Swine IFN cocktail can reduce mortality and lessen the tissue injury caused by African swine-fever-virus-infected piglets

Abstract

African swine fever (ASF), a highly virulent viral infection, poses a significant threat to the global pig industry. Currently, there are no commercially available vaccines against ASF. While the crucial role of interferon (IFN) in combating viral infections is well-established, its impact on the clinical signs and mortality rates of ASF remains unclear. In this study, swine IFN-α2, IFN-γ, and IFN-λ3 were fused with the Fc segment of immunoglobulin G (IgG) and expressed in mammalian cells (293T), and the antiviral efficacy were detected by VSV-3D4/2 and VSV-PK15 systems. Then, the interferon stimulating genes (ISGs) induced by IFNs-hFc in 3D4/2 cells were determined by qRT-PCR. Also, the preventive potential of the interferon (IFN) cocktail (a mixture of IFNα2-hFc, IFNγ-hFc, and IFNλ3-hFc) were evaluated in vivo by 25-day-old piglets. The results showed that the specific activities of IFNα2-hFc, IFNγ-hFc, and IFNλ3-hFc were 2.46 × 10⁷ IU/mL, 4.54 × 10⁹ IU/mL and 7.54 × 10¹⁰ IU/mL, respectively. The IFN-hFc significantly induced the expression of various IFN-stimulated genes (ISGs) in 3D4/2 cells after IFNs-Fc treatment, including IFIT5, Mx1, OASL, ISG12, STAT1, IRF1, PKR, CXCL10, and GBP1. Furthermore, the IFN cocktail treatment reduced the viral load, delayed death, and reduced tissue injury in the piglets infected with ASF virus (ASFV). in conclusion, these results suggest that the IFNs-hFc showed high anti-viral activity, and the IFN cocktail may be potential for the prevention and treatment of ASF.

Keywords: African swine fever; cocktail; interferon; mortality; viral load.

Figure 1

Gene expression in porcine alveolar macrophages (3D4/2) induced by IFNα2-hFc, IFNγ-hFc and IFNλ3-hFc for 12 h and 24 h (A) Gene expression in 3D4/2 induced by IFNα2-hFc. (B) Gene expression in 3D4/2 induced by IFNγ-hFc. (C) Gene expression in 3D4/2 induced by IFNλ3-hFc. It is cultured in the RPMI 1640 medium, including 10% FBS and 5% CO₂, for 24 h The cells are then incubated with IFNα2-hFc, IFNγ-hFc, and IFNλ3-hFc for 12 h and 24 h, respectively. At 12 h and 24 h, cells are collected and treated with TRIzol reagents to extract RNA. The vertical axis shows the relative transcription level (folding change) of the expression of the IFN stimulating gene (ISG), comparing the negative control group (CK) and the treatment group. The folding changes and normalization of RNA levels were measured by the 2-ΔΔCt method. Three replications were made for the q-PCR verification analysis. The bar represents the mean ± SDs (n = 3). Non-parametric testing is used for difference significance analysis. Statistically significant differences are pointed out (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; and ∗∗∗∗p < 0.0001), and the line above the column marks the difference between the two groups.

Figure 2

Effects of IFNα2-hFc, IFNγ-hFc, and IFNλ3-hFc on ISG expression after VSV infected with 3D4/2 cells. (A) ISG expression after VSV infected with 3D4/2 cells by IFNα2-hFc. (B) ISG expression after VSV infected with 3D4/2 cells by IFNγ-hFc. (C) ISG expression after VSV infected with 3D4/2 cells by IFNλ3-hFc.Cell samples are collected at 12 h and 24 h The vertical axis shows the relative transcription level (folding change) of the expression of the IFN stimulating gene (ISG), comparing the positive control group (VSV) and the treatment group. VSV challenges the relative transcription level of IFN-stimulating gene (ISG) expression for 12 h VSV challenges the relative transcription level of IFN stimulating gene (ISG) expression for 24 h The folding changes and normalization of RNA levels were measured by the 2-ΔΔCt method. Three replications were made for the q-PCR verification analysis. The bar represents an average of ± SD (n = 3). Non-parametric testing is used for difference analysis. Significant statistical differences are pointed out (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; and ∗∗∗∗p < 0.0001), and the line above the column marks the difference between the two groups.

Figure 3

Tests were conducted on the body temperature of pigs 28 days after ASFV infection. In ASFV-infected control group, the peak temperature reached 41.7°C, while the lowest temperature stood at 40.1°C. In PBS cohabitation infection control group, the highest body temperature recorded was 42°C and the lowest temperature was 38.9°C. In the IFN cocktail treatment group, the highest temperature was 41.2°C, and the lowest was 38.8°C, with the temperature of 18 DPI surviving pigs starting to drop from 41°C at 17 DPI.

Figure 4

Tests were conducted on the mortality rates of pigs after ASFV infection. In ASFV-infected control group, one pig perished on day 7, achieving a 100% mortality rate on day 9. In PBS cohabitation infection control group, one pig died on day 11 and two pigs died on day 18, resulting in a 100% mortality rate. In the IFN cocktail treatment group, only one pig died on day 18, with a 66.67% survival rate on day 28. Two pigs survived but did not die until 37 DPI.

Figure 5

Virus copies of blood post-ASFV challenge. On the third and seventh days, experimental pigs exhibited a notably greater count of ASFV virus copies in their blood compared to those in the interferon treatment and cohabitation infection groups. (∗∗P < 0.01; n=3).

Figure 6

Clinical tissue lesions. The pig spleen, kidneys, liver, pulmonary portal lymph nodes, mandibular lymph nodes, inguinal lymph nodes, mesenteric lymph nodes, and heart from the ASFV-infected control group, the PBS group cohabitation infection group, and the IFN cocktail treatment group all showed typical pathological changes. The degree of lesions in the large intestine, small intestine, and esophagus in the IFN cocktail treatment group was reduced.

Figure 7

Pathological changes in lymph nodes and spleen. Microscopic examination of sections of the spleen, mandibular lymph nodes, pulmonary portal lymph nodes, inguinal lymph nodes, and mesenteric lymph nodes stained with hematoxylin-eosin (H&E) from the three distinct groups. Arrows mark the pathological alterations observed in the organs. Scale bars represent 100 µm (three fields per pig; three pigs per group).