Discussion
ASF poses a great threat to the pig industry worldwide. Given there is no an effective vaccine available, the disease control relies mainly on rapid diagnosis, culling of infected pigs, and enhancing biosecurity measures on the threaten farms in those affected countries(Dixon et al., 2020). A better understanding of AFSV is key to develop effective control measures. pB602L is an important non-structural protein of ASFV. To date, few studies focused this protein have showed that it plays an essential role during the viral particle assembly and can also be used to develop diagnosis tools(Epifano et al., 2006; C. Gallardo et al., 2009; Gutierrez-Castaneda et al., 2008). The most of the functions of this protein remains unknown. In the current study, we expressed a recombinant pB602L protein and it was then used to immunize mouse for screening mAbs. Eight mouse mAbs were produced and their recognized epitopes were mapped. To our knowledge, this is the first report on the mAbs and epitopes of pB602L and it provides new knowledge on the molecular basis of the antigenicity of this protein.
The mAbs against a viral protein are important biological materials for both basic and applied researches on the virus. Previous studies showed that the antibodies against pB602L were detectable as early as 10 d post infection in pig and ELISA assays developed based on recombinant pB602L proteins showed similar performances with that of the structural proteins p30 or p54, suggesting that pB602L has good antigenicity and is suitable for developing diagnostic tools(C. Gallardo et al., 2009; Gutierrez-Castaneda et al., 2008). However, the molecular basis for pB602L antigenicity remains largely unknown. Therefore, in this study, we developed firstly eight mouse anti-pB602L mAbs. All of them reacted well with recombinant pB602L. We then identified three epitopes (“366ANRERYNY373”, “415GPDAPGLSI423” and “498EMLNVPDD505”) recognized by these mAbs in pB602L. Multiple sequence alignment based on 69 pB602L amino acid sequences showed that all the three epitopes were completely conserved among these ASFVs, suggesting the antigenicity of pB602L probably comes from the conserved regions other than the CVR. This could partially explain, although the existence of CVR in B602L genes of different ASFV isolates (Mai et al., 2021), why a pB602L based ELISA assay worked well in detecting serum antibodies against different ASFV strains(C. Gallardo et al., 2009). Furthermore, it also indicates that a mAb targeting at any of these epitopes could possibly recognize the pB602L of all the currently identified ASFVs.
pB602L plays an essential role in a successful ASFV capsid assembly(Liu et al., 2019), which makes this protein one of the potential targets for anti-ASFV drug screening. However, the current knowledge on this protein is very limited, and much work needs to be done to understand its biological characteristics and the mechanism for functioning as a molecular chaperone of p72 protein. Visualization of the protein expression in ASFV infected cells is of great importance for such studies. To test the potentials of these mAbs for visualizing the pB602L protein in ASFV infected cells and tissues, three mAbs B2A1, B2H10, and B2E12 were used to detect pB602L in ASFV infected cells and tissues. The IFA assay revealed that B2H10, which recognized the Epitope 2, was good in detecting pB602L positive PAM cells, but which were failed by the other two mAbs. We repeated the IFA for three times and consensus results were obtained, indicating that only Group 2 mAbs are suitable for detecting pB602L in ASFV infected PAM by IFA. When these mAbs were employed to detect ASFV infected cells in different tissues that were collected from ASFV (Pig/HLJ/2018) infected pigs, all of them performed well with high specificity and efficacy, indicating that all of them are suitable for immunohistochemical staining. Immunoelectron micrographs provides very detailed information about the location of a target molecule in cellular departments and also commonly used for analyzing behaviors of a molecule. Therefore, we explored the potentials of these mAbs being used for gold-labeling immunoelectron microscopy. Since the high electron density particles were presented only in ASFV assembly factories, indicating that all the three mAbs detected pB602L specifically. Surprisingly, some gold particles presented on viral particles or the membrane of vesiculas. Given pB602L is a nonstructural protein of ASFV, therefore, these special distributions of the protein should be further studied in the future.