Identification of a novel equine rotavirus B from Missouri in 2025, which is distantly related to group B rotaviruses

ABSTRACT Despite the wide circulation of group B rotaviruses in animals and humans, rotavirus B was not found in horses until 2021 in Kentucky, USA. Little is known about the genetic diversity and evolution of rotavirus B in this new host. Here, we described a divergent equine rotavirus B (ERVB) from a foal with idiopathic diarrhea in Missouri (designated MO-ERVB-25). Full-genome sequencing and phylogenetic analysis revealed that MO-ERVB-25 is distantly related to all previously known group B rotaviruses, including ERVB strains identified in 2021 in Kentucky (KY-ERVB-21). The mean nucleotide substitution rate of the VP4 segment was estimated as 1.44 × 10 −3 substitutions/site/year, which is a magnitude higher than the VP7 segment. We further demonstrated that MO-ERVB-25, like human RVB, binds chemically neutral LacNAc-containing complex N-linked glycans, while KY-ERVB-21 recognizes negatively charged polysialic acid (polySia) with α−2,8-linkage, which is a functional receptor for unrelated enteric human adenovirus 52. Using the AlphaFold-enabled structure prediction, we showed that both MO-ERVB-25 and human RVB VP8* lack a novel C-terminal α2 helix that is only observed in the KY-ERVB-21 VP8*, which may help explain the difference in glycan binding specificities. Finally, screening of foal samples by the reverse transcription quantitative PCR assay identified 4.2% of idiopathic foal diarrhea samples (6 out of 140) positive for MO-ERVB-25. These results suggest that MO-ERVB-25 represents a new rotavirus B subgroup that circulates in foals. Its circulation, together with KY-ERVB-25 subgroup, raises the possibility of reassortment and antigenic shift as mechanisms of rotavirus B evolution in equines. IMPORTANCE Equine RVB (ERVB) has emerged as a significant cause of foal diarrhea since 2021. Our study describes a highly divergent RVB strain identified in a foal with severe diarrhea in 2025. This ERVB variant exhibited low amino acid identity compared with previously reported ERVB strains. The novel ERVB strain escaped routine diagnostic detection. Sequence analysis demonstrated that this new strain grouped with bovine, porcine, caprine, and equine RVB but formed a distinct, independent branch. In addition, glycan array studies revealed the substantial difference in glycan recognition between new and previously described ERVB strains, which is likely determined by the presence or absence of a novel C-terminal α2 helix in the VP8* receptor-binding domain. These results suggest that this novel ERVB variant may co-circulate with existing ERVB strains in horses, which may facilitate rapid viral evolution through antigenic diversity, a property previously reported in rotaviruses from other hosts.