Abstract

The nonstructural protein 4 (Nsp4) of porcine reproductive and respiratory syndrome virus (PRRSV), a 3C-like serine protease, functions significantly in viral polyprotein cleavage and immune evasion. We constructed eight hybridoma cell lines stably secreting monoclonal antibodies (mAbs) against the Nsp4 protein in this study. Furthermore, eight novel antigenic epitopes within Nsp4 were identified using a panel of expressed overlapping truncated proteins. Among these, the epitopes recognized by mAbs 10G5 and 1C11, corresponding to amino acid sequences 49 SGVGFNQ 55 and 123 ITEAGELV 130 , were highly conserved in PRRSV-2, which was applicable for PRRSV-2 strain detection. MAb 8H2 and 10E11 targeted the linear epitope 73 WQGVAPK 79 . Notably, three mAbs targeting epitopes within 123–142aa can simultaneously recognize both PRRSV-1 and PRRSV-2 strains, indicating their potential for broad detection across PRRSV types. Furthermore, mAb 1C11 enabled Nsp4 expression detection during the early stage of PRRSV infection. This mAb also exhibited a blocking effect against PRRSV-positive sera, suggesting the participation of Nsp4 in the humoral immune response of the host early in infection. Additionally, mAb 1C11 could suppress PRRSV replication in vitro . To sum up, the present study provide robust evidence to understand the structural and antigenic properties of the PRRSV Nsp4 protein, providing potential reference for developing Nsp4-specific early antigen detection methods.