Determinants of yellow fever pathogenesis in humans

项目摘要/摘要 黄热病(YF)是一种通过受感染的埃及伊蚊叮咬传播的致命疾病。虽然 一种有效的YF疫苗是在20世纪30年代发明的，它不适合大规模生产，而且有 不良事件发生率高(包括死亡)。结果，青年党继续杀害大约30,000人 每年。此外，由于伊蚊媒介的重新出现，东南部的大部分地区 亚洲、大洋洲，甚至欧洲和北美都面临着越来越大的YF病毒传入风险 (YFV)，这将是一场公共卫生灾难。在人类中，YF表现为一种典型的病毒性出血热 (VHF)，造成严重的凝血异常(即凝血障碍)和终末器官损害。这形成了鲜明的对比 在小鼠身上发生的情况，在那里YFV感染基本上是良性的，特点是明显缺乏 凝血障碍/出血特征。决定YF地区VHF发病的宿主因素 人类是未知的。这个项目的目的是在细胞和分子水平上确定这些因素。 目前认为YF的VHF是由肝细胞感染引起的。然而，未发布的数据 从巴西YF患者收集的数据表明，仅有肝细胞破坏不足以解释 在YF中观察到凝血障碍。目标1将检验以下假设：免疫细胞感染在 在YF的VHF发展中发挥着重要的(以前被低估的)作用。这一目标将利用YFV 已植入人肝细胞或人免疫细胞的转基因小鼠的感染-2 被认为在VHF疾病过程中发挥主要作用的细胞群-允许研究YFV 在这种抗YF的宿主中隔离感染这些细胞群。一系列凝血试验 将在感染的小鼠身上进行实验，以确定在YFV期间发生凝血障碍的机制 感染。每天从感染YFV的猕猴身上采集血液样本-这是研究YFV的黄金标准动物模型 对YF-VHF的研究也将被分析，因为这将是确定时间和原因的关键 关键事件之间的关系，如血栓形成，凝血因子耗竭，和肝损伤。在目标2中， CRISPR-Cas9全基因组敲除筛查将利用人和小鼠、肝细胞和 Kupffer细胞系，以确定使人类(和灵长类)对YFV唯一易感的因素。这个 限制YFV在小鼠宿主中复制的因素-假设是干扰素刺激的基因 (ISG)根据初步数据--也将被确定。将使用以下工具验证来自此屏幕的“Hits” 体外靶向基因敲除和反式互补。这将确定以下关键的主机因素 YFV的生命周期，并可能导致新的YF敏感小鼠模型的产生。这项研究将 产生信息和生物工具，可专门用于抗击黄热病，更广泛地用于抗击VHF。 该奖项还将帮助首席研究员Adam Bailey博士，医学博士，博士，确立自己作为 独立研究人员，并使他能够从事高回报的项目，否则是不可行的。

PROJECT SUMMARY/ABSTRACT Yellow fever (YF) is a deadly disease transmitted by the bite of infected Aedes aegypti mosquitoes. Although an effective vaccine for YF was invented in the 1930s, it is not amenable to large-scale production and has a high rate of adverse events (including death). As a result, YF continues to kill approximately 30,000 people each year. Moreover, due to the re-emergence of Aedes mosquito vectors, substantial portions of Southeast Asia, Oceania, and even Europe and North America are at increasing risk for introduction of the YF virus (YFV), which would be a public health disaster. In humans, YF presents as a classic viral hemorrhagic fever (VHF), causing severe coagulation abnormalities (i.e. coagulopathy) and end-organ damage. This contrasts with what happens in mice, where YFV infection is largely benign and characterized by a distinct lack of coagulopathy/hemorrhagic features. The host factors that determine the development of VHF in YF in humans are unknown. This project aims to identify these factors at both cellular and molecular levels. Currently, VHF in YF is thought to be driven by the infection of hepatocytes. However, unpublished data collected from Brazilian YF patients suggests that hepatocyte destruction alone is insufficient to explain the coagulopathy observed in YF. Aim 1 will test the hypothesis that the infection of immune cells plays a major (and previously underappreciated) role in the development of VHF in YF. This Aim will utilize YFV infection of transgenic mice that have been engrafted with human hepatocytes or human immune cells – two cell populations that are thought to play a major role in the VHF disease process – allowing the study YFV infection of these cell populations in isolation in this otherwise YF-resistant host. A range of coagulation tests will be performed on infected mice to determine the mechanism by which coagulopathy develops during YFV infection. Blood samples drawn daily from YFV-infected macaques – the gold-standard animal model for studying VHF in YF – also will be analyzed, as this will be essential for determining the timing and causal relationships between key events such as clot formation, clotting factor depletion, and liver damage. In Aim 2, a CRISPR-Cas9 genome wide knock-out screen will be performed utilizing human and mouse, hepatocyte and Kupffer cell lines, to identify the factors that render humans (and primates) uniquely susceptible to YFV. The factors that restrict YFV replication in the murine host – hypothesized to be an interferon-stimulated gene (ISG) based on preliminary data – also will be identified. ‘Hits’ from this screen will be validated using targeted gene knock-out and trans-complementation in vitro. This will identify host factors that are essential in the YFV life cycle, and may lead to the generation of new YF-susceptible mouse models. This research will generate information and biological tools that can be used to combat YF specifically, and VHF more generally. This award will also help the principal investigator, Dr. Adam Bailey, MD, PhD, establish himself as an independent researcher and enable him to pursue high-reward projects that would otherwise not be feasible.