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Transcriptomic analysis of bovine monocytes in response to non-cytopathic bovine viral diarrhea virus infection

Background: Monocytes are significant players in the detection of invading pathogens, particularly in pathogen defense. Bovine Viral Diarrhea Virus (BVDV) can cause a persistent infection and immune suppression if animals are infected with an non-cytopathic (ncp) biotype. However, its exact role in ncp BVDV1-infected bovine monocytes remains poorly understood. Results: RNA sequencing (RNA-seq) was used to investigate the effect of ncp BVDV1 infection on the transcriptional profile of bovine monocytes. Compared with the non-infected cells, 9959 and 7977 differentially expressed gene (DEGs) were identified at 2 and 24 h hpi, respectively. These DEGs were associated with signal transduction, immune response, apoptotic process, cellular process , binding and cellular component. The differential expression profiles of select the type I interferon signaling pathway , interferon (IFN)-stimulated genes (ISGs), and genes involved in the innate immune response, including IRF7, DDX3X, TLR13, DDX58(RIG-I), MVAS, TLR9, TRAF6, IRF1, IFIT1, STAT1, ISG20, TRIM25, MX1,NLRX1, CYLD, SIKE1 and ZAP70 were confirmed by real-time quantitative PCR and consistent with the RNA-seq data. Conclusion: Our transciptome anslysis provides useful initial data towards better understanding of the infection mechanisms used by ncp BVDV1, while highlighting the potential molecular relationships occurring between the virus and the host’s immune response.

Background Bovine viral diarrhea virus (BVDV) is a small positive-sense single-stranded enveloped RNA virus that is part of the Pestivirus genus of the Flaviviridae family and a viral pathogen of cattle. BVDV infection is found in cattle of all ages and breeds worldwide, with a huge economic impact due to production and reproductive losses [1,2]. BVDV can also be categorized under two biotypes based on their growth characteristics in cell cultures. The rare cytopathic (CP) biotype will damage tissue cultures and the much more common noncytopathic (ncp) will not [3,4].The pathogenesis of the disease caused by BVDV is complex and involves persistent infection (PI) and immune suppression with the ncp biotype during early gestation, followed by an acute infection by a cp biotype [5].
In recent years, the understanding that the vertebrate innate immune system represents the first defense against pathogens has emerged. The synthesis and secretion of type I interferons (IFN-I) is the first step in the induction of cellular innate immune responses [6] to viral infections or doublestranded RNA (dsRNA). dsRNA is a common pathogen-associated molecular pattern (PAMP) that is recognized by a variety of pattern recognition receptors (PRRs). Currently identified PRRs can be divided into the Toll-like receptors (TLRs), the retinoic acid-inducible gene I (RIG-I) receptor-like receptors (RLRs), and the NOD-like receptors (NLR). All three PRRSs recognize the viral PAMPs [7].Each of the three receptors cooperates with their adaptor proteins to deliver information in the presence of viral PAMPs and interferons. The Toll-like receptors affect the Total Recordable Injury Frequency (TRIF) adaptor protein, the Cyclic GMP-AMP synthase (cGAS) receptor effects the Stimulator of interferon genes (STING) adaptor protein, and the RIG-I receptor interacts with the Mitochondrial antiviral-signaling (MAVS) adaptor protein. These connecting molecules recruit and activate downstream signaling pathways to produce corresponding inflammatory cytokines, chemokines, and Type I (IFN-α/β) and III (IFN-g) interferons [8,9]. Many studies have confirmed that innate immune evasion affects the survival and adaptive immune response of virus. [10].
Previous studies have revealed many crucial discrepancies in the results of ncp and cp biotypes in variety of cell types. ncp BVDV does not induce IFN synthesis in cultured cells in vitro, suggesting that this could be a defense mechanism to evade innate host immunity that might be critical for establishing persistent infections [11,12,13]. However, the molecular mechanisms by which BVDV evades host cell immunity remains unclear. RNA-seq has recently been developed for transcriptomics analysis of BVDV infection in MDBK cells. Research was performed with microarrays that showed gene expression changes in host cells infected with ncp BVDV compared with cp BVDV [14,15]. However, it was reported that a large number of genes related to BVDV invasion and replication mechanisms still need to be characterized to better understand the interaction between BVDV pathogenetic mechanisms and host immune defenses. Although ncp BVDV is thought to play an important role in BVDV-infected bovine monocytes [16][17][18][19], little is known about how BVDV disturbs host cell gene expression profiles. During this research, we showed that BVDV infected bovine monocyte gene expression profiles, which provided meaningful information from genetic analyses that could not be discerned using biochemical methods.

Animals
Nine conventionally reared, healthy BVDV-free cows from the Shihezi University Animal Experimental Station were used. All animal experiments were approved by the Shihezi State University Institutional Animal Care and Use Committee. The blood samples of all animals were confirmed to be free of BVDV, infectious bovine rhinotracheitis virus, bovine parainfluenza virus, and Mycoplasma bovis infection using enzyme linked immunosorbent assay (ELISA) kits (Idexx Labs Inc., USA) and by reverse transcription PCR (RT-PCR) or PCR for viral nucleic acid detection [18].All animal serums were negative identified by Bovine viral diarrhoea virus(BVDV) antibody test kit(Idexx Labs Inc., USA)

BVDV stocks and infection
The ncp BVDV1 strain was named BVDV-LC,which isolated from the feces of a cattle with diarrhea, (GenBank accession NO. MK102095, previously identified in our lab), and was classified as genotype 1q and non-cytopathic (ncp). For titration of virus stock, four replicates of 10-fold serially diluted virus (starting from 1/10) were inoculated on MDBK cell monolayer in 96-well culture plates. After 48 h incubation, the culture plates were fixed at 4 °C for 30 min with ice cold absolute ethyl alcohol and subjected to immunofluorescence staining with fluorescein isothiocyanate (FITC)-conjugated polyclonal anti-BVDV (VMRD, USA) antibody (diluted 1:1000). The fluorescence signals were observed undera fluorescence microscopy (ZEISS) and viral titers was expressed as the 50% tissue culture infective dose (TCID50)/mL by Reed-Muench method.
For infection of bovine monocytes, virus dilutions were made in DMEM 4Mm L-glutamine, 4.5 g/l glucose, 1.5 g/l sodium bicarbonate and 10% horse serum. 5×10 6 monocytes were added to each well of a 6 well tissue culture plate and adsorbed with ncp BVDV1 biotypes at the same MOI of 0.1 for 2 h.

RNA extraction
After infection 2 h and 24 h, at least 10 7 cells were pooled in one tube. Cells were harvested and prepared for RNA extraction. The monocytes were rinsed three times with ice-cold PBS, and whole RNA was extracted by DNase digestion using the RNeasy mini kit following the manufacturer's instructions (Qiagen). RNA was quantified using a NanoDrop spectrophotometer (Thermo Fisher) and stored at -80℃. For RNA-seq analysis, the RNA quality was assessed with an Agilent Bioanalyzer (Agilent Technologies).

RNA sequencing and analysis
The RNA elimination was performed using Ribo-Zero rRNA removal kit (Epicenter, USA) following the manufacturer's instructions. Illumina sequencing libraries were prepared using the TruSeq Stranded The sequence data of the reference genome was checked against the NCBI database. The filtered mass reads were aligned with the reference genomic sequence using Bowtie 2. Relative transcript abundance was metered as fragments per kilobase per million mapped reads (FPKM). Genes were clustered into functionally relevant groups using the eggNOG (Genetic Evolution Genealogy: Unsupervised Orthologous Group) database and the metabolic pathways were analyzed using the KEGG (Kyoto Encyclopedia of Genes and Genomes) database. Visualization of mapping outcomes and differentially expressed gene (DEG) analysis was performed using the CLRNASeqTM program (ChunLab, Korea). ncp BVDV1 expression and infected monocyte gene expression were contrasted with gene expression from the inoculum and uninfected monocytes. The fold change in gene expression was calculated from three different sample types.

Real-Time Quantitative Reverse Transcription PCR (RT-qPCR)
DEGs were examined by RT-qPCR to confirm the accuracy of the sequencing data. Primers were made using Primer 5.0 software ( Table 1). cDNA was synthesized from 2 μg of total RNA using a reverse transcription kit (Fisher Scientific, USA) and by following the manufacturer's instructions. Gene expression was quantified using a LightCycler 480 PCR platform (Roche Applied Science, Indianapolis, IN, USA). The RT-qPCR procedure began with an initial step of 10 minutes at 95 °C followed by 45 cycles at 95 °C for 30 seconds, 58 °C for 30 seconds, and 72 °C for 30 seconds. Gene transcription levels were quantified relative to GAPDH gene expression using the relative cycle threshold (ΔCT) method.

Statistical analysis
All data are shown as means ± SE. Differences were evaluated using ANOVA followed by the Student's t-test. Statistical significance was defined as P < 0.05.

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 postinfection ( 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 ( Table S3, Additional file 4: Table S4).
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

Discussion
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 [5]. Although previous research [18] 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 BVDV1 infected monocytes for 2 and 24 hours, involved immune responses, suggesting that ncp BVDV1 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 BVDV1 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 BVDV1 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 BVDV1 viral particles [12,13]. On the other hand, upregulated DEGs, such as NLRX1, CYLD, SIKE1, and RNF125, were shown to interfere with host immune responses in previous documents .NLRX1 acts as a negative regulator of MAVS-mediated antiviral response by inhibiting virus-induced RLH (RIG-like helicase)-MAVS interaction [23][24][25].CYLD (Cylindromatosis) is a deubiquitin enzyme. CYLD removes the K63-connected ubiquitin chain of a specific substrate through its own deubiquitin activity and negatively regulates multiple signal pathways including NF-kappa B [26].SIKE1 (Suppressor of IKBKE 1) was classified as an endogenous inhibitor . Inhibits TLR3-mediated activation of interferonstimulated response elements (ISRE) and the IFN-beta promoter. May act by disrupting the interactions of IKBKE or TBK1 with TICAM1/TRIF, IRF3 and DDX58/RIG-I [27,28].
RNF125 is an E3 ubiquitin protein ligase that mediates ubiquitin and subsequent proteasome degradation of target proteins. As a negative regulator of type I interferon production, it can mediate DDX 58 / RIG-I ubiquitination in "Lys-181", resulting in DDX 58 / RIG-I degradation [29].
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 [27]. SIKE1 directly interacts with TBK1 to inhibit the TBK1-AKT signalling pathway, Which could further lead to blocked or delayed antiviral responses. 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 RT-qPCR 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 BVDV1 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 [23]. RNF125 mediates DDX58/RIG-I ubiquitination inducing DDX58/RIG-I degradation of Lys-181 [29]. SIKE1, IKK-epsilon, and TBK1 genes are physiological inhibitors that act on viral and TLR3triggered 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 [27,28].
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 [41]. In this research, a significant decrease in ZAP70, TNFSF11, V-TCR, and CARD11 was regulated in ncp BVDV1-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 [44]. TNFSF11 is a necessary regulator of T cell and dendritic cell interactions and might regulate T cell-dependent immune responses [45]. Most serine/threonine kinase-expressing genes are increased upon ncp BVDV1 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.

Conclusions
In conclusion, the lack of knowledge on how BVDV evades host immune responses causes improper control of this disease. Our studies have identified IRF7, DDX3X,, TLR13, TLR9, STING, and IFIT1 as key downregulatory molecules in ncp BVDV1 infected monocytes. Conversely, NLRX1, CYLD, SIKE1, ATG12, and RNF125 act as upregulatory molecules. There also appears to be negative regulation of IFN production. On the basis of previously reported studies and current information, we feel that the underlying role of these genes are to defend against ncp BVDV1 infection. More research is needed to clarify the mechanism by which ncp BVDV1 upregulates and downregulates these various molecules and to determine the role of these genes in the pathogenesis of BVDV, which will thus enhance our understanding of BVDV replication and pathogenesis.

Availability of data and materials
The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

Authors' contributions
YH and CC contributed to the study conception and design. YH, JH and JY wrote the manuscript. CC, CX and FZ checked and revised it. YH, JY, XM, XH, XZ and TG performed the experiments and analysed the data. All authors read and approved the final version of the manuscript.

Ethics approval
This study was carried out in accordance with the recommendations of National Standards for

Conflicts of interest
The authors have no conflicts of interest to declare.