Identification of a putative novel rotavirus A VP4 genotype in bats from an urban Atlantic Forest, in the metropolitan region of Rio de Janeiro State, Southeastern Brazil

Abstract Cross-species transmission occurs in rotaviruses, and human infection by animal strains is possible. We investigated the occurrence and genetic diversity of Rotavirus A (RVA) in bats from urban Atlantic Forest remnants under intense anthropogenic pressure in Rio de Janeiro, Brazil. Bats were captured between September 2020 and March 2022, totaling 240 samples (rectal swabs and feces) from 16 species. The RVA infection rate was 1.25% (3/240; Artibeus lituratus and Carollia perspicillata ) using RT-qPCR targeting the NSP3 gene. A complete genomic constellation was attempted for positive samples. Phylogenetic analyses were performed, and homology-based three-dimensional modeling was applied to proteins encoded by genes showing low similarity to GenBank sequences. Partial VP4 sequences were successfully obtained from all three RVA-positive samples. Molecular characterization identified distinct genotypes: Gx-P[26]-I5, G20-P[unclassified*]-I13-T15, and G20-P[unclassified*]-I13-R13-C13-M12-A23-N13-T15-Ex-H15. The latter two harbored a putative novel VP4 genotype (P[unclassified*]) with <75% nucleotide identity to known genotypes and formed a distinct, well-supported phylogenetic clade. The Gx-P[26]-I5 strain showed highest identity to porcine RVA strains, representing the first detection of VP4 genotype P[26] in bats. Except for VP4, both G20P[unclassified*] strains were most closely related to a human G20P[28] strain from Suriname, suggesting a shared genomic constellation and possible reassortment between bat and human viruses. 3D structural analyses revealed conserved VP4 conformation among animal and human strains, suggesting structural compatibility across host species. These findings highlight bats as reservoirs of diverse RVA lineages and their role in viral evolution, reassortment, and zoonotic emergence, reinforcing the need for continuous One Health surveillance.