Th17 cell differentiation induced by cytopathogenic biotype BVDV-2 in bovine PBLCs

Fig. 1. Heat map and volcano plot of the DGEs between control and BVDV-2 infected group at 12 h. a Heat map display the red represent the significantly up-regulated genes and the green represent the significantly down-regulated. b volcano plot show the red represent the significantly up-regulated genes and significantly down-regulated genes in blue

Fig. 2. Verification of the mRNA level of some DEGs by qRT-PCR. a The results of RNA-Seq. b The results of qRT-PCR. GAPDH was used as endogenous internal reference gene and the relative content was calculated according to the 2-ΔΔCt method

Fig. 3. GO annotation for the DEGs between control and BVDV-2 infected bovine PBLCs. Horizontal axis represents the number of genes annotated on GO terms. Vertical axis represents the GO terms of BP, CC, and MF. Red represent the genes of up-regulated and the blue represent the down-regulated genes on different GO categories

Fig. 4. KEGG pathway enriched pathway analysis of DEGs with top 20 enrichment scores [28]

Fig. 5. KEGG interaction network analysis. The significantly differences 16 signaling pathways were represented in red and the DEGs in these pathways were represented in blue

Fig. 6. Protein-protein interaction network analysis of DEGs between control and BVDV-2 infected bovine PBLCs. Parts of DEGs interaction relationships were identified with significant values of p < 0.05 by using cytoscape_v3.6.1 software. The selected DEGs were represented in red circular and the green circular represents the genes with complex interaction network, the black solid line indicates the connection of each genes

Fig. 7. Th17 cells differentiation was detected by flow cytometry. a Percentages of Th17 cells labeled by the CD4 and IL17A monoclonal antibodies. b The bar chart shows the percentage of Th17 cells in control and GS2018 infection group at 6 h, 12 h and 24 h, respectively. Data are representative of three independent experiments and presented as means ± SDs. (ns, not significant; * p < 0.05; **p < 0.01)

Fig. 8. The expression of IL17A was detected by qPCR and Western blot in PBLCs. a qPCR detection at 12 h, 24 h, 48 h and 72 h, respectively. b Western blot detection at 12 h, 24 h and 48 h after GS2018 infection, respectively. The grayscale analysis of western blot result shows in bar chart. Data are representative of three independent experiments and presented as means ± SDs. (ns, not significant; * p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001)

Fig. 9. Overexpression of IL17A inhibited the replication of GS2018. The pEGFP-N1-IL17A and pEGFP-N1 (NC) fluorescence signal was detected by laser confocal microscope at 24 h (a). The expression of IL17A (b) and 5'UTR of GS2018 (c) were detected by qPCR. The BVDV-2 E2 (d) were detected by western blot. The grayscale analysis of western blot result shows in bar chart. Data are representative of three independent experiments and presented as means ± SDs. (ns, not significant; * p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001)