High host specificity of alphacoronaviruses in Nearctic, insectivorous bats

Bats are reservoir hosts for a number of coronaviruses, some of which may pose spillover risks for humans and other animals. Surveillance for bat coronaviruses in temperate regions remains limited and represents an important blind spot for emerging pathogen preparedness and bat conservation. We detected two alphacoronaviruses in big brown bats ( Eptesicus fuscus ) and little brown myotis ( Myotis lucifugus ) in the province of Ontario, Canada. These viruses are closely related to other coronaviruses circulating in bats in North America and Asia and also related to human and swine coronaviruses. We found unexpected diversity in the spike gene of these highly similar coronaviruses. High homology in the receptor-binding domain (RBD) was maintained in viruses derived from the same species of bat, but markedly lower in those derived from other species. RBD in silico structural analysis of closely related coronaviruses suggests that the viruses we detected are less likely to use bat APN (30 bat species) or ACE2 (20 bat species), or human DPP4 or TMPRSS2 as putative receptors or attachment factors. To gain early insights into interferon antagonism, we also functionally characterized the accessory protein ORF3 from both bat viruses and discovered that ORF3 inhibited both IFNβ production and signaling. Taken together, our study provides insights into coronavirus diversity in Nearctic, insectivorous bats in a previously under-sampled region. This work provides a baseline for more in-depth surveillance to better characterize the transmission dynamics of endemic coronaviruses in free-ranging wildlife, and for exploring the evolutionary relationships between coronaviruses and their hosts.