Development of an Indirect ELISA to Detect African Swine Fever Virus pp62 Protein-Specific Antibodies

African swine fever (ASF) is a highly detrimental viral disease caused by African swine fever virus (ASFV). The occurrence and prevalence of this disease have become a serious threat to the global swine industry and national economies. At present, the detection volume of African swine fever is huge, more sensitive and accurate detection techniques are needed for the market. pp62 protein, as a protein in the late stage of infection, has strong antigenicity and a high corresponding antibody titer in infected pigs. In this study, the CP530R gene was cloned into expression vector pET-28a to construct a prokaryotic expression plasmid, which was induced by IPTG to express soluble pp62 protein. Western blot analysis showed that it had great reactivity. Using the purified recombinant protein as an antigen, an indirect ELISA method for detecting ASFV antibody was established. The method was specific only to ASFV-positive serum, 1:1600 diluted positive serum could still be detected, and the coefficients of variation (CV) of the intra assay and inter assay were both <10%. It turns out that the assays had excellent specificity, sensitivity, and repeatability. This provides an accurate, rapid, and economical method for the detection of ASFV antibody in clinical pig serum samples.

African Swine Fever Virus MGF360-14L Negatively Regulates Type I Interferon Signaling by Targeting IRF3

African swine fever (ASF) is a devastating infectious disease caused by African swine fever virus (ASFV). The ASFV genome encodes multiple structural and non-structural proteins that contribute to evasion of host immunity. In this study, we determined that the viral non-structural protein MGF360-14L inhibits interferon-β (IFN-β) promoter activity induced by cGAS-STING signaling. MGF360-14L was also found to downregulate expression of the IRF3 protein and promote its degradation through ubiquitin-meditated proteolysis. Moreover, MGF360-14L was shown to interact with and destabilize IRF3 by facilitating E3 ligase TRIM21-mediated K63-linked ubiquitination of IRF3. Overall, our study revealed that MGF360-14L promotes degradation of IRF3 through TRIM21, thereby inhibiting type I interferon production. These findings provide new insights into the mechanisms underlying ASFV immune evasion.

Efficient inactivation of African swine fever virus by a highly complexed iodine combined with compound organic acids

African swine fever (ASF) is a highly contagious disease with high morbidity and mortality caused by African swine fever virus (ASFV). Cleaning and disinfection remain one of the most effective biosecurity measures to prevent and control the spread of ASFV. In this study, we evaluated the inactivation effects of highly complexed iodine (HPCI) combined with compound organic acids (COAs) against ASFV under different conditions. The results showed that the inactivation rates of the disinfectants on the reporter ASFV increased in dose- and time-dependent manners, the best inactivation effects were obtained when the compatibility ratio of HPCI and COAs was 5:1 at 25°C. Furthermore, there were no significant differences by comparing the efficacy of HPCI combined with COAs (HPCI+COAs) in inactivating wild-type ASFV and the reporter ASFV (P > 0.05). ASFV of 104.0 TCID50/mL was completely inactivated by 0.13% HPCI (0.0065% effective iodine), 0.06% COAs or 0.13% HPCI+COAs (approximately 0.0054% effective iodine), respectively, while 106.0 TCID50/mL ASFV was completely inactivated by 1.00% HPCI (0.05% effective iodine), 0.50% COAs or 1.00% HPCI+COAs (0.042% effective iodine), respectively. Therefore, HPCI+COAs had synergistic effects to inactivate ASFV. This study demonstrated that HPCI+COAs could rapidly and efficiently inactivate ASFV and represent an effective compound disinfectant for the control of ASF.

Lateral Flow Assay for the Detection of African Swine Fever Virus Antibodies Using Gold Nanoparticle-Labeled Acid-Treated p72

African swine fever is a widespread and highly contagious disease in the porcine population, which is caused by African swine fever virus (ASFV). The PCR and ELISA detection methods are the main conventional diagnostic methods for ASFV antigen/antibody detection in the field. However, these methods have limitations of expensive equipment, trained technicians, and time-consuming results. Thus, a rapid, inexpensive, accurate and on-site detection method is urgently needed. Here we describe a double-antigen-sandwich lateral-flow assay based on gold nanoparticle-conjugated ASFV major capsid protein p72, which can detect ASFV antibody in serum samples with high sensitivity and specificity in 10 min and the results can be determined by naked eyes. A lateral flow assay was established by using yeast-expressed and acid-treated ASFV p72 conjugated with gold nanoparticles, which are synthesized by seeding method. A high coincidence (97.8%) of the assay was determined using clinical serum compared to a commercial ELISA kit. In addition, our lateral flow strip can detect as far as 1:10,000 diluted clinically positive serum for demonstration of high sensitivity. In summary, the assay developed here was shown to be rapid, inexpensive, accurate and highly selective. It represents a reliable method for on-site ASFV antibody detection and may help to control the ASFV pandemic.