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

 描述(申请人提供)：传染病对人类发病率和死亡率有很大影响。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，这给研究和开发有效的临床疗法带来了挑战。我们将集中研究丙型肝炎病毒(丙型肝炎病毒)，这是一种宿主范围较窄的典型嗜肝病毒，并推导出影响跨物种感染结果的细胞内源性免疫反应模式。80%的丙型肝炎病毒感染会导致慢性感染，并可能导致肝硬化和肝细胞癌。丙型肝炎病毒的宿主取向不完全明确，几乎仅限于人类和黑猩猩。我们先前已经证明，两个宿主因子CD81和OCLN的氨基酸序列之间的差异(人和鼠之间)阻止了小鼠对丙型肝炎病毒的摄取。虽然从人类到啮齿动物的各种物种的肝细胞中都支持丙型肝炎病毒的组装和释放，但抗病毒的先天反应似乎限制了非人类物种中的丙型肝炎病毒RNA的复制。我们假设，抗病毒基因产物的动力学、多样性和大小因物种而异会影响病毒的趋向性。在这里，我们建议开发和利用一个新的平台来系统地定义影响感染结果的这种特定物种的反应模式。我们的目标是利用从非人类灵长类物种收集的真皮成纤维细胞来产生诱导多能干细胞(IPSCs)，并以一种循序渐进的方式将这些iPSCs分化为肝细胞样细胞(HLCs)。我们将从人类、黑猩猩、矮黑猩猩、大猩猩、猩猩和恒河猴标本中鉴定和比较HLC中的病毒感染。在这些物种中，对支持病毒生命周期至关重要的关键宿主因子的同源基因在很大程度上是保守的，敏感性的差异可能归因于抗病毒反应模式的差异和/或丙型肝炎病毒无法对抗细胞固有防御。我们将丙型肝炎病毒感染依赖的荧光重定位(HDFR)报告与激光捕获显微解剖(LCM)相结合，从不同物种的丙型肝炎病毒感染的HLC中分离感染细胞，并对它们进行转录图谱分析。通过比较和对比感染频率高和低的物种的数据，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。这一合作项目的成功有赖于在丙型肝炎病毒病毒学和病毒嗜性(普洛斯实验室)和干细胞和肝细胞生物学(施瓦茨实验室)方面的互补专业知识。我们相信，这种系统病毒学方法将为抗病毒反应模式的进化保守提供丰富的见解，并阐明抗病毒免疫对限制物种趋向性的影响。由于我们为其收集材料的许多灵长类物种濒临灭绝，不能(再)用于生物医学研究，我们将建立的新兴的IPSC资源库也将为其他需要访问通常无法访问的灵长类物种/组织的研究人员提供一个有利的平台。

 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.