Tracking SARS-CoV-2 one molecule at a time: Spatiotemporal investigation of coronavirus replication dynamics and host response in single cells in vitro and in vivo

项目摘要 目前的流行病突出表明，我们对复制战略的认识存在根本性差距 冠状病毒，以及这些病毒如何在生物体和细胞水平上受到宿主的影响。有一个 迫切需要了解SARS-CoV-2感染和宿主细胞反应如何触发如此多样化的一系列免疫反应。 病理学，以及病毒变异、宿主遗传学和潜在前提条件所起的作用。作为研究 SARS-CoV-2经常利用基于人群的检测，这些检测在感染后几小时到几天进行， 细胞和空间变异性丢失。此外，主人的反应是一种交际空间过程 到细胞间不同的信号梯度。因此，总体的平均值掩盖了异质性， 空间分离，并由于缺乏敏感性而错过最早的病毒和宿主行为。 为了填补这一空白，我们开发了实验和计算方法来量化单个病毒体 进入，第一次复制事件的建立，以及病毒RNA的产生和宿主反应， 细胞，同时保持样品的空间完整性。这个项目的长期目标是将这部小说 方法来深入了解SARS-CoV-2生物学，与使用传统策略收集的生物学不同。这 知识将为COVID-19疾病结果谱提供新的见解，并有助于指导未来 治疗策略 为此，单分子原位分析，包括单分子荧光原位杂交， （smFISH）和多重抗错FISH（MERFISH）将被应用于SARS-CoV-2的研究。目标1将 定量SARS-CoV-2进入、复制和传播以及宿主在不同组织细胞中的转录反应 起源这些数据将用于开发一个随机计算模型，以解决早期死亡的决定因素。 病毒复制和由此产生的细胞反应。目标2将检查宿主突变或预先存在的 影响I型干扰素（IFN）反应并与严重COVID-19相关的疾病，如 以及新出现的病毒变种Aim 3将使用小鼠模型在体内模拟患者合并症 SARS-CoV-2感染，确定宿主反应的功能和空间后果，包括IFN 以及其他细胞因子的表达。我们将进一步利用这些体内模型， 理解为什么发病机理和疾病结果取决于接种剂量和年龄的差异 动物总之，我们的多学科方法利用系统级病毒学的技术和信息， 空间转录组学、宿主遗传学、计算生物学和先天免疫提供了一种强有力的手段， 探索对理解临床结果和告知救生干预措施至关重要的问题。

Project Summary The current pandemic has highlighted fundamental gaps in our knowledge about the replication strategies of coronaviruses, and how these are affected by the host at both the organismal and cellular level. There is a pressing need to understand how SARS-CoV-2 infection and host-cell responses trigger such a diverse set of pathologies, and the roles played by viral variation, host genetics and underlying preconditions. As studies of SARS-CoV-2 frequently utilize population-based assays that look hours to days post infection, information on cellular and spatial variability are lost. Furthermore, host responses are communicative spatial processes subject to signaling gradients that vary between cells. Thus, averages over populations obscure heterogeneity and spatial separations, and miss the earliest viral and host behaviors due to lack of sensitivity. To fill this gap, we developed experimental and computational approaches to quantify individual virion entrance, establishment of the first replicative events, and production of viral RNAs and host responses in single cells, all while maintaining sample spatial integrity. This project’s long-term objective is to apply this novel approach to gain insights into SARS-CoV-2 biology distinct from those gleaned using traditional strategies. This knowledge will provide new insight into the spectrum of COVID-19 disease outcomes and help guide future therapeutic strategies. To this end, single-molecule in situ analyses, including single molecule fluorescence in situ hybridization (smFISH) and multiplexed error-robust FISH (MERFISH) will be applied to the study of SARS-CoV-2. Aim 1 will quantify SARS-CoV-2 entry, replication and spread, and host transcriptional responses in cells of varying tissue origin. These data will be used to develop a stochastic computational model to address the determinants of early viral replication and the resulting cellular response. Aim 2 will examine the effect of host mutations or pre-existing conditions that affect the type I interferon (IFN) response and have been associated with severe COVID-19, as well as emerging viral Variants of Concern. Aim 3 will model patient comorbidities in vivo using mouse models of SARS-CoV-2 infection, identifying the functional and spatial consequences of host responses, including IFN and other cytokine expression, in the respiratory tract and lung. We will further utilize these in vivo models to understand why pathogenesis and disease outcome differ depending on the inoculum dose and the age of the animal. Together, our multidisciplinary approach utilizing techniques and information from systems-level virology, spatial transcriptomics, host genetics, computational biology, and innate immunity provides a powerful means of probing questions central to understanding clinical outcome and informing life-saving interventions.