Characterization of the monoclonal antibody and the immunodominant B-cell epitope of African swine fever virus pA104R by using mouse model.

The African swine fever virus (ASFV) structural protein pA104R is the only histone-like protein encoded by eukaryotic viruses. pA104R is an essential DNA-binding protein required for DNA replication and genome packaging of ASFV, which are vital for pathogen survival and proliferation. pA104R is an important target molecule for diagnosing, treating, and immune prevention of ASFV. This study characterized monoclonal antibodies (mAbs) against pA104R and found them to recognize natural pA104R in ASFV strains with different genotypes, showing high conservation. Confirmation analyses of pA104R epitopes using mAbs indicated the presence of immunodominant B-cell epitopes, and further characterization showed the high antigenic index and surface accessibility coefficients of the identified epitope. Furthermore, the pA104R protein functions through the polar interactions between the binding amino acid sites; however, these interactions may be blocked by the recognition of generated mAbs. Characterizing the immunodominant B-cell epitope of the ASFV critical proteins, such as pA104R, may contribute to developing sensitive diagnostic tools and vaccine candidate targets.IMPORTANCEAfrican swine fever (ASF) is a highly pathogenic, lethal, and contagious viral disease affecting domestic pigs and wild boars. As no effective vaccine or other treatments have been developed, the control of African swine fever virus (ASFV) relies heavily on virus detection and diagnosis. A potential serological target is the structural protein pA104R. However, the molecular basis of pA104R antigenicity remains unclear, and a specific monoclonal antibody (mAb) against this protein is still unavailable. In this study, mAbs against pA104R were characterized and found to recognize natural pA104R in ASFV strains with different genotypes. In addition, confirmation analyses of pA104R epitopes using mAbs indicated the presence of immunodominant B-cell epitopes, and further characterization showed the high antigenic index and surface accessibility coefficients of the identified epitope. Characteristics of the immunodominant B-cell epitope of ASFV proteins, such as pA104R, may contribute to developing sensitive diagnostic tools and identifying vaccine candidate targets.

African Swine Fever Virus pF778R Attenuates Type I Interferon Response by Impeding STAT1 Nuclear Translocation.

African swine fever virus (ASFV) is an extensive and intricate double-stranded DNA virus with approximately 100% lethality in domestic swine. There is no effective vaccine to combat this virus, and this has led to substantial economic losses in the swine industry. ASFV encodes various proteins that impede interferon-based immune defenses in the host by employing diverse mechanisms. However, the roles of most of these proteins remain unknown. Therefore, understanding the immune evasion mechanisms employed by ASFV may facilitate the development of effective measures against the virus. In this study, we discovered a negative regulation of the type I interferon (IFN) response by the ASFV ribonuclease reductase large subunit pF778R. This novel type Ⅰ IFN response antagonist significantly inhibits IFN-α-induced interferon-stimulated response element promoter activation, precludes the upregulation of various interferon-stimulated genes, and prevents STAT1 nuclear translocation. Mechanistically, pF778R did not affect the protein levels of crucial molecules in the JAK/STAT signaling pathway or engage in direct interactions. However, pF778R expression impedes type I IFN responses mediated by the JAK/STAT signaling pathway. Further investigations revealed that pF778R did not interfere with STAT1 phosphorylation or dimerization, but it inhibited IFN signaling by weakening the nuclear accumulation of activated STAT1. The critical role of the ASFV protein pF778R in evading IFN-I-mediated innate immunity highlights a unique mode of ASFV evasion and provides insights into the pathogenic mechanism of the virus.

African swine fever virus pA104R protein acts as a suppressor of type I interferon signaling.

This study evaluates the role of the late viral protein, pA104R, in African swine fever virus immunosuppression. ASFV-encoded pA104R is a putative histone-like protein that is highly conserved throughout different virulent and non-virulent isolates. Previous studies have demonstrated that pA104R plays a vital role in the ASFV replication cycle and is a potential target for antiviral therapy. Here, we demonstrated that pA104R is a potent antagonist of type I interferon signaling. IFN-stimulated response element activity and subsequent transcription of co-transfected and endogenous interferon-stimulated genes were attenuated by pA104R treatment in HEK-293 T cells. Immunoprecipitation assay and reciprocal pull-down showed that pA104R does not interact directly with STAT1, STAT2, or IRF9. However, pA104R could inhibit IFN signaling by attenuating STAT1 phosphorylation, and we identified the critical amino acid residues (R/H69,72 and K/R92,94,97) involved through the targeted mutation functional assays. Although pA104R is a histone-like protein localized to the nucleus, it did not inhibit IFN signaling through its DNA-binding capacity. In addition, activation of the ISRE promoter by IRF9-Stat2(TA), a STAT1-independent pathway, was inhibited by pA104R. Further results revealed that both the transcriptional activation and recruitment of transcriptional stimulators by interferon-stimulated gene factor 3 were not impaired. Although we failed to determine a mechanism for pA104R-mediated IFN signaling inhibition other than attenuating the phosphorylation of STAT1, these results might imply a possible involvement of epigenetic modification by ASFV pA104R. Taken together, these findings support that pA104R is an antagonist of type I interferon signaling, which may interfere with multiple signaling pathways.