2.1 Clinical outcomes and pathology
The clinical signs recorded were intermittent fever, high body temperature (> 40°C), depression, and anorexia. The pigs in the low dose group did not show any ASF clinical signs or gross pathology throughout the experiment and were euthanized at the termination of the experiment (29 dpi: Fig. 1). From 7 dpi, all locally-adapted pigs in the medium and high dose groups showed acute ASF clinical signs, including depression, anorexia, recumbence, accelerated and labored breathing, diarrhea, and slight ataxia, and were euthanized humanely from 7 dpi (n = 1 in high dose), 9 dpi (n = 2; 1 each from high and medium dose), 10 dpi (n = 3; 2 from high dose and 1 from medium dose), 11 dpi (n = 1 from medium dose) and 17 dpi (n = 3 from medium dose). The locally-adapted pigs have black pigment, and as a result, skin lesions could not be scored. We also observed recumbence, reduced feed intake, weight loss from 4 dpi, and foul-smelling watery diarrhea. The highest temperature (39.6°C) was recorded at 7 dpi from the first pig that succumbed at 7 dpi from the high dose group (Supplementary Table 1 and Fig. 1- survival analysis). From 9 dpi, all of the remaining locally-adapted pigs showed febrile temperature reactions (40.5°C to 41.4°C). Survival rates between high and medium doses were not significantly different (p = 0.075) from that between the high and low (p = 0.013) and medium compared to that of low (p = 0.017), which were significant. All the spleen samples in the infection groups collected post-euthanasia tested positive by ASFV qPCR, while ASFV positivity by PCR was observed from 4 dpi in the high- and medium dose groups. Postmortem examination of the organs showed hemorrhagic lymph nodes and fluid accumulation in the abdominal, thoracic, and pericardial cavities.
2.2 RNA-Seq data quality check, mapping to pig host and ASFV pathogen reference genomes
Total RNA from 10 porcine splenic tissues was used for RNA-seq (3 each from medium, high, and low dose groups, and one animal from the control group). The total number of reads attained from the 10 locally-adapted pigs is summarized in Table 1. After QC, deduplication, and trimming off of multi-mapped reads, we retained an average of 45.3 M reads from the low dose pig samples, 12.89 M reads from the medium dose pig samples, and 9.66 M reads from the high dose pig samples (Table 1). There were no significant variations in the number of reads between the replicates.
Table 1
Mapping statistics to the pig and the ASF virus reference genomes.
Study group | High dose | Medium dose | Low dose | Control |
Number of biological replicates | 3 | 3 | 3 | 1 |
Total trimmed reads | 9,660,832 | 12,889,691 | 45,343,542 | 30,355,538 |
No of reads mapped to pig host | 5,763,796 | 3,493,725 | 42,818,622 | 27,560,673 |
No of genes mapped to pig host (% of coding genes mapped) | 14,544 (93.56%) | 13,755 (88.5%) | 15,543 (99.99%) | 15,527 (99.88%) |
No of reads mapped to ASFV pathogen | 14,302 | 13,363 | 12,381 | 0 |
No of genes mapped to ASFV pathogen (% of coding genes mapped) | 172 (98.86%) | 174 (99.43%) | 167 (95.43%) | 0 |
Following the trimming of adaptors, deduplication, and removal of poor-quality reads, on average, 72.58% of the reads mapped to the pig genome (Fig. 2A). Pig HB_1066 (Accession No. xxx) from the medium dose group had very low mapping rates to the pig reference genome (5.75%) and was removed from the analysis. On average, of the trimmed data, 0.15% (min 0.01% and max 0.5%) of the reads mapped to the ASFV genome (Fig. 2B). The pigs challenged with the high- and medium doses had higher ASFV mapping rates, with pig HB_1069 (Accession No. xxx) from the medium dose group having the highest ASFV mapping rate of 0.5% (Fig. 2B).
2.3 Differential gene expression in locally-adapted pig spleen tissues
Compared to the control, we detected the expression of 15,543, 13,755 and 14,544 known pig genes in the low, medium, and high dose groups, respectively (Table 1 and Fig. 3). The complete list of pig genes detected by RNA-Seq is in Supplementary Table S2. We observed a wide variation in the gene count in transcripts per Million (TPM) of the expressed genes in the three infection groups, possibly due to differences in sample collection time points since these pigs reached humane end-point at different times (see survival curve, Fig. 1). We then selected genes with at least a 2-fold increase in gene expression relative to the control genes in at least two pigs as the top expressed pig genes. A summary of select differentially expressed genes (DEGs) is shown in Table 2. Of the 3055 DEGs detected in the high dose group, 1711 were upregulated while 1344 were downregulated (Fig. 3A), while the medium dose group had 896 DEGs upregulated and 875 downregulated. In the low dose group, 105 genes were upregulated while 23 were downregulated (Fig. 3A). In the high and medium dose groups, the top-upregulated pig host genes were genes found to be associated with response to infection due to a highly pathogenic ASFV isolate. These DEGs could be divided into five groups, namely a) genes found on the macrophages, b) genes associated with natural killer cells, c) genes involved in ASFV infection, d) genes linked with the lymphocytes, e) other genes not linked to virus infection or immunity such as DRAM2, and SOGA1, are reported to be associated with autophagy. Other genes such as TIMP1, LTF, CHP2, FOSL1, and FOXF1 play critical roles in viral pathogenesis [Table 2] (50, 51).
Additionally, the Forkhead box (FOX) family of transcription factors (FOXF1, FOXS1, FOXM1, FOXK1, FOXP4, and FOXC1) play a role in cell differentiation and proliferation and are implicated in cancer and drug resistance (52). These FOX transcription factors were all upregulated in the medium and high dose groups but not detected in the low dose group. The CSF3R is a type 1 cytokine receptor that binds the granulocyte colony-stimulating factor (G-CSF). G-CSF is a cytokine that is required for granulocyte proliferation and differentiation. IFITM3 was significantly upregulated (over 3-fold), and it plays a critical role in immunity during viral infection in that it directly engages and shuttles incoming virus particles to lysosomes. We also detected the expression of ATF4, a transcription factor that upregulates genes involved in amino acid import, antioxidative stress response, and regulation of autophagy (53, 54)
Regarding genes associated with resistance/tolerance to ASF (25), the RELA proto-oncogene, NF-kB subunit, or transcription factor p65 (RELA) was detected at higher counts (2846 TPM) in the low dose group than the high and medium doses at 257 and 269 TPM, respectively (Supplementary Table S2). RELA was downregulated at -1.94-fold (padj = 1.98E-04) and − 2.34-fold (padj = 4.71E-05) in the high- and medium dose groups but was not differentially expressed in the low dose group.
In terms of the genes associated with macrophage regulation (55), there was downregulation of the macrophage surface marker gene, CD164 (decreased by 1.15-fold) in pigs that survived a low dose ASFV challenge. HMOX1 codes for heme oxygenase-1, and its expression decreased by 1.89-fold and 1.46-fold in the high- and medium dose, respectively. The macrophage-associated genes were also upregulated in the medium and high dose groups, including S100A4/A6/A8/A9/A13/A16 that play a role in Calcium-binding, innate immune response, and leukocyte migration associated with inflammatory response (56–58). The angiopoietin-like protein (ANGPTL1) genes were highly differentially expressed at 1.61-fold and 5.02-fold in high- and medium dose groups. CD244 is involved in the activation of NK cells leading to cell-mediated cytotoxicity (59) was downregulated in the high and medium dose groups by over 1.65 and 4.7-fold, respectively. Another differentially downregulated gene in medium and high dose groups was CD36 at 3.89 and 4.91-fold change, respectively. CD36 is a scavenger receptor expressed in multiple cell types, mediates lipid uptake, immunological recognition, inflammation, molecular adhesion, and apoptosis, and binds thrombospondin-1 (TSP-1), resulting in attenuation of angiogenesis and induction of apoptosis/blocking the vascular endothelial growth factor receptor 2 (EGFR2) pathway in tumor microvascular endothelial cells (60). We detected an upregulation of TSP-1 in the high and medium dose groups by 1.92- and 3.25-fold change, respectively.
Antimicrobial peptides (AMPs) are molecules that possess a broad-spectrum activity against bacteria, fungi, protozoa, and viruses found in insects, amphibians, and mammals with antimicrobial, immunomodulatory and regulatory activity gut microbiota (61). We detected AMPs of pharmaceutical value, such as protegrin-4 (PG4), a peptide isolated from porcine leucocytes (62). NPG4 was significantly upregulated in the medium dose (7.1-fold) and high dose (3.44-fold). The other upregulated genes were those involved in the host response to virus infection, such as PPP1R15A (protein phosphatase 1 regulatory subunit 15A), which codes for GADD34 complex, a host protein involved in the dephosphorylation of P-eIF2α (Eukaryotic Initiation Factor 2) by an interferon-induced double-stranded RNA-activated protein kinase (PKR) in a prominent cellular antiviral pathway (63, 64). Another essential gene expressed in macrophages is the SpiC, which we found to be the most downregulated gene in the high- and medium dose groups at 6.15-fold and 7.19-fold, respectively. SpiC plays a role in the downregulation of pro-inflammatory cytokines while promoting iron efflux by regulating ferroportin expression in activated macrophages (65).
CD163, a hemoglobin-specific receptor found on the cell surface of macrophages, is implicated in iron recycling and inflammatory response (66, 67), was downregulated. In contrast, RSG16, which restricts the pro-inflammatory response of monocytes (68), was significantly upregulated by over 4-fold in the medium and high dose groups. Expression of Prostaglandin E synthase (PGES) was upregulated in the medium and high dose groups. PGES plays a crucial role in inflammation by converting prostaglandin (PG) H2 to PGE2 (69). The matrix metalloproteinase (MMP) family catalyze proteolytic activities that result in the breakdown of the extracellular matrix (70). MMPs thus play a key role in tumor invasion, neoangiogenesis, and metastasis (70). The matrix metallopeptidase 17 (MMP17; or MT4-MMP) was highly upregulated (4.1-fold) in the low dose group compared to the high dose (1.84-fold). MMP8 was upregulated in the medium and high dose groups (5.96- and 3.87-fold, respectively).
Table 2
List of select pig host genes detected in ASFV-infected pigs. Genes were selected based on function and mean fold change for three dose groups. The highest expressed fold change value between medium and high dose groups was recorded.
Gene | log2 Fold Change | adjusted p-value | Gene Product | Function | Reference |
Genes linked with monocyte macrophages |
CD163 | -1.85 | 1.76E-02 | CD163 antigen | A hemoglobin-specific receptor is found on the cell surface of macrophages involved in iron recycling and inflammatory response. | (66,67) |
RGS16 | 4.18 | 9.93E-19 | Regulator of G Protein Signaling 16 | Restricts pro-inflammatory response of monocytes | (68) |
HOX-1 | -2.13 | 2.06E-05 | Heme oxygenase-1 | Pro-oxidant and pro-inflammatory effects | (71,72) |
S100A8 | 7.03 | 3.01E-09 | Calcyclin | Binds CD68 | (73) |
CSF3R | 2.11 | 1.29E-02 | Granulocyte colony-stimulating factor (G-CSF) | Involved in proliferation and differentiation of granulocytes | (74) |
TSP-1 | 3.25 | 6.58E-04 | Thrombospondin 1 | Inhibits angiogenesis, regulates antitumor immunity and regulates extracellular proteases and growth factors. | (75) |
NK/T cell-associated genes |
IFIT2 | 1.87 | 2.62E-04 | Interferon-induced protein with tetratricopeptide repeats 2 | IFN-induced antiviral protein which inhibits expression of viral messenger RNAs that lack 2'-O-methylation of the 5' cap | (76) |
IFITM3 | 3.05 | 2.80E-09 | interferon induced transmembrane protein 3 | Engages and shuttles the virus particles to lysosomes for clearance from the cells | (77) |
Genes associated with ASFV infection |
PPP1R15A | 2.59 | 7.49E-05 | GADD34 | Guides dephosphorylation of eIF2a by PP1 | (63) |
SPIC | -6.87 | 2.02E-09 | Spi-C Transcription Factor | Restrains inflammation and iron metabolism in macrophages | (65) |
NUPR1 | 5.73 | 6.21E-15 | Nuclear protein 1 | Suppresses programmed cell death by apoptosis and programmed necrosis | (78) |
Genes associated with lymphocytes (B, T cells and NK cells) |
CD244 | -5.01 | 8.73E-03 | CD244 molecule | Involved in NK cell stimulation and NK cell-mediated cytotoxicity | (59) |
CYFIP2 | -3.05 | 1.06E-12 | Cytoplasmic FMR1 interacting protein 2 | Involved in T-cell adhesion and p53/TP53-dependent induction of apoptosis | (79) |
IL-6 | 5.67 | 1.50E-26 | Interleukin 6 | Promotes virus survival and exacerbation of clinical disease | (80) |
Other genes |
DRAM2 | -2.39 | 9.44E-06 | Damage-regulated autophagy modulator 2 | Oncogenic regulator: promotes cell metastasis and proliferation in cancer cells | (51) |
SOGA1 | 1.41 | 1.47E-03 | Suppressor of glucose, autophagy associated 1 | Implicated in autophagy | (50,81) |
TIMP1 | 5.27 | 2.17E-12 | Tissue inhibitor matrix metallopeptidase inhibitor 1 | Promotes tumorigenesis and metastasis of colon cancer and is a potential prognostic indicator for colon cancer | (82) |
LTF | 5.68 | 3.39E-08 | Lactotransferrin | Sequestering iron and antimicrobial activity | (83) |
CHP2 | -5.85 | 7.17E-05 | Calcineurin like EF-hand protein 2 | Regulates cell pH by controlling plasma membrane-type Na+/H + exchange activity; involved in carcinoma progression | (84,85) |
FOSL1 | 7.55 | 2.94E-11 | FOS like 1, AP-1 transcription factor subunit | Regulators of cell proliferation, differentiation, and transformation | (86) |
FOXF1 | 2.37 | 3.23E-04 | Forkhead box F1 | FOXF1 transcription factor promotes lung regeneration | (87) |
PG-4 | 7.11 | 2.84E-07 | Protegrin-4 | Antimicrobial peptide | (62) |
PGES-1 | 4.22 | 1.02E-04 | Prostaglandin E synthase | Plays a crucial role in inflammation by converting prostaglandin (PG) H2 to PGE2 | (88) |
Nine cytokines showed significant differential expression from the high and medium dose groups (Table 3), eight of these were upregulated, and one was down-regulated. Interleukin-6 (IL-6), VEGFA and IL27 were among the most upregulated genes by 5.67-, 3.81- and 2.2-fold, respectively. Seventeen cytokine receptors were differentially expressed, with IL1RL1 being the most highly differentially expressed (5-fold), followed by TNFRSF11A (2.67-fold). TNFRSF9 (TNF receptor superfamily member 9), also known as 4-1BBL or CD137, was most down-regulated by up to 3.11-fold in the medium dose. The interleukin cytokines IL6, IL27, and IL17B, were upregulated in the medium and high dose groups resulting in hemorrhagic fever by cytokine storm. There was an up-regulation of the proinflammatory cytokines involved in the apoptotic processes such as TNF (1.5-fold change), members of the TWEAK family such as TNFSF12 (1.53-fold change), and TNFSF13 (1.63-fold change). The IFN-ω has cross-species antiviral activities and was significantly downregulated in the high dose group (1.62-fold change). The immune-suppressive cytokine, IL27, was significantly upregulated in the high dose (2.2-fold) and medium dose groups (2.61-fold). We detected upregulation of the Interleukin-1 receptor (IL-1R) in the medium and high dose groups by over 5-fold in the high dose group and 4.01-fold in the medium dose group. We detected expression of interleukin-1 receptor-associated kinases (IRAKs) in the high dose group only where IRAK1 and IRAK3 were downregulated while expression of IRAK4 was upregulated (Table 3).
Table 3
Differentially expressed cytokines and their receptors. TND = transcript not detected.
Gene group | Gene | Description | Differential expression in High dose | Differential expression in medium dose | Differential expression in Low dose |
Log2 Fold change | Adjusted p-value | Log2 Fold change | Adjusted p-value | Log2 Fold change | Adjusted p-value |
Cytokines | IL6 | Interleukin 6 | 5.67 | 1.50E-26 | 4.01 | 4.76E-11 | TND | TND |
IL17B | Interleukin 17B | 1.56 | 2.94E-02 | TND | TND | TND | TND |
IL16 | Interleukin 16 | TND | TND | -2.08 | 4.17E-03 | TND | TND |
IL27 | Interleukin 27 | 2.20 | 4.28E-03 | 2.61 | 2.87E-03 | TND | TND |
IFN-ω | Interferon omega 6 | -1.62 | 3.22E-02 | TND | TND | TND | TND |
TNFSF12 | TNF superfamily member 12 | 1.53 | 1.01E-04 | 1.66 | 6.77E-05 | TND | TND |
TNFSF13 | TNF superfamily member 13 | TND | TND | 1.63 | 3.63E-03 | TND | TND |
HBEGF | Heparin binding EGF like growth factor | 1.83 | 4.07E-03 | 1.76 | 9.45E-03 | TND | TND |
VEGFA | Vascular endothelial growth factor A | 3.81 | 1.66E-13 | 2.83 | 1.12E-06 | TND | TND |
Cytokine receptor families | IL1RL1 | Interleukin 1 receptor like 1 | 5.00 | 1.11E-09 | 4.62 | 2.58E-07 | TND | TND |
IL10RA | Interleukin 10 receptor subunit alpha | -1.32 | 1.64E-03 | -2.27 | 1.49E-06 | TND | TND |
IL15RA | interleukin 15 receptor subunit alpha | 1.54 | 4.86E-02 | TND | TND | TND | TND |
IL17RE | Interleukin 17 receptor E like | 1.64 | 2.14E-02 | TND | TND | TND | TND |
IL21R | Interleukin 21 receptor | -2.61 | 1.44E-06 | -2.78 | 3.62E-05 | TND | TND |
IL27RA | Interleukin 27 receptor subunit alpha | TND | TND | -1.48 | 4.36E-03 | TND | TND |
IL31RA | Interleukin 31 receptor A | -2.31 | 3.59E-03 | -2.75 | 5.42E-03 | TND | TND |
TNFRSF11A | TNF receptor superfamily member 11a | -2.67 | 5.45E-04 | -2.05 | 1.24E-02 | TND | TND |
TRAF7 | TNF receptor associated factor 7 | 1.59 | 4.75E-05 | TND | TND | TND | TND |
TNFRSF13B | TNF receptor superfamily member 13B | 2.34 | 8.13E-07 | TND | TND | TND | TND |
TNFRSF18 | TNF receptor superfamily member 18 | 1.99 | 3.68E-03 | TND | TND | TND | TND |
TNFRSF9 | TNF receptor superfamily member 9 | -1.70 | 3.90E-02 | -3.16 | 9.69E-03 | TND | TND |
TNFRSF21 | TNF receptor superfamily member 21 | -1.92 | 1.19E-03 | -1.42 | 2.09E-02 | TND | TND |
TNFRSF13C | TNF receptor superfamily member 13C | TND | TND | -2.33 | 7.88E-03 | TND | TND |
IRAK1 | Interleukin 1 receptor associated kinase 1 | 1.73 | 2.26E-06 | TND | TND | TND | TND |
IRAK3 | Interleukin 1 receptor associated kinase 3 | -0.87 | 3.75E-02 | TND | TND | TND | TND |
IRAK4 | Interleukin 1 receptor associated kinase 4 | -1.22 | 1.67E-02 | TND | TND | TND | TND |
In the spleen, chemokines have an essential role in modulating adaptive immune response by promoting the initial priming of lymphocytes and guiding their differentiation and phenotype. There were nine differentially expressed chemokines (Table 4). The C-C ligand 2 (CCL2) and C-X-C motif chemokine ligand 2 (CXCL2), CXCL10, CCL23, CCL4, and CXCL8 were significantly upregulated in the medium dose group, while in the high dose group, CCL2, CCL4, CXCL2 and CXCL10 were significantly upregulated. CCL21, CCL26 and CXCL13 were significantly downregulated in both the high and medium dose groups (Table 4). CCL4 and CXCL10, the chemo-attractants for immune response, were upregulated in the medium and high dose groups, while CXCL10 was downregulated in the low dose group. CCL2 was the most upregulated chemokine (4.03- and 4.11-fold), while CCL26 and CXCL13 were the most downregulated in the high (5.28- and 2.5-fold) and CCL21 and CCL26 in the medium dose groups (4.30- and 4.17-fold). CXCL2 and CXCL8 are involved in recruiting neutrophils. The ELR+ (glutamic acid – leucine – arginine) CXC chemokines CXCL2 were significantly upregulated in the medium (2.68-fold) and high (3.44-fold) dose groups. CXCR2 signaling is essential for neutrophil release from the bone marrow into the blood.
Table 4
Differentially expressed chemokines and their chemotactic activities. TND = transcript not detected.
Chemokine | Key immune function | Differential expression in High dose | Differential expression in medium dose | Differential expression in Low dose | Chemotactic activity |
log2FoldChange | padj | log2FoldChange | padj | log2FoldChange | padj |
CCL2 | Inflammatory monocyte trafficking | 4.03 | 3.76E-10 | 4.11 | 1.12E-08 | TND | TND | Classical monocyte, Natural killer cells |
CCL4 | Macrophage and natural killer cell migration; T cell–dendritic cell interactions | 1.69 | 2.30E-02 | TND | TND | TND | TND | Nonclassical-Monocyte, Natural killer cells |
CCL21 | T cell and dendritic cell homing to lymph node | TND | TND | -4.30 | 6.14E-04 | | | Neutrophils, CD8 + T cells, dendritic cells |
CCL26 | Eosinophil and basophil migration | -5.28 | 2.51E-04 | -4.17 | 3.45E-03 | TND | TND | Th2 type T lymphocytes |
CXCL2 | Neutrophil trafficking | 3.44 | 9.55E-06 | 2.68 | 1.70E-03 | TND | TND | Neutrophils, CD8 + T cells, monocytes/macrophage, natural killer cells |
CXCL8 | Neutrophil trafficking | TND | TND | 2.42 | 1.22E-02 | TND | TND | Neutrophils, CD8 + effector T cells, monocytes/macrophage, natural killer cells |
CXCL10 | Th1 response; Th1, CD8, NK trafficking | 1.62 | 8.29E-03 | 2.85 | 4.93E-05 | -2.56 | 1.61E-03 | CD8 + T cells, T helper cells (Th1), natural killer cells |
CXCL13 | B cell and follicular helper (Tfh) cell positioning in lymph node | -2.52 | 2.35E-03 | -1.67 | 4.13E-02 | TND | TND | B cells |
Antigen processing and presenting cells were downregulated in the medium and high dose groups. The differentially expressed genes involved in MHC antigen processing and presentation are listed in Table 5. The expression of SLA-DMB, SLA-DQA, SLA-DRA, SLA-DRB, and SLA-DOB were down-regulated in the medium and high dose groups. The cathepsin S was downregulated among the medium and high dose groups, compromising the antigen presentation by MHC class II molecules. Cathepsin S (CTSS) gene is involved in processing antigens before loading to MHC class II and was significantly downregulated (2.10-fold change). The proteasome activators (PSMC, PSMD, PSME, PSMF) were upregulated in the medium and low dose groups.
Table 5
Differentially expressed genes involved in MHC antigen processing and presentation. TND = transcript not detected.
Gene | Description | Differential expression in High dose | Differential expression in medium dose | Differential expression in Low dose |
log2Fold Change | padj | log2Fold Change | padj | log2Fold Change | padj |
SLA-DQB1 | SLA-DQ beta1 domain | -1.36 | 4.51E-03 | -1.64 | 9.01E-04 | TND | TND |
SLA-DQA | MHC class II histocompatibility antigen SLA-DQA | -1.85 | 8.57E-04 | -1.19 | 3.97E-02 | TND | TND |
SLA-DRA | MHC class II DR-alpha | -1.62 | 5.34E-03 | TND | TND | TND | TND |
SLA-DRB1 | MHC class II histocompatibility antigen SLA-DRB1 | TND | TND | -1.20 | 4.72E-02 | TND | TND |
SLA-DOB | MHC class II, DO beta | TND | TND | -2.37 | 2.33E-03 | TND | TND |
SLA-DMB | MHC class II, DM beta | -1.67 | 1.84E-03 | -1.27 | 2.28E-02 | TND | TND |
SEL1L3 | SEL1L family member 3 | 1.59 | 4.11E-02 | TND | TND | TND | TND |
SEL1L | SEL1L adaptor subunit of ERAD E3 ubiquitin ligase | -2.13 | 1.43E-05 | -1.47 | 5.07E-03 | TND | TND |
PSME4 | Proteasome activator subunit 4 | -0.96 | 7.83E-03 | TND | TND | TND | TND |
PSMD4 | Proteasome 26S subunit, non-ATPase 4 | TND | TND | 1.29 | 3.23E-03 | TND | TND |
PSMD3 | Proteasome 26S subunit, non-ATPase 3 | 1.32 | 4.70E-03 | TND | TND | TND | TND |
PSMC5 | Proteasome 26S subunit, ATPase 5 | TND | TND | 0.89 | 2.78E-02 | TND | TND |
PSMC3 | Proteasome 26S subunit, ATPase 3 | TND | TND | 1.16 | 3.35E-02 | TND | TND |
PSMC1 | Proteasome 26S subunit, ATPase 1 | TND | TND | 1.10 | 2.25E-02 | TND | TND |
PMSF1 | Proteasome inhibitor subunit 1 | 0.98 | 3.08E-02 | 1.10 | 1.61E-02 | TND | TND |
CTSS | Cathepsin S | -2.10 | 5.96E-06 | -1.88 | 2.60E-04 | TND | TND |
ADRM1 | ADRM1 26S proteasome ubiquitin receptor | 1.20 | 1.60E-02 | TND | TND | TND | TND |
In total, we detected 10 autophagy-related genes in the medium and high dose study groups and only one was detected in the low dose group. Seven of these were downregulated, namely ATG4C, DRAM2, DCT, EPG5, APAF1, NBR1 and FUNDC1 (Table 6). Autophagy-associated cell death is inhibited by the nuclear protein 1 (NUPR1), a transcriptional regulator gene that was significantly upregulated in the high and medium dose groups by over 5-fold (Tables 2 and 6). In the high and medium dose groups, we also detected the upregulation of pro-apoptosis and an autophagy inducer gene, BCL2 interacting protein 3 (BNIP3). Twelve (12) other autophagy and cell death regulating genes were detected, of which 10 were upregulated, including FAIM2, GAS2L1, GAS2L2, GAS8, MAD1L1, MAD1L2, GADD45G, GADD45B, GAS7, and NUPR1. Five of these were significantly upregulated (FAIM2, GAS2L2, GAS8, MAD1L1, and GADD45G). GAS2L3 (Growth arrest-specific 2 like 3) and APAF1 were downregulated, with GAS2L3 being significantly downregulated (Table 6).
Table 6
Differentially expressed genes associated with autophagy. TND = transcript not detected.
Autophagy-related Genes | Gene | Description | Differential expression in High dose | Differential expression in medium dose | Differential expression in Low dose |
log2 Fold Change | padj | log2 Fold Change | padj | log2 Fold Change | Padj |
ATG4C | Autophagy related 4C cysteine peptidase | -1.39 | 1.64E-02 | TND | TND | TND | TND |
ATG4D | Autophagy related 4D cysteine peptidase | 1.10 | 4.64E-02 | TND | TND | TND | TND |
BNIP3 | BCL2 interacting protein 3 | 3.09 | 2.93E-09 | 2.53 | 1.72E-05 | TND | TND |
DCT | Dopachrome tautomerase | TND | TND | -1.94 | 4.41E-02 | TND | TND |
DRAM2 | DNA damage regulated autophagy modulator 2 | -2.39 | 9.44E-06 | -1.58 | 5.40E-03 | TND | TND |
EPG5 | Ectopic P-granules autophagy protein 5 homolog | TND | TND | -1.38 | 2.98E-02 | TND | TND |
FXR2 | FMR1 autosomal homolog 2 | TND | TND | 1.01 | 1.50E-02 | TND | TND |
NBR1 | NBR1 autophagy cargo receptor | -1.25 | 1.41E-03 | TND | TND | TND | TND |
SOGA1 | Suppressor of glucose, autophagy associated 1 | 1.41 | 1.47E-03 | TND | TND | TND | TND |
SSNA1 | SS nuclear autoantigen 1 | 1.23 | 2.04E-02 | 1.13 | 3.81E-02 | TND | TND |
FUNDC1 | FUN14 domain containing 1 | TND | TND | TND | TND | -0.96 | 3.69E-02 |
Autophagy and cell death regulating genes | GAS2L2 | Growth arrest-specific 2 like 2 | 4.93 | 1.26E-06 | TND | TND | TND | TND |
GAS2L1 | Growth arrest specific 2 like 1 | 1.31 | 4.10E-02 | 1.39 | 2.98E-02 | TND | TND |
GAS2L3 | Growth arrest specific 2 like 3 | TND | TND | -2.32 | 2.45E-02 | TND | TND |
GAS7 | Growth arrest specific 7 | 1.86 | 5.02E-05 | TND | TND | TND | TND |
GAS8 | Growth arrest specific 8 | 3.74 | 2.60E-15 | 1.52 | 4.38E-03 | TND | TND |
MAD1L1 | Mitotic arrest deficient 1 like 1 | 3.30 | 2.53E-08 | TND | TND | TND | TND |
MAD1L2 | Mitotic arrest deficient 1 like 2 | 1.64 | 9.99E-03 | TND | TND | TND | TND |
GADD45G | Growth arrest and DNA damage inducible gamma | 2.25 | 1.39E-02 | 2.11 | 3.35E-02 | TND | TND |
GADD45B | Growth arrest and DNA damage inducible beta | 1.73 | 1.33E-02 | 2.00 | 8.46E-03 | TND | TND |
FAIM2 | Fas apoptotic inhibitory molecule 2 | TND | TND | 3.96 | 2.87E-03 | TND | TND |
NUPR1 | Nuclear protein 1, transcriptional regulator | 5.13 | 8.83E-14 | 5.73 | 6.21E-15 | TND | TND |
APAF1 | Apoptotic peptidase activating factor 1 | -1.49 | 6.29E-03 | TND | TND | TND | TND |
In total, we detected 57 signal transduction and transcription genes required for macrophage activation in the medium and high dose groups (Supplementary Table S3). Twenty-seven of them were upregulated, with 9 being significantly upregulated (> 2-fold change). Of the 30 downregulated signal transduction and transcription genes, nine (USP34, USP44, USP45, USP37, MAP2K6, MAP3K2, IRF4, MEF2C, and MEF2B) were significantly downregulated (> 2-fold change). Two CCAAT enhancer-binding proteins (or CEBPs) were upregulated in the medium and high dose groups, namely CEBPD and CEBPB. TAB3 expression was detected, and five other key immune transcription factors (FOSB, FOSL1, FOSL2, IRF7, JUNB, IRF4, and IRF8) were all upregulated except for IRF4 and IRF8, which were downregulated in the high and medium dose groups (Supplementary Table S3). Also detected was the expression of ten ubiquitin-specific peptidases (USP) and three suppressors of cytokine signaling (SOCS), namely USP1, USP14, USP20, USP24, USP25, USP33, USP34, USP35, USP37, USP4, USP44, USP45, USP47, USP48, USP7, USP9X, SOCS3, SOCS4 and SOCS7. The expression of SOCS3, SOCS7, USP14, USP20, and USP35 was upregulated, while the rest were downregulated in the high dose group.
In total, we detected the expression of 20 mitogen-activated protein kinase (MAPK), namely MAP2K3, MAP2K4, MAP2K6, MAP3K1, MAP3K10, MAP3K14, MAP3K2, MAP3K5, MAP3K6, MAP3K9, MAP4K2, MAP4K4, MAPK1, MAPK6, MAPK7, MAPK8, MAPK8IP3, MAPK9, MAPKAP1, and MAPKAPK2. Of these, MAPK7, MAP3K6, MAP3K10, MAP4K4, MAP2K3, MAP3K14, MAP3K9, MAPK8IP3, MAP3K5 and MAPKAPK2 were upregulated, while MAP3K1, MAP2K4, MAPK1, MAPK6, MAPK8, MAPK9, MAP3K2, MAP2K6, MAP4K2, and MAPKAP1 were downregulated. MAPK cascades are signaling pathways that regulate cellular processes, such as proliferation, differentiation, apoptosis and stress responses crucial for cancer development and progression (89).
Small nucleolar RNAs (SnoRNAs) exhibit oncogenic and tumor-suppressive actions vital in lung cancer tumorigenesis and progression by participating in the invasion of growth suppressors and cell death, activation of invasion and metastasis angiogenesis, and continued proliferative signaling (90). In this study, we detected differential expression of several small SnoRNAs in all three study groups at 10.32- and − 2.54-fold across the groups. 42, 33, and 30 SnoRNAs were differentially expressed in the high, medium and low dose groups, respectively. The downregulated SnoRNAs were 2 in the high dose group and 3 in the low dose group. The small nucleolar RNA, C/D box 45A, was downregulated in both the medium and high dose groups.
2.4 ASFV gene expression in infected spleen tissues
In total, we detected the expression of 172 (high dose), 175 (in medium dose), and 167 (low dose) known ASFV genes and multigene families (MGFs) in the spleen samples analyzed from the locally-adapted pigs (Table 1). The complete list of ASFV genes detected is found in Supplementary Table S4. A total of 44 known MGFs were detected in reference to the Georgia/2007 ASFV genome Supplementary Table S5. We observed a wide variation in the gene count in Transcripts per Million (TPM) of the expressed genes in the different pigs studied, probably due to variations in the time the pigs were euthanized and variations in the number of infected macrophages at the time of euthanasia. In ASFV, higher gene counts of MGFs were detected in pigs from the high- and medium dose groups (Supplementary Table S5). The uncharacterized protein (C84L), viral DNA polymerase (G1211R), polyprotein pp220 (CP2475L), and a hypothetical protein, ASFV_G_ACD_00190, were the top four genes in the low dose group with 204, 123, 109, and 101 TPM being detected. In the medium dose group, MGF_100-1L, Uncharacterized protein (E184L), and the structural protein p72 (B646L) were expressed in 2097, 1533, and 1271 TPM, respectively. The top 10 genes with the highest counts in TPM were MGF 100-1L, E184L, B646L, B385R, I196L, MGF 360-4L, NP1450L, MGF 360-1La, I215L, and K145R (Table 7). The gene L83L interacts with the host IL-1R and was detected in high dose (13 TPM), medium dose (45 TPM) and low dose (6 TPM) [Supplementary Table S4]. E184L, B646L, and MGF_100-1L were expressed at 1004, 1001, and 862 TPM in the high dose group. The top three genes in the medium and high dose were the same, with differences in the number of TPM.
Table 7
Top 20 ASFV genes by counts in TPM
Gene | Gene counts (TPM) High dose | Gene counts (TPM) Medium dose | Gene counts (TPM) Low dose |
A151R | 265 | 653 | 4 |
B354L | 305 | 631 | 81 |
B385R | 436 | 1185 | 18 |
B646L | 1001 | 1271 | 18 |
C962R | 395 | 701 | 47 |
CP312R | 341 | 623 | 15 |
E184L | 1004 | 1533 | 18 |
E301R | 313 | 719 | 10 |
H339R | 259 | 588 | 16 |
I196L | 516 | 1155 | 10 |
I215L | 369 | 781 | 73 |
K145R | 434 | 735 | 43 |
M448R | 260 | 651 | 21 |
MGF 100-1L | 862 | 2097 | 20 |
MGF 360-15R | 286 | 563 | 34 |
MGF 360-1La | 297 | 864 | 5 |
MGF 360-21R | 229 | 696 | 21 |
MGF 360-4L | 376 | 989 | 21 |
MGF 360-6L | 226 | 689 | 40 |
NP1450L | 648 | 917 | 53 |
MGFs demonstrate divergence in sequence, indicating they have evolved over long periods and thus offer a selective advantage to the virus (91). This study detected several MGFs and the top 10 MGFs detected were MGF_100-1L, MGF_360-4L, MGF_360-1La, MGF_360-6L, MGF_360-21R, MGF_360-15R, MGF_100-3L, MGF_505-4R, MGF_505-1R and MGF_110-7L (Supplementary Table S5). These MGFs were detected at very low gene counts (in TPM) in the low dose group (Supplementary Table S5; Fig. 4) compared to the medium and high dose groups (Fig. 4A). The highest gene counts in the low dose group were for MGF 505-4R (62 TPM), MGF 360-6L (40 TPM), and MGF 360-15R (34 TPM). The highest gene counts in the high and medium dose groups were for MGF 100-1L (862, 2097 TPM), MGF 360-4L (376,2097 TPM), and MGF_360-1La (297, 864 TPM).
We then selected genes with a 2-fold increase in gene expression relative to the control genes in the control animal (Table 8). The highest mean expression levels were detected in E184L, MGF 100-1L and NP1450L, all in the high and medium dose groups. MGF 100-1L was recently shown to be highly expressed in ASF surviving pigs (92). We detected ASFV structural, non-structural, and host regulatory genes and genes of unknown function (Supplementary Table S3). We detected MGF 360–15R (A276R) expression that blocks early innate immune responses by inhibiting the induction of IFN-β (93). Also detected were ASFV structural genes such as CP204L, which encodes p30, an immunodominant phosphoprotein of the virion and a preferred target for ASFV serological detection of infection. Another structural protein also detected is P72 (B646L), a major capsid protein involved in virus entry and a major molecular marker for distinguishing and genotyping ASFV isolates. Additionally, the structural protein P54 (E183L) was also expressed. P54 binds the LC8 chain of dynein, and is involved in virus entry, and is also required to recruit envelope precursors. We detected the other structural genes, such as KP177R (p22), A04R (histone-like), A151R (pA151R), and EP402R, which are similar to the pig host CD2 protein that is required to bind the host red blood cells to infected cells and extracellular particles resulting in haemadsorption to infected cells (16). EP402R had fewer gene counts in the low dose group (36 TPM) compared to the medium (234 TPM) and high dose (129) groups. The expression of ASFV A238L was repressed in the high and medium dose groups compared to the low dose groups with gene counts of 18, 2, and 0 TPM, respectively. Another ASFV gene critical in host-pathogen interaction is A224L, an apoptosis inhibitor, which was detected at high amounts in the high and medium dose groups (172 and 303 TPM, respectively) compared to the low dose groups (13 TPM); however, it was not differentially expressed (Supplementary Table S4).
Additional genes differentially expressed were those that code for immunodominant ASFV proteins, namely E184L, CP312R, K205R, and K145R (45). Other genes detected in higher counts in the medium and high dose groups are associated with late ASFV Infection, such as NP1450L and EP1242L (94). Another late ASFV infection gene detected was S273R, which codes for SUMO-1-specific proteases, that cleave the viral polyproteins pp62 and pp220 (95). S273R was detected at very low gene counts (< 20 TPM) in all the study pigs (Supplementary Table S4).
Table 8
Differentially expressed ASFV genes in high and medium dose groups. TND = transcript not detected.
Gene | Differential expression in High dose | Differential expression in medium dose | Differential expression in Low dose |
log2FoldChange | Padj | log2FoldChange | padj | log2FoldChange | padj |
B385R | 2.29 | 4.97E-02 | 2.49 | 4.25E-02 | TND | TND |
B646L | 2.21 | 4.89E-02 | 2.00 | 4.47E-02 | TND | TND |
B962L | 2.28 | 4.97E-02 | 2.14 | 4.80E-02 | TND | TND |
C84L | -2.30 | 4.43E-02 | -2.62 | 2.45E-02 | TND | TND |
C962R | 2.38 | 4.97E-02 | 2.65 | 4.25E-02 | TND | TND |
CP312R | 2.29 | 4.97E-02 | 2.41 | 4.44E-02 | TND | TND |
E184L | 3.19 | 4.43E-02 | 3.24 | 2.45E-02 | TND | TND |
E301R | 2.22 | 4.97E-02 | 2.47 | 4.25E-02 | TND | TND |
EP1242L | 2.27 | 4.97E-02 | 2.17 | 4.80E-02 | TND | TND |
G1211R | -1.71 | 4.97E-02 | TND | TND | TND | TND |
H339R | TND | TND | 2.37 | 4.44E-02 | TND | TND |
I196L | 2.53 | 4.97E-02 | 2.66 | 4.25E-02 | TND | TND |
K145R | 2.48 | 4.97E-02 | 2.48 | 4.25E-02 | TND | TND |
K205R | TND | TND | 2.18 | 4.80E-02 | TND | TND |
K78R | 2.64 | 4.89E-02 | TND | TND | TND | TND |
MGF 100-1L | 2.98 | 4.43E-02 | 3.14 | 2.45E-02 | TND | TND |
MGF 360-15R | TND | TND | 2.18 | 4.80E-02 | TND | TND |
MGF 360-1La | TND | TND | 2.24 | 4.80E-02 | TND | TND |
MGF 360-4L | 2.27 | 4.97E-02 | 2.54 | 4.25E-02 | TND | TND |
MGF 360-6L | TND | TND | 2.14 | 4.80E-02 | TND | TND |
NP1450L | 2.81 | 4.89E-02 | 2.72 | 4.25E-02 | TND | TND |
2.5 Overlaps in differential gene expression in the spleen
Of the 4,954 DEGs, overlaps in differential gene expression between the low-, medium-, and high dose groups were detected. In the pig host, 992 (20%) genes were shared between medium and high dose groups, and 22 (< 1%) genes were shared between high and low dose groups (Fig. 4B; Supplementary Table S7). A total of 63 (1%) DEGs were shared between the three study groups. Some of the uniquely expressed genes in the pig host in the high dose group were CCL4, Calnexin, GAS2L2, IFN-ω, IRAK1, IRAK3, IRAK4, MAD1L1, MAD1L2, NBR1, SLA-DRA, ATG4C, ATGD and SOGA1 (Fig. 4A). In the medium dose group, CCL21, CCL26, CXCL8, FAIM2, GAS2L3, SLA-DRB1, SLA-DOB, and TNFSF13 were among the uniquely expressed genes. While in the low dose group, the expression of CD164, FUNDC1, HIG1A, SNORA12, and SNORD49A were unique to this study group. In the ASFV pathogen, 14 DEGs were shared between the medium and high dose groups (Fig. 4C). Two genes were unique to the high dose (K78R and G1211R), while the medium dose group had 5 unique ASFV DEGs (MGF 360-1La, MGF 360-6L, MGF 360-15R, K205R, and H339R).
2.6 Mapping pig genes to KEGG pathways
The pig and human gene atlases were used to link host gene expression with cells and tissues. The pathways containing the most significant number of genes represented were functionally characterized using the DAVID gene enrichment tool to report KEGG pathways. The medium dose group had the highest number of significantly enriched pathways (n = 11; Table 9). The pathways with the highest gene counts were linked to immune response functions primarily associated with the host immune response to viral infection. In the medium dose group, pathway hsa04060, cytokine-cytokine receptor interaction, had the highest number of highly upregulated genes (n = 24), and the upregulated genes were ssc04657 (IL-17 signaling pathway), ssc04061, (viral protein interaction with cytokine and cytokine receptor) and ssc04657 (IL-17 signaling pathway). The above three pathways contained highly upregulated genes that represented the class 1 helical cytokines (IL6, IL27, CSF3 and IL15RA), IL17-like cytokines (IL17B and ILA7RA), the CC- and CXC-subfamily of chemokines (CCL2, CXCL2, CXCL10, CCL23, CCL4, CXCL8, and CXCL13) and TNF family (TWEAK and TNFR1).
Table 9
The KEGG significantly enriched pathways in the three infective doses (high, medium, and low).
| ID | Description | p.adjust | qvalue | Count |
High vs. control | ssc04610 | Complement and coagulation cascades | 8.42E-03 | 8.17E-03 | 12 |
ssc05323 | Rheumatoid arthritis | 2.89E-02 | 2.80E-02 | 12 |
ssc04657 | IL-17 signaling pathway | 3.37E-02 | 3.27E-02 | 12 |
Medium vs. control | ssc04060 | Cytokine-cytokine receptor interaction | 4.12E-04 | 3.81E-04 | 24 |
ssc05323 | Rheumatoid arthritis | 4.12E-04 | 3.81E-04 | 14 |
ssc04061 | Viral protein interaction with cytokine and cytokine receptor | 4.12E-04 | 3.81E-04 | 13 |
ssc05340 | Primary immunodeficiency | 1.37E-03 | 1.26E-03 | 9 |
ssc04640 | Hematopoietic cell lineage | 1.40E-03 | 1.29E-03 | 13 |
ssc05144 | Malaria | 1.68E-03 | 1.56E-03 | 10 |
ssc04080 | Neuroactive ligand-receptor interaction | 1.80E-02 | 1.66E-02 | 17 |
ssc04672 | Intestinal immune network for IgA production | 1.80E-02 | 1.66E-02 | 8 |
ssc05142 | Chagas disease | 1.80E-02 | 1.66E-02 | 13 |
ssc04657 | IL-17 signaling pathway | 1.80E-02 | 1.66E-02 | 11 |
ssc04514 | Cell adhesion molecules | 2.28E-02 | 2.11E-02 | 14 |
Low vs. control | ssc04610 | Complement and coagulation cascades | 5.04E-03 | 4.60E-03 | 3 |
ssc05171 | Coronavirus disease - COVID-19 | 7.38E-03 | 6.73E-03 | 4 |
ssc04611 | Platelet activation | 1.38E-02 | 1.26E-02 | 3 |
ssc04613 | Neutrophil extracellular trap formation | 1.38E-02 | 1.26E-02 | 3 |
In the surviving low dose group, four pathways were significantly enriched in which all represented genes were significantly downregulated: ssc04610 (complement and coagulation cascades), ssc05171 (Coronavirus disease, COVID-19), ssc04611 (platelet activation), ssc04613 (neutrophil extracellular trap formation). The following genes were significantly downregulated in the complement and coagulation cascade, platelet activation and neutrophil extracellular trap formation pathway: FGG (fibrinogen gamma chain) plays a crucial role in pathophysiologic processes, such as inflammation and thrombosis (95) was highly downregulated by 5.47-fold. Other genes in the complement and coagulation cascades were FGA (fibrinogen alpha chain) downregulated at 4.96-fold and fibrinogen beta chain suppressed by over 3.7-fold. In the COVID-19 pathway, the CXCL10 chemokines were highly downregulated in the low dose group by over 2.56-fold.
The Hippo signaling pathway that controls animal organ size through cell proliferation and apoptosis regulation, including cell proliferation, apoptosis, and various stress responses (96), was significantly enriched in the medium and high dose groups. The tumor promotor called Hippocalcin 1 (HPCAL1) was differentially expressed by 1.54 and 1.93-fold change, respectively, in the medium and high dose groups, resulting in the observed organomegaly (96) among pigs in the high and medium dose groups following a postmortem. The hypoxia-inducible pathways were also significantly enriched, such as the HIF-1 signaling pathway that mediates adaptive responses to oxygen deprivation (97), typical in ASF infection.
In the low dose group, the enriched pathways were in response to viral carcinogenesis, thermogenesis, antigen processing and presentation, protein synthesis and metabolic activities (Supplementary Figures S2 A, B and C). All the genes in the KEGG enriched terms in the low dose group were downregulated. HIG1 hypoxia inducible domain family member 1A (HIGD1A) gene was downregulated in the surviving low dose group only by 1.02-fold.
2.7 Functional annotation
When comparing the enriched GO terms, we detected 139 terms, of which 115 represented the biological processes (CC), 3 were cellular components (CC), and 21 molecular functions (MF) [Supplementary Table S6]. The GO terms: response to external stimulus (GO:0009605), extracellular region (GO:0005576), cellular response to an organic substance (GO:0071310) had the highest gene counts in the three infective groups studied. The GO terms response to external stimulus (GO:0009605), extracellular region (GO:0005576), cellular response to an organic substance (GO:0071310), defense response (GO:0006952), the biological process involved in interspecies interaction between organisms (GO:0044419), response to external biotic stimulus (GO:0043207), response to other organisms (GO:0051707), and inflammatory response (GO:0006954) were detected in the high and medium dose groups only. In the low dose, there were less than 5 gene counts represented in these GO terms: extracellular region (GO:0005576), signaling receptor binding (GO:0005102), extracellular space (GO:0005615), cytokine receptor binding (GO:0005126), heme-binding (GO:0020037), hydrogen peroxide metabolic process (GO:0042743), tetrapyrrole binding (GO:0046906). The last three terms were detected only in the low dose group.
The host gene expression within cells and tissues was linked using the pig and human gene atlases. The transcriptome profiles were primarily associated with immunity, consistent with the upregulation of genes in monocytes, macrophages and lymphocytes. In the high dose group, only three molecular mechanisms were significantly enriched for transmembrane signaling receptor activity and molecular transducer activity (Figs. 5A and B). Interestingly, the genes represented here were downregulated (Fig. 5C), including signal transduction and transcription regulatory molecules shown in Supplementary Table S3. The cytokine regulators and signal transduction molecules are critical in stimulating apoptosis and inflammation (26, 37).
The enriched terms were immune responses to infection and biotic stimulus in the medium dose group (Fig. 6). The genes represented by these terms were predominantly upregulated, with a few being downregulated (Fig. 6C). The upregulated genes for the immune response include LTF, CCL3L, TNFS13, IL6, and TGFB3, all over 3-fold, while CD244 was downregulated by 4.7-fold (Fig. 6C).
Another significantly activated gene in the low dose group was CD164 (endolyn), which encodes a transmembrane sialomucin. This cell adhesion molecule regulates the proliferation, adhesion, and migration of hematopoietic progenitor cells and is also a significant contributor to tumorigenesis in normal human cells (55). The encoded protein by CD164 also interacts with the C-X-C chemokine receptor type 4 and may regulate muscle development (98). CD164 is enhanced by the FOXK2 gene (also known as interleukin enhancer-binding Factor 1) transcriptional regulator involved in glucose metabolism, aerobic glycolysis, and autophagy. When all GO terms were considered, we detected suppression of a set of genes involved in protein translation, phosphatase activity, and replication peptide metabolic process (Fig. 7).