Fecal metatranscriptomics and virus isolation reveal picornavirus diversity and evolution in Japanese wild boars and pigs.

To elucidate the enteric virome and its relationship between wild boars and domestic pigs, metatranscriptomic analysis and virus isolation using a swine testis (ST) cell line were conducted on fecal samples collected from 300 wild boars and 363 pigs in Japan between 2017 and 2023. Inoculation onto ST cells induced cytopathic effects (CPEs) in samples from 47 wild boars and 178 pigs. Deep sequencing of fecal supernatants and CPE-positive culture supernatants revealed numerous viral sequences belonging to the order Picornavirales (picornaviruses; PVs), which became the focus of this study. A total of 42 (6 from wild boars and 36 from pigs) and 247 (39 from wild boars and 208 from pigs) PV sequences were obtained from fecal supernatants (wild-type; wt) and cell culture supernatants (tissue culture-adapted; tc), respectively. Among 67 porcine teschovirus (PTV) strains detected, 66 (3 from wild boars and 63 from pigs) were isolated in cell culture, except for one B1 serotype strain. The 63 porcine isolates were classified into 11 serotypes, and intertypic homologous recombination events were identified. Of the three wild boar strains, one showed high similarity to Japanese porcine strains, whereas another strain differed significantly, suggesting independent evolution. A total of 73 enterovirus G (EV-G) strains (25 from wild boars and 48 from pigs) were classified into nine genotypes. Genotypes G17 and G6 were predominant in both wild boar and pig populations, suggesting a close relationship between the two hosts. Papain-like cysteine protease (PL-CP) sequences were detected in six genotypes, and tc-type PL-CP-positive EV-G strains of genotypes G2, G8, and G18 are reported here for the first time. Although all previously reported G17 strains possess PL-CP, eight of thirteen G17 strains from wild boars in 2023 lacked PL-CP, highlighting the need to monitor future population dynamics. A total of 126 porcine sapelovirus (PSV) strains (14 from wild boars and 112 from pigs) were identified, most of which (13 and 107, respectively) were isolated in cell culture. PSV strains formed region-specific clusters, indicating geographic associations between wild boar and pig populations. Multiple genetically diverse groups coexisted within single districts, and numerous homologous recombination events were detected among them. Porcine kobuvirus (PKV) was detected in wild boars in Japan for the first time. For porcine sapovirus (SaV), genogroup GIII was the most frequently detected. The sole SaV strain identified from a wild boar clustered with genotype GXI but showed low VP1 sequence similarity to other GXI strains. The 3'-terminal region of this strain exhibited high identity to a Chinese GVII.5 strain, suggesting past cross-species transmission and homologous recombination events. These findings indicate that while some PVs in wild boars may have evolved independently, others share close genetic relationships with strains from pigs. PVs appear to be diversifying within wild boar and pig populations, with homologous recombination serving as a key driver of their genetic variability.