Impact of species-specific host responses on restriction of hepatotropic viruses

 描述（由申请人提供）：传染病对人类发病率和死亡率有显著影响。许多在人类中引起疾病的病原体表现出非常狭窄的宿主范围，这对研究和开发有效的临床疗法都提出了挑战。我们将把我们的研究重点放在丙型肝炎病毒（HCV），一个典型的嗜肝病毒与一个狭窄的主机范围，并得出细胞内在的免疫反应模式，影响跨物种的感染结果。80%的HCV感染导致慢性感染，并可导致肝硬化和肝细胞癌。HCV具有不完全确定的宿主嗜性，几乎仅限于人类和黑猩猩。我们以前已经证明，两个宿主因子，CD 81和闭合蛋白（OCLN）的氨基酸序列之间的差异（在人类和小鼠之间）阻断小鼠对HCV的摄取。虽然HCV组装和释放在从人类到啮齿动物的各种物种的肝细胞中得到支持，但抗病毒先天反应似乎限制了非人类物种中的HCV RNA复制。我们推测，种属特异性差异的动力学，多样性和规模的抗病毒基因产物影响病毒的向性。在这里，我们建议开发和利用一个新的平台，系统地定义这种物种特异性的反应模式有助于感染的结果。我们的目标是利用来自非人灵长类物种的真皮成纤维细胞的集合来产生诱导多能干细胞（iPSC），并以逐步的方式将这些猿猴iPSC分化为肝细胞样细胞（HLC）。我们将描述和比较来自人类，黑猩猩，倭黑猩猩，大猩猩，猩猩和恒河猴标本的HLC中的病毒感染。对于支持病毒生命周期至关重要的宿主因子的直系同源物在这些物种中很大程度上是保守的，并且易感性的差异可能归因于抗病毒反应模式的差异和/或HCV无法拮抗细胞内在防御。我们将HCV感染依赖性荧光重定位（HDFR）报告基因与激光捕获显微切割（LCM）结合，从不同物种的HCV感染的HLC中分离感染的细胞，并对其进行转录组学分析。通过比较和对比高和低感染频率物种的数据，我们将能够确定与有效的抗病毒控制相关的物种特异性特征。该合作项目的成功取决于HCV病毒学和病毒嗜性（Ploss实验室）以及干细胞和肝细胞生物学（Schwartz实验室）的互补专业知识。我们相信，这种系统病毒学的方法将提供丰富的见解进化保护的抗病毒反应模式，并揭示了抗病毒免疫的影响，以限制物种嗜性。由于我们收集材料的许多灵长类物种濒临灭绝，不能用于生物医学研究，我们将建立的新兴iPSC库也将成为其他需要访问通常无法访问的灵长类物种/组织的研究人员的一个支持平台。

 DESCRIPTION (provided by applicant): Infectious diseases contribute significantly to human morbidity and mortality. Many pathogens that cause disease in humans, exhibit a very narrow host range, which poses challenges both for study and for developing effective clinical therapies. We will focus our studies on hepatitis C virus (HCV), an archetypal hepatotropic virus with a narrow host range and derive cell-intrinsic immune response patterns that impact infection outcome across species. Eighty percent of HCV infections result in chronic infection and can lead to cirrhosis and hepatocellular carcinoma. HCV has an incompletely defined host tropism limited almost exclusively to humans and chimpanzees. We have previously demonstrated that differences (between human and mouse) between the amino acid sequences of two host factors, CD81 and occludin (OCLN) block HCV uptake in mice. While HCV assembly and release is supported in hepatocytes across a broad variety of species ranging from humans to rodents, antiviral innate responses appear to limit HCV RNA replication in non-human species. We hypothesize that species-specific differences in the kinetics, diversity and magnitude of antiviral gene products influence viral tropism. Here, we propose to develop and utilize a novel platform to systematically define such species-specific response patterns contributing to infection outcome. We aim to capitalize on a collection of dermal fibroblasts from non-human primate species to generate induced pluripotent stem cells (iPSCs) and differentiate these simian iPSCs in a stepwise manner to hepatocyte like cells (HLCs). We will characterize and compare viral infections in HLCs derived from human, chimpanzee, bonobo, gorilla, orangutan and rhesus monkey specimens. Orthologs of essential host factors critical for supporting the viral life-cycle are largely conserved across these species and differences in susceptibility can likely be attributed to differences in antiviral response patterns and/or HCV's inability to antagonize cell-intrinsic defenses. We will couple a HCV infection-dependent fluorescence relocalization (HDFR) reporter with laser capture microdissection (LCM) to isolate infected cells from HCV infected HLCs from different species and subject them to transcriptomic profiling. By comparing and contrasting data from species with high and low infection frequencies, we will be able to determine species-specific signatures associated with effective antiviral control. The success of this collaborative project depends on the complementary expertise in HCV virology and viral tropism (Ploss lab) and stem cell and hepatocyte biology (Schwartz lab). We are convinced that this systems virology approach will provide rich insights into evolutionary conservation of antiviral response patterns and shed light on the impact of antiviral immunity to restrict species tropism. Since many of the primate species for which we collect materials are endangered and cannot be used (anymore) in biomedical research, the emerging iPSC repository we will build will also be an enabling platform for other researchers needing access to usually inaccessible primate species/tissues.