Molecular Biology Of Hepatitis C Virus

丙型肝炎病毒的分子生物学

• 批准号: 6503690
• 负责人: Robert H. Purcell
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6503690
• 关键词: Callithricidae; Pan; biotechnology; enzyme linked immunosorbent assay; hepatitis C; hepatitis C virus; hepatitis vaccine; human therapy evaluation; human tissue; immunotherapy; interferons; microorganism immunology; molecular cloning; neutralizing antibody; patient oriented research; vaccine development; virus genetics

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, have been 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. With the reagents we have developed (infectious cDNA clones that are infectious by in vivo transfection; titered pools of polyclonal and monoclonal HCV representing different strains, subgenotypes and genotypes) we are pursuing a collaborative study of the immunopathogenesis of HCV infections. We have demonstrated that resolution versus progression to chronicity is a function of the host, not the virus, since infections with monoclonal viruses have yielded both results. We have shown that humoral immunity appears not to be important in control of the virus, at least in the control of infection or in preventing reinfection. We have demonstrated that sterilizing immunity can be achieved by repeated infection of chimpanzees, but that this sterilizing immunity is strain-specific. We are currently examining the role of CD4 versus CD8 cells in the control of HCV infection. When taken together, these studies will provide an in-depth analysis of humoral versus cellular immune responses to HCV infection in chimpanzees, a surrogate of man. 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. In other studies we have deleted the hypervariable region 1 (HVR1) of the E2 protein of HCV, the region that contains a neutralization epitope. Surprisingly, the deletion mutant virus was viable but attenuated when transfected into chimpanzees. We are also performing a mutational analysis of the gene that encodes a small protein designated "p7". An analogous protein is found in related pestiviruses. We have demonstrated that p7 in HCV is critical for replication and we are now mapping the critical region of the gene. 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 are currently using the GBV-B tamarin system to study characteristics of the virus that it shares with HCV, a virus 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. However, the transfected genome expresses large quantities of structural proteins in susceptible cells; such cells are a useful substrate for immunofluorescence studies. Similar studies are being carried out with dengue 4 virus and HCV. 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 the 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. Similar studies have been carried out in patients chronically infected with HCV and who were undergoing therapy with interferon. Patients could be separated into four groups, based upon their response to interferon therapy: a) long-term responders, b) those who relapsed following cessation of treatment, c) those who responded but had a break-through of viral replication while still on therapy and d) those who failed to respond (nonresponders). As with acutely infected HCV patients, distinctive patterns of dynamic change in the sequence and heterogeneity of HCV clones obtained early in therapy were predictive of outcome. Long-term responders demonstrated a marked decrease in heterogeneity of HCV, resulting in eradication of the virus. Relapsing patients also demonstrated a change in heterogeneity and a decrease in viral titer but a new dominant strain usually emerged following cessation of therapy. Nonresponders maintained the same dominant strain throughout therapy, suggesting that an interferon-resistant strain already existed before therapy. Patients who experienced a breakthrough during therapy had patterns that were similar to those of nonresponders, suggesting that this was a mixed group of patients. These findings may be useful in predicting the outcome of therapy with interferon early in the course of treatment.

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，已经在黑猩猩中进行了生物扩增，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。已从感染单克隆丙型肝炎病毒（通过体内转染感染性cDNA的RNA转录物获得）的黑猩猩中制备了感染性池;这些池已在其他黑猩猩中进行了感染性滴度。使用我们开发的试剂（通过体内转染具有感染性的感染性cDNA克隆;代表不同毒株、亚型和基因型的多克隆和单克隆HCV的滴定池），我们正在进行HCV感染免疫发病机制的合作研究。我们已经证明，缓解与进展为慢性是宿主的功能，而不是病毒，因为单克隆病毒感染产生了两种结果。我们已经证明，体液免疫似乎在控制病毒方面并不重要，至少在控制感染或预防再感染方面并不重要。我们已经证明，通过反复感染黑猩猩，可以获得绝育免疫，但这种绝育免疫是菌株特异性的。我们目前正在研究CD 4与CD 8细胞在控制HCV感染中的作用。当两者结合在一起，这些研究将提供一个深入的分析，在黑猩猩，人类的替代品HCV感染的体液与细胞免疫反应。此外，感染性的HCV的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。在其他研究中，我们删除了HCV E2蛋白的高变区1（HVR 1），该区域含有中和表位。令人惊讶的是，缺失突变病毒是可行的，但在转染到黑猩猩时减弱。我们还在对编码一种名为“p7”的小蛋白质的基因进行突变分析。在相关的瘟病毒中发现了类似的蛋白质。我们已经证明了HCV中的p7对复制至关重要，我们现在正在绘制该基因的关键区域。 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。我们目前正在使用GBV-B绢毛猴系统来研究它与HCV共有的病毒的特征，HCV是一种必须在黑猩猩中研究的病毒。 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，但在没有辅助病毒的情况下，所得的病毒产物不能组装成感染性病毒。然而，转染的基因组在易感细胞中表达大量的结构蛋白;这些细胞是免疫荧光研究的有用底物。目前正在对登革4型病毒和HCV进行类似的研究。 我们已经确定了从输血后感染的患者中回收的HCV分离株的遗传异质性。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。类似的研究也在慢性HCV感染患者中进行，这些患者正在接受干扰素治疗。根据患者对干扰素治疗的反应，可将其分为四组：a）长期应答者，B）停止治疗后复发者，c）应答但仍在治疗期间病毒复制突破者和d）未应答者（无应答者）。与急性感染的HCV患者一样，在治疗早期获得的HCV克隆的序列和异质性的动态变化的独特模式可预测结果。长期应答者表现出HCV异质性显著降低，导致病毒根除。复发患者也表现出异质性的变化和病毒滴度的降低，但通常在停止治疗后出现新的优势毒株。在整个治疗过程中，无应答者保持相同的优势菌株，这表明在治疗前已经存在干扰素耐药菌株。在治疗期间经历突破的患者的模式与无应答者相似，表明这是一组混合患者。这些发现可能有助于在治疗过程中早期预测干扰素治疗的结果。