Spread and Release of Measles in the Airways

麻疹在呼吸道中的传播和释放

基本信息

批准号：
10408155
负责人：
PATRICK L SINN
金额：
$ 49.8万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-07-01 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10408155
关键词：
Address Alveolar Animal Model Animals 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 Persons Physiological Process Proteins Reporting Research Role Rupture Sentinel Series Sneezing 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 innovation insight nectin nonhuman primate pathogen pathogenic virus preference programs receptor respiratory virus response transmission process treadmill vector

项目摘要

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.

项目摘要：人类是唯一具有传染性麻疹病毒（MEV）的天然储藏。因此，一个批判 MEV研究的挑战是识别代表性人类模型系统。几十年来，梅夫是被认为是通过气道细胞的顶部表面进入人类宿主的，这是基于研究的误解在永生的细胞系中。来自人类供体的气道上皮细胞的分化良好的原发性培养（HAE）提供了人类气道的更生理相关的模型。使用HAE，我们发现Mev 专门进入基底外侧膜。该观察结果导致了一个全新的范式梅夫进入人类主持人。除基底外侧外，我们还观察到HAE的MEV感染导致保留完整质膜的传染中心的形成，与在永生的细胞中观察到的合胞体。传染中心与合成中心有两种重要方式不同：1）感染中心在感染后3-4天停止生长，2）传染中心消失了〜10天后使细胞层完好无损。为什么传染心中心停止增长以及它们如何消失在HAE中神秘，是该应用程序的重点。我们假设呼吸道中的传染中心形成上皮是最终放大过程中至关重要的一步，然后释放给下一个主机。在AIM 1中，我们定义气道中的先天免疫反应途径。我们在12个时间点量化了14个抗病毒前哨基因从6小时到2周不等。另外，传染中心的激光捕获用于隔离感染细胞从上皮片中的未感染细胞和深度测序用于映射细胞响应梅夫。在AIM 2中，我们解决了MEV如何从HAE释放的方式。初步数据表明传染中心从HAE中脱落，完整（而不是破裂）。脱落的时间和频率将是定义。细胞增殖和细胞死亡途径的作用将通过细胞标记来探测以辨别如何可以释放大型传染中心，但上皮完整性得到了维护。 MEV介导的细胞骨架修改很可能与传染中心发行有关，我们使用 F-肌动蛋白跑步机的抑制剂。在AIM 3中，我们假设流失感染中心是生理的 MEV交付的相关向量。我们将将与细胞相关的和无细胞的MEV传递到恒河的气道猕猴并量化感染的时间顺序。这个目标有可能重塑基本 MEV的教条传播到主持人。总而言之，这些研究使用适当的模型系统来研究MEV进入，传播和腔释放。我们的研究将阐明最多的机制传染性的人类呼吸道病毒在发布给下一个宿主之前经历了最终的扩增步骤。