Identifying quantitative trait loci that regulate enterovirus D68 pathogenesis using the Collaborative Cross

项目摘要 肠道病毒是人类最普遍的病原体之一。传统上，这些病毒与 随着消化道内细胞的感染，宿主之间的口粪传播和 罕见的神经系统并发症，包括急性弛缓性肌无力（AFM）。然而，这种病毒的许多成员 包括人鼻病毒和肠道病毒D-68（EV-D 68）的家族是呼吸道病原体，复制 在上呼吸道和下呼吸道的上皮细胞内，并通过气溶胶在宿主之间传播 2014年，人们认识到EV-D 68感染也可导致AFM。其后每两年 随着EV-D 68感染的爆发，感染EV-D 68的儿童人数一直在上升， AFM确诊病例数。尽管有强有力的流行病学证据表明，该病毒是一种病原体， AFM的代理人，这种关联已受到质疑，由于无法分离感染性病毒从 EV-D 68致瘫痪患儿脑脊液。总之，这些观察结果证明， EV-D 68感染的社会经济影响以及未满足的医疗需求，以更好地了解 EV-D 68生物学和新疗法的开发。尽管发展了多种 EV-D 68感染的动物模型，没有系统复制与EV-D 68相关的病理谱; 因此，这些模型不足以用于疫苗和抗病毒疗法的开发和测试 治疗或预防感染或AFM的发展。我们假设，有限的遗传多样性， 在许多体内感染性疾病模型中使用的近交系小鼠限制了感染性疾病动物模型的建立， EV-D 68生物学。协同杂交（CC）小鼠的遗传多样性增强， 人类群体以及相关表型中的微妙之处，在null小鼠中缺失。的目标 本研究拟建立EV-D 68小鼠病理模型， 基因座的遗传多态性，其产物定量调节对病毒性疾病的易感性。以识别 对EV-D 68感染敏感的CC小鼠，支气管上皮的气液界面培养物和 器官型脑切片培养物将从随机遗传的8天和28天大的小鼠产生， 背景和感染三个分离的EV-D 68，并评估感染性病毒的产生 通过噬斑测定。我们还将确定被感染的细胞类型，并可视化组织损伤 通过H&E染色和间接免疫荧光，使用针对 病毒蛋白VP 1和呼吸道和神经元细胞类型特异性蛋白。目标1中确定的小鼠 将在目标2中杂交以最高和最低效率复制的EV-D 68。F1分析 F_2代决定了EV-D 68易感性的遗传方式，F_2代决定了EV-D 68易感性的遗传方式 将描述调节EV-D 68感染的数量性状基因座（QTL）。使用CC小鼠将促进 开发反映与EV-D 68感染相关的病理学谱的动物模型。

Project Summary Enteroviruses are among the most prevalent human pathogens. Traditionally, these viruses were associated with infection of cells within the alimentary tract, oral-fecal transmission between hosts and the development of rare neurological complications including acute flaccid myelitis (AFM). Yet, numerous members of this virus family including human rhinoviruses and enterovirus D-68 (EV-D68) are respiratory pathogens, replicating within the epithelial cells of the upper and lower respiratory tracts, and transmitted between hosts by aerosols In 2014 it was recognized that EV-D68 infection can also result in AFM. With each subsequent biennial outbreak of EV-D68 infection, the number of children infected with EV-D68 has been rising as well as the number of confirmed cases of AFM. Despite strong epidemiologically evidence that the virus is an etiologic agent of AFM, this association has been questioned due the inability to isolate infectious virus from the cerebrospinal fluid of children with EV-D68 induced paralysis. Together, these observations attest to the socioeconomic impact of EV-D68 infection as well as the unmet medical need for better an understanding of EV-D68 biology and the development of novel therapeutics. Notwithstanding the development of multiple animal models of EV-D68 infection, no system replicates the spectrum of pathologies associated with EV-D68; consequently, these models are insufficient for the development and testing of vaccines and antiviral therapies to treat or prevent infection or the development of AFM. We hypothesize that the limited genetic diversity of inbred mice used in many in vivo models of infectious disease limits the establishment of an animal model of EV-D68 biology. The enhanced genetic diversity of mice of the collaborative cross (CC) emulates that of the human population as well as the subtleties in associated phenotypes that are missing in null mice. The goal of the proposed research is to establish a mouse model of EV-D68 pathology and identify naturally-occurring genetic polymorphisms in loci whose products quantitatively regulate susceptibility to viral disease. To identify CC mice susceptible to EV-D68 infection, air-liquid interface cultures of the bronchial epithelium and organotypic brain slice cultures will be generated from 8- and 28-day old mice of randomized genetic backgrounds and infected with three isolates of EV-D68, and the production of infectious virus will be assessed by plaque assay. We will also identify the cell types that are infected by, and visualize tissue damage resulting from, virus infection by H&E staining and indirect immunofluorescence using antibodies generated against the viral protein VP1, and proteins specific to respiratory and neuronal cell types. The mice identified in Aim 1 in which EV-D68 replicated with the highest and lowest efficiency will be crossed in Aim 2. Analysis of the F1 generation will determine the mode by which EV-D68 susceptibility is inherited, while that of the F2 generation will describe the quantitative trait loci (QTL) that modulate EV-D68 infection. Use of CC mice will promote the development of an animal model that reflects the spectrum of pathologies associated with EV-D68 infection.