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

一次跟踪一个分子 SARS-CoV-2：体外和体内单细胞中冠状病毒复制动态和宿主反应的时空研究

• 批准号: 10446423
• 负责人: Charles M Rice
• 金额: $ 63.76万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-11 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10446423
• 关键词: 2019-nCoV; Address; Affect; Age; Animals; Autoantibodies; Behavior; Biological Assay; Biology; COVID-19; COVID-19 pandemic; Cells; Cellular biology; Cessation of life; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Computational Biology; Computer Models; Coronavirus; Data; Data Set; Disease; Disease Outcome; Dose; Encapsulated; Environment; Epidemiologic Monitoring; Escape Mutant; Event; Fluorescent in Situ Hybridization; Future; Gene Expression; Genetic; Genetic Transcription; Genetic Variation; Genomics; Genotype; Glean; Hamsters; Heterogeneity; Hour; Human Genetics; Image; Immune response; Immunity; In Situ; In Vitro; Individual; Infection; Information Systems; Interferon Type I; Interferons; Intervention; Investigation; Kinetics; Knock-in; Knock-out; Knowledge; Lead; Life; Link; Lung; Measures; Methods; Modeling; Molecular; Monkeys; Mus; Mutation; Natural Immunity; Nature; Outcome; Pathogenesis; Pathology; Pathway interactions; Patient-Focused Outcomes; Patients; Persons; Phenotype; Play; Population; Predisposition; Process; Production; Respiratory System; Risk; Role; SARS-CoV-2 infection; Sampling; Savings; Severity of illness; Signal Transduction; Symptoms; System; Techniques; Testing; Therapeutic; Time; Tissues; Vaccines; Variant; Viral; Virion; Virus; Virus Diseases; Virus Replication; base; cell type; clinically relevant; comorbidity; cytokine; fitness; genetic variant; human disease; in vivo; in vivo Model; insight; interdisciplinary approach; mouse genetics; mouse model; mutant; neutralizing antibody; novel strategies; pandemic disease; population based; response; severe COVID-19; single molecule; spatiotemporal; transcriptomics; variants of concern; viral RNA; virology; virus genetics

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.

项目概要 当前的大流行凸显了我们对复制策略的认识存在根本差距 冠状病毒，以及它们如何在有机体和细胞水平上受到宿主的影响。 迫切需要了解 SARS-CoV-2 感染和宿主细胞反应如何引发如此多样化的反应 病理学，以及病毒变异、宿主遗传学和潜在先决条件所发挥的作用。 SARS-CoV-2 经常利用基于人群的检测来观察感染后数小时至数天的情况，有关信息 此外，宿主反应是交流空间过程的主题。 因此，群体的平均值掩盖了异质性和差异性。 空间分离，并且由于缺乏敏感性而错过了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、第一个复制事件的建立、病毒 RNA 的产生和宿主反应在单个 细胞，同时保持样本空间完整性。该项目的长期目标是应用这种新颖的方法。 方法来深入了解 SARS-CoV-2 生物学，与使用传统策略收集的数据不同。 知识将为了解 COVID-19 疾病结果提供新的见解，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) 和多重抗差错 FISH (MERFISH) 将应用于 SARS-CoV-2 的研究。 量化不同组织细胞中 SARS-CoV-2 的进入、复制和传播以及宿主转录反应 这些数据将用于开发随机计算模型，以解决早期的决定因素。 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 (IFN) 反应并与严重 COVID-19 相关的病症，如 以及新兴病毒变种 Aim 3 将使用小鼠模型对患者体内合并症进行建模。 SARS-CoV-2 感染，确定宿主反应的功能和空间后果，包括 IFN 以及其他细胞因子在呼吸道和肺部的表达，我们将进一步利用这些体内模型来研究。 了解为什么发病机制和疾病结果因接种剂量和年龄而异 我们的多学科方法共同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强大的手段 探究对于了解临床结果和为挽救生命的干预措施提供信息至关重要的问题。