Validation of a naturally-occurring animal model for SARS-CoV-2 infection

本项目的总体目标是验证SARS-CoV-2期间病毒适合性和免疫致病机制。 家猫的感染，为下游的翻译研究建立基线。主要目标(具体目标) 项目如下： 目的1.评价SARS-CoV-2在家猫体内的感染动力学和病毒适合性。我们将使用水滴数字聚合酶链式反应 (DdPCR)定量血液、鼻拭子和呼吸道组织中SARS-CoV-2RNA的绝对拷贝数，以便 描述家猫急性感染期间病毒复制的动力学，并将这些变化与共同表达的 呼吸道组织中的病毒抗原和ACE2受体。我们还将使用病毒扩增子测序组件来评估 猫宿主遗传分化的可能性(即病毒是否在家猫感染期间进化)。 假设：SARS-CoV-2感染表达ACE2的猫呼吸道细胞，导致遗传上的渐进性复制 类似于人类新冠肺炎的保守病毒元件和病变 目的2.确定免疫功能障碍导致新冠肺炎疾病进展的关键因素。我们将使用scRNASeq、Flow 细胞术，和多重免疫分析，以确定在急性SARS-CoV-2感染期间免疫图谱的变化。我们会 将免疫学参数的变化与病毒复制动力学(目标1)和临床疾病进展顺序进行比较 以(I)确定免疫功能的紊乱如何影响临床疾病进展和(Ii)确定新的 免疫调节靶点，以指导更有效的治疗或候选疫苗。 假设：猫重症新冠肺炎的进展类似于人类疾病，并与(I)CD_4~+和CD_8~+T细胞相关 细胞缺陷和(Ii)促炎症细胞因子的表达(IL-6、IL-1β、肿瘤坏死因子α)

The overall objective of this project is to validate mechanisms of viral fitness and immunopathogenesis during SARS-CoV-2 infection in domestic cats to establish baselines for downstream translational studies. Major goals (specific aims) for this project are as follows: Aim 1. Evaluate in vivo infection kinetics and viral fitness of SARS-CoV-2 in the domestic cat. We will use droplet digital PCR (ddPCR) to quantify absolute copy numbers of SARS-CoV-2 RNA in blood, nasal swabs, and respiratory tissues in order to characterize viral replication kinetics during acute infection in domestic cats, and compare these changes with co-expression of viral antigen and ACE2 receptors in respiratory tissues (IHC). We will also use virus amplicon sequencing assembly to evaluate the potential for genetic divergence in the feline host (i.e. does the virus evolve during infection in domestic cats). Hypotheses: SARS-CoV-2 infects ACE2-expressing feline respiratory cells, resulting in progressive replication of genetically conserved virus elements and lesions analogous to human COVID-19 Aim 2. Identify key factors of immune dysfunction contributing to COVID-19 disease progression. We will use scRNASeq, flow cytometry, and multiplex immunoassays to define shifts in the immune profile during acute SARS-CoV-2 infection. We will compare changes in immunological parameters with viral replication kinetics (Aim 1) and clinical disease progression in order to (i) define how perturbations of immune function impact clinical disease progression and (ii) identify novel immunomodulatory targets to guide more effective therapies or vaccine candidates. Hypothesis: Progression of severe COVID-19 in cats is analogous to human disease and correlated with (i) CD4+ and CD8+ T cell deficiencies and (ii) pro-inflammatory cytokine expression (IL-6, IL-1β, TNFα,)