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African swine fever virus pB318L, a trans-geranylgeranyl-diphosphate synthase, negatively regulates cGAS-STING and IFNAR-JAK-STAT signaling pathways [1]

['Xiaohong Liu', 'National African Swine Fever Para-Reference Laboratory', 'State Key Laboratory Of Veterinary Biotechnology', 'Harbin Veterinary Research Institute', 'Chinese Academy Of Agricultural Sciences', 'Harbin', 'Hefeng Chen', 'Guangqiang Ye', 'Hongyang Liu', 'Chunying Feng']

Date: 2024-05

African swine fever (ASF) is an acute, hemorrhagic, and severe infectious disease caused by the ASF virus (ASFV). ASFV has evolved multiple strategies to escape host antiviral immune responses. Here, we reported that ASFV pB318L, a trans-geranylgeranyl-diphosphate synthase, reduced the expression of type I interferon (IFN-I) and IFN-stimulated genes (ISGs). Mechanically, pB318L not only interacted with STING to reduce the translocation of STING from the endoplasmic reticulum to the Golgi apparatus but also interacted with IFN receptors to reduce the interaction of IFNAR1/TYK2 and IFNAR2/JAK1. Of note, ASFV with interruption of B318L gene (ASFV-intB318L) infected PAMs produces more IFN-I and ISGs than that in PAMs infected with its parental ASFV HLJ/18 at the late stage of infection. Consistently, the pathogenicity of ASFV-intB318L is attenuated in piglets compared with its parental virus. Taken together, our data reveal that B318L gene may partially affect ASFV pathogenicity by reducing the production of IFN-I and ISGs. This study provides a clue to design antiviral agents or live attenuated vaccines to prevent and control ASF.

African swine fever virus (ASFV) causes a highly lethal swine disease in many countries, severely affecting the pig industry. Until now, African swine fever has caused substantial economic losses to the world pig industry because of the lack of commercial vaccines and drugs. Therefore, there is an urgent need for efficient commercialized vaccines to prevent and control the disease. It has been reported that the ability of ASFV to escape host antiviral immune responses is closely related to its pathogenicity, and deletion of ASFV virulence-related gene(s) may contribute to the development of attenuated live vaccines. In this study, we found that pB318L reduces the production of IFN-I induced by cGAS-STING and ISGs induced by IFN-α. As a virulence-related gene, interruption of B318L significantly attenuated ASFV pathogenicity. Our findings provide a new clue to understand the functions of ASFV-encoded pB318L and its role in viral infection-induced pathogenesis, which might help design antiviral agents or live attenuated vaccines to control ASF.

Funding: This study was supported by the National Natural Science Foundation of China (grant No. 32322081) (LH), the National Natural Science Foundation of China (grant No. U21A20256) (CW), the National Natural Science Foundation of China (grant No. 32270156) (LH), the National Key Research and Development Program of China (grant No. 2021YFD1801300) (LH), the National Key Research and Development Program of China (grant No. 2021YFD1800100) (CW), the Central Public-interest Scientific Institution Basal Research Fund (grant No. 1610302022013) (LH), and Innovation Program of Chinese Academy of Agricultural Sciences (grant No. CAAS CSLPDCP-2023002) (LH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

In this study, we found that pB318L suppresses the production of IFN-I and the expression of ISGs by regulating cGAS-STING and JAK-STAT signaling pathways. ASFV interrupting the B318L gene (ASFV-intB318L) significantly reduces the ASFV virulence in piglets compared with its parental ASFV HLJ/18 isolate in vivo. Taken together, our findings reveal that ASFV B318L gene is a key virulence-related gene that affects the pathogenicity of ASFV, and it functions by reducing the production of IFN-I and ISGs via targeting STING and IFNAR1/2.

ASFV B318L gene is an ASFV late gene, which encodes the pB318L protein with 318 amino acids (aa) with a molecular weight of 36 kDa. Previous studies showed that ASFV pB318L is a GGPPS [ 17 , 18 ], containing four highly conserved regions unique to these enzymes [ 19 ]. So far, the ASFV B318L gene is the only prenyltransferase gene of virus origin identified [ 18 ]. Moreover, pB318L is expressed in the later stage of infection, which is necessary for the assembly and release of virus particles [ 20 ]. It showed that the geranylation of protein plays a vital role in regulating host antiviral immune responses [ 21 ].

Prenylation is one of the post-translational lipid modifications of many membrane proteins, which plays a crucial role in cellular signaling transduction during viral infections [ 12 ]. The mevalonate pathway is one of the essential pathways of cell metabolism, which regulates cholesterol synthesis and prenylation of proteins [ 13 ]. The prenylation pathway and the mevalonate pathway are inseparable, and mevalonate is metabolized to isoprenyl pyrophosphate (IPP), which is the direct source for the synthesis of farnesyl diphosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). This process is catalyzed by farnesyl diphosphate synthase (FPPS) and GGPP synthase (GGPPS). Farnesyltransferase (FTase) catalyzes the FPP to the target protein, simultaneously releasing a free diphosphate. Geranyltransferase type 1 and type 2 (GGTsae-I and GGTase-II) use GGPP as a substrate [ 14 ]. FTase and GGTase-I recognize the C-terminal CaaX motif in the target proteins, where C is the modified cysteine site [ 15 ]. Prenylation can increase the affinity of proteins to membranes, thereby regulating their localization and transport [ 16 ].

The innate immune response is the first line of host defense against the invasion of pathogenic microorganisms. Upon pathogenic infection, pattern recognition receptors (PRRs) in host cells recognize pathogen-related molecular patterns (PAMPs) and initiate the production of IFNs, ISGs, inflammatory cytokines, and other antiviral proteins to eliminate the pathogens [ 5 , 6 ]. During ASFV infection, cyclic GMP-AMP synthase (cGAS) recognizes the viral genomic DNA and catalyzes the cyclization reaction of ATP and GTP to form cyclic-GMP-AMP (cGAMP) [ 7 ]. Subsequently, cGAMP binds to the intracellular stimulator of IFN genes (STING), causing a conformational change of STING, which then translocates from the endoplasmic reticulum (ER) to the Golgi apparatus to recruit and phosphorylate TANK binding kinase 1 (TBK1) [ 8 , 9 ]. The phosphorylated TBK1 further recruit interferon regulatory factor 3 (IRF3) to promote its phosphorylation and activation [ 9 ]. The activated IRF3 forms a dimer and then translocates to the nucleus to regulate the production of type I interferons (IFN-I). The secreted IFN-I binds to its receptors (IFNAR1 and/or IFNAR2), resulting in the phosphorylation of two JAK kinases (JAK1 and TYK2), which subsequently phosphorylate signal transducers and activators of transcription 1/2 (STAT1/2). The phosphorylated STAT1/2 interacts with IFN regulatory factor 9 (IRF9) to form a heterotrimeric complex, interferon-stimulated gene factor 3 complex (ISGF3), which then enters the nucleus and binds to the IFN-stimulated response elements (ISREs) to induce the expression of many ISGs such as ISG15, ISG54, and ISG56. These ISGs maintain a potent antiviral response in ASFV-infected cells [ 10 , 11 ].

African swine fever (ASF) is an acute and severe infectious disease caused by ASF virus (ASFV), which can induce approximately 100% mortality in domestic pigs [ 1 ]. The ASF epidemic was first reported in Kenya in 1921 [ 2 ] and became widespread in Africa, Europe, and East Asia. At present, ASF is a severe plague in Asian countries, including China, Vietnam, and Korea, and has caused huge economic losses. ASFV is the only member of the ASFV family. The ASFV genome varies from 170–193 kb in length, containing more than 150 open reading frames (ORF) encoding 150–200 viral proteins. Some ASFV proteins are required for viral replication [ 3 ], and several proteins antagonize the host’s innate immune responses [ 4 ]. However, the functions of most ASFV-encoded proteins are still not known.

Results

ASFV pB318L reduces IFN-I production dependent on its enzymatic activity It has been reported that pB318L is a geranylgeranyl diphosphate synthase [18]. Lovastatin is an inhibitor of HMG-CoA reductase, which reduces the production of mevalonate, thereby inhibiting the prenylation modification. Lonafarnib is an inhibitor of FTase, and GGTI-286 is an inhibitor of GGTase-I. To test whether ASFV pB318L negatively regulates IFN-I production through its geranylgeranyl diphosphate synthase activity, HEK293T cells were transfected with IFN-β luciferase reporter and plasmids expressing STING and pB318L as indicated in the presence of three prenylation inhibitors, respectively. We found that both Lovastatin and GGTI-286 could rescue the suppression of cGAS-STING-induced IFN-β promoter activity by pB318L, while Lonafarnib couldn’t (Fig 5A). These results suggest that the geranylgeranyl diphosphate synthase activity of pB318L is required for its suppressing IFN-I production. PPT PowerPoint slide

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TIFF original image Download: Fig 5. Asp129 of ASFV pB318L affects its inhibition of STING phosphorylation and translocation. (A) HEK293T cells were transfected with an IFN-β luciferase reporter, a Renilla-TK reporter, and plasmids expressing HA-cGAS and HA-STING, together with a plasmid expressing Flag-pB318L. After 24 h, the cells were treated with Lovastatin, Lonfarnib, GGTI-286 for 12 h, then the luciferase activities were detected. (B) HEK293T cells were transfected with an IFN-β luciferase reporter, a Renilla-TK reporter, and plasmids expressing HA-cGAS and HA-STING, together with increased amount (100 ng, 200 ng, 400 ng) of a plasmid expressing Flag-pB318L-WT or Flag-pB318L-Mut, the luciferase activities were detected after 24 h. (C-D) HEK293T cells were transfected with plasmids expressing HA-cGAS and HA-STING, together with increase amount (100 ng, 200 ng, 400 ng) of a plasmid expressing Flag-pB318L-WT (100 ng, 200 ng, 400 ng) or Flag-pB318L-Mut, the mRNA levels of Ifnb1 and Isg56 were analyzed by qPCR. (E) HEK293T cells were transfected with plasmids expressing Flag-pB318L-D129A and HA-STING. Co-IP analysis was performed to detect the interaction between pB318L-D129A and STING after 24 h. (F-G) CRL2843 cells were transfected with plasmids expressing HA-STING and GFP-pB318L or GFP-pB318L-D129A as indicated. At 24 hpt, the cells were stimulated with cGAMP (10 μg/mL) for another 12 h. The subcellular localization of STING was visualized by immunofluorescence microscopy. Scale bars, 20 μm (F). The fluorescence intensity of STING was analyzed using the Zeiss processing system (G). (H-I) HeLa cells were transfected with a plasmid expressing Flag-pB318L or Flag-pB318L-D129A for 24 h, and then treated with the STING agonist for another 6 h. The cells were collected and lysed, and the phosphorylation of STING was detected by Western blotting (H). Quantitation of p-STING/STING ratio was analyzed with Image J (I). Data are representative of three independent experiments with three biological replicates (mean ± s.d.). Ns, not significantly, ** p < 0.01, *** p < 0.001 (one-way ANOVA). https://doi.org/10.1371/journal.ppat.1012136.g005 Based on the amino acids of the active center of the homologous structure of pB318L [25], we analyzed the active center of pB318L and predicted that D129, D135, and D212 may affect the enzyme activity. To map the key amino acid residues that are related to pB318L enzymatic activity on reducing IFN-І production, three plasmids expressing ASFV pB318L mutants such as pB318L-D129A, pB318L-D135A, and pB318L-D212A, were generated. The results showed that except for pB318L-D129A, pB318L-WT and all the other mutants reduced both the promoter activity of IFN-β (Fig 5B) and the mRNA expression levels of Ifnb1 and Isg56 (Fig 5C and 5D) induced by expressed cGAS-STING, which indicates that D129 of ASFV pB318L plays an important role in reducing IFN-I production. Additionally, we found that although pB318L-D129A still interacted with STING, but it lost its inhibition of the Golgi apparatus translocation and phosphorylation of STING (Fig 5E–5I). RhoA is a type of Rho GTPase that has been shown to be isoprenylated [26]. To demonstrate that Asp129 is required for the enzyme activity of pB318L, the effect of B318L-WT and pB318L-D129A on the isoprenylation of RhoA was detected. We found that pB318L promoted the isoprenylation of RhoA; however, the effect of B318L-D129A is relatively weak (S3H Fig), suggesting that Asp129 affects but not determines the enzyme activity of pB318L. Taken together, our findings suggest that the enzymatic activity of pB318L is related to its function in suppressing IFN-I production, and the Asp129 plays a key role in the process.

ASFV pB318L reduces the phosphorylation and translocation of STATs We noticed that ASFV pB318L also reduces the mRNA of Isg54 and Isg56 in HEK293T cells (Fig 2E and 2F). We next explored whether pB318L affects the expression of ISGs during ASFV infection. PAMs were infected with ASFV-WT or ASFV-intB318L for 24 h (MOI = 1) and then treated with IFN-α for an additional 12 h. The mRNA levels of several ISGs were detected by qPCR. We found that ASFV-WT infection strongly suppressed the mRNA levels of Isg56, Isg15, Mx1, and Oas2 induced by IFN-α. In contrast, the inhibitory effect was obviously attenuated upon ASFV-intB318L infection (Fig 6A–6D). To exclude the possibility that the enhanced ISG expressions during ASFV-intB318L infection were due to the higher viral load compared to the ASFV-WT, we detected the mRNA expression of ASFV p72 protein. The results showed that the mRNA level of ASFV p72 in ASFV-intB318L-infected PAMs was significantly lower than that of ASFV-WT in the presence of IFN-α (S4 Fig). Similarly, overexpressed pB318L significantly reduced both the promoter activities of ISRE, ISG54, and ISG56 (Fig 6E–6G) and the mRNA levels of Isg56, Isg15, and Mx1 triggered by IFN-α in a dose-dependent manner (Fig 6H–6J). PPT PowerPoint slide

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TIFF original image Download: Fig 6. ASFV pB318L reduces the production of ISGs. (A-D) PAMs were infected with ASFV-WT or ASFV-intB318L for 24 h (MOI = 1) and then treated with IFN-α (1 μg/mL) for another 12 h. The mRNA levels of Isg56 (A), Isg15 (B), Mx1 (C), and Oas2 (D) were analyzed by qPCR. (E-G) HEK293T cells were co-transfected with ISRE-luciferase (E), ISG54-luciferase (F), ISG56-luciferase (G) reporters, and a Renilla-TK reporter along with different doses of a plasmid (0, 100, 200, 400 ng) expressing Flag-pB318L. At 24 hpt, the cells were treated with IFN-α (1 μg/mL) for another 12 h. The cells were collected to detect the luciferase activity. The expressions of pB318L and GAPDH were analyzed by Western blotting. (H-J) Different doses of a plasmid (0, 100, 200, 400 ng) expressing Flag-pB318L were transfected into HEK293T cells for 24 h, and then the cells were stimulated with IFN-α (1 μg/mL) for 12 h. The mRNA levels of Isg56 (H), Isg15 (I), and Mx1 (J) were analyzed by qPCR. Data are representative of three independent experiments with three biological replicates (mean ± s.d.). * p < 0.05, ** p < 0.01, *** p < 0.001 (one-way ANOVA). https://doi.org/10.1371/journal.ppat.1012136.g006 To examine whether the phosphorylation and subcellular localization of STAT1 and STAT2 are affected by the ASFV pB318L, PAMs were infected with ASFV-WT or ASFV-intB318L and then treated with IFN-α. We found that ASFV-WT infection significantly reduced the phosphorylation and nuclear translocation of STAT1 and STAT2 induced by IFN-α. ASFV-intB318L infection also obviously suppressed the phosphorylation and nuclear translocation of STAT1 and STAT2; the inhibitory effect was significantly lower than that of ASFV-WT infection (Fig 7A–7D). Consistently, ectopically expressed pB318L also significantly reduced the phosphorylation (Fig 7E and 7F) and nuclear translocation (Fig 7G–7J) of STAT1 and STAT2 in vitro. These results indicate that ASFV pB318L reduces STAT1/2 phosphorylation and nuclear translocation during ASFV infection. PPT PowerPoint slide

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TIFF original image Download: Fig 7. ASFV pB318L reduces phosphorylation and translocation of STATs. (A-B) PAMs were infected with ASFV-WT (MOI = 1) or ASFV-intB318L (MOI = 1) for 24 h and then treated with or without IFN-α (1 μg/mL) for 12 h. The cells were collected to test the expressions of STAT1, STAT2, pB318L, p30, GAPDH, and the phosphorylation of STAT1 and STAT2 (A). Quantitation of p-STAT1 and p-STAT2 ratio was analyzed with image J (B). (C-D) PAMs were infected with ASFV-WT (MOI = 1) or ASFV-intB318L (MOI = 1) for 24 h and then treated with IFN-α (1 μg/mL) for 12 h. The cells were harvested and processed using nuclear and cytoplasmic extraction reagents to extract the cytoplasmic and nuclear fractions and detect the distribution of STAT1 and STAT2 in the cytoplasm and nucleus, respectively (C). Quantitation of STAT1 and STAT2 in the nuclear ratio was analyzed with Image J (D). (E-F) HEK293T cells were transfected with a plasmid expressing Flag-pB318L or empty vector for 24 h. Then, the cells were treated with IFN-α (1 μg/mL) for 30 or 60 min. The cells were collected to analyze the phosphorylation of STAT1 and STAT2 (E). Quantitation of p-STAT1 and p-STAT2 ratios were analyzed with Image J (F). (G-H) CRL-2843 cells were transfected with a plasmid expressing HA-pB318L or an empty vector for 24 h, and the cells were stimulated with IFN-α (1 μg/mL) for 60 min. The localizations of STAT1 or STAT2 and pB318L were detected by immunofluorescence microscopy. Scale bars, 20 μm (G). The green fluorescence intensities of the images were analyzed using the Zeiss processing system (H). (I-J) HEK293T cells were transfected with a plasmid expressing Flag-pB318L or an empty vector for 24 h. Then, the cells were treated with IFN-α (1 μg/mL) for 60 min. The cells were harvested and processed using nuclear and cytoplasmic extraction reagents to detect the distribution of STAT1 and STAT2 in the cytoplasm and nucleus, respectively (I). Quantitation of STAT1 and STAT2 ratios were analyzed with Image J (J). Data are representative of three independent experiments with three biological replicates (mean ± s.d.). * p < 0.05, 0.001 < ** p < 0.01, *** p < 0.001, (one-way ANOVA). https://doi.org/10.1371/journal.ppat.1012136.g007

The Inhibition of ISGs production by ASFV pB318L depends on its enzymic activity To detect whether the inhibition of ISGs production by pB318L still depends on its enzymatic activity, HEK293T cells were pretreated with inhibitors Lovastatin, GGTI-286 or Lonafarnib, then stimulated with IFN-α, and the ISG56 reporter activity was detected. The results showed that both Lovastatin and GGTI-286, but not Lonafarnib, strongly rescued the inhibitory effect of pB318L on ISG56 promoter activity induced by IFN-α (Fig 9A). Consistently, pB318L-D129A, but not pB318L-D135A and pB318L-D212A, completely lost its inhibitory effect on the promoter activity and the upregulation of mRNA expression of Isg56 (Fig 9B and 9C). In addition, we noticed that pB318L-D129A still interacted with IFNAR1/IFNAR2, but it failed to reduce the phosphorylation and nuclear translocation of STAT1 and STAT2 induced by IFN-α (Fig 9D–9I). Taken together, our results suggest that inhibition of IFN-α-mediated ISGs production by pB318L is related to its GGPPS enzymatic activity and that Asp129 plays a role in supporting the pB318L enzyme function. PPT PowerPoint slide

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TIFF original image Download: Fig 9. Asp129 of ASFV pB318L affects its inhibition of ISGs expression. (A) HEK293T cells were transfected with an ISG56 luciferase reporter and a Renilla-TK reporter, together with a plasmid expressing Flag-pB318L. After 24 h, the cells were treated with IFN-α (1 μg/mL) for 12 h, and then treated with Lovastatin, Lonfarnib, GGTI-286 for another 12 h. After that, the luciferase activities were analyzed. (B) HEK293T cells were transfected with an ISG56 luciferase reporter and a Renilla-TK reporter, together with increased amounts (100 ng, 200 ng, 400 ng) of a plasmid expressing Flag-pB318L-WT (100 ng, 200 ng, 400 ng) or Flag-pB318L-Mut. After 24 h, the cells were treated with IFN-α (1 μg/mL) for 12 h, and the luciferase activities were then detected. (C) HEK293T cells were transfected with increased amounts (100 ng, 200 ng, 400 ng) of a plasmid expressing Flag-pB318L-WT or Flag-pB318L-Mut, the mRNA level of Isg56 was analyzed by qPCR. (D-E) HEK293T cells were transfected with plasmids expressing Flag-pB318L-D129A and HA-IFNAR1 or HA-IFNAR2 as indicated. Co-IP analysis was performed to detect the interaction between pB318L-D129A and IFNAR1/IFNAR2 after 24 h. (F-G) HEK293T cells were transfected with a plasmid expressing Flag-pB318L or Flag-pB318L-D129A for 24 h, and then treated with IFN-α (1 μg/mL) for another 60 min. The cells were collected and lysed, and the phosphorylation levels of STAT1 and STAT2 were detected by Western blotting (F). Quantitation of p-STAT1 and p-STAT2 ratios were analyzed with Image J (G). (H-I) HEK293T cells were transfected with plasmids expressing HA-pB318L or HA-pB318L-D129A for 24 h, and the cells were then stimulated with IFN-α (1 μg/mL) for another 12 h. The cells were harvested and processed using nuclear and cytoplasmic extraction reagents to extract the cytoplasmic and nuclear fractions and detect the distribution of STAT1 and STAT2 in the cytoplasm and nucleus, respectively (H). Quantitation of STAT1 and STAT2 in nuclear ratio was analyzed with Image J (I). Data are representative of three independent experiments with three biological replicates (mean ± s.d.). Ns, not significantly, * p < 0.05, ** p < 0.01, *** p < 0.001, (one-way ANOVA). https://doi.org/10.1371/journal.ppat.1012136.g009

The reading frame interruption of B318L gene attenuates the pathogenicity of ASFV HLJ/18 To examine the role of the B318L gene in ASFV pathogenicity, SPF pigs were incubated with 102.5 HAD 50 ASFV-WT or ASFV-intB318L, respectively. All pigs infected with ASFV-WT developed a fever at 4 days post inoculation (dpi), were depressed, and had a reduced appetite at 5 dpi. ASFV-WT-infected pigs started to die from 7 dpi, and all died within 11 dpi (Fig 10A–10C). In comparison, only two pigs infected with ASFV-intB318L developed a fever at 5 dpi, and then showed significant depression of spirit and loss of appetite at 10 dpi (Fig 10A and 10B). ASFV-intB318L infected pigs started to die from 12 dpi, and 60% of pigs continued to survive until 21 dpi (Fig 10A and 10C). The spleens, tonsil, submandibular lymph nodes, and inguinal lymph nodes from pigs challenged with ASFV-WT showed extensive bleeding, while these tissues from pigs challenged with ASFV-intB318L showed no obvious lesions (Fig 10D). PPT PowerPoint slide

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TIFF original image Download: Fig 10. ASFV-intB318L is attenuated in pigs. (A-C) Body survival (A), temperatures (B), and clinical score (C) of pigs intramuscularly (IM) inoculated with PBS (n = 3), 102.5 HAD 50 ASFV-intB318L (n = 5) or ASFV-WT (n = 5). (D) Tissue lesions in the spleens, tonsils, submaxillary lymph nodes, and inguinal lymph nodes of pigs infected with ASFV-WT or ASFV-intB318L. (E) qPCR analysis of ASFV genomic DNA copy number in blood samples obtained from pigs at 0, 1, 4, 7, 11, 16, and 21 days after being infected with either ASFV-intB318L or ASFV-WT. (F) qPCR analysis of ASFV genomic DNA copy number in the tissues as indicated obtained from pigs that were infected with either ASFV-intB318L or parental ASFV-WT. (G) Histopathological section of thymus, spleen, submaxillary lymph nodes, inguinal lymph nodes, and bronchial lymph nodes. (H-K) qPCR analysis of mRNA levels of Ifnα(H), Ifnb1(I), Isg15 (J), and Mx1 (K) in the spleen, tonsil, thymus, submaxillary lymph node, bronchial lymph node, and gastrohepatic lymph node obtained from pigs mock infected or infected with either of ASFV-intB318L or parental ASFV-WT. (L-M) The protein levels of IFN-α and IFN-β in peripheral serum samples from ASFV-intB318L or ASFV-WT-challenged pigs were monitored at 0, 1, 4, and 7 dpi by ELISA. Data represent three independent experiments with three biological replicates (mean ± s.d.). Ns, not significantly, * p < 0.05, ** p < 0.01, *** p < 0.001, (one-way ANOVA). https://doi.org/10.1371/journal.ppat.1012136.g010 Pigs in ASFV-WT inoculated group presented viremia at 4 dpi, while only two pigs in ASFV-intB318L-challenged group presented viremia at 10 dpi (Fig 10E). Viral DNA copy numbers in organs/tissues including heart, liver, lung, spleen, kidney, tonsil, and six lymph nodes (mediastinal, mesenteric, inguinal, submandibular, bronchial, and gastrohepatic) detected in ASFV-intB318L-infected pigs were significantly lower than that detected from the ASFV-WT-infected group (Fig 10F). Histopathological sections showed massive necrosis and decrease of lymphocytes in thymus, bronchial lymph nodes, inguinal lymph nodes and submandibular lymph nodes and increased hematocrit in spleen in ASFV-WT infection group. However, these pathological phenomena were significantly milder in the ASFV-intB318L infection group (Fig 10G). To compare the innate immune responses of pigs inoculated with ASFV-WT or ASFV-intB318L, the mRNA levels of IFN-I and ISGs in the immune tissues as indicated were detected by qPCR. The results showed that the mRNA levels of Ifnα, Ifnb1, Mx1, and Isg15 in the tissues of pigs challenged with ASFV-intB318L were significantly higher than those in the tissues of pigs challenged with ASFV-WT (Fig 10H–10K). The protein levels of IFN-α and IFN-β in peripheral serum samples were also monitored at 0, 1, 4, and 7 dpi using ELISA. The results showed that the secretions of IFN-α and IFN-β in the ASFV-intB318L-inoculated group were 2.0 and 2.5 folds higher than those in the ASFV-WT-inoculated group at 7 dpi, respectively (Fig 10L and 10M).

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