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 目前的大流行突显了我们对复制战略的认识存在根本差距 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是如何引发如此多样化的 病理学，以及病毒变异、宿主遗传学和潜在的先决条件所起的作用。作为研究的对象 SARS-CoV-2经常使用基于人群的检测，在感染后几小时到几天内进行，信息关于 细胞和空间的变异性消失了。此外，宿主反应是交际性空间过程的主体 到不同细胞之间不同的信号梯度。因此，总体平均值掩盖了异质性和 空间分离，由于缺乏敏感度而错过了病毒和宿主的最早行为。 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粒子 进入，第一复制事件的建立，以及病毒RNA和宿主反应的产生 单元格，同时保持样本空间的完整性。该项目的长期目标是将这部小说 深入了解SARS-CoV-2生物学的方法，有别于使用传统策略收集的信息。这 知识将为新冠肺炎疾病的预后提供新的洞察力，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)和多路错误稳健FISH(MerFish)将被应用于SARS-CoV-2的研究。目标1将 量化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。这些数据将被用来开发一个随机计算模型，以解决早期 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 影响I型干扰素应答并与严重新冠肺炎相关的条件，如 以及新出现的令人担忧的病毒变种。AIM 3将使用小鼠模型在体内模拟患者的合并症 确定包括干扰素在内的宿主反应的功能和空间后果 和其他细胞因子的表达，在呼吸道和肺部。我们将进一步利用这些体内模型来 了解为什么发病机制和疾病结果因接种剂量和年龄的不同而不同 动物。总之，我们的多学科方法利用了系统级病毒学的技术和信息， 空间转录学、宿主遗传学、计算生物学和天然免疫提供了一种强大的手段 探讨对了解临床结果和提供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.