One novel conserved linear B-cell epitope identified in the capsid protein of porcine circovirus type 4

Porcine circovirus type 4 (PCV4) is an emerging pathogen that poses a significant potential threat to the global swine industry. The capsid (Cap) protein is the primary target for host immune responses and a critical component for diagnostic and vaccine development. However, the lack of specific monoclonal antibodies (mAbs) and defined antigenic determinants has hindered both diagnostic accuracy and pathogenesis research. In this study, we generated a highly specific, high affinity mAb, designated 6C3 (IgG2b/κ, K_D = 0.29 nM) against the PCV4 dCap protein. This mAb recognizes PCV4 Cap without cross-reactivity toward PCV2, PCV3 or other swine viruses. Epitope mapping identified a minimal linear B-cell epitope spanning residues ¹¹⁹LDGD¹²². Homology modeling predicted that this motif is situated on a surface-exposed random coil loop of the PCV4 Cap. Furthermore, Alanine scanning mutagenesis revealed that residues L¹¹⁹, G¹²¹, and D¹²² were indispensable for antibody binding. Comprehensive sequence alignment revealed that the ¹¹⁹LDGD¹²² motif is completely conserved across all known global PCV4 strains but remains distinct from other PCVs due to pronounced sequence variations at the G¹²¹ and D¹²² positions. The development of mAb 6C3 and the characterization of this novel, highly conserved, and PCV4 specific epitope provide powerful tools for accurate serological differentiation, establishing a robust foundation for targeted diagnostic assays and future vaccine design.