Immunopathogenesis of SARS-CoV-2 infection

我们已经撰写了一份动物研究提案，并已获得NIAID动物护理和使用委员会的批准。我们还没有开始研究，因为BSL3和ABSL3设施要到2020年夏天才开放。我们正在等待BSL3审查委员会批准我们的提案。在第一年，C57BL/6基因的WT和huACE2转基因小鼠将被SARS-CoV和SARS-CoV-2临床分离株感染。在第二年，一株适应小鼠组织培养繁殖的SARS-CoV-2菌株（mSARS-CoV-2）也将与最初的SARS-CoV-2临床分离株进行比较，以确定它是否对小鼠ACE2具有更大的亲和力，并且对表达小鼠ACE2的C57BL/6 WT小鼠具有更大的毒力。对于每种类型的实验，将测试四种病毒剂量，以确定是否可以为每种病毒开发无症状、轻度毒力或较高毒力的模型。根据小鼠的其他传染病模型，将对女性和男性进行检测，预计在冠状病毒疾病的临床进展和组织病理学方面存在差异，并在感染SARS-CoV和SARS-CoV-2的人类中观察到性别差异。因此，他们将被视为单独的队列，但在下表中的数字将被合并。在本实验和所有后续实验中，临床疾病进展将通过咽拭子、血液和粪便中的病毒恢复来评估；感染前及感染后多个时间间隔的血清抗体滴度、血象、血液化学及血清细胞因子；以及身体外观，体重减轻，进展到死亡和感染后一个月的存活。感染后最迟一个月，动物将通过二氧化碳和颈椎脱臼安乐死。根据我们的经验，为了观察生存的显著差异，每个实验需要20只小鼠/队列，由于实验之间的生存差异，我们需要重复实验3次。这些数字应该足以观察到其他临床数据的显著差异。由于预计这些对照之间的差异较小，因此需要较少的未感染小鼠。在本实验和后续实验中，将作为E栏管理的动物在下表中用黄色突出显示。

We have written an Animal Study Proposal that has been approved by the NIAID Animal Care and Use Committee. We have not yet begun research because the BSL3 and ABSL3 facilities were not open until summer 2020. We are awaiting approval of our proposal by the BSL3 review committee. In the 1st Year, WT and huACE2 transgenic mice on a C57BL/6 background will be infected with clinical isolates of SARS-CoV and SARS-CoV-2. In the 2nd Year a strain of SARS-CoV-2 that has been adapted to mouse tissue culture propagation (mSARS-CoV-2) will also be compared with the original clinical isolate of SARS-CoV-2 to determine whether it has greater affinity for mouse ACE2 and greater virulence in C57BL/6 WT mice expressing mouse ACE2. For each type of experiment four virus doses will be tested to determine if models for asymptomatic, mild virulence or higher virulence can be developed for each virus. Both females and males will be tested and are expected to have differences in clinical progression of coronaviral disease and histopathology, based on other infectious disease models in mice, and differences between the sexes observed in humans infected with SARS-CoV and SARS-CoV-2. As a result, they will be treated as separate cohorts but are combined in the numbers in the table below. In this and all subsequent experiments, clinical disease progression will be assessed by virus recovery from throat swabs, blood and stool; serum antibody titers, hemograms, blood chemistries and serum cytokines before and at multiple intervals after infection; as well as physical appearance, weight loss, and progression to moribundity and survival for a month after infection. Animals will be euthanized by CO2 and cervical dislocation at the latest one-month post-infection. In our experience, to observe significant differences in survival we will need 20 mice/cohort in each experiment and repeat the experiment 3 times due to the variance in survival between experiments. These numbers should be sufficient to observe significant differences in the other clinical data as well. Fewer non-infected mice will be needed since there is expected to be less variance among these controls. Animals to be managed as Column E are highlighted in yellow in the tables below for this and subsequent experiments.