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。 HCV的各种基因型的原型菌株，包括在本实验室中发现的一些，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等研究中的挑战接种物。HCV的全长cDNA克隆（基因型1a，1b和2a）已经构建并转录RNA，用于通过体内肝转染将丙型肝炎病毒传播给黑猩猩。用HCV感染性cDNA克隆转染的黑猩猩，正在被跟踪以确定感染的自然史。 已从感染单克隆HCV（通过感染性cDNA的RNA转录物体内转染获得）的黑猩猩中制备了感染性样本池;已对这些样本池在其他黑猩猩中的感染性进行了滴定。此外，HCV感染性cDNA克隆的可用性首次允许基因组区域的突变分析。 例如，从全长克隆中缺失3 ′ NCR的各个部分，并通过肝内转染将所得缺失突变体克隆接种到黑猩猩中。NCR的某些区域已被鉴定为对HCV的体内复制至关重要。 我们已经构建了GB病毒-B（GBV-B）的感染性cDNA克隆，GBV-B是一种与HCV最接近的猴病毒。 此外，我们还制备了GBV-B的挑战池，并测定了它们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HCV共有的病毒的特征，这必须在黑猩猩中进行研究。在其他研究中，我们已经构建了嵌合基因组从感染性的cDNA克隆的HCV和牛病毒性腹泻病毒。 这些基因组可以在转染的细胞中复制，但所产生的病毒产物不能组装成感染性病毒在没有辅助病毒。我们已经确定了HCV分离株的遗传异质性，这些分离株从输血后感染的患者中回收。 测定了从暴发性肝炎患者、急性肝炎后恢复期患者和进展为慢性丙型肝炎患者获得的多个克隆的包膜蛋白1和2的高变区和相邻部分的序列。 在感染的前几周内，观察到克隆序列动态变化的独特模式。 暴发性或缓解性肝炎患者的克隆序列几乎没有变化，而进展为慢性肝炎的患者的克隆序列有许多变化。 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