Porcine Circovirus Modulates Swine Influenza Virus Replication in Pig Tracheal Epithelial Cells and Porcine Alveolar Macrophages

Figure 1. PCV2b and SwIV H1N1 co-localization in both NPTr and iPAM 3D4/21 co-infected cells. IFA was performed at 24 h post-infection to detect PCV2b (red) and SwIV H1N1 (green) in co-infected cells. The cells were infected with 0.05 MOI of PCV2b and passaged 2–3 times. Then, PCV2b-infected cells were infected with 1 MOI of SwIV H1N1. Nuclear staining with DAPI is shown in blue. Co-localization pictures were realized with ImageJ. The red arrows point to single cells expressing both PCV2b and SwIV H1N1 antigens. Statistical analyses were carried out using a two-way ANOVA followed by Sidak’s multiple comparison test (GraphPad Prism software, version 7.00). *** p < 0.001.

Figure 2. PCV2b co-infection effects on SwIV replication in infected NPTr and iPAM 3D4/21 cells. The SwIV titer was determined in MDCK cells using the Spearman–Kärber method and expressed in tissue culture infectious dose 50% per mL (TCID50/mL). The PCV2b/SwIV H1N1 (A) and PCV2b/SwIV H3N2 (C) co-infection experiment in NPTr cells, and PCV2b/SwIV H1N1 (B) and PCV2b/SwIV H3N2 (D) co-infection experiment in iPAM 3D4/21 cells were repeated 3 times. Data are presented with standard deviation (SD) values. Statistical analyses were carried out using two-way repeated-measures ANOVA (GraphPad Prism software, version 7.00). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 3. SwIV co-infection effects on PCV2b replication in infected NPTr and iPAM 3D4/21 cells. PCV2b quantification in co-infected NPTr (A) and iPAM 3D4/21 (B) cells was performed using qPCR assay and expressed as PCV2b genome copies per mL of sample. The experiments were repeated 3 times. Mock-infected cells were PCV2 qPCR-negative (with Ct > 36) and the results are not illustrated in the figure. Data are presented with standard deviation (SD) values. Statistical analyses were carried out using two-way repeated-measures ANOVA (GraphPad Prism software, version 7.0.0).

Figure 4. Cell viability was determined in single-infected and co-infected PCV2b/SwIV cells at 24 hpi (A,B), 48 hpi (C,D) and 72 hpi (E,F) in NPTr (A,C,E) and iPAM 3D4/21 cells (B,D,F). The experiments were repeated three times. The data represent percentage of cell viability in infected cells with respect to mock-infected cells and are presented with standard deviation (SD) values. Statistical analyses were carried out using one-way ANOVA followed by Tukey’s multiple comparison test (GraphPad Prism software, version 7.0.0). Different superscripts indicate a statistically significant difference (p < 0.05) between groups.

Figure 5. PCV2b/SwIV co-infection effects on the modulation of cytokines’ mRNA expression. The mRNA expressions of IL-6, IL-8 and IL-10 in NPTr cells (A) and iPAM 3D4/21 (B) as well as of IFN-α, IFN-β and IFN-γ in NPTr cells (C) and iPAM 3D4/21 (D) were determined using RT-qPCR assays. The 2−ΔΔCt method was used to calculate the fold change of cytokine mRNA expression in infected cells with respect to mock-infected cells at 24 h post-infection. The experiments were repeated at least three times. All data are presented with standard deviation (SD) values. Statistical analyses were carried out using an ordinary two-way ANOVA followed by Tukey’s multiple comparison test (GraphPad Prism software, version 7.0.0). Different superscripts indicate a statistically significant difference (p < 0.01) between groups within the same tested cytokine mRNA.

Figure 6. Venn diagram of differentially expressed genes (DEGs) in PCV2b/SwIV co-infected cells. Mock-infected cells were used as control to identify DEGs in NPTr (A) and iPAM 3D4/21 (B) single-infected and co-infected cells. The numbers in overlapping areas represent the number of DEGs shared among the different virus infection experimental groups. A false discovery rate (FDR) < 0.05 and a fold change cut-off of 1.5 was used to identify the DEGs.

Figure 7. KEGG pathway enrichment analysis of the DEGs identified in single-infected and co-infected cells. Enriched KEGG pathways after PCV2b, SwIV H1N1 and PCV2b/SwIV H1N1 infection in NPTr cells ((A,C,E), respectively) and in iPAM 3D4/21 cells ((B,D,F), respectively) are illustrated. The FDR value < 0.05 was considered for statistically significant enriched pathways.