Elucidating Virus:Host Interactions of Human Parainfluenza Virus 3 in a Novel Human Lung Tissue Explant Model

项目总结： 人类副流感病毒3型(HPIV3)在美国构成了一个主要的公共卫生问题，因为它是 年，五岁以下儿童住院的第二大原因是高死亡率 免疫功能受损的成年人。HPIV3的研究传统上是使用细胞系、小型啮齿动物进行的 模型和人类呼吸道上皮模型，所有这些模型都有能力提供 对人类肺部疾病的洞察。为了解决这一知识缺口，我们开发了一种新的肺 采用全人肺组织外植体的模型。在初步数据中，我们利用了最近开发的绿色荧光蛋白- 标记的HPIV3感染人肺组织，表明HPIV3可以在该系统中旺盛地复制，如 通过流式细胞仪检测GFP阳性细胞的存在来评估。我们假设，对 人类肺组织模型中的HPIV3感染将阐明新的病毒与宿主的相互作用并提供见解 HPIV3在人类中的发病机制。在目标1中，我们将使用GFP标记的HPIV3感染人肺 组织来鉴定感染的细胞类型并评估感染引起的转录变化 单细胞RNA测序。在目标2中，我们将通过以下方式表征HPIV3引起的炎症反应 用多重ELISA法检测趋化因子/细胞因子的诱导，并确定这些因子/细胞因子的诱导程度 导致发病的炎症介质是由感染细胞和/或旁观者细胞产生的。这些 数据将为HPIV3如何导致疾病提供新的见解，并有可能识别新的 免疫治疗或抗病毒靶标。这个项目提供了一个独特的培训机会，因为它与分子 克隆、传统病毒学技术、尖端单细胞RNA测序技术、翻译 在深入指导下进行研究和高维数据分析，指导如何成为成功的 学院派科学家。

PROJECT SUMMARY: Human parainfluenza virus 3 (HPIV3) constitutes a major public health concern in the United States as it is the second largest cause of hospitalization in children under the age of five and responsible for high mortality in immunocompromised adults. Studying HPIV3 has traditionally been conducted using cell lines, small rodent models and human airway epithelial models, all of which have limitations with regards to their ability to provide insights into human disease in the lung. To address this gap in knowledge, we have developed a novel lung model using whole human lung tissue explants. In preliminary data, we utilized a recently developed GFP- tagged HPIV3 to infect human lung tissue and show that HPIV3 can replicate robustly in this system, as assessed by the presence of GFP-positive cells detected by flow cytometry. We hypothesize that evaluation of HPIV3 infection in this human lung tissue model will elucidate novel virus:host interactions and provide insights into the pathogenesis of HPIV3 in humans. In Aim 1, we will use GFP-tagged HPIV3 to infect human lung tissues to identify the infected cell types and evaluate the transcriptional changes induced by infection using single cell RNA sequencing. In Aim 2, we will characterize the inflammatory response induced by HPIV3 by examining the induction of chemokines/cytokines using multiplex ELISA, and identify the extent to which these inflammatory mediators that induce morbidity are produced from infected cells and/or bystander cells. These data will provide novel insights into how HPIV3 causes disease and have the potential to identify novel immunotherapeutic or antiviral targets. This project offers a unique training opportunity, as it meshes molecular cloning, traditional virology techniques, cutting edge single cell RNA sequencing technologies, translational research and high dimensional data analysis with thorough mentorship on how to become a successful academic scientist.