Identification of a novel equine rhinitis B virus detected in horse from Japan.

Abstract: Equine rhinitis B virus (ERBV), a member of the Picornaviridae family, is associated with mild to moderate respiratory illness in horses, yet its genomic diversity remains incompletely characterized. In this study, we performed metagenomic analysis on a rectal swab sample from a diarrheic foal confirmed to be positive for rotavirus A. Unexpectedly, we identified a highly divergent ERBV strain with a 9,448-nucleotide genome encoding a 2,721-amino-acid polyprotein. Sequence analysis revealed only 62.5-63.1% identity in the polyprotein and 47.1-49.8% in the VP1 region compared to known ERBV serotypes, suggesting a novel genotype. The genome exhibited typical picornavirus features, including a type II internal ribosome entry site (IRES), but also unique elements such as an 87-amino-acid insertion in the leader proteinase region and atypical cleavage motifs. A strain-specific RT-qPCR assay was developed alongside a broadly reactive assay targeting ERBV1-3. Screening of 37 rectal swab samples from horses revealed a 10.8% positivity rate using the new assay, with clustered cases from the same farm in 2022 and an additional case in 2024. No samples were positive using the broad-range primers, indicating potential gaps in surveillance. These findings highlight the importance of metagenomics in pathogen discovery and underscore the need for updated molecular tools to monitor genetically distinct ERBV strains and assess their clinical significance.