Transcriptome Profiling Reveals That the African Swine Fever Virus C315R Exploits the IL-6 STAT3 Signaling Axis to Facilitate Virus Replication

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  Figure 1. Sample data pattern analysis. (A) 3D4/21 cells were transfected with 2 μg of either pcDNA3.1-HA (NC) or pcDNA3.1-HA-C315R (C315R) for 24 h. Successful expression of C315R was confirmed by Western blotting. (B) Sample-to-sample distance analysis. The correlation between samples was assessed by listing the identified genes and their respective quantities. A deeper color indicates a high correlation with minimal distance between the samples, while a lighter color reflects a low correlation with greater distance. (C) Repeatability scatter plot. The degree of correlation between samples is represented by the proximity of points to the diagonal line with fewer deviations indicating higher correlation.

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  Figure 2. Changes in differentially expressed genes (DEGs) between cells transfected with the plasmid expressing HA-tagged C315R or the empty vector. (A) The total number of differentially expressed genes (DEGs) identified in the comparison between NC and C315R groups. (B) Volcano plot depicting global DEGs between the NC and C315R samples. Significantly upregulated genes are shown in blue, while downregulated genes are shown in red. (C) Heatmap clustering of all DEGs across samples, illustrating the overall expression patterns. Pink and blue colors indicate up- and down- regulation in Log2 cpm values, respectively. Significantly upregulated genes are shown in pink, while downregulated genes are shown in blue. Each column in the graph represents a sample, each row represents a gene, and the expression of genes in different samples is represented by different colors, with pinker colors indicating higher expression and bluer colors indicating lower expressio. (D) Heatmap clustering of the top 25 DEGs, highlighting the most significantly altered genes between the NC and C315R groups. Pink and yellow colors indicate up- and down- regulation in Log2 cpm values, respectively. Significantly…

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  Figure 3. GO, KEGG, and Reactome enrichment of DEGs. (A) Bar chart depicting the Gene Ontology (GO) term enrichment of differentially expressed genes (DEGs), categorized into Biological Processes (BPs), Molecular Functions (MFs), and Cellular Components (CCs). (B) Bar chart showing the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of DEGs, highlighting significant pathways involved in cellular processes. JAK STAT related factors were significantly enriched. (C) Bar chart illustrating Reactome pathway enrichment of DEGs, emphasizing key signaling and regulatory pathways associated with the DEGs. IL-6 related factors were significantly enriched.

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  Figure 4. Protein–protein interaction (PPI) network of significant DEGs. The PPI network was constructed using the STRING database to visualize interactions among differentially expressed genes (DEGs) with |Log2FC| ≥ 2 and high confidence (0.70). The network highlights key hub genes, including CDC20, CDCA8, H2AC6, HSPA5, IL-6, CCL2, UBB, and CENPA, which are implicated in cellular responses to chemical stimuli, the regulation of transcription, and DNA damage response, underscoring their potential roles in mediating C315R’s effects on host cell processes, IL-6 and H2AC6 also play important roles in the protein protein interaction network.

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  Figure 5. Validation of gene expression differences. (A) RT-qPCR validation of significantly upregulated differentially expressed genes (DEGs) between the NC and C315R groups. (B) RT-qPCR validation of significantly downregulated DEGs between the NC and C315R groups. Data were shown as mean ± SD based on three independent experiments. Statistical significance is denoted by asterisks (* p < 0.05; ** p < 0.01; *** p < 0.001 determined by two-tailed Student’s t-test. ns, no significance). (C) KEGG pathway enrichment chord diagram illustrating the top 10 DEGs and their involvement in relevant signaling pathways. (D) Immunolocalization images of lung and spleen tissues from ASFV-infected or mock-infected pigs, which was stained for H2AC6. The immunostaining was performed using 5% (w/v) DAB as a substrate, and all sections were counterstained with hematoxylin to highlight tissue morphology.

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  Figure 6. ASFV promotes IL-6 expression to activate STAT3 phosphorylation by C315R. (A) RT-PCR analysis of IL-6 mRNA expression in 3D4/21 cells infected with ASFV of different MOIs at different time points post infection, calculated using the 2−ΔΔCT method. The data were analyzed using GraphPad Prism 8.0.2 software. The data were shown as mean ± SD based on three independent experiments. Statistical significance is denoted by asterisks (* p < 0.05; ** p < 0.01; **** p < 0.0001 determined by two-tailed Student’s t-test. ns, no significance). (B) Immunoblotting of whole cell lysates from 3D4/21 cells infected with ASFV at different time points and MOIs, probing for pSTAT3 (Ser727), STAT3, IL-6, p30, p72, and the cytosolic markers α-β-tubulin. (C) Confocal microscopy of 3D4/21 cells infected with ASFV at 24 hpi, showing colocalization of p30 (red) and pSTAT3 (Ser727) (green). Nuclei were counterstained with DAPI (blue). Scale bar: 25 μm. (D) Fluorescence intensity analysis of individual cells in C. (E) RT-PCR analysis of IL-6 expression in 3D4/21 cells transfected with either the empty vector or pcDNA3.1-HA-C315R, calculated by the 2−ΔΔCT method. (F) Immunoblotting of lysates from 3D4/21…

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  Figure 7. Inhibition of STAT3 phosphorylation reduces ASFV replication and DNA synthesis. (A) Cytotoxicity of Stattic was assessed using the CCK8 assay, which was represented as the average of three independent experiments. The data were analyzed using GraphPad Prism 8.0.2 software. (B) 3D4/21 cells were pretreated with Stattic or DMSO (control) for 4 h, followed by ASFV infection. IL-6 and p30 mRNA levels were analyzed by RT-PCR and calculated using the 2−ΔΔCT method. Data were shown as mean ± SD based on three independent experiments. Statistical significance is denoted by asterisks (* p < 0.05; ** p < 0.01 determined by two-tailed Student’s t-test. ns, no significance). (C) Immunoblotting of 3D4/21 cell lysates treated with Stattic or DMSO for 4 h, probing for pSTAT3 (Ser727), STAT3, p30, and the cytosolic marker β-Tubulin. (D) PAMs pretreated with Stattic and infected with ASFV showed a significant reduction in viral yields from 5.37 to 2.00 HAD50/mL. The data were analyzed using GraphPad Prism 8.0.2 software. (E) 3D4/21 cells pretreated with Stattic or DMSO were infected with GFP-ASFV (green) for 24 h. Fluorescence images were captured using a fluorescence microscope (scale bar: 300…

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  Figure 8. Model of C315R-mediated activation of the IL-6 STAT3 signaling axis facilitating ASFV infection. C315R promotes the transcription of the pro-inflammatory cytokine IL-6, which in turn activates STAT3 phosphorylation (pSTAT3). Phosphorylated STAT3 translocate to the nucleus, where it enhances the transcription of viral genes, thereby supporting ASFV replication.