RNA sequence analysis of ncp BVDV1-infected monocytes
The results show that more differential expression, either up- or down-regulated was seen at 2 h post-infection ( hpi), the expression of 9959 genes was significantly changed compared with those of the controls, of which 4968 genes were upregulated, and 4991 genes were down-regulated (Fig.1A, Additional file 1:Table S1). At 24 hpi, the expression of 7977 genes was significantly different from that of the controls, with 4184 genes up-regulated and 3793 genes down-regulated (Fig.1A, Additional file 2:Table S2). These significantly altered genes were involved in signal transduction, immune response, apoptotic process, cellular process , binding and cellular component, with most of the differential genes being involved in signal transduction.
The DEGs were filtered to determine which genes were present at both of the time points (2 hpi and 24 hpi). At 2 hpi time point, 9959 genes responded to ncp BVDV1 infection, and at 24 hpi time point, 7977 genes responded; however, only 5709 genes were common at both time points (Figure. 1A). Two libraries were packaged and analyzed against the non-redundant NCBI database using the BLAST program. Next, we mapped and annotated the genetic libraries using the Gene ontology (GO) database. To identify pathways involved in immune activation after ncp BVDV1 infection of bovine monocytes, transcript records were further analyzed. The GO terms classify the function of BVDV-infected cell transcripts, offering 4014 transcripts involved in molecular functions, 4476 involved in biological processes, both and 3341 involved in discretely cellular processes (Figure. 1B). Analysis revealed that differentially expressed genes are involved in a variety of biological processes, including immune responses and immune system procedures, cell death, macromolecular localization, apoptosis, cell death regulators, antigen processing and presentation, protein localization, and others (Figure. 1C, Additional file 3:Table S3, Additional file 4:Table S4).
To further define DEGs function, KEGG pathway/enrichment analysis was performed. DEGs were significantly enriched in 20 pathways(Fig.1D and E). A total of 126 and 113 DEGs show Ribosome pathway enrichment in 2 hpi and 24 hpi, respectively. This pathway was mainly responsible for gene expression regulation and protein translation. Many significantly DEGs were involved in immune responses such as with the TNF signaling, B cell receptor signaling pathway, Fc epsilon RI signaling pathway, T cell receptor signaling pathway and NF-κB signaling pathway (Fig.1D and E, Additional file 5:Table S5, Additional file 6:Table S6). These DEGs, which include TRIM25, TRAF6, TRAF3, IL-1β, CD14, BCL2, TICAM1/2, TNIP3, TNFSF13B, ZAP70, TRAF2, TRAF2, TNFSF11, PIDD1, CARD11, LYN, BLNK, BCAP, BCL-10, CD81, CD19, MEK1/2, AP1, BCL10, Ikkα, Ikkγ, Ikkβ, PD-1, CD45, LCK, CBL, ZAP70, LAT, etc, play an important role in activating or inhibiting innate and adaptive immunity (Table 2, Additional file 1:Table S1, Additional file 2:Table S2). These DGEs were further analyzed (GO: 0006955) (Figure.2A), A list of genes that were differentially expressed between 2 hpi and 24 hpi is shown in Table 2. Most of the upregulated genes were related to the immune system. Upregulated genes included IL6 (3.1 fold), TNFSF13B (2.5 fold), CD14 (2.8 fold), IFI27 (5.7 fold), RNF125 (1.8 fold) and MAPK12 (2.4 fold), and downregulated genes included IRF7 (1.08 fold), TLR13 (2.2 fold), ZAP70 (2.9 fold), TLR8 (1.5 fold) , TLR9 (1.5 fold), IFITM1 (1.8 fold), and IGHG2 (2.9 fold). Some genes (eg, IFI30, JUN, CD40, LTBP2, IKBKB, CCL5, and MAPK11) were up-regulated at 2 hpi and down-regulated at 24 hpi. Other genes, such as EIF2AK2, DHX58, DDX3X, IFI6, TIRAP, STING, and IFIT1 were downregulated at 2 hpi and upregulated at 24 hpi (Table 2).
Through careful examination of the information set (Figure. 2B and Table 2), several ISGs [20] mainly produced by type I interferons, were differentially expressed in ncp BVDV1-infected cells. Figure.2A and B show stratified heat maps of all of the up or down regulated genes in ncp BVDV1-infected cells, and they were all associated with immune responses and type 1 IFN signaling. Overall, the RNA-seq analysis indicated that ncp BVDV1 infection resulted in different expression of genes related to inherited immunity, type I IFNs, stimulatory cytokines, and cell signaling pathways, leading to the creation of antiviral responses in monocytes.
Partial validation of the DEGs involved in the type 1 IFN pathway by RT-qPCR
Sequences found using RNA-seq analysis were validated with RT-qPCR to assess if mRNA expression was upregulated or downregulated in relation to the genes and to the immune responses. The genes were identified if they were recognized as being involved in type I IFN responses, were reported to be related to innate antiviral immune responses [21, 22], and if they were common to both infected groups. Using GAPDH as an internal control, the transcription levels of 31 DEGs were measured (between the 2 and 24 hour time points) with RT-qPCR with the primers listed in Table 1. In conclusion, the RNA-seq and RT-qPCR results from bovine monocytes at 2 and 24 hpi were fairly consistent (Figure. 3). IRF7, TLR13, ZAP70, IFITM1 were all down-regulated 2 hours and 24 hpi with ncp BVDV1 virus. DDX3X, IFIT1, STING were down-regulated 2 hpi and up-regulated 24 hpi. The expression of IKBKB, TRAF1, IRF5 was up-regulated 2 hpi and down-regulated 24 hpi. EF2AK2, IFI27, IFI30, MX1, MX2 and other genes were up-regulated sharply 2 hpi compared with 24 hpi.