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：
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
项目类别：
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

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，一个关键的 MeV 研究的挑战是识别代表性的人体模型系统。几十年来，MeV 人们认为通过气道细胞的顶端表面进入人类宿主，这是基于研究的误解在永生化细胞系中。来自人类供体的气道上皮细胞的分化良好的原代培养物（HAE）提供了与人体气道更加生理相关的模型。使用 HAE，我们发现 MeV 完全进入基底外侧膜。这一观察结果带来了一个全新的范式 MeV进入人类宿主。除了基底外侧进入之外，我们还观察到 HAE 的 MeV 感染会导致保留完整质膜的感染中心的形成与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不同之处：1) 感染中心在感染后 3-4 天停止生长，2) 感染中心在约 10 天后消失保持细胞层完整。为什么传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至关重要的一步。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 个时间点量化了 14 个抗病毒前哨基因 6小时至2周不等。此外，利用激光捕获感染中心来分离受感染的细胞来自上皮片内未感染的细胞，并使用深度测序来绘制细胞反应兆伏。在目标 2 中，我们讨论了如何从 HAE 中释放 MeV。初步数据表明，传染中心完整地从 HAE 中脱落（而不是破裂）。脱落的时间和频率定义的。将通过细胞标记来探究细胞增殖和细胞死亡途径的作用，以了解细胞增殖和细胞死亡途径的作用大型感染中心可以被释放，但上皮完整性得以维持。 MeV介导的细胞骨架修改可能在机制上涉及感染中心的释放，我们使用 F-肌动蛋白跑步机抑制剂。在目标 3 中，我们假设棚内传染中心在生理上是 MeV 传递的相关向量。我们将向恒河猴气道输送细胞相关和无细胞 MeV 猕猴并量化感染的时间过程。这一目标有可能重塑基本面 MeV 如何在主机之间传播的教条。总之，这些研究使用适当的模型系统来研究 MeV 进入、扩散和管腔释放。我们的研究将阐明最重要的机制传染性人类呼吸道病毒在释放到下一个宿主之前要经历最后的扩增步骤。