Spread and Release of Measles in the Airways

项目总结： 人类是极具传染性的麻疹病毒(MeV)的唯一天然宿主。因此，一个关键的 MEV研究面临的挑战是识别具有代表性的人体模型系统。几十年来，MeV一直是 认为通过呼吸道细胞的顶端表面进入人体是一种基于研究的误解 在永生细胞系中。人供体呼吸道上皮细胞分化良好的原代培养 (HAE)提供了一种更具生理学意义的人体呼吸道模型。使用HAE，我们发现MeV 排他地进入基底膜。这一观察导致了一种全新的范式，即 MEV进入人类宿主。除了基底外侧进入，我们观察到HAE的MeV感染导致 感染中心的形成，它保留完整的质膜，与 在永生化细胞中观察到的合胞体。感染中心与合胞体在两个重要方面不同：1) 感染后3-4天感染中心停止生长，2)感染中心在10天后消失 保持细胞层完好无损。为什么感染中心停止增长以及它们是如何在HAE中消失的仍然是一个问题 神秘，也是本应用程序的重点。我们假设感染中心是在呼吸道形成的 在释放给下一宿主之前，上皮细胞是最终扩增过程中至关重要的一步。在目标1中，我们定义 呼吸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。此外，激光捕获感染中心用于分离受感染的细胞 来自上皮片内未受感染的细胞，并使用深度测序来映射细胞响应 MEV。在目标2中，我们讨论了如何将MeV从HAE中释放出来。初步数据显示，感染中心 从HAE上完好无损地脱落(而不是破裂)。剥离的时间进程和频率将是 已定义。将通过细胞标记来探索细胞增殖和细胞死亡途径的作用，以识别如何 可以释放较大的感染中心，但保持上皮完整性。MEV介导的细胞骨架 修改很可能在机械上涉及到感染中心的释放，我们使用 F-肌动蛋白踏板运动的抑制剂。在目标3中，我们假设脱落的感染中心是生理上的 用于MeV传递的相关媒介。我们将把细胞相关的和无细胞的MeV输送到恒河猴的呼吸道 并量化感染的时间进程。这一目标有可能重塑一个根本性的 关于MeV如何在宿主之间传播的教条。总而言之，这些研究使用适当的模型系统来 研究MeV进入、扩散和腔内释放。我们的研究将阐明大多数 传染性人类呼吸道病毒在释放给下一宿主之前经历了最后的扩增步骤。

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.