Determinants of yellow fever pathogenesis in humans

人类黄热病发病机制的决定因素

• 批准号: 10326721
• 负责人: Adam Lee Bailey
• 金额: $ 32.1万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-10 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10326721
• 关键词: Determinants; yellow; fever; pathogenesis; humans

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 However, unpublished data collected from Brazilian YF patients suggests that hepatocyte destruction alone is insufficient to explain the coagulopathy observed in YF.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.

项目总结/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， 不良事件发生率高（包括死亡）。结果，YF继续杀害大约3万人， 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、甚至欧洲和北美的YF病毒传入风险正在增加 (YFV)这将是一场公共卫生灾难。在人类中，YF表现为典型的病毒性出血热 (VHF)引起严重的凝血异常（即凝血病）和终末器官损伤。此相反 在小鼠中发生的情况，YFV感染在很大程度上是良性的，其特征是明显缺乏免疫反应。 凝血病/出血特征。决定YF VHF发展的主要因素 人类是未知的。该项目旨在在细胞和分子水平上识别这些因素。 目前，YF中的VHF被认为是由肝细胞感染驱动的。然而，未公布的数据 从巴西YF患者中收集的数据表明，单靠肝细胞破坏不足以解释 在YF中观察到凝血病。目的1将检验免疫细胞的感染在免疫系统中起作用的假设。 在YF甚高频的发展中发挥了重要作用（以前未得到充分重视）。该目标将利用YFV 已植入人肝细胞或人免疫细胞的转基因小鼠的感染-两种 被认为在VHF疾病过程中起主要作用的细胞群-允许研究YFV 这些细胞群在该另外的YF抗性宿主中被隔离感染。一系列凝血试验 将在感染的小鼠上进行，以确定在YFV期间发生凝血病的机制 感染每天从YFV感染的猕猴中抽取血液样本- 研究YF中的VHF-也将进行分析，因为这对于确定时间和因果关系至关重要 关键事件之间的关系，如凝块形成，凝血因子消耗和肝损伤。在目标2中， CRISPR-Cas9全基因组敲除筛选将利用人和小鼠、肝细胞和 Kupffer细胞系，以确定使人类（和灵长类动物）对YFV唯一易感的因素。的 限制YFV在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-也将确定。来自此屏幕的“点击”将使用 体外靶向基因敲除和反式互补。这将确定主机的因素是必不可少的， YFV的生命周期，并可能导致新的YF易感小鼠模型的产生。这项研究将 生成信息和生物工具，可用于具体打击YF，更广泛地说，是VHF。 该奖项也将帮助主要研究者，亚当贝利博士，医学博士，博士，建立自己作为一个 独立研究人员，使他能够追求高回报的项目，否则是不可行的。