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，已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攻击疫苗等。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。利用我们开发的试剂（通过体内转染具有传染性的感染性cDNA克隆；代表不同菌株、亚基因型和基因型的多克隆和单克隆HCV滴度池），我们正在进行HCV感染的免疫发病机制的合作研究。我们已经证明，缓解与进展到慢性是宿主的功能，而不是病毒的功能，因为单克隆病毒感染产生了这两种结果。我们已经表明，体液免疫在控制病毒方面似乎并不重要，至少在控制感染或防止再感染方面并不重要。我们已经证明，绝育免疫可以通过反复感染黑猩猩来实现，但这种绝育免疫是菌株特异性的。我们目前正在研究CD4和CD8细胞在控制HCV感染中的作用。综合起来，这些研究将提供对黑猩猩（人类的替代品）对HCV感染的体液免疫与细胞免疫反应的深入分析。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。在其他研究中，我们删除了HCV E2蛋白的高变区1 (HVR1)，该区域包含一个中和表位。令人惊讶的是，这种缺失突变病毒是可以存活的，但在将其转染到黑猩猩体内后却减弱了。我们还对编码一种名为“p7”的小蛋白质的基因进行突变分析。在相关的鼠疫病毒中发现了类似的蛋白质。我们已经证明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。我们目前正在使用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，丙型肝炎病毒必须在黑猩猩身上进行研究。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。然而，转染后的基因组在易感细胞中表达大量的结构蛋白；这种细胞是免疫荧光研究的有用底物。正在对登革热4型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，测定了包膜蛋白1和2的高变区和邻近部分的序列。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。在慢性丙型肝炎病毒感染者和正在接受干扰素治疗的患者中也进行了类似的研究。根据患者对干扰素治疗的反应，可将患者分为四组：a)长期反应者；b)停止治疗后复发者；c)有反应但在治疗期间病毒复制取得突破者；d)无反应者（无反应者）。与急性感染HCV患者一样，在治疗早期获得的HCV克隆序列的动态变化和异质性的独特模式可预测结果。长期应答者表现出HCV异质性的显著降低，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降低，但通常在停止治疗后出现新的优势菌株。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。在治疗过程中经历突破的患者的模式与无反应的患者相似，这表明这是一个混合的患者群体。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.