Identification of a conserved surface-exposed B-cell epitope on PCV4 cap protein and preparation of its specific monoclonal antibody.

Porcine circovirus 4 (PCV4), which emerged in 2019 in China, has spread widely worldwide and caused enormous economic losses to the swine industry. However, the lack of knowledge regarding the antigenic epitope characteristics of PCV4 renders its detection and prevention challenging, thus more in-depth studies are necessary. In this study, we prepared PCV4 Cap which is capable of self-assembling into virus-like particles (VLPs), and further obtained monoclonal antibody 2D3 (mAb 2D3) by immunizing mice with the VLPs. Subsequently, the minimal epitope motif targeted by 2D3 was accurately pinpointed as 179WWISMAD185 at the C-terminus through gradient truncated protein assays and alanine scanning mutagenesis. Furthermore, conservation analysis indicated that this epitope is highly conserved within the PCV4 family, and mAb 2D3 showed no cross-reaction with other unrelated proteins. In addition, homology modeling indicated that this epitope is anchored at the outermost "peak" of the VLPs and fully exposed on their surface. In summary, the high conservation and surface accessibility of this epitope provide a solid theoretical basis for development of PCV4 diagnostic reagents and peptides vaccine, as well as an ideal target for PCV4-based vaccine design.