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.

Modified Public Health Relevance Section Most emerging human viruses come from wildlife, and coronaviruses in particular represent a significant threat to public health and biosecurity in the US and globally, as was demonstrated by the SARS, MERS, and COVID-19 outbreaks. This project seeks to understand what factors allow bat-origin coronaviruses, including close relatives to SARS, to jump into the human population by studying their evolutionary diversity, the patterns of spillover in people that live in high-risk communities, and analyzing characteristics of bat coronaviruses that could allow them to emerge. This work will produce reagents and genetic sequences that can be used to test vaccines and therapeutics to fight future pandemics, and hotspot maps that can be used to target surveillance and control measures to prevent their emergence.