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。各种HCV基因型的原型株，包括本实验室发现的一些原型株，已在黑猩猩中进行了生物学扩增，包装和分发用作被动和主动免疫预防研究等中的攻击接种物。HCV的全长cDNA克隆（基因型1a、1b和2a）已被构建并转录用于通过体内肝转染将丙型肝炎传播给黑猩猩的RNA。 正在对转染 HCV 传染性 cDNA 克隆的黑猩猩进行跟踪以确定感染的自然史。已从感染单克隆 HCV 的黑猩猩中制备了感染库（通过体内感染性 cDNA 的 RNA 转录本转染而得）；这些病毒已在其他黑猩猩中进行了感染滴度测定。利用我们开发的试剂（通过体内转染具有传染性的传染性 cDNA 克隆；代表不同毒株、亚基因型和基因型的多克隆和单克隆 HCV 滴度池），我们正在开展 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，而不是病毒的功能，因为单克隆病毒的感染已经产生了这两种结果。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如此。我们已经证明，通过反复感染黑猩猩可以实现绝育免疫力，但这种绝育免疫力是品系特异性的。我们目前正在研究 CD4 细胞与 CD8 细胞在控制 HCV 感染中的作用。综合起来，这些研究将深入分析黑猩猩（人类的替代品）对 HCV 感染的体液免疫和细胞免疫反应。 此外，HCV感染性cDNA克隆的出现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，并将所得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对于 HCV 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 (HVR1)，该区域包含中和表位。令人惊讶的是，缺失突变病毒是有活力的，但在转染到黑猩猩体内后减毒。我们还对编码名为“p7”的小蛋白质的基因进行突变分析。在相关瘟病毒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，我们现在正在绘制该基因的关键区域。 我们构建了 GB 病毒-B (GBV-B) 的感染性 cDNA 克隆，GB 病毒是与 HCV 最接近的猴病毒。此外，我们还准备了 GBV-B 攻击池，并测定了它们在狨猴中的感染滴度。我们目前正在使用 GBV-B 狨猴系统来研究它与 HCV 共有的病毒特征，而 HCV 是一种必须在黑猩猩身上进行研究的病毒。 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细胞中复制，但在没有辅助病毒的情况下，所得病毒产物无法组装成感染性病毒。然而，转染的基因组在易感细胞中表达大量结构蛋白；此类细胞是免疫荧光研究的有用底物。目前正在对登革热 4 病毒和丙型肝炎病毒进行类似的研究。 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、从急性肝炎后恢复的患者以及从进展为慢性丙型肝炎的患者获得的多个克隆的高变区和邻近部分的包膜蛋白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则有很多变化。因此，HCV 感染的结果可以在感染的最初几周内预测。在慢性丙型肝炎病毒感染且正在接受干扰素治疗的患者中也进行了类似的研究。根据对干扰素治疗的反应，患者可以分为四组：a）长期反应者，b）停止治疗后复发的人，c）有反应但仍在治疗期间病毒复制突破的人，d）没有反应的人（无反应者）。与急性感染的 HCV 患者一样，治疗早期获得的 HCV 克隆序列动态变化和异质性的独特模式可以预测结果。长期反应者表现出丙型肝炎病毒的异质性显着降低，从而导致病毒被根除。复发患者也表现出异质性的变化和病毒滴度的下降，但在治疗停止后通常会出现新的优势毒株。无反应者在整个治疗过程中保持相同的优势菌株，这表明在治疗前就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反应者相似，表明这是一组混合的患者。这些发现可能有助于在治疗过程的早期预测干扰素治疗的结果。

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.