Host Factors Controlling Neuroinvasive Flavivirus Pathogenesis

摘要 黄病毒，如Powassan病毒（POWV）、西尼罗河病毒（WNV）和日本脑炎病毒（JEV） 是由蜱虫和蚊子传播的。黄病毒感染的结果是异质性的，只有一个 进展为神经侵袭性疾病（例如脑炎、脑膜炎或麻痹）的亚组。我们假设 宿主遗传变异，特别是抗病毒反应基因的变异，有助于不同的疾病结果 黄病毒感染后。重组近交系小鼠的协作杂交（CC）组提供了一种理想的 系统发现免疫介导的控制黄病毒发病机制的新机制，因为这些 小鼠在可再现的遗传背景上表现出扩大的免疫表型范围。我们感染了17个 CC株系感染POWV，并鉴定了多个高感株系，包括CC 071和CC 015，以及一个单感株系。 抗性系CC 045。POWV、WNV和JEV的大部分表型一致，但也有部分株系的表型一致。 表现出病毒特异性抗性，这意味着存在病毒特异性和泛黄病毒机制， 控制对神经侵入性黄病毒的抗性。我们建议使用CC来确定病毒和 本发明的目的在于研究POWV发病机制的免疫学特征并鉴定有助于POWV抗性的宿主基因。 目的1：明确POWV在CC小鼠中致病的病毒学和免疫学特征。我们发现CC045 与CC071小鼠相比，小鼠表现出相当的病毒血症，但脑病毒载量较低，表明POWV 抵抗可能是由于神经侵袭减少。我们将评估脑病毒载量和浸润的白细胞， 在POWV感染后的敏感和抗性CC系。我们将评估血脑屏障通透性， 基线和响应病毒感染和炎症刺激。我们将从 敏感和抗性CC系，并测量POWV和其它黄病毒的复制。 目的2：定位POWV的数量性状基因座，评价与POWV相关的宿主因子的抗病毒活性 阻力为了鉴定决定POWV感染结果的多态性宿主基因，我们产生了 敏感和抗性品系（CC 071 x CC 045和CC 015 x CC 045）的两个F2杂交，并评估致死率 POWV感染后，每次杂交约300只F2小鼠中的病毒载量，以及约120只F2小鼠中的CNS病毒载量。我们将绘制 在两个杂交组合中检测与POWV抗性相关的QTL，并在显著QTL下研究候选基因。 目的3：区分CC小鼠泛黄病毒和病毒特异性限制因子。我们会感染 另外的CC系与JEV的杂交以鉴定与POWV或WNV相比对JEV具有差异抗性的系。 我们将评估脑病毒载量和浸润的白细胞后，乙脑病毒感染。我们将产生F2后代 并定位了与JEV抗性相关的QTL。 这些研究将有助于深入了解POWV的致病机制，并揭示其多态性。 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 为今后研究控制黄病毒致病机制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.