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Peer-reviewedCC BY 4.0Journal of Virology

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

  • Jiwen Sun1,2,3
  • Jian Wang1,3
  • Kun Li1,3
  • Delong Wei1,3
  • Jiaoyang Li1,3
  • Pu Sun1,3
  • Dong Li1,3
  • Pinghua Li1,3
  • Yimei Cao1,3
  • Lijie Tang2
  • Zengjun Lu1,3
  • Jing Zhang1,3
  1. 1State Key Laboratory of Animal Disease Control and Prevention, College of Veterinary Medicine, Lanzhou University, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China
  2. 2College of Veterinary Medicine, Northeast Agricultural University, Harbin, China
  3. 3Gansu Province Research Center for Basic Disciplines of Pathogen Biology, Lanzhou, China
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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 (92LSDSGRISYTVE103) 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 (92LSDSGRISYTVE103) 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.

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Cite

BibTeXRIS
BibTeX
@article{sun2026generation,
  title = {{Generation and characterization of a PRRSV-2 DIVA marker virus by deletion of a novel epitope in the nucleocapsid protein}},
  author = {Jiwen Sun and Jian Wang and Kun Li and Delong Wei and Jiaoyang Li and Pu Sun and Dong Li and Pinghua Li and Yimei Cao and Lijie Tang and Zengjun Lu and Jing Zhang},
  journal = {Journal of Virology},
  year = {2026},
  month = sep,
  doi = {10.1128/jvi.01259-26},
  url = {https://doi.org/10.1128/jvi.01259-26},
  note = {CC BY 4.0},
}
RIS
TY  - JOUR
TI  - Generation and characterization of a PRRSV-2 DIVA marker virus by deletion of a novel epitope in the nucleocapsid protein
AU  - Jiwen Sun
AU  - Jian Wang
AU  - Kun Li
AU  - Delong Wei
AU  - Jiaoyang Li
AU  - Pu Sun
AU  - Dong Li
AU  - Pinghua Li
AU  - Yimei Cao
AU  - Lijie Tang
AU  - Zengjun Lu
AU  - Jing Zhang
PY  - 2026
DA  - 2026/09/24
JO  - Journal of Virology
DO  - 10.1128/jvi.01259-26
UR  - https://doi.org/10.1128/jvi.01259-26
AB  - 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 (92LSDSGRISYTVE103) 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 (92LSDSGRISYTVE103) 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.
SN  - 0022-538X
SN  - 1098-5514
ER  -