Genetic investigation of SARS-CoV-2 infection in oral and nasal tissues

项目摘要/摘要 新冠肺炎疫情引发了一场全球性的健康和经济危机。虽然大流行正在逐渐 由于大规模接种疫苗而有所减轻，这场灾难的完全控制受到几个持续存在的问题的阻碍 包括：(1)抗药性或难以获得疫苗接种；(2)出现新的更具传染性的病毒变种；(3) 一些患者出现“长冠状病毒”症状；以及(Iv)对病毒进入机制的了解不完全。 因此，确定其他阻断感染的方法并提高对感染的认识仍然很重要。 持续的症状，以便能够更有效地预防和/或治疗这些症状。SARS-CoV-2感染 通过空气中的飞沫传播，味道和气味的丧失或改变在疾病早期出现 进展，可在没有其他症状的情况下发生，并可作为COVID-19的诊断工具， 表明口腔和鼻腔上皮细胞是主要的病毒目标。然而，这些上皮细胞的功能证据 缺乏原发感染部位，以及化学感觉症状的基础，以及为什么这些症状在 疾病的恢复，仍然知之甚少。特别是，新冠肺炎是否与味觉异常有关 继发于嗅觉障碍、相关口腔上皮感染和/或唾液分泌改变，或由 味蕾或神经的直接或继发性感染，目前尚不清楚。在活体中解决这些问题，请访问 机械层面需要健壮的、基因可操控的模型系统。老鼠提供了一个极好的 用于人类疾病建模的基因系统，但不能被野生型SARS-CoV-2强势感染 因为它的刺突蛋白不能有效地与小鼠的ACE2受体结合。现有人类(H)ACE2- 表达hACE2的小鼠或者以转基因的非内源性方式表达hACE2，或者表现出相对较弱的表达 敲击小鼠ACE2等位基因导致临床疾病消失后hACE2的表达 SARS-CoV-2感染，限制了它们在机制研究中的使用。拟议的研究将利用强大的新技术 支持致死性SARS-CoV-2感染并允许组织特异性的hACE2条件性敲击小鼠系 Cre介导的hACE2功能的丧失和获得。使用这些工具，我们将：(I)确定hACE2是否 口腔和鼻腔上皮细胞表达是致命性新冠肺炎病所必需和/或足够的；和 阐明新冠肺炎病味觉障碍的口腔和鼻腔细胞机制。组合在一起 通过人体组织验证，这些研究将确定口腔和鼻部细胞在新冠肺炎病和 通过靶向这些细胞揭示预防或治疗疾病的策略。

Project Summary/Abstract The COVID-19 pandemic has produced a global health and economic crisis. While the pandemic is gradually being lessened by mass vaccinations, full control of this disaster is hampered by several ongoing problems including: (i) resistance or poor access to vaccination; (ii) emergence of new more infectious viral variants; (iii) “long COVID” symptoms in some patients; and (iv) incomplete understanding of the mechanisms of viral entry. Thus, it remains important to identify additional methods of blocking infection and to improve understanding of long-lasting symptoms so that these can be prevented and/or treated more effectively. SARS-CoV-2 infection is transmitted via airborne droplets, and loss or alterations of taste and smell appear early in disease progression, can occur in the absence of other symptoms, and serve as a diagnostic tool for COVID-19, suggesting oral and nasal epithelia as primary viral targets. However, functional evidence for these epithelia as primary infection sites is lacking, and the basis for chemosensory symptoms, and why these can persist after disease recovery, remains poorly understood. In particular, whether COVID-19-associated taste abnormalities are secondary to anosmia, infection of associated oral epithelia, and/or altered salivary secretion, or result from direct or secondary infection of taste buds or nerves, is currently unclear. Addressing these questions in vivo at the mechanistic level requires robust and genetically manipulable model systems. Mice provide an excellent genetic system for human disease modeling, but cannot be robustly infected by wild-type SARS-CoV-2 because its spike protein does not efficiently bind the mouse ACE2 receptor. Existing human (h)ACE2- expressing mice either express hACE2 in a transgenic, non-endogenous manner, or exhibit relatively weak expression of hACE2 following knockin to the mouse Ace2 allele resulting in absence of clinical disease after SARS-CoV-2 infection, limiting their use in mechanistic studies. The proposed studies will utilize powerful new hACE2 conditional knockin mouse lines that support lethal SARS-CoV-2 infection and permit tissue-specific Cre-mediated loss and gain of hACE2 function. Using these tools, we will: (i) determine whether hACE2 expression in oral and nasal epithelia is required and/or sufficient for lethal COVID-19 disease; and (ii) elucidate the oral and nasal cellular mechanisms underlying taste dysfunction in COVID-19 disease. Combined with human tissue validation, these studies will define the role of oral and nasal cells in COVID-19 disease and reveal strategies to prevent or treat the disease by targeting these cells.