The pig experiment and experimental programs used in this study have been approved by the Ethics Committee of the Chinese Center for Disease Control and Prevention (IAS2022–157). Pig experiments were carried out in the animal biosafety level 3 (ABSL-3) laboratory of the Chinese Center for Disease Control and Prevention.

Porcine alveolar macrophages (3D4/2) and pig kidney cells (PK15) were purchased from ATCC, China. Human renal epithelial cells (293T) and vesicular stomatitis virus (VSV-GFP) are stored in our laboratory. African swine fever virus (ASFV/CADC_HN09) is preserved in the China Center for Animal Disease Control and Prevention ABSL-3 laboratory.

According to the gene sequences of pig IFN-α2 (MH538099.1), IFN-γ (NM_213948), IFN-λ3 (NM_001166490.1), and hFc (AF150959.1), the IFN sequences were synthesized and connected with hFc genes. There were no termination codons at the ends of IFN-α2, IFN-γ, and IFN-λ3, and the EcoRI enzyme tangent and Kozak sequence were introduced upstream of these IFNs; the thrombin lysis site and NheI enzyme incision point were introduced downstream of the hFc gene; and the codon was optimized according to the preference of mammal codon. The expression plasmid pMal-IFNα2-hFc, pMal-IFNγ-hFc, and pMal-IFNλ3-hFc were transfected into the 293T cells and the cells were cultured for 7 days. The expression of these IFNs was verified using SDS-PAGE, Western Blot, and mass spectrometry. Proteins were purified by using affinity chromatography and were identified by SDS-PAGE and Western Blot, and the protein concentrations were detected by the Bradford method.

To assess the antiviral activity of IFNα2-hFc, IFNγ-hFc, and IFNλ3-hFc expressed by mammalian cells, the cytopathic inhibition method of VSV-3D4/2 and VSV-PK15 systems were used in vitro. The 3D4/2 cells were prepared into a uniform cell suspension with 1 × 10⁶/mL, then transferred into the 96-well cell culture plate, 100 μL per well, and cultured in a 37°C and 5% CO₂ incubator for 24 h. All the initial concentrations of the IFNs were 10 μg/μL, and then, they were diluted four times the ratio, totaling eight dilution gradients. Synchronously, blank control, negative control (cells), and positive control (VSV-GFP 100TCID₅₀) were set up. Each dilution of IFN-hFc to be tested was added to the corresponding well, 100 μL per well, and each dilution was repeated in six wells, and cultured in 37°C and 5% CO₂ incubators for 24 h. The liquid was removed and washed twice with PBS; then, 100 μL of 100TCID₅₀ VSV-GFP virus solution (1,640 + 2% FBS) was added to each well, and cultured in a 37°C and 5% CO₂ incubator for 48 h. Once cytopathy (CPE) appeared in positive control wells, the liquid was removed, and washed twice with PBS; the 100 μL of CCK-8 working fluid (10% V/V) was added and cultured in a 37°C and 5% CO₂ incubator for 2 h, and then, the value of OD₄₅₀ was measured. The antiviral activities of IFNs-hFc were calculated according to the Reed–Muench method (Reed and Muench, 1938).

To determine the expression of interferon stimulating genes (ISGs) induced by IFNs-hFc (IFNα2-hFc, IFNγ-hFc, and IFNλ3-hFc, at 1 μg/mL) in porcine alveolar macrophages (3D4/2), quantitative real-time polymerase chain reaction (qRT-PCR) was used. The porcine alveolar macrophages (3D4/2), 2 × 10⁵/mL, were grown in a six-cell culture plate, 1 mL per well, and cultivated in a 37°C and 5% CO₂ incubator for 24 h (set at 0 hours). Then, the cells were treated with IFNs-hFc for 12 h and 24 h, and then, total RNAs were extracted with TRIzol reagent (Invitrogen, Thermo Fisher Scientific, United States), and cDNAs were transcripted with HiScript III RT SuperMix (Vazyme, China Cat. No. R323–01). qRT-PCR was carried out with 2×Color SYBR Green qPCR Master Mix [Vazyme, China (Cat. No. Q712–03)], and detected on the Roche Light Cycler480 (Switzerland) fluorescence quantitative PCR instrument. The 2^-△△CT was calculated with the Cycle Threshold (CT value) of the ISG and the beta-actin (β-actin) gene.

This study used 25-day-old piglets that were negative for ASFV, classical swine fever virus (CSFV), pseudorabies virus (PRV), porcine parvovirus (PPV), porcine circovirus 1/2 (PCV1/2), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine epidemic diarrhea virus (PEDV). The piglets were randomly divided into three groups (IFN cocktail treatment group, ASFV-infected control group, and PBS cohabitation infection control group), and each group contained three pigs ( Table 1 ). They were raised in the ABSL-3 and were intramuscularly injected with 100 HAD₅₀ of ASFV/CADC_HN09. The IFN cocktail (100,000 IU/kg per ingredient, 2 mL/pig) was intramuscularly injected 3 days before challenge (-1, -2, -3) and 1, 2, 3, 4, 5, 13, 14, 15, 16, and 17 days post-infection (DPI) in the IFN cocktail treatment group. After the challenge, the body temperature and clinical symptoms were recorded every day in each group, and the mouth, nose, and anal swabs were collected on 0, 3, 7, 11, 14, 18, 21, 25, and 28 DPI. Blood was collected on 0, 3, 7, 14, 21, and 28 DPI. To compare the difference in viral load, the genome DNA of ASFV in the mouth, nose, anal swabs, and blood of pigs in each group after treatment were extracted, and the TaqMan RT-qPCR of the ASFV P72 gene was carried out as described (Fan et al., 2020). Necropsy was performed after death, and tissue was collected for histological examination.

Statistical analyses were performed using GraphPad Prism8.0 (GraphPad Software Inc.). Comparisons between groups were performed using the Student’s t-test. The data were expressed as the mean ± standard deviation (SD). Differences with p < 0.05 were considered statistically significant.

IFNα2-hFc, IFNγ-hFc, and IFNλ3-hFc were purified by protein A chromatographic columns. The concentrations of IFNα2-hFc, IFNγ-hFc, and IFNλ3-hFc were 1.51 mg/mL, 4.3 mg/mL, and 8.1 mg/mL, respectively, as detected by the Bradford method. Meanwhile, the viral inhibitory activity values for 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, as detected by the VSV-3D4/2 and VSV-PK15 systems ( Table 2 ).

To investigate whether IFNα2-hFc, IFNγ-hFc, and IFNλ3-hFc fusion proteins induce ISG expression, 3D4/2 cells were treated with IFNα2-hFc, IFNγ-hFc, and IFNλ3-hF for 12 h and 24 h, then, the transcription levels of IFIT5, Mx1, OASL, ISG12, STAT1, IRF1, PKR, CXCL10, and GBP1 were measured via qRT-PCR. The results showed that IFNα2-hFc ( Figure 1A ), IFNγ-hFc ( Figure 1B ), and IFNλ3-hFc ( Figure 1C ) could successfully trigger a range of ISG expressions.

To investigate the antiviral properties of IFNα2-hFc, IFNγ-hFc, and IFNλ3-hFc, 3D4/2 cells were prepared at 2 × 10⁵/mL, cultivated in a six-well plate, 1 mL per well, and cultured in a 37°C and 5% CO₂ incubator for 24 h. Subsequently, IFNs were added and continuously cultured for 24 h, and then, 100TCID₅₀ VSV-GFP were added for 12 h and 24 h. The transcription levels of IFIT5, Mx1, OASL, ISG12, STAT1, IRF1, PKR, CXCL10, GBP1, ISG56, and APDBEC3 genes in 12 h and 24 h were measured using qRT-PCR. The results indicated that IFNα2-hFc ( Figure 2A ), IFNγ-hFc ( Figure 2B ), and IFNλ3-hFc ( Figure 2C ) triggered a variety of antiviral gene expressions combated with VSV infections. The expressions of major antiviral genes such as Mx1, OASL, and PKR, were higher in 12 h than in 24 h.

To examine whether the IFN cocktail can fight ASFV, the pigs were segmented into three distinct groups: the IFN cocktail treatment group, the ASFV-infected control group, and the PBS cohabitation infection control group. After infection, daily temperature ( Figure 3 ) and clinical symptoms were monitored. The mortality rates for each category were documented for a period ranging from 0 to 28 days ( Figure 4 ). After the ASFV challenge, the temperature of pigs in the ASFV-infected control group increased rapidly to 40.5°C at 3 DPI and reached 41.7°C at 5 DPI, while the lowest temperature stood at 40.1°C. One pig died at 7 DPI, and all pigs died until 9 DPI, with a mortality rate of 100%. The temperature of pigs in the PBS cohabitation infection control group increased to 40°C at 7 DPI and reached the highest of 42°C at 10 DPI and the lowest temperature was 38.9°C. One pig died at 7 DPI, and all pigs died until 18 DPI, with a mortality rate of 100%. Comparatively, the temperature of pigs in the IFN cocktail treatment group increased to 40.5°C at 9 DPI, and reached the highest of 41.2°C at 16 DPI, and the lowest was 38.8°C; one pig died at 18 DPI. Fortunately, the temperature of the other two pigs in the IFN cocktail treatment group dropped from 17 DPI to 28 DPI and survived, with a survival rate of 66.7% until 28 DPI, and died until 37 DPI. The results indicated that the treatment of IFN cocktail could effectively relieve the clinical symptoms of ASF, and delay the death of pigs, and as such, the IFN cocktail could be used as an emergency treatment for ASF.

To further evaluate the effects of interferon cocktail on viral load in the infected pigs, the oral, nasal, and anal swab samples were collected at 0, 3, 7, 11, 14, 18, 21, 25, and 28 DPI, and the genome DNA of ASFV in the mouth, nose, anal swabs, and in the blood of pigs in each group after treatment were extracted, and the TaqMan qRT-PCR of the ASFV P72 gene was carried out, as described (Fan et al., 2020).

The results ( Tables 3 , 4 ) showed that viruses were released in all pigs at 3 DPI. From beginning to death, viruses were released from the oral cavity, nasal cavity, and anus. The viral load of the PBS group cohabitation infection group and ASFV-infected control group was higher than that of the interferon treatment group, and the oral and nasal viral loads of these two control groups were significantly different. Only at 3DPI did the interferon-treated group exhibit higher levels of oral and nasal viruses than the cohabitation infection group. The viral loads in the oral, nasal cavity, and anal regions were detected at 7, 11, 18, and 21 DPI, while the viral loads of the interferon group were lesser than those in the PBS group cohabitation infection group, and ASFV-infected control group, which had notable differences. Two pigs survived in the interferon treatment group. The viral load was detected in the blood of all pigs ( Figure 5 ). The results ( Table 5 ) showed that during the 3 DPI and 7 DPI tests, two pigs in the ASFV-infected control group had viremia. There was no viremia in the IFN cocktail treatment group except for one pig only at 14 DPI.

To observe whether there are differences in tissue lesions and pathological changes in dead pigs between the IFN cocktail treatment group, ASFV-infected control group, and the PBS group cohabitation infection group, the tissue lesions and the pathological changes of each tissue and organ were observed and recorded including the spleen, kidney, pulmonary portal lymph nodes, mandibular lymph nodes, inguinal lymph nodes, mesenteric lymph nodes, liver, lungs, intestines, esophagus, tonsils, and heart. Clinical tissue lesions ( Figure 6 ) showed that the pigs displayed a range of bleeding patterns, encompassing areas on their back, abdomen, limbs, mouth, and nose, accompanied by spleen blockage, growth, lymph node blockage, kidney surface hemorrhage, renal pelvis and calendula hemorrhaging, lung and liver edge blockages, tonsil expansion and blockage, mucous tissue surface hemorrhage, and peripheral fluid accumulation. Compared with the pigs in the ASFV-infected and PBS cohabitation infection control group pigs, tissue damage, tissue swelling, bleeding, and hemorrhagic necrotic spots were significantly reduced by the IFN cocktail treatment. Compared to the ASFV-infected control group and the PBS group cohabitation infection group, the degree of lesions in the intestines and esophagus in the IFN cocktail treatment group was reduced. Only the tonsils of one pig were enlarged and congested, and the tonsils of the other two pigs were not significantly swollen and congested, indicating that the IFN cocktail can reduce the pathological damage of the mucous membrane system of pigs.

The analysis and scrutiny of histopathological samples ( Figure 7 ) indicated that after the death of pigs in the ASFV infection control, PBS cohabitation infection control, and IFN cocktail groups presented diffused hemorrhage in the spleen tissue, and the lymphocytes in the white pulp have deteriorated, turning necrotic and fragmented, resulting in a discontinuity. The bleeding zone presented an increased quantity of red blood cells and iron-rich hematin. As to lymphatic tissue (mandibular lymph nodes, pulmonary portal lymph nodes, inguinal lymph nodes, and mesenteric lymph nodes) observed in animals after euthanasia, the three distinct groups indicated that the membrane was intact and smooth; the membrane and portal connective tissue extended into the lymph node parenchyma, forming a trabecular structure; the lymphocyte composition was reduced; the lymph node structure was obscure; and the red blood cells around the cortical and medullary lymph nodes were accumulated. There were no obvious differences in tissue damage in the spleen, mandible, hilum, groin, and mesenteric lymph nodes.