Inhibition of African Swine Fever Virus Replication by Porcine Type I and Type II Interferons

FIGURE 1. PoIFN-α and PoIFN-γ inhibit the replication of African swine fever virus (ASFV) in primary alveolar macrophages (PAMs). (A) IFN inhibited virus replication. Primary alveolar macrophages were stimulated with PoIFN-α and PoIFN-γ at the indicated concentrations for 12 h in 24-well plates, and then the cells were infected with ASFV strain SY18 for 1 h at an MOI of 1. After removing the virus, PAMs were further cultured for 72 h prior to sample collection. (B) Indirect immunofluorescence analysis of anti-P30 expression in PAMs. The cells were exposed to a concentration of 1000 U/mL PoIFN-α, PoIFN-γ, or those combined for 12 h prior to infection with ASFV strain 18 for 1 h at an MOI of 1, maintained for 72 h after adsorption without PoIFN-α or PoIFN-γ, washed, and fixed with 4% paraformaldehyde. P30 expression was detected with a rabbit polyclonal antibody and FITC-conjugated AffiniPure Goat Anti-Rabbit IgG (H + L), and the nucleus was stained with DAPI. Scale bars represent 75 μm. (C) The antivial of IFNs against ASFV by immunoblot analysis. The PAM cells were treated with IFNs and then were infected with SY18 and collected for indirect immunofluorescence 48 h after infection. Next…

FIGURE 2. Gene expression in primary alveolar macrophages (PAMs) induced with PoIFN-α and PoIFN-γ. Fresh PAMs were isolated from healthy 35-day-old pigs and cultured in RPMI 1640 medium with 10% FBS and 5% CO2 for 12 h. Then, the cells were treated with PoIFN-α or PoIFN-γ for 12 h. After the 12 h, the cells were harvested and lysed in TRIzol Reagent for RNA extraction. The vertical axis shows the relative transcription level (fold-change) of IFN-stimulated gene (ISG) expression comparing the negative control and treated groups. The fold-changes were measured by the 2–ΔΔCt method, and RNA levels were normalized. Three replicates were performed for q-PCR verification analyses. Bars represent the means ± SDs (n = 3). A non-parametric test was used for the difference significance analysis. Statistically significant differences are indicated (∗P < 0.05; ∗∗∗P < 0.01; ∗∗∗P < 0.001) and the line above the column marks the two groups with differences.

FIGURE 3. African swine fever virus (ASFV) P72 Ct values at the indicated time points in PoIFN-α- and PoIFN-γ-treated surviving pigs after ASFV infection. Groups included PBS (Untreated), low-dose PoIFN-α and PoIFN-γ combined (LDI, 1 × 106 U/kg), and high-dose PoIFN-α and PoIFN-γ combined (HDI, 1 × 107 U/kg). (A) The average Ct value of the P72 gene was recorded for each group. (B) The increase in the ΔCt value of the P72 gene was determined for each group. Data are representative of the mean ± SDs (n = 3). A non-parametric test was used for the difference significance analysis. (C) Gene expression in pig peripheral blood lymphocyte induced with PoIFN-α and PoIFN-γ. Anticoagulant peripheral blood was drawn from pig and lysed in TRIzol Reagent for RNA extraction. The vertical axis shows the relative transcription level (fold-change) of IFN-stimulated gene (ISG) expression comparing the negative control and treated groups. The fold-changes were measured by the 2−ΔΔCt method, and RNA levels were normalized. Three replicates were performed for q-PCR verification analyses. Bars represent the means ± SDs (n = 3). A non-parametric test was used for the difference significance analysis.…