Divergent spike protein with a remodeled receptor-binding interface in an unclassified bat-associated alphacoronavirus from Brazil

Background Bats harbor the greatest known diversity of alphacoronaviruses (alphaCoVs), yet this diversity remains poorly sampled in the Neotropics. Characterizing divergent bat-associated alphaCoVs is central to understanding coronavirus (CoV) evolution, Spike protein diversification, and the molecular basis of host adaptation. We characterized the virome profiles of samples from Brazilian bats and jaguars, identified previously undescribed bat-associated alphaCoVs, and modeled the Spike protein of one unclassified alphaCoV for which a complete Spike gene was recovered. Methods Individual samples were first screened for CoVs using a pan-CoVs PCR assay, and for influenza A virus (IAV) and rabies virus (RABV). CoV amplicons were subsequently sequenced using the Sanger method. Samples were then pooled, and metatranscriptomic sequencing was performed on bat tissues ( Molossus molossus and Uroderma bilobatum ) and whole-blood samples from jaguar ( Panthera onca ). Viral contigs were assembled, manually curated, and analyzed using maximum-likelihood and Bayesian phylogenetic inference, nucleotide similarity profiling, and recombination screening. The complete Spike protein was modeled with AlphaFold3 and compared with experimentally determined alphaCoV Spike structures by structural superposition, electrostatic surface mapping, and prediction of complexes with the human receptors angiotensin-converting enzyme 2 (ACE2) and aminopeptidase N (APN). All inferences are based on sequence and structural analysis; no virus isolation, receptor-binding assay or cell-entry experiment was performed. Results AlphaCoV sequences were detected in M. molossus and U. bilobatum bats, from which 13 partial alphaCoV contigs were recovered and analyzed individually. Phylogenetic analyses placed most of these sequences within divergent bat-associated alphaCoV clades that could not be assigned to any recognized ICTV species or subgenus; one short gastrointestinal tract-derived sequence from U. bilobatum instead clustered within the clade corresponding to the recognized subgenus Amalacovirus. The recovered Spike gene was highly divergent from all characterized alphaCoVs, with no evidence of recent recombination. Despite this divergence, the predicted Spike structure retained the overall receptor-binding domain (RBD) fold, while exhibiting extensive remodeling of the receptor-binding motif and of surface electrostatic properties. AlphaFold3 predicted sterically compatible complexes with both ACE2 and APN, but the predicted interfaces were formed by residue sets and geometries distinct from those of experimentally determined human alphaCoV complexes. Non-CoV sequences recovered from jaguar whole blood were reported as taxonomic matches of uncertain provenance; they do not establish infection, and the absence of CoV sequences in blood does not establish that these animals were uninfected. Conclusions These findings extend the known diversity of bat-associated alphaCoVs in Brazil and identify sequences that could not be assigned to any recognized subgenus of the genus Alphacoronavirus. The conservation of the RBD fold, despite extensive remodeling of the receptor-binding interface, is consistent with a model in which the receptor-binding surface of alphaCoVs can diversify while the underlying structural scaffold is retained. Because the structural analyses are entirely computational, they generate hypotheses for experimental testing rather than evidence of human receptor usage. Overall, these findings provide new insights into alphaCoV evolution and offer a framework for investigating how extensive remodeling of the Spike receptor-binding interface can occur while preserving the underlying structural scaffold.