Fig 1: 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 HAD50 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).
Fig 2: 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).
Fig 3: ASFV pB318L reduces type I IFN production and ISGs expression.(A-C) HEK293T cells were transfected with an IFN-β- (A), NF-κB- (B) or ISG56- (C) luciferase reporter, a Renilla-TK reporter, and plasmids expressing HA-cGAS and HA-STING, together with increase amounts (0, 100, 200, and 400 ng) of a plasmid expressing Flag-pB318L. Luciferase activities were analyzed at 24 hpt. (D-F) HEK293T cells were transfected with plasmids expressing HA-cGAS and HA-STING, together with increased amounts (0, 100, 200, and 400 ng) of a plasmid expressing Flag-pB318L. The mRNA levels of Ifnb1, Isg56, and Isg54 were analyzed by qPCR after 24 h. The expressions of cGAS, STING, pB318L, and GAPDH were detected by Western blotting. (G-H) HeLa cells were transfected with a plasmid expressing HA-pB318L for 24 h and then treated with poly (dA:dT) for another 24 h. The cells were collected and lysed, and the phosphorylation of TBK1 and IRF3 was detected by Western blotting (G). Quantitations of p-IRF3 and p-TBK1 ratio were analyzed with image J (H). 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).
Fig 4: Interruption of B318L enhances type I IFN production.(A-E) PAMs were mock infected or infected with ASFV-WT or ASFV-intB318L for 6, 12, or 24 h (MOI = 1). The cells were collected to detect the mRNA levels of Ifna (A), Ifnb1 (B), Isg56 (C), Mx1 (D), and ASFV genomic copies (G) by qPCR, and the cell supernatants were collected to detect the secreted IFN-α (E) and IFN-β (F) by ELISA. 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).
Fig 5: ASFV pB318L reduces STING-mediated IFN production.(A-C) HEK293T cells were transfected with an IFN-β luciferase reporter, a Renilla-TK reporter, a plasmid expressing HA-STING (A), HA-TBK1 (B), or HA-IRF3-5D (C), together with increase amounts (0, 100, 200, and 400 ng) of a plasmid expressing Flag-pB318L. The luciferase activities were detected after 24 h. (D-F) HEK293T cells were transfected with a plasmid expressing HA-STING (D), HA-TBK1 (E), or HA-IRF3-5D (F), together with increased amounts (0, 100, 200, and 400 ng) of a plasmid expressing Flag-pB318L. The mRNA levels of Ifnb1 were analyzed by qPCR. The expressions of STING, TBK1, IRF3-5D, pB318L, and GAPDH were detected by Western blotting. Data are representative of three independent experiments with three biological replicates (mean ± s.d.). Ns, not significantly, *** p < 0.001 (one-way ANOVA).
Supplier Page from Biorbyt for Porcine IFN beta ELISA Kit (Ready to Use)
Application Notes: Pre-diluted detection reagents and enhanced stabilityserum, plasma, tissue homogenates, cell lysates, cell culture supernates or other biological fluids.