Divergent Alphacoronavirus Lineages Identified in Alabama Bats, USA

Bats are crucial reservoirs for coronaviruses, yet surveillance in the southeastern United States remains limited. Using carcasses submitted from public health surveillance for rabies, we screened 170 non-rabid bat carcasses from Alabama using a tiered real-time PCR framework targeting broad Alpha- and Betacoronavirus, alongside specific Sarbecovirus and Merbecovirus lineages. Overall, coronavirus prevalence was 3.5% (6/170), with all positives resolving as Alphacoronavirus. Detections spanned six counties and three bat taxa: Tadarida brasiliensis, Myotis spp., and Eptesicus fuscus, with the latter accounting for most cases (4/6; 66.7%). Sequence and phylogenetic analyses revealed two distinct evolutionary lineages. One isolate, ID-51 (E. fuscus), exhibited relative conservation, sharing 95.3% nucleotide and 93.0% amino acid identity with known references, forming a distinct sibling branch. Conversely, a highly divergent cluster of five isolates (ID-06, -145, -147, -169, -175) displayed remarkably low nucleotide similarities (83.7%–84.3%) to GenBank references despite maintaining high amino acid conservation (98.3%–100%). Topological shifts between nucleotide and amino acid phylogenies demonstrated genetic variation within this novel cluster. These findings underscore a previously uncharacterized genetic diversity of coronaviruses in regional US wildlife, highlighting the necessity of expanded local genomic surveillance.