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Volume 31 Issue 3
June 2016
Article Contents
Citation: Zixin Ni, Fan Yang, Weijun Cao, Xiangle Zhang, Ye Jin, Ruoqing Mao, Xiaoli Du, Weiwei Li, Jianhong Guo, Xiangtao Liu, Zixiang Zhu, Haixue Zheng. Differential gene expression in porcine SK6 cells infected with wild-type and SAP domain-mutant foot-and-mouth disease virus [J].VIROLOGICA SINICA, 2016, 31(3) : 249-257.  http://dx.doi.org/10.1007/s12250-015-3709-x

Differential gene expression in porcine SK6 cells infected with wild-type and SAP domain-mutant foot-and-mouth disease virus

  • Corresponding author: Zixiang Zhu, zhuzixiang@126.com Haixue Zheng, haixuezheng@163.com
  • ORCID: 0000-0002-4093-9683,0000-0001-6850-1379; 
  • Received Date: 26 December 2015
    Accepted Date: 03 March 2016
    Published Date: 08 April 2016
  • Foot-and-mouth disease virus (FMDV) is the causative agent of a highly contagious disease in livestock. The viral proteinase Lpro of FMDV is involved in pathogenicity, and mutation of the Lpro SAP domain reduces FMDV pathogenicity in pigs. To determine the gene expression profiles associated with decreased pathogenicity in porcine cells, we performed transcriptome analysis using next-generation sequencing technology and compared differentially expressed genes in SK6 cells infected with FMDV containing Lpro with either a wild-type or mutated version of the SAP domain. This analysis yielded 1,853 genes that exhibited a ≥ 2-fold change in expression and was validated by real-time quantitative PCR detection of several differentially expressed genes. Many of the differentially expressed genes correlated with antiviral responses corresponded to genes associated with transcription factors, immune regulation, cytokine production, inflammatory response, and apoptosis. Alterations in gene expression profiles may be responsible for the variations in pathogenicity observed between the two FMDV variants. Our results provided genes of interest for the further study of antiviral pathways and pathogenic mechanisms related to FMDV Lpro.
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    Differential gene expression in porcine SK6 cells infected with wild-type and SAP domain-mutant foot-and-mouth disease virus

      Corresponding author: Zixiang Zhu, zhuzixiang@126.com
      Corresponding author: Haixue Zheng, haixuezheng@163.com
    • 1. State Key Laboratory of Veterinary Etiological Biology, National Foot and Mouth Diseases Reference Laboratory, Key Laboratory of Animal Virology of Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China
    • 2. College of Veterinary Medicine, China Agricultural University, Beijing 100083, China

    Abstract: Foot-and-mouth disease virus (FMDV) is the causative agent of a highly contagious disease in livestock. The viral proteinase Lpro of FMDV is involved in pathogenicity, and mutation of the Lpro SAP domain reduces FMDV pathogenicity in pigs. To determine the gene expression profiles associated with decreased pathogenicity in porcine cells, we performed transcriptome analysis using next-generation sequencing technology and compared differentially expressed genes in SK6 cells infected with FMDV containing Lpro with either a wild-type or mutated version of the SAP domain. This analysis yielded 1,853 genes that exhibited a ≥ 2-fold change in expression and was validated by real-time quantitative PCR detection of several differentially expressed genes. Many of the differentially expressed genes correlated with antiviral responses corresponded to genes associated with transcription factors, immune regulation, cytokine production, inflammatory response, and apoptosis. Alterations in gene expression profiles may be responsible for the variations in pathogenicity observed between the two FMDV variants. Our results provided genes of interest for the further study of antiviral pathways and pathogenic mechanisms related to FMDV Lpro.

    • Foot-and-mouth disease virus (FMDV) is a positive-stranded RNA virus capable of infecting a variety of domestic and wild biungulate species (Pega et al., 2013). The highly contagious foot-and-mouth disease (FMD) is caused by FMDV and is perhaps the most important limiting factor in the trade of animals and animal products (Barasa et al., 2008; Perry et al., 2007; Rufael et al., 2008), with outbreaks usually resulting in large economic losses for the local livestock industry. FMDV belongs to the Aphthovirus genus of the Picornaviridae family, and its genome is a single-stranded, positive-sense RNA that encodes a polyprotein. The polyprotein is post-translationally cleaved by three viral proteinases, leader (Lpro), 2A, and 3Cpro, into precursors and mature viral structural (VP4, VP2, VP3, and VP1) and nonstructural proteins (Lpro, 2A, 2B, 2C, 3A, 3B, 3Cpro, and 3D) (Racaniello, 2007).

      Translation of the FMDV polyprotein begins at two different AUG start codons separated by 84 nucleotides, resulting in two alternative forms of Lpro designated as Labpro and Lbpro (Clarke et al., 1985; Piccone et al., 1995). During its evolution, FMDV has consistently counteracted against host immune systems to facilitate its survival and replication; several mechanisms have evolved to antagonize host immune responses, with Lpro reported to play significant pathogenic roles (Grubman et al., 2008). Lpro is a well-characterized, papain-like proteinase (Medina et al., 1993; Piccone et al., 1995) that can self-cleave from the nascent polyprotein. Host translation-initiation factor eIF-4G can also be cleaved by Lpro, greatly reducing host cap-dependent mRNA translation without affecting viral cap-independent protein synthesis, which is a characteristic of most picornavirus infections (de Los Santos et al., 2009; Devaney et al., 1988; Kirchweger et al., 1994; Zhu et al., 2010). Additionally, Lpro inhibits dsRNA-induced type Ⅰ interferon (IFN) transcription by inhibiting the expression of IFN-regulatory factor 3/7 (Wang et al., 2010).

      In eukaryotic cells, the SAP domain (scaffold-attachment factors A and B, apoptotic chromatin-condensation inducer in the nucleus, and protein inhibitor of activated STAT proteins) is a putative DNA-binding domain found in diverse nuclear proteins (Aravind et al., 2000). The SAP domain consists of 35 amino acids, including conserved hydrophobic and charged residues (Aravind et al., 2000), and is found in a number of chromatin-associating proteins, such as scaffold-attachment factors, DNA-repair proteins, RNA-processing complexes, and proto-oncogene proteins (Ahn et al., 2003; Aravind et al., 2000; Bohm et al., 2005; Kipp et al., 2000).

      A conserved SAP domain was also identified in the Lpro-coding region of FMDV (de Los Santos et al., 2009). Genetically engineered FMDV strains lacking the Lpro-coding region (leaderless viruses) or possessing a mutated Lpro SAP domain exhibited attenuated viral replication in infected cattle and swine (Chinsangaram et al., 1998; Diaz-San Segundo et al., 2012; Zhu et al., 2010). SAP-domain mutants carrying I55A and L58A substitutions abolish Lpro retention in the nuclei of FMDV-infected cells and subsequently prevent FMDV-related degradation of nuclear factor-kappa B (NF-κB) (de Los Santos et al., 2007), resulting in upregulation of several cytokines, chemokines, and IFN-stimulated genes (ISGs) (de Los Santos et al., 2009). Additionally, inoculation of swine with FMDV containing a mutated SAP domain induced early protection against disease (Diaz-San Segundo et al., 2012), and transcriptome analysis of embryonic bovine kidney cells (EBKs) infected with SAP-mutated FMDV showed enhanced expression of various IFN-related genes as compared with EBK cells infected with FMDV containing a wild-type SAP domain (de Los Santos et al., 2009).

      FMDV pathogenesis presents particular features depending on the host (Pega et al., 2013). In different species, the viral entry routes, primary infective and replicative sites, and, consequently, the associated symptoms and immune responses elicited showed clear differences (Alexandersen et al., 2003). The different host responses triggered by FMDV also correlated with viral replication, thereby affecting viral propagation in different cells. In EBK cells, an intact Lpro SAP domain was correlated with type Ⅰ IFN responses (de Los et al., 2006; Zhu et al., 2010). Here, we compared the pathogenic characteristics of FMDV containing wild-type or mutant SAP domains in swine PK15 and SK6 cells and found that the SAP-mutant variant exhibited decreased pathogenicity in both cell lines, with a more pronounced decrease observed in SK6 cells, relative to the wild-type SAP variant.

      To analyze the different transcription profiles induced by SAP-domain status, FMDV containing either wild-type or mutant SAP variants was used to infect SK6 cells, followed by comparative transcriptome analysis using next-generation sequencing (NGS) technology to systematically observe the differences in gene expression and host response. Of 20, 421 genes detected, differentially enhanced or repressed expression was observed in 1, 670 and 183 genes, respectively, with many associated with antiviral responses involving transcription, immune response, inflammation, apoptosis, and cytokines or chemokine production. Our results indicated that the FMDV Lpro SAP domain was significantly correlated with FMDV viral pathogenicity in SK6 cells, and that the differential expression of various host genes is dependent upon infection with FMDV containing an intact Lpro SAP domain.

    • An engineered chimeric virus, rA-FMDV, which was previously constructed by Zheng et al., was used as a candidate vaccine (Zheng et al., 2013). The rA-FMDV was constructed by replacing the P1 gene in the O/CHA/99 strain (GenBank accession number: AF506822) with the P1 gene from the A/HuBWH/CHA/2009 strain (GenBank accession number: JF792355). A SAP-mutant virus, rA-SAP-FMDV, was previously constructed by Zheng et al. by introducing the I55A and L58A mutations into the SAP domain of Lbpro in rA-FMDV (unpublished data). The schematic representation of the mutation information is shown in Figure 1A. SK6 cells were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM; Invitrogen, Carlsbad, USA) and cultured in the medium supplemented with 10% fetal bovine serum (FBS) at 37℃ under 5% CO2.

      Figure 1.  (A) Schematic of the I55A and L58A mutations (red, italics) in the Lpro SAP domain. (B) Different pathogenicity observed between rA-FMDV and rA-SAP-FMDV in SK6 and PK15 cells. SK6 and PK15 cells were infected with rA-FMDV or rA-SAP-FMDV at similar MOIs, and the viral TCID50 was detected and recorded. Results are presented as the mean ± standard error from three independent experiments.

    • SK6 and PK15 cells were washed with phosphate-buffered saline and infected with FMDV at a multiplicity of infection (MOI) of 1 at 37℃. After a 1-h adsorption period, the supernatant was removed, and the cells were incubated at 37℃ with DMEM containing 0.5% FBS. The cells used for transcription profile analyses were harvested at 6-h post-infection, because a minimal cytopathic effect (CPE) was observed at ~6 h, enabling a more complete identification of differentially expressed genes. The TCID50 assay was performed in 96-well plates according to standard procedures, and cells were cultured until CPEs were clearly observed (3-5 days). The TCID50 value was calculated using the Reed-Muench method (Reed et al., 1938).

    • Total RNA was extracted from SK6 cells using TRIzol Reagent (Invitrogen) according to manufacturer protocol. Two micrograms of total RNA was used to synthesize the first strand of cDNA using M-MLV reverse transcriptase (Invitrogen), and the synthesized cDNA were subjected to qPCR analysis performed using SYBR Premix Ex Taq (Takara, Kyoto, Japan) according to manufacturer protocol. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase was used as an internal control. Primer sequences used in this study are listed in Supplementary Table S1. The results were obtained from three independent experiments.

    • A sample-pooling strategy was performed in this study. The rA-FMDV-and rA-SAP-FMDV-infected samples were used as mixture samples, with each sample prepared by mixing four different dishes of virus-infected cells. After total-RNA extraction and DNase Ⅰ treatment, magnetic beads conjugated with oligo (dT) were used to isolate mRNA. The mRNA was divided into short fragments, and cDNA was synthesized using the mRNA fragments as templates. The synthesized cDNA was purified and resolved with elution buffer [10 mmol/L Tris-Cl (pH 8.5)] for end repair and single-nucleotide (adenine) addition. Subsequently, the treated fragments were connected using adapters and subjected to agarose gel electrophoresis, and suitable fragments were selected as templates for PCR amplification. The quality of the obtained library was verified using an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) and an ABI StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) for quantification and qualification. The library was sequenced using an Illumina HiSeqTM 2000 (Illumina, San Diego, CA, USA), and the raw data was deposited as a National Center for Biotechnology Information (NCBI) BioProject (accession reference: PRJNA269140).

    • To identify the host genes associated with FMDV pathogenicity in SK6 cells, the original data were filtered and screened for differentially expressed genes. The NCBInr database was selected as the analytical database, and genetic data containing adaptors and low-quality reads were excluded. The short oligonucleotide analysis package (SOAPaligner/SOAP2) (Li et al., 2008) was used to quickly and accurately align the reads generated by the Illumina/Solexa Genome Analyzer (Illumina); and the reads per kilobase transcriptome per million mapped reads [RPKM; RPKM=106C/(N×L/103), where C represents the number of reads uniquely aligned to the gene of interest, N is the total number of reads that are uniquely aligned to all genes, and L is the number of bases in the gene of interest (Mortazavi et al., 2008)] was calculated. The RPKM method is able to eliminate the influence of different gene lengths and sequencing discrepancies on the calculation of gene expression. Therefore, the calculated gene expression can be used for comparing differences in gene expression among samples. A method to calculate the significance of digital gene-expression profiles was used for analysis of differentially expressed genes (Audic et al., 1997). We used a false discovery rate (FDR) ≤ 0.001 (mascot FDR calculation: http://www.matrixscience.com/help/decoy_help.html) and an absolute value of the Log2Ratio ≥ 1 as the thresholds to judge the significance of differences in gene expression. The screened differentially expressed genes were further analyzed by Gene Ontology (GO) and pathway-enrichment analysis. The GO database (http://www.geneontology.org/) and GO TermFinder software (http://smd.stanford.edu/help/GO-TermFinder/GO_TermFinder_help.shtml/) were used to perform GO analysis, and the Kyoto Encyclopedia of Genes and Genomes database (Kanehisa et al., 2008) was used for pathway-enrichment analysis.

    • The mutated region in the SAP domain of rA-SAP-FMDV was confirmed by sequencing analysis. A low MOI leads to infection of a percentage of cells, resulting in these infected cells signaling adjacent, uninfected cells via cytokines in order to activate antiviral genes, including secreted innate immune proteins. To study the signal transduction pathways and proteins involved, infections in this study were performed at 1 MOI. To compare the replication status of rA-FMDV and rA-SAP-FMDV in porcine PK15 and SK6 cells, the cells were infected with equal concentrations of rA-FMDV or rA-SAP-FMDV. The samples were collected 12-h post-infection, and the titers determined by TCID50 assay. The results showed that rA-FMDV replicated more quickly relative to rA-SAP-FMDV in both PK15 and SK6 cells (Figure 1B), indicating that the SAP mutation decreased FMDV replication in porcine PK15 and SK6 cells, with a larger decrease observed in SK6 cells.

    • To explore the differentially expressed genes involved in the altered pathogenicity observed in SK6 cells infected with FMDV containing the SAP mutation, rA-FMDV-infected and rA-SAP-FMDV-infected SK6 cells were collected at 6-h post-infection, and transcriptome analysis was performed. After a stringent quality check and filtering of the data (FDR ≤ 0.001 and fold-change ≥ 2), 20, 421 genes were detected, with 1, 853 differentially expressed genes identified. A total of 1, 670 and 183 genes were differentially upregulated and downregulated, respectively, between rA-SAP-FMDV-and rA-FMDV-infected SK6 cells (Supplementary Figure S1, S2). The expression of 117 transcription factor-related genes involved in 12 biological processes, 114 immune regulation-related genes participating in 40 immune-regulatory processes, 69 cytokine-related genes, including 20 involved in cytokine-production and -secretion processes, 12 inflammatory response-related genes, and 19 apoptosis-related genes were significantly altered (Table 1, Supplementary Table S2-S5). The distinctively different expression profiles of these genes may explain the decreased pathogenicity observed following infection with rA-SAP-FMDV. An analysis of the available literature indicated that the majority of the differentially expressed genes correlating with antiviral responses included (Table 2): 1) genes involved in transcriptional regulation (EIF4A2, EIF5B, EIF3J, NFKBIA, and NFKBIZ); 2) genes involved in the regulation of immune response (IFIT1, ITCH, IL7R, JAK2, LTB, TNFSF10, IL7, BLM, IFIT1, IL18, IL6, and FOS); 3) cytokine-related genes (IL1, IL6, IL20, TNF, CCL2, CCL20, CXCL10, CXCL2, CCL3L1, CCL4, CCL5, and CXCL11); 4) genes involved in the regulation of inflammation and chemokine production (TNF, CCL5, IL1A, IL6, IL6ST, CCL2, and ITCH); and 5) genes involved in apoptosis (BLM, CASP3, BRCA2, PMAIP1, CD38, MAP3K5, CUL5, TNFSF10, and XIAP).

      FunctionTotal gene numberUp-or down-regulated gene number
      Up-regulatedDown-regulated
      transcription factor-related genes1171098
      immune regulation-related genes11410410
      cytokine-related genes69627
      inflammatory response-related genes12111
      apoptosis-related genes19181

      Table 1.  Summary of differential expressed genes

      GeneFoldGene descriptionFunction
      EIF4A22.23Eukaryotic initiation factor 4A-ⅡRNA helicase activity; adenyl ribonucleotide binding
      EIF5B3.11Eukaryotic translation initiation factor 5BTranslation factor activity, nucleic acid binding
      EIF3J2.47Eukaryotic translation initiation factor 3 subunit
      J-like isoform 1
      Translation factor activity, nucleic acid binding
      NFKBIA3.83NF-kappa-B inhibitor alphaTranscription factor binding
      NFKBIZ3.47NF-kappa-B inhibitor zetaTranscription cofactor activity, protein binding
      IFIT13.74Interferon induced protein with tetratricopeptide repeats 1RNA binding, protein binding
      ITCH2.87Itchy E3 ubiquitin protein ligaseChemokine receptor binding, ubiquitin protein ligase activity
      IL7R4.32Interleukin 7 receptorCytokine receptor activity
      JAK22.27Janus kinase 2Kinase binding, cytokine receptor, protein kinase activity
      LTB13.15Lymphotoxin-betaTumor necrosis factor receptor superfamily bindin
      TNFSF103.67Tumor necrosis factor superfamily member 10Cation binding, tumor necrosis factor receptor binding
      IL72.31PREDICTED: interleukin-7 isoform 3Cytokine receptor binding
      BLM3.15Bloom syndrome proteinATP-dependent DNA helicase activity, double-stranded DNA binding
      IL182.27Interleukin 18Receptor binding, cytokine activity
      IL611.05Interleukin 6Cytokine receptor binding, cytokine activity
      FOS3.78FBJ osteosarcoma oncogeneNucleic acid binding transcription factor activity, protein dimerization activity
      IL13.11Interleukin 1Cytokine activity
      IL2027.75Interleukin 20Cytokine receptor binding, cytokine activity
      TNF20.89Tumor necrosis factorTumor necrosis factor receptor superfamily binding, sequence-specific DNA binding
      CCL25.73C-C motif chemokine ligand 2Kinase activity, chemokine receptor binding
      CCL207.62C-C motif chemokine ligand 20Cytokine activity, chemokine receptor binding
      CXCL105.89Chemokine (C-X-C motif) ligand 10Protein kinase regulator activity, cytokine activity
      CXCL22.38Chemokine (C-X-C motif) ligand 2Cytokine activity, chemokine activity
      CCL3L16.17C-C motif chemokine ligand 3 like 1CCR chemokine receptor binding, chemokine activity
      CCL415.34C-C motif chemokine ligand 4Cytokine activity, chemokine activity
      CCL52.58C-C motif chemokine ligand 5CCR chemokine receptor binding, chemokine activity, protein tyrosine kinase activator activity
      CXCL116.17C-X-C motif chemokine ligand 11Heparin binding, chemokine activity
      IL1A3.11Interleukin-1 alpha precursorTransition metal ion binding, cytokine receptor binding
      IL6ST3.58Interleukin 6 signal transducerCiliary neurotrophic factor receptor activity, cytokine receptor binding
      CASP32.08Caspase 3Endopeptidase activity, cyclin-dependent protein kinase regulator activity
      BRCA25.42Breast cancer 2Structure-specific DNA binding, histone acetyltransferase activity
      PMAIP12.78Phorbol-12-myristate-13-acetate-induced protein 1Protein binding
      CD382.51Cluster of differentiation 38Transferase activity, NAD(P)+ nucleosidase activity
      MAP3K52.23Mitogen-activated protein kinase kinase kinase 5Metal ion binding, phosphatase binding, apoptotic protease activator activity
      CUL53.23Cullin 5Signal transducer activity, enzyme binding
      XIAP2.59X-linked inhibitor of apoptosisTransition metal ion binding, cysteine-type endopeptidase inhibitor activity
      Note: A minimum of twofold change (P < 0.0001, Q < 0.0001) was used as the standards for selecting genes of interest.

      Table 2.  List of genes that displayed significant differential expression at WT and SAP mutant FMDV-infected SK6 cells

    • To further confirm and validate the transcriptome analysis results, we performed qPCR analysis to determine the reproducibility of the differential gene expression. A selected group of genes for which we had an established method available in our laboratory (with established primers and melting/annealing temperatures previously) were chosen for analysis. Six upregulated genes (CCL4, CCL2, IL6, IL7, IL18, and EGR1) and four downregulated genes (SRPX2, CREB5, RASAL1, and RIN2) were analyzed, and qPCR results confirmed the differential expression identified between rA-SAP-FMDV-and rA-FMDV-infected cells. As shown in Figure 2, the qPCR results corresponded with transcriptome analysis results and, while some fold-change differences were observed between results from each method, similarities in the overall expression profiles were revealed.

      Figure 2.  Validation of differentially expressed genes identified by transcriptome analysis through qPCR detection. Six upregulated and four downregulated genes were detected in an independent infection experiment undertaken in order to validate transcriptome analysis results. The expression profiles of the 10 selected genes were consistent between the transcriptome-analysis and qPCR-detection results. Results are presented as the mean ± standard error from three independent experiments.

    • Infection with FMDV containing the SAP-domain mutation altered gene expression in SK6 cells, thereby affecting various biological processes and signal transduction pathways, and resulting in blocked viral replication and decreased pathogenicity. To systematically analyze the functional characterization of the differentially expressed genes and pathways associated with the FMDV Lpro SAP domain, GO analysis and pathway annotation were conducted. The results indicated that the differentially expressed genes were involved in metabolism, cell cycle processes, and cellular component organization or biogenesis processes (Supplementary Figure S3), and functional analysis revealed that many of these genes were involved in nucleotide binding and functions associated with nucleic acids (Supplementary Figure S4). Cellular component annotation results are shown in Supplementary Figure S5.

      Pathway analysis indicated that 35 pathways were altered in rA-SAP-FMDV-infected cells as compared with rA-FMDV-infected cells, including regulation of actin cytoskeleton formation, endocytosis, phagosome formation, chemokine-signaling pathways, the cell cycle, the retinoic acid-inducible gene 1-like receptor signaling pathway, the NF-κB signaling pathway, and the nucleotide-binding oligomerization domain-like receptor signaling pathway (Supplementary Figure S6). The potential host targets for Lpro and the protein-protein interaction pathways involved are shown in Figure 3. These findings suggested that expression of these genes might potentially result in decreased rA-SAP-FMDV replication.

      Figure 3.  The potential host targets of Lpro and the involved protein-protein interaction pathways.

    • FMDV has the ability to manipulate various host-cell signal transduction pathways by subverting gene expression. FMDV Lpro is a host-cell antagonist that interferes with host gene expression, promotes viral propagation, and subverts host immune systems by targeting eIF-4G, IRF3, IRF7, and NF-κB (de Los Santos et al., 2009; Kirchweger et al., 1994; Wang et al., 2010). The FMDV Lpro SAP domain is a putative DNA-binding domain found in diverse eukaryotic nuclear proteins (Aravind et al., 2000; de Los Santos et al., 2009) and inhibits host innate immune response (de Los Santos et al., 2009). The mutation of two amino acids (I55A and L58A) in the Lbpro SAP domain alters Lpro subcellular localization and function (de Los Santos et al., 2009; Diaz-San Segundo et al., 2012).

      In a previous study, we constructed a chimeric virus, rA-FMDV, as a candidate vaccine (Zheng et al., 2013). To decrease rA-FMDV pathogenicity and develop a potential live attenuated vaccine, we constructed rA-SAP-FMDV containing a mutated SAP domain (unpublished data). Here, we compared the different pathogenic characteristics of rA-FMDV and rA-SAP-FMDV in PK15 and SK6 cells, and showed that rA-SAP-FMDV was less pathogenic relative to rA-FMDV in both cell lines, suggesting that rA-SAP-FMDV may have potential as a vaccine strain based on its failure to disrupt cellular responses that inhibit viral replication.

      Decreased rA-SAP-FMDV replication was more evident in SK6 cells as compared with PK15 cells. To analyze the differentially expressed genes correlated with the altered viral replication observed in SK6 cells, transcriptome analysis was performed using NGS technology. The results indicated differential expression of 1, 853 genes between infected and non-infected SK6 cells, with these findings subsequently confirmed by qPCR analysis. These findings suggested that mutation of the FMDV Lpro SAP domain might adversely affect the ability of FMDV to inhibit host gene expression during infection, resulting in reduced viral pathogenicity.

      Among the differentially expressed genes upregulated in rA-SAP-FMDV-infected cells, EIF4A2, EIF5B, and EIF3J are involved in the initiation of host translation by aiding in the recruitment of protein and mRNA components to ribosomes (Cheyssac et al., 2006; ElAntak et al., 2007; Kyono et al., 2002; Meijer et al., 2013; Unbehaun et al., 2007). Swine infected with FMDV containing a mutated SAP domain developed a strong neutralizing-antibody response as early as 2-days post-inoculation as compared with those infected with wild-type FMDV (Diaz-San Segundo et al., 2012). The upregulation of these translation factors possibly resulted in the enhancement of neutralizing-antibody production. Furthermore, our analysis revealed upregulation of other genes, including those involved in metabolic and cellular-response processes (Supplementary Figure S3).

      FMDV infection can induce degradation of NF-κB (de Los Santos et al., 2007); however, NF-κB activity was significantly enhanced in cells infected with FMDV containing a mutated SAP domain (Zhu et al., 2010). NFKBIA and NFKBIZ are involved cytokine production through NF-κB regulation (Ninomiya-Tsuji et al., 1999; Yamazaki et al., 2001). In this study, we found that the expression of NFKBIA and NFKBIZ was upregulated in rA-SAP-FMDV-infected cells, which may have altered the subsequent expression of NF-κB-induced cytokines to ensure a robust immune response (Figure 3). CCL4 and IL7, both involved in cellular immune and inflammatory responses, were also upregulated in these cells. Additionally, the upregulation of many other genes involved in immune response, inflammation, chemokine production, and apoptosis was also observed.

      Upregulation of EGR1 and IL6 expression was also observed in rA-SAP-FMDV-infected cells. EGR1 mediates cell proliferation, differentiation, inflammation, and apoptosis (Han et al., 2015), and may interact with p53 or FOS to regulate apoptosis or transcription (Figure 3). IL6 is involved in inflammation, B cell maturation, and suppression of viral replication (Dienz et al., 2012). Here, upregulation of IL6 expression possibly enhanced acute-phase response and suppressed rA-SAP-FMDV replication. Our results suggested that Lpro may attenuate EGR1 and IL6 expression and adversely affect regulation of cell proliferation, transcription, differentiation, inflammation, immune response, and apoptosis to promote viral replication. Furthermore, mutation of the Lpro SAP domain might impair this antagonistic effect, thereby inhibiting viral replication (Figure 3). The resulting upregulation of these genes likely reinforced the antiviral activity of the cells, directly resulting in the decreased pathogenicity observed following rA-SAP-FMDV infection. Conversely, downregulation of SRPX2, which is involved in anti-apoptotic activity, was observed in rA-SAP-FMDV-infected SK6 cells (Figures 2 and 3). This indicated an alternative pathway for suppressing viral replication through the promotion of cell death. Intact Lpro likely inhibits the initiation of apoptosis, with our results suggesting that mutation of the Lpro SAP domain impaired this inhibitory effect.

      A previous study found that in EBK cells infected with a FMDV containing the SAP-domain mutation, NF-κB was the primary factor responsible for the differential transcription of many upregulated genes associated with innate immune response (Zhu et al., 2010). Here, we found that the differential gene expression observed in rA-SAP-FMDV-infected SK6 cells resulted from altered expression of genes involved in transcription and immune-related regulation. However, IFN-stimulated genes, such as ISG15, ISG20, MX1, GBP1, and OAS1, which were differentially expressed in EBK cells infected with a FMDV containing the SAP-domain mutation, were not observed in this study. This is possibly due to SK6 cells being deficient in type Ⅰ IFN production (Ruggli et al., 2003), although the enhancement of various type Ⅰ IFN-independent genes and pathways determined in this study were implied to perform crucial antiviral effects.

      In summary, we reported that a FMDV Lpro SAP-domain mutant exhibited decreased replication ability in SK6 cells as compared with wild-type FMDV. Transcriptome analysis suggested that the altered expression of genes involved in transcription, immune response, cytokine and chemokine production, inflammation, and apoptosis were the primary reasons for the observed decrease in pathogenicity. Our results provided insight into the pathogenic mechanisms associated with the FMDV Lpro SAP-domain and suggested that mutation of region provides a strategy for the development new FMDV-related vaccines having impaired host-antagonistic ability.

    • We thank Dr. Jinwen Liu for providing valuable technical assistance and suggestions. This work was supported by grants from the National Science and Technology Ministry (2015BAD12B04), National Natural Sciences Foundation of China (No. 31302118, 31502042 and 31402179), the Gansu Science Foundation for Distinguished Young Scholars (no. 145RJDA328), the International Atomic Energy Agency (16025/R0) and the Key technologies R & D program of Gansu Province (1302NKDA027).

    • The authors declare that they have no conflict of interest. This article does not contain any studies with human oranimal subjects performed by any of the authors.

    • ZXN, FY, ZXZ and HXZ designed the research, ZXN, FY, ZXZ, XD, WWL and WJC performed the experiments. XLZ, YJ, JHG and XTL provided experiment support. ZXN, ZXZ and HXZ wrote the manuscript. All authors have read and approved the final manuscript for submission.

      Supplementary figures/tables are available on the website of Virologica Sinica: www.virosin.org; link.springer. com/journal/12250.

    • Gene Primers(5′→3′)
      CCL4 Forward: CACCTCCTGCTGCTTCACATA
      Reverse: CAGACCTGCCTGCCCTTTT
      CCL2 Forward: GTCACCAGCAGCAAGTGTCCT
      Reverse: ATGTGCCCAAGTCTCCGTTTA
      IL6 Forward: GACAAAGCCACCACCCCTAA
      Reverse: CTCGTTCTGTGACTGCAGCTTATC
      IL7 Forward: GGGATGGATGAAACAGAAGG
      Reverse: GCTACTGGCAACAGAACAAGG'
      IL18 Forward: GCACCTCAGACCGTATTTATT
      Reverse: CATCATGTCCAGGAACACTTC
      EGR1 Forward: TCAACACCACCTACCAGTCCCA
      Reverse: GATCTTGGTATGCCTCTTGCGTT
      SRPX2 Forward: AACGTGGTATGCAGGTTCAGG
      Reverse: GTAGTCACAGCGGGAGTCAAGA
      CREB5 Forward: TATCTTCCCTGCTACATCTTCACA
      Reverse: AACGCAGCCTTCAACCTCATT
      RASAL1 Forward: GTGAAAGTGGACGACGAGGTGG
      Reverse: GGGAAGCGTGTCTTCTTGATGG
      RIN2 Forward: CCTTGAAGTTGCCTTATGCTGTTT
      Reverse: GCTACGTTCCCATGTGGGTGAT
      GAPDH Forward: ACATGGCCTCCAAGGAGTAAGA
      Reverse: GATCGAGTTGGGGCTGTGACT

      Table S1.  The qPCR primers used in this study

      Gene function description Up-regulated genes Down-regulated genes
      Gene number Gene name list Gene number Gene name list
      Transcription factor complex 9 ING2, CNOT7, TAF13, TAF9B, MNAT1, RB1, NFYB, GTF2A1, TAF2 0 -
      Transcription factor binding 22 HES1, KLF4, LOC100154750, GTF2A1, LOC100739605, MDFIC, MTDH, MED13, JMJD1C, PRDM5, LOC100626982, NFYB, EGR2, NRIP1, NFKBIA, SIRT1, HMGB1, CAND1, EIF4A2, EIF4A2, EIF5B, EIF3J 0 -
      Protein binding transcription factor activity 34 FGF2, JMY, SP100, IFNB1, NPAT, MTDH, CIR1, SCAI, USP16, NFE2, GABPA, SIRT1, C1D, LOC102161761, LOC102160069, TRIP11, LOC100154750, MYSM1, RB1, SS18, LOC100739605, LOC100622863, TAF9B, TMF1, MED13, NMI, COPS2, LOC102166221, TBL1XR1, CASP8AP2, NRIP1, AEBP2, KMT2E, SKIL 3 DYRK1B, PIR, TFCP2L1
      Transcription factor binding transcription factor activity 34 FGF2, JMY, SP100, IFNB1, NPAT, MTDH, CIR1, SCAI, USP16, NFE2, GABPA, SIRT1, C1D, LOC102161761, LOC102160069, TRIP11, LOC100154750, MYSM1, RB1, SS18, LOC100739605, LOC100622863, TAF9B, TMF1, MED13, NMI, COPS2, LOC102166221, TBL1XR1, CASP8AP2, NRIP1, AEBP2, KMT2E, SKIL 3 DYRK1B, PIR, TFCP2L1
      Nucleic acid binding transcription factor activity 60 KLF4, ZHX1, EGR4, ZNF24, LOC102159255, TFAM, NFIA, EGR2, GABPA, GATA3, NFE2L2, HDAC1, HDAC2, HES1, PAXBP1, ZC3H8, TAF13, CREBRF, GATA2, TOPOⅡ, TOP2B, RBPJ, MYNN, EHF, DMTF1, ZNF84, ZBTB38, AHR, HMGB1, ZNF197, CNOT7, GCFC2, BTAF1, LOC102163244, SLC30A9, NPAT, LOC100737142, CIR1, ZNF189, ETV1, FOXN2, TBX21, SIX4, ZEB2, NFYB, EGR1, ZNF287, ARID4A, RB1, BTG2, FOS, LOC100622863, BLZF1, LOC100520527, LOC100515279, NR1D2, SCML2, HIF1A, LOC100626982, POU3F2 5 ARNT2, NR4A2, RCAN1, ZNF71, TFCP2L1
      Ligand-activated sequence-specific DNA binding RNA polymerase Ⅱ transcription factor activity 1 NR1D2 1 NR4A2
      Sequence-specific DNA binding transcription factor activity 5 HES1, BTG2, HIF1A, NR1D2, FOXN2 1 NR4A2
      Sequence-specific DNA binding RNA polymerase Ⅱ transcription factor activity 4 BTG2, HIF1A, NR1D2, FOXN2 1 NR4A2
      Positive regulation of sequence-specific DNA binding transcription factor activity 8 MALT1, TNF, KRAS, TAB3, CHUK, LOC100620995, NFKBIA, MTDH 0 -
      Regulation of sequence-specific DNA binding transcription factor activity 10 MALT1, ITCH, TNF, KRAS, TAB3, CHUK, LOC100620995, NFKBIA, LOC100153617, MTDH 1 LOC100515993
      Negative regulation of sequence-specific DNA binding transcription factor activity 3 NFKBIA, LOC100153617, ITCH 1 LOC100515993
      Regulation of transcription factor import into nucleus 2 NFKBIA, TNF 0 -

      Table S2.  Differentially expressed genes involved in transcription-related functions

      Gene function description Up-regulated genes Down-regulated genes
      Gene number Gene name list Gene number Gene name list
      Negative regulation of immune effector process 4 SOCS5, IFIT1, ITCH, IL7R 1 TGFB3
      Production of molecular mediator of immune response 8 MALT1, LOC100621191, TNF, XRCC4, LOC100620995, LOC100739713, IL7R, MSH2 0 -
      Immune system process 94 BMPR1A, IL18, CASP3, CXCL10, SEMA3C, TGFBR1, MASP2, IFNB1, IFIT1, CCL5, ROCK1, S100A12, LOC100739781, LOC100625180, LOC100518921, CD79A, XRCC4, CD84, GATA3, IL7R, DPP8, HES1, LOC102161761, ZC3H8, CSF2, TNF, TBK1, TAB3, PIK3CA, IRG6, LOC100736872, RBPJ, BRCA2, ATM, CCL3L1, LOC100620995, AHR, HMGB1, APC, MSH2, SLAMF7, CCL20, S100A9, ADAM10, KRAS, JAK2, LTB, TNFSF10, IL7, BLM, LOC100622217, IL1RAP, SIX4, LOC100622970, LOC100738058, RTKN2, NCK1, MAP3K8, EGR1, CHUK, IL1A, LOC100739713, ANGPT1, KITLG, LOC102163753, LOC100621191, LOC100627112, RB1, MSH3, CXCL2, UBD, BMI1, FOS, LOC100628215, STXBP3, MALT1, CCL2, MTAP, TNFSF15, CFH, CSF1, CD38, APOBEC1, NFKBIA, RICTOR, CCL4, KMT2E, CXCL11, ITGA6, LOC100739007, IL6ST, SKIL, IL6, LOC100620730 6 SUSD2, GRB7, MAP2K6, GPX2, LOC102166152, LOC100522330
      Cytokine production involved in immune response 4 MALT1, LOC100620995, LOC100621191, TNF 0 -
      Somatic recombination of immunoglobulin genes involved in immune response 3 LOC100739713, MSH2, XRCC4 0 -
      Somatic diversification of immunoglobulins involved in immune response 3 LOC100739713, MSH2, XRCC4 0 -
      Immunoglobulin production involved in immunoglobulin mediated immune response 3 LOC100739713, MSH2, XRCC4 0 -
      Regulation of immune system process 48 LOC100737466, IL18, CASP3, ITCH, ADAM10, MASP2, IFNB1, IFIT1, CCL5, EDN1, IL7, BLM, LOC100622217, CD79A, TBX21, LOC100622970, LOC100738058, NCK1, MAP3K8, CHUK, IL7R, SOCS5, LOC100627112, LOC100737558, TNF, TBK1, RB1, TAB3, PIK3CA, FOS, ELMOD2, LOC100628215, MALT1, CCL2, HIF1A, ATM, ACVR2A, CFH, CSF1, CD38, RICTOR, LOC100620995, NFKBIA, KMT2E, DDX58, LOC100739007, IL6, APC 5 TNFSF4, MAP2K6, SEMA7A, TGFB3, LOC102161418
      Somatic diversification of immune receptors via germline recombination within a single locus 5 LOC100739713, HMGB1, MSH3, MSH2, XRCC4 0 -
      Immune response 19 IL18, LOC100621191, TNF, MASP2, IFNB1, IFIT1, BMI1, IL7, STXBP3, S100A12, MALT1, CCL2, CFH, XRCC4, LOC100620995, LOC100739713, GATA3, IL6ST, MSH2 1 GPX2
      Adaptive immune response 10 MALT1, IL18, TNF, MASP2, XRCC4, LOC100620995, LOC100739713, GATA3, IL6ST, MSH2 0 -
      Somatic diversification of immune receptors 5 LOC100739713, HMGB1, MSH3, MSH2, XRCC4 0 -
      Immune system development 29 KITLG, ZC3H8, RB1, MSH3, TGFBR1, BMI1, JAK2, LTB, RBPJ, BLM, LOC100628215, LOC100739781, MALT1, BRCA2, ATM, LOC100518921, CD79A, SIX4, MTAP, RTKN2, XRCC4, EGR1, CHUK, LOC100739713, HMGB1, IL7R, APC, ANGPT1, MSH2 ; 1 LOC102166152
      Myeloid cell activation involved in immune response 2 S100A12, STXBP3 0 -
      Somatic diversification of immune receptors via somatic mutation 2 LOC100739713, MSH2 0 -
      Adaptive immune response based on somatic recombination of immune receptors built from immunoglobulin superfamily domains 9 MALT1, IL18, TNF, MASP2, XRCC4, LOC100620995, LOC100739713, GATA3, MSH2 0 -
      Regulation of production of molecular mediator of immune response 2 TBX21, TNF 2 SEMA7A, TGFB3
      Negative regulation of immune system process 5 CASP3, SOCS5, IFIT1, ITCH, IL7R 1 TGFB3
      Regulation of immune effector process 11 LOC100737466, SOCS5, TBX21, ITCH, TNF, IFNB1, IFIT1, DDX58, ELMOD2, IL6, IL7R 2 TGFB3, SEMA7A
      Immunoglobulin mediated immune response 5 LOC100739713, TNF, MASP2, MSH2, XRCC4 0 -
      Positive regulation of immune system process 28 IL18, SOCS5, LOC100627112, ITCH, TBK1, ADAM10, TAB3, MASP2, IFNB1, PIK3CA, FOS, CCL5, EDN1, BLM, LOC100622217, TBX21, CD79A, CFH, LOC100622970, LOC100738058, NCK1, MAP3K8, CD38, RICTOR, CHUK, LOC100620995, NFKBIA, IL6 2 TNFSF4, MAP2K6
      Regulation of cytokine production involved in immune response 0 - 2 SEMA7A, TGFB3
      Innate immune response 2 CCL2, IFIT1 0 -
      Activation of innate immune response 8 LOC100622970, TBK1, TAB3, LOC100738058, CHUK, LOC100620995, NFKBIA, FOS 1 MAP2K6
      Innate immune response-activating signal transduction 8 LOC100622970, TBK1, TAB3, LOC100738058, CHUK, LOC100620995, NFKBIA, FOS 1 MAP2K6
      Immune effector process 14 S100A12, STXBP3, MALT1, CFH, TNF, MASP2, XRCC4, APOBEC1, LOC100620995, IRG6, KMT2E, LOC100739713, GATA3, MSH2 0 -
      Positive regulation of innate immune response 8 LOC100622970, TBK1, TAB3, LOC100738058, CHUK, LOC100620995, NFKBIA, FOS 1 MAP2K6
      Regulation of innate immune response 8 LOC100622970, TBK1, TAB3, LOC100738058, CHUK, LOC100620995, NFKBIA, FOS 1 MAP2K6
      Regulation of immune response 17 SOCS5, TBK1, TAB3, MASP2, PIK3CA, FOS, TBX21, CD79A, LOC100622970, CFH, LOC100738058, NCK1, CD38, CHUK, LOC100620995, NFKBIA, IL6 3 TGFB3, MAP2K6, SEMA7A
      Immune response-activating cell surface receptor signaling pathway 7 NCK1, CD38, PIK3CA, NFKBIA, LOC100620995, CHUK, CD79A 0 -
      Immune response-regulating cell surface receptor signaling pathway 7 NCK1, CD38, PIK3CA, NFKBIA, LOC100620995, CHUK, CD79A 0 -
      Activation of immune response 14 CD79A, CFH, LOC100622970, TBK1, TAB3, MASP2, LOC100738058, NCK1, CD38, PIK3CA, CHUK, LOC100620995, NFKBIA, FOS 1 MAP2K6
      Immune response-activating signal transduction 12 CD79A, LOC100622970, TBK1, TAB3, LOC100738058, NCK1, CD38, PIK3CA, CHUK, LOC100620995, NFKBIA, FOS 1 MAP2K6
      Immune response-regulating signaling pathway 12 CD79A, LOC100622970, TBK1, TAB3, LOC100738058, NCK1, CD38, PIK3CA, CHUK, LOC100620995, NFKBIA, FOS 1 MAP2K6
      Positive regulation of immune response 14 CD79A, CFH, LOC100622970, TBK1, TAB3, MASP2, LOC100738058, NCK1, CD38, PIK3CA, CHUK, LOC100620995, NFKBIA, FOS 1 MAP2K6
      Humoral immune response mediated by circulating immunoglobulin 2 TNF, MASP2 0 -
      Humoral immune response 3 CFH, TNF, MASP2 0 -
      Cell activation involved in immune response 2 S100A12, STXBP3 0 -
      Leukocyte activation involved in immune response 2 S100A12, STXBP3 0 -
      Positive regulation of immune effector process 3 SOCS5, TBX21, IL6 0 -

      Table S3.  Differentially expressed genes involved in immune regulation

      Gene function description Up-regulated genes Down-regulated genes
      Gene number Gene name list Gene number Gene name list
      Cytokine receptor binding 25 KITLG, CSF2, IL20, ITCH, TNF, TGFBR1, IFNB1, CCL5, JAK2, LTB, TNFSF10, IL7, LOC100739781, CCL2, IL1RAP, TNFSF15, CSF1, CASP8AP2, IL1A, LIFR, LOC100739007, IL6ST, IL6, LOC100620730, ANGPT1 3 TGFB3, TNFSF4, LOC100515993
      Cytokine activity 9 CCL2, CCL20, CXCL10, CXCL2, CCL3L1, CCL4, CCL5, CXCL11, LOC100620730 0 -
      Cytokine receptor activity 5 IL1RAP, LIFR, IL12RB2, IL6ST, IL7R 0 -
      Positive regulation of cytokine production 18 CSF2, LOC100737466, IL18, TNF, TBK1, IFNB1, POLR3G, NOX1, IL12RB2, MALT1, HIF1A, LOC100622970, LOC100620995, IL1A, DDX58, GATA3, IL6, IL6ST 1 TNFSF4
      Regulation of cytokine production 26 CSF2, LOC100737466, IL18, ITCH, TNF, TBK1, IFNB1, IFIH1, POLR3G, NOX1, IL12RB2, LTB, RNF125, ATG5, MALT1, HIF1A, LOC100625180, TNFSF15, LOC100622970, ZNF287, LOC100620995, IL1A, GATA3, DDX58, IL6ST, IL6 5 LOC780431, TGFB3, TNFSF4, SEMA7A, ACP5
      Response to cytokine stimulus 17 CASP3, SP100, TNF, ADAM10, UBD, IFIT1, IFIT3, RPS6KB1, EDN1, ACSL4, CCL2, IFIT2, EGR1, CD38, LIFR, HMGB1, IL6 2 LOC100515993, LOC102161418
      Cytokine production involved in immune response 4 MALT1, LOC100620995, LOC100621191, TNF 0 -
      Regulation of cytokine secretion 3 IL1A, TNFSF15, TNF 1 TNFSF4
      Negative regulation of cytokine production 9 ATG5, LOC100737466, ITCH, LOC100622970, TNF, TBK1, IFIH1, DDX58, RNF125 3 TGFB3, TNFSF4, ACP5
      T cell cytokine production 2 MALT1, LOC100620995 0 -
      Cytokine production 9 MALT1, IL18, HIF1A, IL1RAP, LOC100621191, TNF, LOC100620995, IL12RB2, JAK2 1 ACP5
      Negative regulation of cytokine-mediated signaling pathway 2 CCL5, IL6ST 0 -
      Negative regulation of response to cytokine stimulus 2 CCL5, IL6ST 0 -
      Cytokine-mediated signaling pathway 9 EGR1, CCL2, IFIT1, LIFR, IFIT3, SP100, IL6, TNF, IFIT2 1 LOC102161418
      Cellular response to cytokine stimulus 9 EGR1, CCL2, IFIT1, LIFR, IFIT3, SP100, IL6, TNF, IFIT2 1 LOC102161418
      Positive regulation of cytokine biosynthetic process 6 LOC100625180, IL1A, LTB, LOC100622970, TNF, TBK1 0 -
      Regulation of cytokine production involved in immune response 0 - 2 SEMA7A, TGFB3
      Regulation of cytokine biosynthetic process 7 LOC100625180, IL1A, LTB, IL6, LOC100622970, TNF, TBK1 0 -
      Regulation of cytokine-mediated signaling pathway 4 SOCS1, CCL5, JAK2, IL6ST 0 -
      Regulation of response to cytokine stimulus 4 SOCS1, CCL5, JAK2, IL6ST 0 -

      Table S4.  Differentially expressed genes involved in cytokine-related functions

      Gene function description Up-regulated genes Down-regulated genes
      Gene number Gene name list Gene number Gene name list
      Chronic inflammatory response 8 TNF, PTGER3, PLA2G4A, CCL5, IL1A, IL6, IL6ST, PTGS2 0 -
      Acute inflammatory response 11 HIF1A, TNF, PTGER3, IFNB1, PLA2G4A, CCL5, IL1A, HMGB1, IL6, IL6ST, PTGS2 1 GPX2
      Inflammatory response 1 TNF 1 GPX2
      Inflammatory response to antigenic stimulus 8 TNF, PTGER3, PLA2G4A, CCL5, IL1A, IL6, IL6ST, PTGS2 0 -
      Chemokine receptor binding 4 CCL2, CCL5, ITCH, LOC100620730 0 -
      CCR chemokine receptor binding 2 CCL2, CCL5 0 -
      Regulation of execution phase of apoptosis 5 BRCA2, LOC100624979, LOC100739713, PMAIP1, MSH2 0 -
      Induction of apoptosis 13 CD38, LOC100738156, LOC100622580, MAP3K5, CUL5, TNFSF10, XIAP, TNF, BLM, BCLAF1, CASP3, CASP4, CASP8AP2 1 LOC102161387

      Table S5.  Differentially expressed genes involved in inflammation, chemokine production-, and apoptosis-related functions

      Figure S1.  GO analysis of the immune-related genes indicating altered expression in rA-SAP-FMDV-infected SK6 cells as compared with rA-FMDV-infected SK6 cells. The X-axis indicates the correspondent number of genes.

      Figure S2.  GO analysis of the transcription factor-, inflammatory response-, apoptosis-and cytokine-related genes showing altered expression in rA-SAP-FMDV-infected SK6 cells as compared with rA-FMDV-infected SK6 cells. The X-axis indicates the correspondent number of genes.

      Figure S3.  Biological processes derived from GO annotations for the differentially expressed genes. The X-axis indicates the correspondent number of genes.

      Figure S4.  Molecular functions derived from the GO annotation for differentially expressed genes. The X-axis indicates the correspondent number of genes.

      Figure S5.  Annotations of cellular components associated with the differentially expressed genes. The X-axis indicates the correspondent number of genes.

      Figure S6.  Pathway annotations for the differentially expressed genes. The X-axis indicates the correspondent number of genes.

    Figure (9)  Table (7) Reference (40) Relative (20)

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