Virological characteristics of SARS-CoV-2-related coronaviruses dynamically circulating in Southeast Asia

Following the emergence of SARS-CoV-2 in humans, the scientific community has ramped up its efforts to better understand the biology, ecology and spillover risk of SARS-CoV-2-related viruses (SC2r-CoVs), primarily circulating in horseshoe bats. Here, we present a holistic approach to going from field sampling to experimental, phylogenetic, and ecological characterization of novel SC2r-CoVs. We sampled acuminate horseshoe bats from an artificial cave in Thailand and identified two SC2r-CoV lineages co-circulating in the same population. Our sampling identifies the first co-infection of an ACE2- and a non-ACE2-using SC2r-CoV, raising important implications about the interactions between related coronaviruses that utilize different entry receptors. Despite the new ACE2-using virus’s ability to bind to human ACE2, it exhibits reduced fusogenicity and replication in vitro, and lower pathogenicity and transmissibility in hamsters compared to SARS-CoV-2, highlighting the latter’s key biological differences to its bat-infecting relatives. Finally, we implemented a state-of-the-art phylogeographic approach to trace the movement of the bat SC2r-CoVs presented here across their evolutionary history. We show that some of their genomic segments recently moved from Laos to Thailand, reinforcing the notion that SC2r-CoVs continuously travel across their host ranges in Southeast Asia. The multi-faceted approach we employ here provides comprehensive insights into the biology and ecology of the viruses closest to SARS-CoV-2 and paves the way for effectively assessing the spillover potential of animal viruses.