCV体内复制的关键区域。我们构建了与HCV最接近的猴病毒GB病毒- b （GBV-B）的感染性cDNA克隆。此外，我们还准备了GBV-B攻毒池，并确定了这些攻毒池在绢毛猴中的感染性滴度。我们计划使用GBV-B绢毛猴系统来研究它与丙型肝炎病毒共有的病毒特征，这必须在黑猩猩中进行研究。在其他研究中，我们从HCV和牛病毒性腹泻病毒的感染性cDNA克隆构建了嵌合基因组。这些基因组可以在转染的细胞中复制，但在没有辅助病毒的情况下，所产生的病毒产物不能组装成感染性病毒。我们已经确定了从输血后感染的患者中恢复的HCV分离株的遗传异质性。从暴发性肝炎患者、急性肝炎恢复期患者和进展为慢性丙型肝炎患者获得的多个克隆中，测定了高变区和邻近包膜蛋白1和2的序列。在感染的最初几周内，观察到克隆序列的动态变化的独特模式。暴发性或消退性肝炎患者的克隆序列变化不大，而进展为慢性肝炎患者的克隆序列变化较多。因此，HCV感染的结果可以在感染的最初几周内预测

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