Development and application of truncated cap protein-based indirect ELISA for screening antibodies against porcine...

Porcine circovirus type 3 (PCV3) is an emerging pathogen linked with reproductive failure, respiratory disease, dermatitis, nephropathy, and multisystemic inflammation, posing growing threats to the swine industry worldwide. Although molecular evidence of PCV3 circulation has been reported in India, there is no published information on its seroprevalence, leaving the extent of exposure and population immunity largely unknown. Serological tools are critical for epidemiological surveillance and monitoring vaccine responses; however, validated assays for PCV3 remain scarce. In the present study, we developed and evaluated an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of PCV3-specific antibodies, based on a recombinant truncated Cap protein expressed in Escherichia coli. The antigenic fragment of the Cap gene, excluding the N-terminal nuclear localization signal, was used as coating antigen and assay parameters like optimal antigen concentration, serum dilution, and conjugate conditions, were standardized. The developed ELISA exhibited high specificity, with no cross-reactivity to antisera against other common porcine viruses, and the relative diagnostic sensitivity and specificity were estimated to be 97.0 % and 94.5 %, respectively. The assay also demonstrated strong repeatability and reproducibility and was validated at four different laboratories with κ- values indicating perfect agreement Application of the assay to field sera revealed widespread seropositivity (64.25 %), underscoring its utility for epidemiological surveillance and sero-monitoring of PCV3. This study provides the first indigenous ELISA with potential application in India, and is the first report indicating widespread seropositivity of PCV3 in India.