Successful cell culture isolation and experimental pathogenicity evaluation of porcine bocavirus G3

Since its initial discovery in Swedish pigs in 2009, porcine bocavirus (PBoV) has been detected across Asia, Europe, Africa, and North America. However, the pathogenic potential of PBoV has remained uncertain due to the lack of suitable cell culture systems for viral propagation. In this study, we report the first successful isolation of a Chinese PBoV strain (BK19) from diarrheic piglets in Hunan Province using trypsin-supplemented LLC-PK1 cells. The isolate was characterized through immunofluorescence assay, electron microscopy, plaque formation, and growth kinetics. Whole genome sequencing revealed 43.4-95.7% nucleotide identity with known PBoV strains, with phylogenetic analysis classifying BK19 within the G3 genogroup. Experimental infection of 5-8, 17-19, and 31-33 days old piglets demonstrated age-dependent pathogenicity, with all groups developing characteristic clinical signs including fever, respiratory distress, and diarrhea lasting 3-4 days. Viral shedding peaked in rectal swabs at 4 days post-infection (dpi), with persistent detection through 14 dpi in 5-8 and 17-19 days old groups. Postmortem examination revealed broad tissue tropism in 5-8 and 17-19 days old piglets and age-dependent pathological lesions in intestinal, pulmonary, lymphoid and renal tissues. Immunohistochemical analyses confirmed viral antigen presence in these tissues in 5-8 days old piglets, which correlated with enhanced proliferation of infected cells. These findings provide definitive evidence that PBoV is a primary pathogen in swine, with particular clinical significance for young piglets. This study establishes crucial tools for further research into PBoV biology and control strategies.