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）是社区获得性病毒肝炎的主要原因。 HCV各种基因型的原型菌株（包括在该实验室中发现的一些基因型）在黑猩猩中在生物学上进行了扩增，包装和分布在被动和主动免疫预防的研究中用作挑战接种。黑猩猩通过体内肝转染。 正在遵循用感染性cDNA克隆转染的黑猩猩，以确定感染的自然病史。感染性池是由感染单克隆HCV的黑猩猩制备的（通过体内转染，用感染性cDNA的RNA转录得出）；这些已被替代其他黑猩猩的感染性。我们开发的试剂（通过体内转染具有感染性的传染性cDNA克隆；我们正在代表不同菌株，亚基型和基因型的多克隆和单克隆HCV池池）进行了对HCV感染免疫原理的协作研究。我们已经证明，分辨率与慢性的进展是宿主的函数，而不是病毒的函数，因为单克隆病毒的感染已经产生了这两种结果。我们已经表明，至少在控制感染或防止再感染方面，体液免疫似乎在控制病毒方面并不重要。我们已经证明，通过反复感染黑猩猩可以实现灭菌免疫力，但这种灭菌免疫是特异性的。我们目前正在研究CD4与CD8细胞在控制HCV感染中的作用。合并后，这些研究将对黑猩猩（人类的替代物）中对HCV感染的体液和细胞免疫反应进行深入分析。 此外，HCV的传染性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的各个部分已从全长克隆中删除，所得的缺失突变克隆通过缩写内转染接种到黑猩猩中。 NCR的某些区域已被确定为HCV体内复制至关重要。在其他研究中，我们删除了HCV的E2蛋白的高变量区域1（HVR1），该区域包含中和表位。令人惊讶的是，删除突变病毒是可行的，但是当转染黑猩猩时会衰减。我们还对编码小蛋白的基因进行了突变分析，称为“ P7”。在相关的Pestivires中发现了类似的蛋白质。我们已经证明，HCV中的P7对于复制至关重要，现在我们正在绘制基因的关键区域。 我们已经构建了GB病毒-B（GBV-B）的传染性cDNA克隆，这是一种猴子病毒，是最接近HCV的猴子病毒。此外，我们已经准备了GBV-B的挑战池，并确定了菜他蛋白中的感染性滴度。我们目前正在使用GBV-B tamarin系统来研究它与HCV共享的病毒的特征，该病毒是一种必须在黑猩猩中研究的病毒。 在其他研究中，我们从HCV和牛病毒腹泻病毒的传染性cDNA克隆中构建了嵌合基因组。这些基因组可以在转染的细胞中复制，但是在没有辅助病毒的情况下，由此产生的病毒产物不能组装成传染病。但是，转染的基因组在易感细胞中表达大量的结构蛋白。这些细胞是用于免疫荧光研究的有用底物。登革热4病毒和HCV也正在进行类似的研究。 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。确定了从患有暴发性肝炎的患者获得的多个克隆，从急性肝炎后恢复疗法的患者以及从第一次急性肝炎的患者中获得的多个患者，从患有暴发性肝炎的患者获得了多个克隆的序列和邻近的部分。暴发或解决肝炎的患者的克隆序列几乎没有变化，而从发展为慢性肝炎的患者的克隆序列序列发生了许多变化。因此，可以在感染的前几周预测HCV感染的结果。在长期感染HCV并接受干扰素治疗的患者中，已经进行了类似的研究。根据他们对干扰素治疗的反应，可以将患者分为四组：a）长期反应者，b）那些在停止治疗后复发的患者，c）那些反应但仍接受病毒复制的人，同时仍接受治疗，而d）那些未能反应的患者（无反应者）。与急性感染的HCV患者一样，在治疗早期获得的HCV克隆序列和异质性的动态变化的独特模式可以预测结果。长期响应者表明，HCV的异质性显着下降，导致该病毒的根除。复发患者还表现出异质性的变化和病毒滴度的减少，但在停止治疗后通常会出现新的显性菌株。在整个治疗过程中，无反应者保持相同的优势菌株，这表明在治疗前已经存在抗干扰素的菌株。在治疗期间经历突破的患者的模式与无反应者类似，这表明这是一群混合的患者。这些发现在治疗过程中早期预测干扰素治疗的结果可能很有用。