First isolation of a novel tottorivirus from wild boar faeces in Japan

Viruses of the genus Tottorivirus (family Picornaviridae ) have been identified in pigs but no isolates have been reported to date. In this study, a novel tottorivirus strain, designated Tottori/tc/Wild Boar/Japan/Ishi-6/2020 (Ishi-6), was isolated for the first time from a faecal sample of a wild boar using swine testis cells, where cytopathic effects (CPEs) were observed. Ultrathin-section transmission electron microscopy revealed virus-like structures arranged around vesicular membranes in infected cells. The Ishi-6 replicated in Porcine Kidney-15 and Cloned Porcine Kidney cells with CPEs but no replication was observed in non-porcine cell lines. Sequence analysis showed that Ishi-6 shared high aa sequence identity with a U.S. pig slurry-derived strain in the polyprotein, especially 2C+3 CD region with the highest identity (95.16%). In contrast, the P1 region was more closely related to a Chinese pig-derived strain (74.18%), while showing low identity to Japanese pig-derived tottorivirus strains. Phylogenetic analysis divided tottoriviruses into three clusters based on the polyprotein sequence, whereas different clustering patterns were observed for the P1 and 2C+3 CD regions. The incongruent clustering of Ishi-6 between these regions suggested recombination, which was further supported by recombination analysis identifying a breakpoint near the VP1–2A junction, involving U.S. pig slurry-derived and Chinese pig-derived strains. Real-time reverse transcription PCR targeting the 5′ UTR of Ishi-6 was performed on faecal samples from 280 wild boars and 70 domestic pigs collected between 2018 and 2023. Nine wild boar samples collected between 2018 and 2022 tested positive, while no positive sample was obtained from pigs. These findings indicate that Ishi-6 is a putative recombinant tottorivirus that may have circulated in Japanese wild boar populations. Tottoriviruses exhibit considerable genetic diversity, likely driven by recombination, and continued surveillance is warranted, including their pathogenicity in pigs.