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

Fig 1. PCV3 identification using IFA, Western blot, transmission electron microscopy, and viral growth kinetic curve. (A) IFA detection (400×) of PCV3. Immunostaining of transfected cells passaged to the third generation with virulence after transfection using monoclonal antibody 2E6 to visualize PCV3-specific nuclear fluorescence (green) and DAPI-stained nuclei (blue). (B) Western blot analysis of PCV3. PK-15 cells were used as a negative control, while PK-15 cells transfected with the eukaryotic plasmid pCAGGS-PCV3-Cap were used as a positive control. The size of the target band for PCV3-Cap was consistent with that of the positive control. (C) Transmission electron microscopy observation (73,000×). Circular virus particles with a diameter of approximately 20 nm are visible under transmission electron microscopy. (D) Viral growth kinetic curve. Viral copy numbers were determined in virulent passaged cells at 12, 24, 36, 48, 60, and 72 h after passaging, with the highest viral copy number at 36 h.

Fig 2. PCV3 replication is associated with microtubule polymerization, and the infection of PK-15 cells with PCV3 and wPCV3 was detected by IFAs. (A) Cell viability after drug treatment was determined using the CCK-8 assay, and there was no significant difference in cell viability between the drug-treated group and the mock-treated group. (B and C) Compared to those in the control group, the viral titer and cap expression in the drug-treated group were significantly decreased in a dose-dependent manner. The data are presented as the means ± SDs. *P < 0.05; **P < 0.01. (D) Cytoplasmic fluorescence (green) was observed in the PCV3- and wPCV3-infected PK-15 cells at 24 h post-infection using a 2E6 monoclonal antibody, while the cell nuclei were stained with DAPI (blue).

Fig 3. Clinical performance and immunological responses. (A) Spleen organ index at 35 d post-infection. (B) AWG within 35 d post-infection. (C) Temperature change curve within 35 d post-infection. (D) Detection of antibody levels within 35 d post-infection using the block ELISA method, expressed as percent inhibition (PI).

Fig 4. Infection characteristics of PCV3. Detection of viral viremia in sera (A) and viral load in tissues (B) using a real-time PCR method. (C) Histopathological lesions in the lungs, lymph nodes (LNs), liver, and intestine 35 d post-infection, with lesion sites indicated by red arrows (400×). (D) At 35 d post-infection, immunohistochemical analysis of the heart, liver, lungs, lymph nodes (LNs), spleen, and intestines was performed. Brown-specific punctate deposits were observed following DAB staining (400×). (E) At 14 and 28 days post-infection, PBMCs were separately stimulated with ConA and VLP-PCV3-Cap. After stimulation with ConA, there was no significant difference in cell proliferation between the infected group and the control group. However, following stimulation with VLP-PCV3-Cap, there was a significant difference between the two groups, and cell proliferation was inhibited. The data are presented as the means ± SDs. *P < 0.05; **P < 0.01.

Fig 5. Single-cell profiling of cell populations in PBMCs. UMAP visualization of cell types in PBMCs from the control group (A) and the PCV3-infected group (B). (C) UMAP visualization comparing the heterogeneity of cell types between the control group and the PCV3-infected group. (D) The distribution of each cell type in the control group and the PCV3-infected group was represented as percentages.

Fig 6. Reclassification and pseudotime analysis of T-cell subsets in PBMCs. UMAP and pie charts showing the cell subpopulations and proportions after T-cell repopulation in control (A) and PCV3-infected (B) PBMCs. (C) The heterogeneity of cell subpopulations between the control and infected groups was demonstrated with UMAP plots after re-clustering, and the number of cells in the different subpopulations is labeled with bar graphs. (D) Fitted-time analysis of the distribution of cell subpopulations across differentiation states. (E) Number of cells between samples in the five differentiation states.