Interspecies transmission and genome heterogeneity of porcine-originated Rotavirus A between domestic pigs and wildlife in the Croatian ecosystem.

Rotavirus A (RVA) is a major cause of non-bacterial gastroenteritis in mammals and birds, with sporadic zoonotic events. Despite well-documented interspecies transmission of porcine-originated RVA strains (poRVAs), the role of wildlife in transmission dynamics remains underexplored. Using a One Health spatiotemporal approach, this study investigated the genetic interconnectedness of poRVAs in domestic pigs and wildlife in Croatia (2018-2021). Fecal samples or rectal swabs from 445 domestic pigs, 441 wild boars, 533 red foxes, and 131 golden jackals underwent RNA extraction, RT-PCR, VP7/VP4 genotyping, and Sanger sequencing. From these, nineteen samples carrying poRVA genotypes in wildlife and matching genotypes in domestic pigs were selected for the NGS and complete RVA genome analysis. Results revealed porcine genogroup 1 constellation, with surface protein genotypes characteristic of porcine hosts in all detected RVA strains. Wildlife-derived poRVAs proved evolutionary related to domestic pig-derived strains, verifying previously hypothesized interspecies transmission. Among VP7 genotypes, G3 dominated in wildlife, G5 in domestic pigs, and G4 showed zoonotic potential. In VP4, P[13] was the most prevalent, while P[23] and P[6] exhibited recombination events. Mixed-genotype RVAs appeared only in domestic pigs encompassing VP7, VP4, and NSP4 gene segments. This study provides insights into RVA host diversity, presenting the first complete RVA genome data from golden jackals and the second from red foxes globally. In addition, it presents the first complete RVA genomes from wild boars outside of Asia to date. Wildlife-derived RVAs showed evolutionary links to domestic pig strains, including zoonotic strains, highlighting the role of wildlife in RVA dissemination. These findings emphasize the need for expanded animal RVA surveillance to better understand environmental transmission, zoonotic risks, and control strategies within a One Health framework.