Isolation of PCV3 from Perinatal and Reproductive Cases of PCV3-Associated Disease and In Vivo Characterization of PCV3 Replication in CD/CD Growing Pigs

Figure 1. In vitro PCV3 growth kinetic curve. PK-15 cell cultures (six replicates per time point) were inoculated with PCV3 isolate ISU27734 passage 3 containing 3.33 × 108 genomic copies per mL (Cq 17.8) or mock-inoculated (infection medium), and incubated at 37 °C with 5% CO2 for 72 h. Time 2 corresponded to cell culture 2 h after virus inoculation. Virus was harvested every 12 h and the genomic copies per mL were assessed by qPCR. Each time point is represented by the mean of six technical replicates and bars represent the standard error (SE) of the mean.

Figure 2. Detection of PCV3 replication in vitro by indirect fluorescent antibody assay (iIFA). PK-15 cells were infected with PCV3 isolate ISU27734 (passage 6). The reactivity PCV3-specific mouse antisera collected from two mice (L and N) was first evaluated and compared to pre-immune sera at different dilutions by ELISA (panel 2A) and IFA (panel 2B). Subsequently, PCV3 infection of PK-15 cells was demonstrated using PCV3-specific mouse hyperimmune serum diluted 1:20 as primary antibody, FITC-labelled goat anti-Mouse IgG (1:100) was used as secondary antibody and DAPI (1:500) as nuclear counterstain. Absence of cellular reactivity was observed in PCV3 infected cells stained with pre-immune mouse serum (panel 2C). Contrary, a strong green intracytoplasmic fluorescent reactivity was observed in PCV3 positive infected cells using PCV3 hyperimmune sera L (panel 2D) or N (panel 2E) as primary antibody for staining. Images were obtained using a fluorescence inverted microscope (IX83, Olympus) at 40× magnification.

Figure 3. PK-15 infected cells show strong intracytoplasmic hybridization signal. (A) An RNA cocktail probe targeting PCV3 ORF2 showed positive signal (red) in individual cells and occasionally clustering cells (20×). (B) Positive cells are characterized by small red dots diffusely distributed in the cytoplasm of epithelial cells (40×). Negative cells in purple.

Figure 4. Phylogenetic analysis based on four complete genome sequences of three PCV3 isolated and 46 reference strains. The solid circles indicate the sequences of four viruses isolated in this study. Maximum parsimony (MP) tree based on the nucleotide sequences of the whole genome was protein reconstructed using Geneious R9 software. The value along the branches represent substitutions per site. The clade of trees was estimated using 1000 replicates of bootstrap (BT) analysis. All PCV3 strains were divided into three clades, PCV3a (orange), PCV3b (blue) and PCV3c (red). PCV3a was further divided into three subclades.

Figure 5. Phylogenetic analysis based on four full-length Cap (ORF2) protein sequences of three PCV3 isolates and 46 reference strains. Solid circles indicate the sequences of four viruses isolated in this study. Maximum parsimony (MP) tree based on the nucleotide sequences of the cap protein reconstructed using Geneious R9 software. The value along the branches represent substitutions per site. The clade of trees was estimated with 1000 replicates of bootstrap (BT) analysis. All PCV3 strains were divided into three clades, PCV3a (orange), PCV3b (blue) and PCV3c (red), based on two amino acids mutation (A24V and R27K) and phylogenetic relationship of cap gene. PCV3a was further divided into three subclades.

Figure 6. Infection dynamics in 6-week-old CD/CD pigs (n = 14) following experimental inoculation with either 2 mL of infection medium (n = 6; negative control group) or 2 mL of PCV3 isolate ISU27734 (n = 8; PCV3 inoculated group). IgG/IgM antibody responses (mean S/p values) detected by PCV3 rORF2 iELISA over the course of the infection (A). Viral loads detected by qPCR (mean log genomic copies per mL) over the course of the infection (B).

Figure 7. Microscopic lesions and RNAscope in situ hybridization (ISH) on tissues collected at necropsy (28 dpi) from 6-weeks-old CD/CD PCV3 inoculated pigs. The myocardium is multifocal infiltrated by small aggregates of lymphocytes and plasma cells. H&E (20×) (A). The tunica adventitia and media of multiple arterioles of the heart are infiltrated by lymphocytes and plasma cells. H&E (20×) (B). PCV3 was confirmed by ISH in the cytoplasm of cardiomyocytes and inflammatory and endothelial cells. H&E counterstain (20×) (C). Virus replication was detected by ISH within smooth muscle of arteries. H&E counterstain (20×) (D).

Figure 8. Microscopic lesions and RNAscope in situ hybridization on tissues collected at necropsy (28 dpi) from 6-weeks-old CD/CD PCV3 inoculated pigs. The cortical intestitu is multifocal infiltrated by small aggregates of lymphocytes and plasma cells. H&E (20×) (A). The tunica adventitia and media of multiple cortical arterioles are infiltrated by lymphocytes and plasma cells. H&E (20×) (B). PCV3 was confirmed by ISH within inflammatory foci (C,D) endothelial cells (D) within smooth muscle of arteries (E) H&E counterstain (20×).