Spread and Release of Measles in the Airways

麻疹在呼吸道中的传播和释放

• 批准号: 10190793
• 负责人: PATRICK L SINN
• 金额: $ 52.08万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10190793
• 关键词: Address; Alveolar; Animal Model; Animals; Antiviral Agents; Antiviral Response; Apical; Apoptosis; Autophagocytosis; Benign; Biological Models; Cell Death; Cell Line; Cell Proliferation; Cell membrane; Cell model; Cell surface; Cells; Child; Childhood; Clinical; Contracts; Coughing; Data; Disease; Environment; Epithelial; Epithelial Cells; Europe; Event; Exanthema; F-Actin; Frequencies; Genes; Giant Cells; Goals; Growth; Herd Immunity; Hour; Human; Infection; Innate Immune Response; Interferons; Irrigation; Knowledge; Label; Laboratories; Lasers; Lead; Link; Lymphatic System; Macaca; Macaca mulatta; Maps; Measles; Measles virus; Mediating; Microscopic; Modeling; Modification; Monitor; Movement; Pathology; Pathway interactions; Physiological; Process; Proteins; Reporting; Research; Role; Rupture; Sentinel; Series; Sneezing; Structure of respiratory epithelium; Surface; Testing; Time; Vaccination; Viral Pathogenesis; Viremia; Virion; Virus; Virus Diseases; Work; airway epithelium; base; basolateral membrane; cell immortalization; contagion; deep sequencing; genetic approach; human model; improved; in vitro Model; in vivo; inhibitor/antagonist; innovation; insight; nectin; nonhuman primate; pathogen; pathogenic virus; preference; programs; receptor; respiratory virus; response; transmission process; treadmill; vector

PROJECT SUMMARY: Humans are the only natural reservoir for the extremely contagious measles virus (MeV). Thus, a critical challenge for MeV study is identification of representative human model systems. For decades, MeV was thought to enter the human host through the apical surface of airway cells, a misconception based on studies in immortalized cell lines. Well-differentiated primary cultures of airways epithelial cells from human donors (HAE) provide a more physiological relevant model of human airways. Using HAE, we found that MeV exclusively enters the basolateral membrane. This observation lead to a completely new paradigm for how MeV enters the human host. In addition to basolateral entry, we observed that MeV infection of HAE results in the formation of infectious centers that retain intact plasma membranes and are substantially different than the syncytia observed in immortalized cells. Infectious centers differ from syncytia in two important ways: 1) infectious centers stop growing 3-4 days post-infection and 2) infectious centers disappear after ~10 days leaving the cell layer intact. Why infectious centers stop growing and how they disappear in HAE remain a mystery and is the focus of this application. We hypothesize that infectious center formation in the respiratory epithelium is a vital step in the final amplification process before release to the next host. In Aim 1, we define the innate immune response pathways in the airways. We quantify 14 antiviral sentinel genes at 12 timepoints ranging from 6 hours to 2 weeks. In addition, laser capture of infectious centers is used to isolate infected cells from uninfected cells within an epithelial sheet and deep sequencing is used to map the cellular response to MeV. In Aim 2, we address how MeV is released from HAE. Preliminary data suggest that infectious centers are shed, intact, from the HAE (rather than rupturing). The timecourse and frequency of shedding will be defined. The roles of cell proliferation and cell death pathways will be probed by cell labeling to discern how large infectious centers can be released yet the epithelial integrity is maintained. MeV mediated cytoskeletal modifications are likely to be mechanistically involved in infectious center release, which we test using inhibitors of F-actin treadmilling. In Aim 3, we hypothesize that shed infectious centers are physiologically relevant vectors for MeV delivery. We will deliver cell-associated and cell-free MeV to the airways of rhesus macaques and quantify the time-course of infection. This aim has the potential to reshape a fundamental dogma of how MeV is spread host-to-host. In summary, these studies use an appropriate model system to study MeV entry, spread, and luminal release. Our research will elucidate mechanisms by which the most contagious human respiratory virus undergoes its final amplification step before release to its next host.

项目概要： 人类是传染性极强的麻疹病毒（MeV）的唯一天然宿主。因此， MeV研究面临的挑战是确定具有代表性的人体模型系统。几十年来，MeV 认为通过气道细胞的顶端表面进入人体宿主，这是基于研究的误解 在永生化细胞系中。人供体气道上皮细胞的高分化原代培养 (HAE)提供了人体气道的更生理相关的模型。使用HAE，我们发现MeV 只进入基底外侧膜。这一观察为我们提供了一个全新的范例， MeV能量进入人体。除了基底外侧进入外，我们观察到HAE的MeV感染导致HAE的细胞凋亡。 感染中心的形成，保留完整的质膜， 在永生化细胞中观察到的合胞体。感染中心与合胞体有两个重要的区别：1） 感染中心在感染后3-4天停止生长，2）感染中心在约10天后消失 留下完整的细胞层。为什么感染中心停止增长以及它们如何在HAE中消失仍然是一个问题。 神秘，是这个应用程序的重点。我们假设呼吸道感染中心的形成 上皮细胞是释放到下一个宿主之前的最终扩增过程中的重要步骤。在目标1中，我们定义 气道中的先天免疫反应途径。我们在12个时间点对14个抗病毒前哨基因进行定量 从6小时到2周不等。此外，激光捕获感染中心用于分离感染细胞 从上皮片层内未感染的细胞中提取，并使用深度测序来绘制细胞反应， 兆电子伏在目标2中，我们解决了如何从HAE释放MeV。初步数据显示， 从HAE脱落，完好无损（而不是破裂）。脱落的时间进程和频率将是 定义了细胞增殖和细胞死亡途径的作用将通过细胞标记来探测， 可以释放大的感染中心，而上皮完整性得以保持。MeV介导的细胞骨架 修改很可能是机械参与传染中心的释放，我们测试使用 F-肌动蛋白研磨的抑制剂。在目标3中，我们假设脱落的感染中心是生理性的， MeV输送的相关矢量。我们将向恒河猴的气道输送细胞相关和无细胞MeV 并量化感染的时间进程。这一目标有可能重塑一个基本的 MeV如何在主机间传播的教条。总之，这些研究使用适当的模型系统， 研究MeV进入、扩散和管腔释放。我们的研究将阐明大多数 传染性人类呼吸道病毒在释放到下一个宿主之前经历最后的扩增步骤。