Indirect ELISA for African swine fever virus serological detection and recombinant porcine reproductive and respiratory syndrome virus-based bivalent vaccine.

African Swine Fever Virus (ASFV) is a highly virulent, hemorrhagic, and highly transmissible pathogen of domestic and wild boars, causing severe economic losses in the global swine industry. To date, no globally recognized, effective vaccines or antiviral therapeutics are available for African swine fever (ASF), making rapid and reliable serological diagnosis a critical component of disease control and prevention strategies. In this study, we developed an indirect enzyme-linked immunosorbent assay (iELISA) based on the ASFV pI215L and pI73R proteins. The corresponding recombinant antigens (rpI215L) and pI73R (rpI73R) were expressed in a prokaryotic system and purified for use as solid-phase coating antigens. When evaluated with 132 clinical swine serum samples and benchmarked against a commercial ID.VET kit, the iELISA demonstrated 98.48% diagnostic accuracy, confirming its robustness for ASFV antibody detection in field samples. In parallel, we constructed recombinant porcine reproductive and respiratory syndrome virus (PRRSV) as live vectors expressing ASFV antigens. Exogenous pI215L and pI73R genes were inserted ORF1b and ORF2a of the attenuated PRRSV vaccine backbone pHuN4-F112, generating rPRRSV-I215L and rPRRSV-I73R, respectively. Virological characterization revealed that these recombinant viruses retained comparable replication kinetics and plaque morphology to the parental strain. Furthermore, animal experiments demonstrated that rPRRSV-I215L elicited simultaneous antibody responses against both PRRSV N protein and ASFV pI215L in immunized pigs. These findings not only provide a reliable and sensitive serodiagnosis tool for ASFV surveillance but also identify promising live vector vaccine candidate for simultaneous immunoprophylaxis against ASFV and PRRS, offering new avenues for integrated disease control in swine populations.