Understanding the Risk of Bat Coronavirus Emergence

修改后的项目摘要/摘要部分 人畜共患冠状病毒(CoV)是对全球健康的重大威胁，SARS、MERS和新冠肺炎的出现证明了这一点。我们的团队确定蝙蝠是SARS冠状病毒的野生动物宿主，我们现在已经发表了数百个新的SARS相关冠状病毒(SARSR-CoV)序列。在我们之前的NIH资助下以及其他人的研究表明，亚洲蝙蝠拥有非同寻常的SARSR-CoV多样性，其中一些可以利用人类ACE2进入细胞，在实验室动物中引起类似SARS的疾病，并可能逃避目前的治疗或疫苗。我们对生态和人类血清学数据的分析表明，该地区每年约有6.6万人感染BAT-SARSR-CoV，公共卫生影响未知。在对我们于2019年首次授予的更新的R01进行的这次修改中，我们计划实现三个目标：目标1.分析我们之前在中国南部采集的蝙蝠SARSR冠状病毒的300多个全基因组/大基因组片段，以确定导致“重组热点”的病毒特性、宿主生物学特性和环境/生态因素。我们将使用宿主范围建模、复杂的系统发生学以及生态、环境和人口数据来评估可能促进重组的因素。我们还将分析我们先前工作中确定的200多个其他BAT冠状病毒RdRp序列的溢出风险，以评估所有BAT冠状病毒的SARSR冠状病毒溢出风险相关性的一般性；目标2.使用我们基于社区和临床的调查数据和存档的新冠肺炎前人类样本来确定推定的SARSR冠状病毒溢出事件、暴露途径和潜在的公共健康后果。我们将用SARSR-CoV ACE2替代病毒中和检测样本，以确定SARSR-CoV和其他结合ACE2的CoV是否感染新冠肺炎前。我们将测试存档的临床症状监测样本，以评估出现流感样疾病和严重急性呼吸道疾病的患者是否有SARSR-CoV感染的PCR或血清学证据；目标3.使用计算机建模和细胞培养来分析新的BAT-CoV、假定的宿主和中间宿主以及人之间的潜在结合相互作用，以验证AIMS 1和2的溢出预测。我们将不使用重组病毒技术，而是使用氨基酸序列分析、RBD与人、BAT和其他可能的宿主ACE2和其他受体的结合建模，以及与非传染性病毒蛋白和伪病毒技术的体外结合分析来估计CoV与人类细胞的结合。然后，我们将检验我们的假设，即与SARS-CoV或SARS-CoV-2具有10-25%尖峰蛋白序列差异的SARSR-CoV能够感染人类细胞，并逃避治疗和疫苗。所有这三个目标的工作将有助于证明我们的BAT CoV计划是一个整合导致CoV出现的病毒学和生态因素分析并为预防未来大流行提供高影响力战略的示范平台。这包括提供关键试剂、治疗干预措施和病毒基因组序列，为大流行和公共卫生做好准备，以应对未来可能爆发的冠状病毒。

Modified Project Summary/Abstract Section Zoonotic coronaviruses (CoVs) are a significant threat to global health, as demonstrated by the emergence of SARS, MERS, and COVID-19. Our group identified bats as the wildlife reservoirs of SARS-CoV, and we have now published hundreds of novel SARS-related CoV (SARSr-CoV) sequences. Work under our previous NIH funding, and by others, has demonstrated that bats in Asia harbor an extraordinary diversity of SARSr-CoVs, some of which can use human ACE2 for cell entry, cause SARS-like illness in laboratory animals, and may evade current therapies or vaccines. Our analysis of ecological and human serological data indicates a median of around 66,000 people are infected by bat-SARSr-CoVs in the region each year, with unknown public health impacts. In this modification to our renewed R01, first awarded in 2019, we plan three aims: Aim 1. Analyze more than 300 full genomes/large genome segments of bat SARSr-CoVs from our prior bat sampling in southern China to identify viral characteristics, host biological traits, and environmental/ecological factors that lead to ‘recombination hotspots’. We will use host range modeling, complex phylogenetics, and ecological, environmental and demographic data to assess factors that may enhance recombination. We will also analyze spillover risk from over 200 other bat CoV RdRp sequences identified in our prior work to assess the generality of correlates of SARSr-CoV spillover risk for all bat-CoVs; Aim 2. Use our community- and clinic-based survey data and archived pre-COVID-19 human samples to identify putative SARSr-CoV spillover events, routes of exposure, and potential public health consequences. We will test samples with a SARSr-CoV ACE2 surrogate virus neutralization to identify pre-COVID-19 infection with SARSr-CoVs and other ACE2 binding CoVs. We will test archived clinic-based syndromic surveillance samples to assess if patients presenting with influenza-like illness and severe acute respiratory illness have PCR or serological evidence of SARSr-CoV infection; Aim 3. Use computer modeling and cell culture to analyze potential binding interactions among novel bat-CoVs, putative reservoir and intermediate hosts, and people, to validate spillover predictions from Aims 1 & 2. Rather than use recombinant virus technology, we will estimate CoV binding to human cells using amino acid sequence analysis, modeling of RBD binding to human, bat & other putative host ACE2 and other receptors, and binding assays with non-infectious viral proteins and pseudovirus technology in vitro. We will then test our hypothesis that SARSr-CoVs with 10-25% spike protein sequence divergence from SARS-CoV or SARS-CoV-2 are able to infect human cells, and evade therapeutics and vaccines. Work from all three aims will help demonstrate proof-of-concept that our bat CoV program is a model platform to integrate analysis of virological and ecological factors contributing to CoV emergence and inform high impact strategies to prevent future pandemics. This includes providing critical reagents, therapeutic interventions, and viral genome sequences for pandemic and public health preparedness to counter future potential coronavirus outbreaks.