Rescue of naïve porcine circovirus type 3 and its pathogenesis in CD pigs

The pathogenesis of naïve porcine circovirus type 3 (PCV3) in pigs is not accurately elucidated due to virus isolation failure in vitro. In this study, naïve PCV3 was successfully rescued and used to infect cesarean-derived (CD) pigs. The PCV3 genome was synthesized, cloned, and inserted into the pBluescript SK vector. PK-15 cells transfected with the recombinant plasmid pSK-PCV3 were passaged, and Cap protein expression was confirmed by immunofluorescence assay (IFA) and western blotting. Virus particles (~20 nm) were observed via transmission electron microscopy (TEM). The viral growth curve was plotted to determine the replication of the virus within the cells. Nocodazole treatment demonstrated that PCV3 replication is dependent on microtubule polymerization in the cell. Cells infected with PCV3 or PCV3-positive clinical samples (wPCV3) showed only cytoplasmic fluorescence. PCV3 can successfully infect CD pigs, resulting in persistent viremia, and increased viral loads in tissues and an antibody delay were observed. Inflammatory lesions were also observed in the lungs, lymph nodes, livers, and intestines of infected pigs. Stimulation of peripheral blood mononuclear cells (PBMCs) with virus-like particles (VLPs) containing the Cap protein significantly inhibited cell proliferation. scRNA-seq revealed a significant reduction in T helper 2 (Th2) cells and the migration of T helper 1 (Th1) cells toward the late differentiation stage following infection. In particular, the decrease in Th2 cells may indicate impaired humoral immunity, leading to delayed antibody production and immunosuppression. Our study is the first to observe PCV3 via TEM and to elucidate its immunosuppressive mechanisms in CD pigs.

This study is of great significance as it successfully rescued naïve PCV3 and, for the first time, observed clear PCV3 viral particles using transmission electron microscopy. The successful infection of CD pigs deepened our understanding of its pathogenic mechanisms. The results revealed key aspects of viral replication, the impact of the virus on immune responses, and associated inflammatory lesions in various tissues. Notably, the study found that the reduction of Th2 cells leads to impaired humoral immunity and delayed antibody production, which may provide valuable insights for vaccine development and swine disease management.