Molecular Characterization and Isolation of Contemporary Porcine Sapoviruses from Swine Farms in the United States

Porcine sapovirus (PoSaV) is an emerging enteric pathogen in swine industry, primarily associated with diarrhea in weaning and post-weaning pigs. To date, eight genogroups of PoSaVs have been identified, with genogroup III (GIII) being the most prevalent worldwide. However, the isolation of field strains in various cell lines has achieved limited success, hindering progress in PoSaV research. We aimed to isolate contemporary PoSaVs from U.S. swine farms using different porcine cell lines. A total of 42 fecal or intestinal samples were collected from 12-28 days old pigs with mild-moderate diarrhea across five farms and screened for the presence of PoSaV using reverse transcription (RT)-PCR. Positive samples were serially passaged in swine testicular (ST) cells, porcine kidney cells (LLC-PK1), and porcine intestinal epithelial cells (IPEC-J2). Virus isolation was confirmed by RT-PCR and immunofluorescence assays, and representative PoSaV genomes were sequenced. The overall PoSaV detection rate was 60 % (25/42), with farm-level prevalence of 80 % (4/5), and all strains classified as GIII. Sequence analysis revealed high similarity among the isolates but notable divergence from classical GIII strains. The success rates of virus isolation were 8 % (2/25) in ST cells, 16 % (8/25) in LLC-PK1 cells and 0 % (0/10) in IPEC-J2 cells. To our knowledge, this is the first report of successful PoSaV propagation in ST cells, providing a new platform for PoSaV research and for vaccine development.