Identification of a monoclonal antibody recognizing a B-cell epitope within the nuclear localization signal of porcine circovirus 4 (PCV4) capsid protein

Porcine circovirus type 4 (PCV4) is an emerging pathogen with a broad geographical distribution, having been detected in both healthy and diseased pigs across China, South Korea, Europe, and Other regions. Similar to PCV2, PCV4 rescued from infectious clones has been shown to be pathogenic in pigs, posing a potential threat to the swine industry. The capsid (Cap) protein, the primary structural and immunogenic component of PCV4, contains a nuclear localization signal (NLS) critical for viral replication. However, the immunogenicity and antigenic epitopes of PCV4 Cap protein remain poorly characterized. In this study, the PCV4 Cap protein was expressed and used to immunize BALB/c mice. Four monoclonal antibodies (mAbs) specific to PCV4 Cap were generated through hybridoma technique and screened by indirect ELISA. Western blot and indirect immunofluorescence assays demonstrated that all four mAbs specifically recognized PCV4 Cap protein expressed in both HEK293T cells and Escherichia coli. Subclass typing revealed that the mAbs possessed IgG1 heavy chains and kappa light chains. Epitope mapping using truncated Cap proteins identified a common B-cell epitope recognized by all four mAbs, located within the NLS region corresponding to amino acid residues 28-43. The mAbs exhibited high specificity for PCV4 Cap, with no cross-reactivity against PCV2 or PCV3 Cap proteins. The generation of these PCV4 Cap-specific mAbs provides valuable tools for developing pathogen-specific detection methods to support epidemiological surveillance of PCV4 and for further investigating NLS-associated viral replication mechanisms, particularly those involving nuclear import and export processes.