The complex and unique nature of BVDV continues to challenge infectious disease researchers,veterinarians,and the cattle industry. The different genotypes and biotypes of BVDV ability to induce persistently infection, as well as its ability to interfere both innate and adaptive immunity of the host, make it difficult for prevention and control. Although Mais evaluated the effect of cp and ncp BVDV infection of bovine monocytes to determine their role in viral immune suppression and uncontrolled inflammation by using proteomics, this study was the first to analyze transcriptionally diversified gene expression after in vitro ncp BVDV infection of bovine monocytes. In this study, using RNA-seq, we demonstrated that most of the DEGs between ncp BVDV infected monocytes for 2 and 24 hours, involved immune responses, suggesting that ncp BVDV infection plays an important role in bovine monocytes. We observed increased levels in a variety of antiviral genes such as IFI27, IFI30, MX1, TRIM25, DDX41, ISG20, STAT1, TRAF6, CD14, CD40, IL1B, IL6, MAPK12, MAPK13, TNFSF13B, and TBKBP1 (Table 2), It seems to play a fundamental role in ncp BVDV infection in monocytes. The expression levels of the IFI27, IFI30, ISG20, STAT1, TRAF6 and TRIM25 genes obtained from the RNA-seq data were confirmed by the real-time PCR mRNA expression data (Table 1 and Figure. 3). These proteins might be upregulated in bovine monocytes because of the ncp BVDV infection. However, we observed that type I IFN genes were reduced such as IRF7, DDX3X, DDX58, TLR13, TLR9, STING, and IFIT1, which was possibly caused by the ncp BVDV viral particles[12, 13]. On the other hand, upregulated DEGs, such as NLRX1, CYLD, SIKE1, ATG12, and RNF125, were shown to interfere with host immune responses in previous documents [23–26].
The DEAD-box proteins are characterized by the conserved sequence Asp-Glu-Ala-Asp (DEAD) is a presumptive RNA unwindase involved in many cellular processes, such as RNA combination and RNA secondary framework changes. This gene codes proteins involved in the RNA helicase-DEAD box protein motif and the caspase employment domain (CARD). It is related to the viral double-stranded (ds) RNA identification and coordination of immune responses [27, 28, 29].
Our research showed that DHX16, DHX34, DHX37, DHX58 (Table 2) were not significantly DEGs after ncp BVDV infection, but DHX58 expression was increased (Figure. 3). In other studies, viral dsRNA stimulation did not induce high expression of DEAD-box proteins, and viral dsRNA was shown to be degraded by some proteases, such as BVDV Erns protein [30, 31]. We found that DHX16, DHX34, DHX37, DHX58 DDX3X, DDX10, DDX51, DDX54, and DDX55 were differentially expressed after the ncp BVDV infection of monocytes. Post-downregulation was not significant (Table 2), but DHX41 expression was significantly increased, suggesting that DDX41 plays an important role in RNA binding and secondary structure changes [32, 33].
Toll-like receptors (TLRs) are one of the most essential innate immune receptors that identify many types of PAMPs from different pathogens and activate downstream immune responses. TLRs are grouped into two groups: TLR1, TLR2, TLR4, TLR5, TLR6, and TLR11, which are concerned with membrane proteins that detect microbial membrane components, while TLR3, TLR7, TLR8, and TLR9 are concerned with nucleic acid recognition of proteins, cells, intracytoplasmic molecules, and microorganisms [34, 35]. Nevertheless, studies showed that infections and administrative mechanisms restrict the TLR13 signaling pathway in mollusks. In this study, TLR8, TLR9, and TLR13 expression in BVDV infected bovine monocytes were downregulated (Table 2 and Figure. 3), which may be the reason for the decreased type 1 IFN expression that was seen [36- 40].
This study found common groups of genes related to important cellular processes at 2 hpi. For example, inhibition of NF-κB activation was induced by upregulated IKKE and SIKE1, negative regulators of the NF-κB signaling pathway, and by downregulated p65/RelA-NF-κB . These inhibitory actions could lead to blocked or delayed antiviral responses (Table 2). These results are interesting because they show that infected living cells exhibit strong immunosuppressive phenotypes. In fact, the results also show downregulation of vital immune response genes, for example, the PIDD1, IKBKG, IKBKB, INFGR2, PRKCQ and EIF2AK2 genes (Table 2). Moreover, the expression of IKKA and SIKE1 transcripts indicates the blocking of NF-κB in infected cells, which were support by the qRT-PCR results (Figure. 3 and Table 2).
Our research demonstrates that there is antiviral regulation into bovine monocytes upon activation, as shown by upregulation of the NLRX1, CYLD, SIKE1, ATG12, and RNF125 genes (Table 2 and Figure. 3). These findings are vital for the understanding of the molecular mechanisms by which ncp BVDV evades innate immunity. NLRX1 is involved in antiviral signaling. By inhibiting the virus-induced RLH (RIG-like helicase)-MAVS interaction, NLRX is a negative regulator of the MAVS-mediated antiviral response . RNF125 mediates DDX58/RIG-I ubiquitination inducing DDX58/RIG-I degradation of Lys–181 . SIKE1, IKK-epsilon, and TBK1 genes are physiological inhibitors that act on viral and TLR3-triggered IRF3 to inhibit TLR3-mediated interferon-stimulated response elements (ISRE) and IFN-β promoters. Activation of the ISRE and IFN-β promoters can be achieved by disrupting the interaction between IKBKE or TBK1 and TICAM1/TRIF, IRF3, and DDX58/RIG-I, which does not inhibit the NF-κB activation pathway .
Type I IFNs are key mediators of host immune responses to viral infections. These IFNs induce the expression of many ISGs of which some could be antiviral. Besides, they regulate innate and adaptive immunity by activating immature DCs, strengthening NK cell responses, and boosting the survival and effector functions of T and B lymphocytes . In this research, a significant decrease in ZAP70, TNFSF11, V-TCR, and CARD11 was regulated in ncp BVDV-infected bovine monocytes. ZAP70 regulates T cell activation by modulating TCR expression on T cell surfaces [42, 43]. CARD11 is involved in the costimulatory signals necessary for T cell receptor (TCR)-mediated T cell activation. CARD11 connects with the DPP4 and guides T cell multiplication and NF-κB activation in a T cell receptor/CD3-dependent manner . TNFSF11 is a necessary regulator of T cell and dendritic cell interactions and might regulate T cell-dependent immune responses . Most serine/threonine kinase-expressing genes are increased upon ncp BVDV infection of monocytes, which is quite interesting because only MAP2K6 is significantly reduced. MAP2K6 plays a vital role in regulating cytokine to cytokine responses, particularly as it relates to the induction of IL–6 production. IL–6 is increased 2 hpi and is decreased by 24 hpi (Table 2 and Figure. 2) [46, 47].