Generation and characterization of a PRRSV-2 DIVA marker virus by deletion of a novel epitope in the nucleocapsid protein

ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) infection causes enormous economic losses to the world pig industry. Currently, there is no DIVA (differentiating infected from vaccinated animals) vaccine for PRRS available on the market. In this study, we successfully produced a porcine monoclonal antibody (C8) against PRRSV nucleocapsid (N) protein using fluorescence-based single-B-cell antibody technology. The C8 monoclonal antibody (mAb) recognizes a novel linear epitope ( 92 LSDSGRISYTVE 103 ) at the C terminus of genotype 2 PRRSV N protein. We developed a competitive enzyme-linked immunosorbent assay for detecting PRRSV N protein antibodies against this epitope using C8 mAb. Furthermore, we successfully rescued one PRRSV mutant, rGSWW15/N∆93. In vitro and in vivo assays showed that the mutant virus replicated efficiently and elicited a specific antibody response to PRRSV as measured by a commercial ELISA, but not to the specific epitope ( 92 LSDSGRISYTVE 103 ) detected by our competitive ELISA (cELISA). Our findings suggest that the recombinant virus, in combination with the cELISA diagnostic tests, enables serological differentiation between animals vaccinated with the marker virus and those infected with the wild-type virus. This study demonstrates the feasibility of developing a PRRSV DIVA vaccine through targeted modification of an N protein epitope, providing a potential strategy to facilitate PRRS control. IMPORTANCE The development of a PRRSV DIVA vaccine is crucial for effectively managing outbreaks in swine populations. DIVA vaccines allow differentiation between vaccinated and naturally infected animals, aiding disease surveillance. In this study, we identified a novel linear epitope (92LSDSGRISYTVE103) on the genotype 2 PRRSV N protein and developed the porcine monoclonal antibody C8 targeting this epitope. A competitive ELISA (cELISA) was then created to detect PRRSV N protein antibodies specific to the C8 epitope. Additionally, we successfully generated an epitope deletion mutant, rGSWW15/N∆93. While in vivo assessments confirmed specific antibody responses via commercial ELISA, these antibodies did not recognize the C8-specific epitope identified in our cELISA. These findings suggest that combining the recombinant virus with cELISA diagnostic tests allows accurate differentiation between vaccinated and wild-type virus-infected animals.