Animal And Culture Models of Viral Hepatitis And Hepatoc

虽然丙型肝炎病毒（HCV）是世界范围内发病率和死亡率的主要原因，但病毒基因表达对感染细胞的影响仍不清楚。努力开发丙型肝炎的培养和动物模型对于了解病毒及其引起的疾病至关重要。我们的合作者先前已经表明，从暴发性肝炎患者中克隆的JFH-1基因型2a株的亚基因组复制子在细胞培养中有效复制。在最近的一项研究中，我们发现全长JFH-1基因组在转染到人肝癌细胞系后有效复制并分泌病毒颗粒。病毒颗粒的密度为约1.17 μ g/ml，平均直径为约55 nm。分泌的病毒对Huh 7细胞具有感染性，并且感染性可以被CD 81特异性抗体以及来自慢性感染患者的免疫球蛋白中和。细胞培养产生的HCV在黑猩猩中具有感染性。该系统为研究病毒的生命周期和开发抗病毒策略提供了强有力的工具。 基于四环素调控系统，我们建立了一个双元转基因模型，其中两个转基因，SV 40 T抗原（TAg）和LacZ的条件表达，可以通过四环素管理在肝脏中紧密调控。tTA和TAg转基因小鼠发展为肝细胞腺瘤和增生，可通过持续四环素给药预防。我们已经采用这种转基因系统，利用该模型系统在肝脏中调节HCV蛋白的表达。对Alb-tTA/TRE-HCV双转基因小鼠的分析表明，四环素给药可以完全抑制肝脏中HCV RNA和蛋白的表达，并且四环素停药可以以可逆的方式诱导肝脏中HCV RNA和蛋白的表达。HCV组成型表达的小鼠在11个月大之前没有肝脏病理学证据，此时脂肪变性发生。相比之下，四环素介导的抑制，然后在2个月大时停药的小鼠在4个月大时出现肝脏炎症伴ALT升高。肝脏脂肪变性在5月龄时也变得明显，这比具有组成性HCV表达的小鼠早得多。在连续接受四环素治疗的小鼠中未观察到肝脏炎症或脂肪变性。由于HCV的表达受到严格调控，该模型类似于自然感染中观察到的那些事件，并为研究丙型肝炎的发病机制提供了有价值的工具。 我们还启动了对黑猩猩的研究，黑猩猩是丙型肝炎唯一的传染性动物模型。我们构建了HCV基因型1b的分子感染性克隆，并研究了四只黑猩猩接种这种单型接种物的详细病毒学、血清学和免疫学参数。其中两只黑猩猩清除了感染，两只成为持续感染。在所有四只黑猩猩的急性期只检测到微弱和短暂的T辅助反应。使用cDNA微阵列分析在感染过程中收集的肝活检样品，以表征一组广泛的宿主基因的表达水平。对于表现出强烈的病毒血症（> 10 E5基因组/ml）和肝损伤证据（典型的急性HCV感染）的三只动物，在感染的前8周期间观察到一组I型干扰素调节基因的诱导，而不管动物最终是否清除了感染。然而，某些与信号转导/细胞生长相关的基因的诱导可能与病毒清除特异性相关。在一只黑猩猩的病毒清除阶段，细胞免疫的肝内激活，特别是CD 8 + T细胞的标志物，是明显的。另一方面，尽管先天I型干扰素反应的诱导同样强烈，但继续慢性感染的两只黑猩猩没有表现出这种适应性免疫反应的证据。这些结果与其他将体外肝脏CD 8 + T细胞功能与清除率相关的报告一致，并且它们进一步支持了强有力的肝内免疫应答对于病毒清除很重要的观点。这种观察到的差异可能在于慢性感染的黑猩猩无法激活从先天免疫向适应性免疫过渡的途径。表现出弱病毒血症（<500个基因组/ml）和快速清除感染（<8周）的第四只动物没有可检测到的I型干扰素调节基因或T细胞应答标志物的诱导。这只黑猩猩的感染与可能经常发生在人类身上的亚临床感染一致。这一结果表明，HCV暴露可以通过先天免疫来控制，如果这一过程在感染的初始阶段有效，则适应性免疫应答可能不是病毒清除所必需的。这些动物模型不仅对解决HCV基因产物的免疫发病机制和细胞病变潜力的问题，而且对研究HCV在体内的复制是有价值的。

Although hepatitis C virus (HCV) is a leading cause of morbidity and mortality worldwide, the effects of viral gene expression on infected cells remain unclear in vivo. Efforts to develop culture and animal models for hepatitis C are critical for the understanding of the virus and the disease it causes. Our collaborators had previously shown that subgenomic replicon of the JFH-1 genotype 2a strain cloned from a patient with fulminant hepatitis replicates efficiently in cell culture. In a recent study, we showed that the full-length JFH-1 genome replicates efficiently and secrets viral particles after transfection into a human hepatoma cell line. The viral particles have a density of about 1.17 g/ml with an average diameter of about 55 nm. The secreted virus is infectious for Huh7 cells and infectivity can be neutralized by CD81-specific antibodies as well as immunoglobulins from chronically infected patients. The cell culture-generated HCV is infectious in chimpanzee. This system provides a powerful tool for studying the viral life cycle and developing anti-viral strategies. Based on the tetracycline regulatory system, we established a binary transgenic model in which the conditional expression of two transgenes, SV40 T antigen (TAg) and LacZ, can be tightly regulated in the liver by administration of tetracycline. Mice with tTA and TAg transgenes developed hepatocellular adenomas and hyperplasia that could be prevented by continuous tetracycline administration. We have adopted this transgenic system to the regulated expression of HCV proteins in the liver using this model system. Analysis of Alb-tTA/TRE-HCV double transgenic mice revealed that the HCV RNA and protein expression in the liver could be completely suppressed by tetracycline administration, and induced in a reversible fashion by tetracycline withdrawal. Mice with constitutive expression of HCV had no evidence of hepatic pathology until 11 months of age, when steatosis developed. In contrast, mice with tetracycline-mediated suppression and then withdrawal at 2 months of age developed hepatic inflammation with ALT elevation by 4 months of age. Hepatic steatosis also became evident at 5 months of age, which occurred much earlier than the mice with constitutive HCV expression. Neither hepatic inflammation nor steatosis was observed in mice that had been continuously subjected to tetracycline treatment. With a tightly regulatable HCV expression, this model resembles those events observed in natural infection and offers a valuable tool to study the pathogenesis of hepatitis C. We have also initiated studies in chimpanzees, which are the only infectious animal model for hepatitis C. We have constructed a molecular infectious clone of HCV genotype 1b and studied the detailed virologic, serologic, and immunologic parameters of four chimpanzees inoculated with this monotypic inoculum. Two of the chimpanzees cleared the infection and two became persistently infected. Only weak and transient T helper responses were detected during the acute phase in all four chimpanzees. Liver biopsy samples collected over the course of infection were analyzed using cDNA microarray to characterize expression levels of a broad set of host genes. For three animals that exhibited a robust viremia (>10E5 genomes/ml) and evidence of liver injury, typical of acute HCV infection, the induction of a set of type I interferon regulated genes was observed during the first 8 weeks of infection, regardless of whether the animal eventually cleared the infection or not. However, the induction of certain genes related to signal transduction/cell growth could be associated specifically with viral clearance. During the viral clearance phase of one chimpanzee, intrahepatic activation of cellular immunity, particularly markers of CD8+ T-cells, was evident. On the other hand, the two chimpanzees that went on to chronic infection exhibited no evidence of such an adaptive immune response, despite the equally strong induction of the innate type I IFN response. These results are consistent with other reports that correlate in vitro hepatic CD8+ T-cell functions with clearance and they further support the notion that a vigorous intra-hepatic immune response is important for viral clearance. This observed difference possibly reside in the inability of the chimpanzee with chronic infection to activate the pathway transitioning from the innate to adaptive immunity. A fourth animal that exhibited a weak viremia (<500 genomes/ml) and quickly cleared infection (<8 weeks) had no detectable induction of type I interferon regulated genes or markers for T-cell response. Infection in this chimpanzee is consistent with a subclinical infection that probably occurs frequently in humans. This result suggests that HCV exposure can be controlled by an innate immunity, and if this process is effective during the initial phase of infection, adaptive immune response may not be necessary for viral clearance. These animal models are valuable not only to address issues of immunopathogenesis and cytopathic potential of HCV gene products but also to study HCV replication in vivo.