Objective A serological detection method targeting the capsid protein of porcine circovirus type 4 (PCV4) was developed for epidemiological studies on the viral disease.

Method The genome of PCV4 FJ2010001 was used as the PCR amplification template with specific primers designed for the viral capsid protein. Full-length coding sequence of the gene was obtained by PCR to clone it into a pET30a(+) prokaryotic expression system. After purification by affinity chromatography, DS-PAGE and western blotting were used to verify antigenicity of the recombinant protein. A capsid protein detection system applying the optimized conditions of 1.25 μg·mL⁻¹ for antigen encapsulation concentration, 1:200 for serum dilution, and 1:10 000 for working concentration of secondary antibody was established. Retrospectively, 1 195 serum specimens collected from the large-scale pig farms in Fujian Province between 2020 and 2024 were tested using the newly developed assay. Cases of PCV4 co-infecting with PCV2 and/or PCV3 were recorded.

Result Efficient soluble expression induced at 15 ℃ for 16 h was shown by the SDS-PAGE analysis of the recombinant capsid protein with a molecular weight of approximately 28.3 kDa. Western blotting confirmed the single band protein to have specific immunoreactivity with PCV4-positive serum. The methodological validation data proved the detection had no cross-reactivity with sera positive for common swine pathogens (e.g., PRV, PRRSV, etc.). The assay provided a minimum detection threshold at 1:3 200 dilution with coefficients of variation less than 10% both within and between batches. The seroepidemiologic surveys for the past 5 years in the province indicated a total positive rate of PCV4 antibodies of 7.62% (91/1195), which peaked in 2021. The rate of co-infection between PCV4 and PCV2 was 26.37% (24/91), while that between PCV4 and both PCV2 and PCV3 was 14.29% (13/91).

Conclusion The newly established indirect ELISA was highly efficient and applicable for epidemiological studies on the viral diseases caused by PCV2, PCV3, and PCV4.