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.