Monoclonal antibodies to the ORF5 product of porcine reproductive and respiratory syndrome virus define linear neutralizing determinants

Complementary DNA encoding the ORF5 gene of a Quebec reference isolate (IAF-Klop) of porcine reproductive and respiratory syndrome virus (PRRSV) was cloned into the prokaryotic expression vectors pGEX-4T and pET21a to produce ORF5-glutathione S-transferase and ORF5-polyhistidine fusion proteins. Five hybridoma cell lines producing monoclonal antibodies (MAbs) to the 25 kDa viral envelope glycoprotein (GP(5)) were obtained from BALB/c mice immunized with the affinity chromatography-purified GST-ORF5 fusion protein. The polypeptide specificity of these anti-PRRSV MAbs, belonging to the IgG1 isotype, was confirmed by Western immunoblotting assays with recombinant and native viral proteins, and by radioimmunoprecipitation using [S-35]methionine-labelled concentrated extracellular virus. All these MAbs showed virus-neutralizing (VN) activity, with VN titres ranging from 1:32 to 1:128. Two MAbs (IAF-1B8 and IAF-8A8) reacted with similar titres with the modified live attenuated vaccine strain ATCC VR-2332, but all five failed to react to the prototype European strain, the Lelystad virus, in VN and indirect immunofluorescence tests. The results obtained suggest that these five anti-PRRSV MAbs are directed to serotype-specific linear neutralizing epitopes which are not affected by the absence of carbohydrate residues.