Development of monoclonal antibody for differentiating porcine reproductive and respiratory syndrome virus and identification of a novel non-structural protein 2 epitope peptide.

Porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein (NP) is the immunodominant region of PRRSV viral proteins. Non-structural protein 2 (Nsp2) and its hypervariable region play an essential role in the differential diagnosis of PRRSV. Western blot and immunofluorescence assay (IFA) analyses found that 2 out of 18 monoclonal antibodies (MAbs) recognized the NP and that 5 of 11 MAbs recognized Nsp2-120aa. IFA data demonstrated that 2 MAbs raised against the NP have a positive reaction to PRRSV; either HP-PRRSV, classic PRRSV or the vaccine strain at 1:100 dilution. Two MAbs raise against Nsp2-120aa also react positively with the classic PRRSV nor HP-PRRSV, but not with the PRRSV vaccine strain TJM-F92. Epitope mapping using truncated proteins identified a novel Nsp2-120aa epitope. In addition, we show that MAb BR/PNsp2-2A20 recognizes a 20 amino acid peptide (707) GRFEFLPKMILETPPPHPCG (727) of Nsp2. Based on our findings, we propose that MAb BR/PNsp2-2A20, raised against Nsp2-120aa of PRRSV, as a candidate specific diagnostic MAb for differentiation of the PRRSV virulent strains infected pig from vaccine strain TJM-F92 inoculated ones. The MAbs developed here have potential for use in diagnostic and research tools, including immunofluorescence assay, enzyme-linked immunosorbent assay and Western blotting.

Effect of Nonstructural Protein 2 Hypervariable Regions in the Replication of Porcine Reproductive and Respiratory Syndrome Virus in Marc-145 Cells.

BACKGROUND: Highly pathogenic (HP) porcine reproductive and respiratory syndrome virus (PRRSV) causes prolonged high fever, red discoloration of the body, blue ears and a high mortality. Previously, we found that the PRRSV vaccine strain TJM contained a deletion of 120 amino acids (aa 628-747) in nonstructural protein 2 (Nsp2). We aimed to explore the replication features of PRRSV after adding the transiently expressed product of these 120 aa in vitro. METHODS: We constructed seven eukaryotic expression plasmids containing different parts of the 120-aa sequence, transfected them into Marc-145 cells and then inoculated the cells with 103 TCID50 TJM per well. We detected virus replication at mRNA and protein level by real-time RT-PCR and Western blotting, respectively, and determined the virus titer. RESULTS: The transiently expressed 120 aa and one of its truncated polypeptides inhibited PRRSV TJM propagation on Marc-145 cells. The complete 120-aa sequence induced a remarkable decrease in PRRSV replication, causing a reduction in structural protein levels between 36 and 48 h after infection. Additionally, aa 628-727 partly reduced the replication of PRRSV on Marc-145 cells. CONCLUSIONS: The 120 aa from Nsp2, especially aa 628-727, play a negative role in PRRSV TJM proliferation.