Host Factors Controlling Neuroinvasive Flavivirus Pathogenesis

摘要 黄病毒，如鲍瓦桑病毒(POWV)、西尼罗河病毒(WNV)和日本脑炎病毒(JEV) 是通过扁虱和蚊子传播的。黄病毒感染的结果是不同的，只有一种 进展为神经侵袭性疾病(如脑炎、脑膜炎或瘫痪)的亚群。我们假设 宿主遗传变异，特别是抗病毒反应基因变异，导致不同的疾病结局 在感染了黄病毒之后。重组近交系小鼠的合作杂交(CC)小组提供了一个理想的 系统发现免疫介导控制黄病毒致病的新机制，因为这些 小鼠在可复制的遗传背景上表现出更广泛的免疫表型。我们感染了17人 鉴定出多个高感品系，包括CC071和CC015，以及一个 抗病品系CC045。POWV、WNV和JEV之间的表型大部分是一致的，但也有一些品系 表现出病毒特异性抗药性，这意味着存在病毒特异性和泛黄病毒机制， 控制对神经侵袭性黄病毒的耐药性。我们建议使用CC来确定病毒和 POWV致病的免疫学特征，并鉴定与POWV抗性有关的宿主基因。 目的1：明确CC小鼠POWV致病的病毒和免疫学特征。我们发现CC045 与CC071小鼠相比，小鼠表现出同等的病毒血症，但脑病毒载量较低，这表明POWV 抵抗可能是由于神经侵袭减少所致。我们将评估脑病毒载量和浸润性白细胞 POWV感染后的感病和抗病CC系。我们将评估血脑屏障通透性在 基线和对病毒感染和炎症刺激的反应。我们将从以下位置生成原生细胞 敏感和抗性的CC品系，并测量POWV和其他黄病毒的复制。 目的2：定位与POWV相关的宿主因子的数量性状基因座并评价其抗病毒活性 抵抗。为了确定决定POWV感染结果的多态宿主基因，我们产生了 感病和抗病品系(CC071×CC045和CC015×CC045)的两个F2杂交组合及其致死性评价 在POWV感染后，每个杂交的大约300只F2小鼠，以及大约120只F2小鼠的中枢神经系统病毒载量。我们将绘制地图 在两个杂交组合中发现与白粉病抗性相关的QTL，并研究显著QTL下的候选基因。 目的3：在CC小鼠体内区分泛黄病毒和病毒特异性限制因子。我们会感染 附加CC品系与JEV，以识别与POWV或WNV相比对JEV具有不同抗性的品系。 我们将评估乙脑病毒感染后的脑病毒载量和浸润性白细胞。我们将产生F2后代 对感病和抗病品系进行定位，定位与乙脑病毒抗性相关的QTL。 建议的研究将为POWV的致病机制提供深入的了解，并揭示多态 影响黄病毒神经侵袭性疾病易感性的宿主免疫机制。这项工作将提供 为未来研究控制黄病毒致病的新型免疫因子奠定了基础。

ABSTRACT Flaviviruses such as Powassan virus (POWV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) are transmitted by ticks and mosquitoes. The outcomes of flavivirus infection are heterogeneous, with only a subset progressing to neuroinvasive disease (e.g. encephalitis, meningitis, or paralysis). We hypothesize that host genetic variation, particularly in antiviral response genes, contributes to differential disease outcome following flavivirus infection. The Collaborative Cross (CC) panel of recombinant inbred mice provides an ideal system to discover novel mechanisms of immune-mediated control of flavivirus pathogenesis because these mice exhibit an expanded range of immune phenotypes on reproducible genetic backgrounds. We infected 17 CC lines with POWV and identified multiple highly susceptible lines, including CC071 and CC015, and a single resistant line, CC045. Most phenotypes were concordant among POWV, WNV, and JEV, but some lines exhibited virus-specific resistance, implying that there are both virus-specific and pan-flavivirus mechanisms that control resistance to neuroinvasive flaviviruses. We propose to use the CC to determine the viral and immunologic features of POWV pathogenesis and to identify host genes that contribute to POWV resistance. Aim 1: Define viral and immunologic features of POWV pathogenesis in CC mice. We found that CC045 mice exhibited equivalent viremia but lower brain viral loads compared to CC071 mice, suggesting that POWV resistance may result from reduced neuroinvasion. We will assess brain viral loads and infiltrating leukocytes in susceptible and resistant CC lines following POWV infection. We will assess blood-brain barrier permeability at baseline and in response to viral infection and inflammatory stimuli. We will generate primary cells from susceptible and resistant CC lines and measure replication of POWV and other flaviviruses. Aim 2: Map quantitative trait loci and evaluate antiviral activity of host factors associated with POWV resistance. To identify polymorphic host genes that determine the outcome of POWV infection, we generated two F2 crosses of susceptible and resistant lines (CC071 x CC045 and CC015 x CC045) and evaluated lethality in ~300 F2 mice per cross following POWV infection, as well as CNS viral loads in ~120 F2 mice. We will map QTL associated with POWV resistance in both crosses and investigate candidate genes under significant QTL. Aim 3: Distinguish pan-flavivirus and virus-specific restriction factors in CC mice. We will infect additional CC lines with JEV to identify lines that are differentially resistant to JEV compared to POWV or WNV. We will evaluate brain viral loads and infiltrating leukocytes following JEV infection. We will generate F2 progeny of susceptible and resistant lines and map QTL associated with JEV resistance. The proposed studies will provide insight into the pathogenic mechanisms of POWV and reveal polymorphic host immune mechanisms that impact susceptibility to flavivirus neuroinvasive disease. This work will provide the foundation for future investigations of novel immune factors that control flavivirus pathogenesis.