Pathogenesis Of Enteric Viral Hepatitis

我们研究了病毒性肝炎的发病机制，以及这些重要病原体毒力和减毒的分子基础。我们已经证明甲型肝炎病毒(HAV)的毒力和减毒主要由两个基因控制：VP1/2A和2C。然而，衰减性突变在体内被强烈选择，导致出现毒力变异。这对甲型肝炎减毒活疫苗的研制具有重要意义。甲型肝炎的发病机制也正在通过基因芯片分析在黑猩猩模型中进行研究。先天反应和后天反应都已被记录下来。有趣的是，甲型肝炎病毒不会像丙型肝炎病毒和HDV感染那样强烈地触发某些干扰素刺激基因的上调。这是令人惊讶的，因为甲型肝炎病毒和丙型肝炎病毒都是单链RNA病毒，具有双链复制形式，而HDV是具有广泛碱基配对的单链病毒，被认为是双链RNA。具体地说，在2009财年和2010财年，我们比较了几只实验感染甲型肝炎的黑猩猩对甲型肝炎病毒感染的先天和获得性免疫反应，包括接受强毒疫苗接种的动物和感染甲型肝炎减毒株的动物。感染强毒株的动物有强大的先天免疫反应(尽管不如感染丙型肝炎病毒的黑猩猩那样强烈)，而在弱化感染中，这种反应不那么明显。有趣的是，在后一组黑猩猩中，先天免疫反应被缩短，但先天免疫反应的检测几乎与检测甲型肝炎病毒感染的特定血清学或分子探针一样敏感。也存在获得性免疫反应，但不如感染丙型肝炎病毒的黑猩猩的获得性免疫反应强。重要的是寻找病毒的控制机制，可能会阻止其中的一些系统。 尽管戊型肝炎在美国很少见，但它是亚洲、中东和北非成年人急性肝炎的最重要原因。像大多数肝炎病毒一样，它在细胞培养中复制很差，甚至根本不复制，并且不能传播给小型实验室动物。我们已经开发了用于在体外研究HEV的复制体；这些工具允许对病毒复制进行详细的分子分析，这可以通过分子工程病毒的感染性克隆在体内得到证实。此外，我们与同事们正在开发小型动物模型(猪的猪HEV，鸡的禽HEV)，利用HEV的非人类灵长类动物模型，为研究戊型肝炎病毒的相对发病机制提供了前所未有的机会。最后，还利用基因芯片对戊型肝炎进行了研究，发现了一种活跃的先天免疫反应，但获得性免疫反应较弱。具体地说，在2009财年和2010财年，我们比较了几只黑猩猩对实验性HEV感染的先天和获得性免疫反应，以及在实验感染丙型肝炎病毒或甲型肝炎病毒的黑猩猩身上观察到的反应。与这些感染相比，HEV感染的特征是适应性免疫反应缩短和略有缩短。鉴于其他观察结果，HEV感染的抗体滴度往往比其他肝炎病毒感染的抗体滴度下降得更快，这一点特别有趣。更具体地说，我们在2010年完成了对多只黑猩猩感染丙型肝炎病毒和戊型肝炎病毒的微阵列分析，比较了人类和黑猩猩基因组序列是否适合解释从感染这两种病毒之一的黑猩猩的一系列临床样本中获得的Affymetrix微阵列数据。我们发现，人类基因组序列在识别上调和下调基因方面更敏感、更特异，可能是因为它比最近测得的黑猩猩基因组更精准。我们还比较了两种分析方法，一种是利用Affymetrix探针组和相关系数的方法，另一种是利用单个完全匹配的探针和t检验分析的方法。这两种分析都显示了宿主对这两种RNA病毒的免疫反应的相似和不同。这些信息将有助于更好地了解病毒性肝炎的发病机制。 2011年，我们完成了几年前就开始在老鼠身上进行的HEV研究。大鼠戊型肝炎病毒的序列出现，使我们能够重新评估大鼠HEV在实验室大鼠中的广泛传播研究。我们获得了部分序列数据，准备了已知传染性滴度的池，进行了发病机制研究，并确定了老鼠病毒的跨物种敏感性。我们的结论是，大鼠HEV在实验室大鼠中不会产生强烈的感染，也不会传播给非人类灵长类动物，这表明它不会对人类构成威胁。 在其他合作研究中，我们测试了被诊断为患有药物相关性肝损伤(DILI)的患者的血清，以寻找HEV感染的证据，发现318名患者中有3%实际上患有戊型肝炎。戊型肝炎患者大多是老年男性(89%为男性，平均年龄67岁)，两人HIV阳性。我们的结论是，怀疑由药物引起的急性肝损伤病例中，有一小部分但很重要的一部分实际上是戊型肝炎。 2012年，我们发现，从免疫抑制个体的慢性感染中获得的HEV是嵌合病毒，含有宿主基因的插入，使病毒能够跨越物种屏障，在通常对病毒具有抵抗力的细胞中生长。此外，我们还发现，HEV的衣壳蛋白在细胞中过表达时，可以有效地包裹病毒RNA并将其传递给NAVE细胞。

We have studied the pathogenesis of viral hepatitis and the molecular basis for virulence and attenuation of these important pathogens. We have shown previously that virulence and attenuation of hepatitis A virus (HAV) are controlled principally by two genes: VP1/2A and 2C. However, attenuating mutations are strongly selected against in vivo, resulting in the emergence of virulent variants. This has important implications for the development of live attenuated hepatitis A vaccines. The pathogenesis of hepatitis A is also being studied in the chimpanzee model by microarray analysis. Both innate and adaptive responses have been recorded. Interestingly, HAV does not trigger as robust an up-regulation of certain interferon stimulated genes as HCV and HDV infections. This is surprising because HAV and HCV are both single-stranded RNA viruses with a double-stranded replicative form and HDV is a single-stranded virus with extensive base pairing that is perceived as double-stranded RNA. Specifically, in FY 2009, and 2010 we compared the innate and adaptive immune responses to HAV infection in several experimentally infected chimpanzees, including animals that received virulent inocula and those that were infected with attenuated strains of HAV. Animals infected with virulent strains had a robust innate immune response (although not as robust as that seen in HCV-infected chimpanzees), whereas the response was less marked in the attenuated infections. Interestingly, innate immune responses were abbreviated in the latter chimpanzees, but detection of the innate immune response was almost as sensitive a diagnostic test of infection as was detection of specific serologic or molecular probes of HAV infection. The adaptive immune responses were also present, but not as robust as the adaptive immune responses in HCV infections of chimpanzees. It will be important to search for viral mechanisms of control that may block some of these systems. Although rare in the United States, hepatitis E is the single most important cause of acute hepatitis among adults throughout Asia, the Middle East and North Africa. Like most of the hepatitis viruses, it replicates poorly or not at all in cell culture and cannot be transmitted to small laboratory animals. We have developed replicons for the study of HEV in vitro; these tools are permitting a detailed molecular analysis of viral replication that can be confirmed in vivo with molecularly engineered infectious cDNA clones of the virus. In addition, with colleagues, we are developing small animal models (swine HEV in swine, avian HEV in chickens) that, with nonhuman primate models of HEV, provide an unprecedented opportunity for studying the comparative pathogenesis of hepatitis E viruses. Finally, hepatitis E has also been studied by microarray and a brisk innate but weak adaptive immune response has been seen. Specifically, in FY 2009 and 2010, we compared the innate and adaptive immune responses of several chimpanzees to experimental HEV infections with those observed in chimpanzees that had been experimentally infected with HCV or HAV. In comparison with those infections, HEV infections were characterized by an abbreviated and somewhat shortened adaptive immune response. This is particularly interesting in light of other observations that antibody titers tend to diminish more rapidly in HEV infections than in infections with the other hepatitis viruses. More specifically, in 2010, we completed a microarray analysis of HCV and HEV infections of multiple chimpanzees, comparing the sequence of human and chimpanzee genomes for suitability in interpreting Affymetrix microarray data obtained from serial clinical samples of chimpanzees infected with one or the other of the two viruses. We found that the human genome sequence was more sensitive and specific for identifying up-regulated and down-regulated genes, probably because it is better curated than the more recently sequenced chimpanzee genome. We also compared two analytic methods, a method utilizing Affymetrix probe sets and correlation coefficients with one utilizing individual perfectly matched probes and t-test analysis. Both analyses demonstrated similarities and differences in the host immune response to these two RNA viruses. This information will be useful for a better understanding of the pathogenesis of viral hepatitis. In 2011 we completed a study of HEV in rats that we had begun some years earlier. The sequence of the rat hepatitis E virus became available and permitted us to reevaluate extensive transmission studies of rat HEV in laboratory rats. We obtained partial sequence data, prepared pools of known infectivity titer, performed pathogenesis studies and determined cross-species susceptibility of the rat virus. We concluded that the rat HEV does not produce a robust infection in laboratory rats and is not transmissible to nonhuman primates, suggesting that it does not pose a threat to humans. In other collaborative studies we tested sera from patients diagnosed as having drug-induced liver injury (DILI) for evidence of HEV infection and found that 3% of 318 patients actually had hepatitis E. The hepatitis E patients were mostly older men (89% male, mean age 67 years) and two were HIV-positive. We concluded that a small but important proportion of cases of acute liver injury suspected to be drug-induced are actually hepatitis E. In 2012 we showed that HEV obtained from chronic infections of immunosuppressed individuals were chimeric viruses containing an insertion of a host gene that permitted the virus to cross species barriers and grow in cells normally resistant to the virus. In addition, we showed that the capsid protein of HEV, when over-expressed in cells, efficiently encapsulated and transmitted viral RNA to nave cells.