Using metagenomic sequencing we identified equine rotavirus group B (ERVB) of ruminant origin in foal diarrhea outbreaks in the 2021 foaling season. To further investigate ERVB occurrence and determine its environmental stability, we collected mare and foal fecal samples from different farms in Central Kentucky during the 2022 foaling season. The RT-qPCR-based analyses showed that ERVB genome was detected in 16.67 % (42/252) of surveyed mare samples and 26.56 % (34/128) of foal samples. Furthermore, 94.12 % (16/17) of collected soil samples and 100 % (13/13) of water samples obtained from the ERVB-positive farm premises also tested weakly positive. In addition, ERVB genome fragments were detected in 58.33 % (7/12) of indoor samples collected from the equipment/barn/hospital wards during the outbreak period. Finally, the seroprevalence study showed 87 % (113/130) of surveyed horse serum samples were positive for ERVB antibodies. Despite unsuccessful attempts in ERVB cultivation, phylogenetic analyses showed that fecal ERVB strains representing 2022 and 2023 foal diarrhea outbreaks, like 2021 strains, were more closely related to ruminant rotavirus B than other viruses. Further sequence analyses revealed that none of the three viral capsid proteins, the primary targets of virus-neutralizing antibodies, exhibited notable mutations among ERVB strains circulated over the past three years. Our data demonstrated that ERVB was widespread in horses on affected farms with extreme stability in the farm environment. These findings continue to support the need for future surveillance of ERVB in horses and the surrounding environment, and the development of effective countermeasures to protect horses against this new viral disease.
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