Identification of cepharanthine as an effective inhibitor of African swine fever virus replication

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  Figure 1.. Screening of antiviral compounds against ASFV in the Selleck natural compounds library. (A) Depiction of the screening process of anti-ASFV compounds. A total of 803 compounds were screened in ASFV infected PAMs (1 MOI) using a cell-based Elisa assay. Two replicates were set for each compound at a concentration of 10 μM, and those with high cytotoxicity were excluded in the second round of screening. (B) The screening results of anti-ASFV compounds. Each dot represents a compound, ranging by infection ratio. (C) The 50% cytotoxicity concentrations (CC50) of candidate anti-ASFV compounds. PAMs (2 × 105 cells/well) were seeded in a 96 well plate overnight, treated with the indicated concentration of DMSO or compounds for 24 h, and then treated with CCK-8 agent for an additional 2 h. The OD450 value was measured for cell viability detection. (D) The Half Maximal Inhibitory Concentration (IC50) of candidate anti-ASFV compounds. PAMs were treated with the indicated concentration of DMSO or compounds for 2 h, followed by a cell-based ELISA assay. Data were calculated through nonlinear regression analysis.

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  Figure 2.. CEP inhibits viral replication during the ASFV entry stage. (A) The chemical structures of CEP. PAMs were pretreated with CEP at the indicated concentration and then infected with or without ASFV (1 MOI). At 24 hpi, the cells were collected for analysis. (B-C) Viral p72 mRNA expression level (B) and intracellular virus titer (C) were measured at 24 hpi in ASFV-infected cells incubated with DMSO or CEP (4 μM). (D) Samples were analyzed by Western blotting assay; ASFV-p72 protein levels were detected with a specific ASFV-p72 monoclonal antibody. (E) PAMs were infected with ASFV (1, 2, or 5 MOI) and then applied using the same method as given in (B). (F) PAMs were treated with DMSO (top panel), 2 μM CEP (middle panel), or 4 μM (bottom panel) CEP for 2 h and then infected with ASFV (1 MOI). After 24 h, the cells were fixed and subjected to an indirect immunofluorescence assay. ASFV-p72 (Green) was detected in each group, and representative images are shown (Scale bar = 50 μm). (G-H) Time-of-addition analysis of CEP against ASFV. (G) Overall scheme for PAMs infection and treatment with CEP (4 μM); the black line refers to the ASFV infection period and the gray line refers to the CEP…

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  Figure 3.. Summary of transcript abundance. (A) Sample treatment process for each group. (B) Principal component analysis (PCA) results comparing 12 transcriptomes from PAMs infected with or without ASFV and treated with or without CEP. (C) Graph showing the number of DEGs between different groups. Up-regulated DEGs are denoted in black and down-regulated DEGs are indicated in gray. (D-E) Transcriptome analysis of ASFV-infected PAMs. (D) Volcano plot of 214 DEGs with q value < 0.05 in the Virus-Mock group comparison. The red color represents upregulated genes in the virus-infected cell with Log2FC > 1 and blue represents downregulated genes with Log2FC < −1. (E) Top upregulated pathways enriched from 182 upregulated genes in the virus-infected cells shown in the bubble plot. The bubble color scales the enrichment score. The size of the bubble scales the count of the enriched genes. The X-axis depicts the enrichment score, while the Y-axis shows the name of the KEGG-enriched pathway.

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  Figure 4.. Transcriptional response of PAMs after CEP treatment. (A-B) Volcano plot (A) and heatmap (B) of a total of 329 DEGs. The red color represents upregulated genes in CEP-treated cells with Log2FC > 1, and the blue color represents downregulated genes with Log2FC < −1. The relative expression in the Z-score mode is shown in the heatmap. Red cells represent upregulated genes, and blue cells represent downregulated genes. (C-D) GO enrichment analysis of CEP-Mock DEGs. (C) Top upregulated pathways enriched from 110 upregulated genes. (D) Top downregulated pathways enriched from 219 downregulated genes in CEP-treated cells are shown in the bubble plot. The bubble color scales the P-value. The size of the bubble scales the count of the enriched genes. The X-axis is equal to the enrichment score, and Y-axis is the name of the GO enriched pathway. (E) Heatmap of pathways of cholesterol biosynthetic process, ATP binding, and innate immune response. Red cells represent upregulated genes; blue cells represent downregulated genes. (F) Network visualization of pathways enriched by 110 upregulated genes in CEP treated cells analysis. (G) Protein-protein network visualization of the gene component.

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  Figure 5.. ASFV infection poorly alters the gene transcription status of CEP-treated PAMs. (A) Venn diagrams show an overlap of DEGs in CEP-Mock and (CEP + Virus)-Mock comparison. (B) Graph depicting the number of DEGs in (CEP + Virus)-CEP. (C-D) Bar diagram displaying the top enrichment results of the same downregulated DEGs (C) and the same upregulated DEGs (D) in CEP-Mock and (CEP + Virus)-Mock comparison. (E) Heatmap showing genes of metabolism of the cholesterol pathway in the Mock, Virus, CEP, and (CEP + Virus) group. Yellow cells represent upregulated genes, while blue cells represent downregulated genes. (F) RT-qPCR showed that the expression of SREBF2, HMGCR, HMGCS1, FASN, and SQLE genes was significantly higher in the CEP-treated group with or without ASFV infection (*P < 0.05, **P < 0.01, ***P < 0.001).

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  Figure 7.. CEP extended the survival time of ASFV-infected pigs in animal challenge experiments. (A) Schematic illustration of the animal experiment. Landrace pigs (42 days old) were randomly assigned into the ASFV-/CEP- group (n = 2), ASFV group (n = 3), and CEP + ASFV group (n = 3). CEP (4.44 mg/kg) or DMSO was injected into the neck muscle at −2, −1, 0, and 1 d. Next, the drug was injected every 48 h before the virus challenge. At 0 d, 50 TCID50 ASFV was inoculated intramuscularly (i.m.) into the ASFV and CEP + ASFV group pigs. The CEP + ASFV group pigs were co-housed with those from the ASFV group from day 0 until the termination of the experiment. (B) Rectal temperatures of animals after inoculation with ASFV. (C) The survival rates of animals after inoculation with ASFV. Statics were calculated using GraphPad Prism 9.0 software. (D) Tissue lesions of pigs in each group. (E) Major organ tissues samples including the lung, heart, and spleen were sectioned (H&E) and histopathologically analyzed (20 μm scale). (F-G) Total nucleic acids were extracted from the tissues or swabs after animal death, and viral loading in the nucleic acids of each sample was detected. (F) Viral loading in the…