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

Qiuhong Wang, The Ohio State University

Bikash Aryal 1, Thomas Petznick 2, Hirotaka Takagi 3, Mingde Liu 1,7, Tomoichiro Oka 4,5, Ganwu Li 6, and Qiuhong Wang 1,7 * 1 Center for Food Animal Health, Department of Animal Sciences, College of Food, Agricultural and Environmental Sciences, The Ohio State University, Wooster, OH 44691, USA 2 ArkCare, Omaha, NE 68132, USA 3 Research Center for Biosafety, Laboratory Animal and Pathogen Bank, National Institute of Infectious Diseases, Musashimurayama, Tokyo 208-0011, Japan 4 Division of Biomedical Food Research, National Institute of Health Sciences, Kawasaki, Kanagawa 210-9501, Japan 5 Department of Virology II, National Institute of Infectious Diseases, Musashimurayama, Tokyo 208-0011, Japan 6 Veterinary Diagnostic Laboratory, Iowa State University, Ames, Iowa, 50011 7 Department of Veterinary Preventive Medicine, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210, USA * Correspondence: wang.655@osu.edu

Molecular Epidemiology & Evolution

Porcine sapovirus (PoSaV) infection is an emerging issue in the swine industry, affecting pigs of all ages, particularly during weaning and post-weaning, causing diarrhea and weight loss. Eight PoSaV genogroups have been identified, with GIII the most prevalent worldwide. However, isolating circulating field strains in cell lines remains challenging. This study aimed to characterize and isolate contemporary PoSaVs from U.S. swine farms using three cell lines. Forty-two clinically suspicious fecal and intestinal samples were collected from diarrheic pigs in late lactation across five farms and tested for PoSaV using RT-PCR and RT-qPCR. Positive samples were then serially passaged in swine testicular (ST) cells, porcine kidney (LLC-PK1) cells, and porcine intestinal epithelial (IPEC-J2) cells. PoSaV replication was confirmed through RT-qPCR and immunofluorescence assays. Additionally, the genomes of representative positive samples were sequenced. The overall PoSaV detection rate was 60% (25/42). Representative PoSaV strains shared 87.5%-87.7% nucleotide identity in the VP1 protein with the GIII/Cowden strain. The success rates for isolating field PoSaV samples were 16% (8/25) in LLC-PK1 cells, 8% (2/25) in ST cells, and 0% (0/10) in IPEC-J2 cells. Replication kinetics of an isolated strain showed earlier replication and a higher peak titer in LLC-PK1 cells (48 hours post-infection; 7.4 ± 0.21 log10 TCID50/mL) compared to ST cells (72 hours post-infection; 5.69 ± 0.23 log10 TCID50/mL).
This study marks the first successful isolation of circulating PoSaV strains in ST cells, paving the way for future research into the virus's biology and disease control and prevention.