Host cell receptor variation and control of viral cross-species transmission

描述(申请人提供)：病毒出现的一种重要形式是病毒从其正常动物宿主向新的动物和/或人类宿主的跨物种传播。我的研究试图确定支配犬细小病毒(CPV)作为一种新的大流行病原体出现的病毒、宿主和进化机制。CPV在20世纪70年代中期出现在狗身上，并于1978年在世界范围内传播，我的赞助商(Parrish)实验室表明，CPV是猫泛白细胞减少症病毒(FPV)的变种，犬感染FPV的关键宿主范围障碍是细胞受体-1转铁蛋白受体(TFR)。只有很少的病毒衣壳变化允许CPV与犬TFR结合并感染犬细胞，这为确定控制跨物种传播、适应和病原体出现的分子相互作用提供了一个很好的模型。现在也很清楚，其他哺乳动物宿主也可以被这些细小病毒感染，这些宿主可能在CPV的大流行中发挥了作用。因此，我提出的研究试图定义在替代动物宿主中发现的病毒变异的范围，确定这些病毒如何适应特定的宿主，并解释病毒与受体之间的相互作用如何决定成功的跨物种感染。目的1：确定CPV作为大流行病原体出现的进化史，并确定不同宿主在这些病毒进化和跨物种传播中的作用。我们将从不同的食肉动物物种中分离并全面鉴定病毒，以确定它们在细小病毒进化中的宿主作用。病毒将通过基因组测序、系统发育分析、抗原性研究和在不同宿主细胞中的复制分析来表征。病毒中关键残基变化的定点突变将用于测试它们对病毒结构和控制宿主范围的影响。目的：对来自不同宿主的TFR进行鉴定和功能测试，以阐明病毒受体序列和结构变异在确定细小病毒感染易感性中的作用。我们将克隆和表达各种肉食动物的TFR，并在微流控设备中利用全内反射荧光来表征病毒(或具有特定变化的病毒突变体)的受体结合特性 (TIRF)显微镜。将检查宿主TFR序列的变异，以确定在宿主进化过程中受体正向选择的证据。在体外不同宿主细胞中生长的病毒将被检查改变受体结合的衣壳变化，在那里我们将使用深度基因组测序来辨别所选病毒变异的速度和类型。这些研究将使我们能够独一无二地确定病毒衣壳和宿主受体之间的相互作用，这些受体介导跨物种的传播和出现。

DESCRIPTION (provided by applicant): An important form of virus emergence is the cross-species transmission of a virus from its normal animal reservoir to a new animal and/or human host. My studies seek to identify the viral, host, and evolutionary mechanisms that governed the emergence of canine parvovirus (CPV) as a new pandemic pathogen. CPV emerged in dogs in the mid-1970s, and spread world-wide in 1978, and my sponsor (Parrish) laboratory showed that CPV is a variant of feline panleukopenia virus (FPV) and that a key host range barrier to FPV infection in dogs was the cell receptor, transferrin receptor type-1 (TfR). Only a few viral capsid changes allowed CPV to bind the canine TfR and infect canine cells, and this provides an excellent model for defining the molecular interactions that govern cross-species transmission, adaptation, and hence pathogen emergence. It is now also clear that other mammalian hosts can be infected by these parvoviruses and that those hosts likely played roles in the pandemic emergence of CPV. My proposed studies therefore seek to define the range of viral variants found in alternative animal hosts, to determine how these viruses adapt to specific hosts, and also to explain how interactions between the viruses and receptors determine successful cross-species infections. Aim 1: To determine the evolutionary history of CPV emergence as a pandemic pathogen and to define the roles of different hosts in the evolution and cross-species transmission of these viruses. We will isolate and comprehensively characterize viruses from various carnivore species to determine their roles as hosts in parvovirus evolution. Viruses will be characterized by genomic sequencing, phylogenetic analysis, antigenic studies, and replication assays in various host cells. Site-directed mutagenesis of key residue changes in the viruses will be used to test for their effects on viral structures and in controlling host range. Aim 2: To characterize and functionally test the TfRs from various hosts to clarify the role of viral receptor sequence and structural variation in determining susceptibility to parvovirus infection. We will clone and express the TfRs from various carnivore species and characterize receptor-binding properties for viruses (or viral mutants with specific changes) in microfluidic devices using total internal reflection fluorescence (TIRF) microscopy. Variation in host TfR sequences will be examined to identify evidence of positive selection of the receptors during the evolution of the hosts. Viruses grown in different host cells in vitro will be examined for capsid changes that alter receptor binding, where we will use deep genome sequencing to discern the rate and types of viral variation selected. These studies will allow us to uniquely determine the interplay between the viral capsid and host receptors that mediate cross-species transmission and emergence.