Localization and fine mapping of antigenic sites on the nucleocapsid protein N of porcine reproductive and respiratory syndrome virus with monoclonalantibodies

The purpose of this study was to analyze the antigenic structure of the nuc leocapsid protein N of the Lelystad virus isolate of porcine reproductive a nd respiratory syndrome virus (PRRSV) and to identify antigenic differences between this prototype European isolate and other North American isolates. To do this, we generated a panel of monoclonal antibodies (mAbs) directed against the N protein of Lelystad virus and tested them in competition assa ys with other N-specific mAbs described previously (Drew et al., 1995; Nels on et ai., 1993; van Nieuwstadt el ai., 1996). Four different competition g roups of mAbs were identified. Pepscan analysis with solid-phase dodecapept ides was used to identify specific antigenic regions in the N protein that were hound by the mAbs. In this pepscan analysis, we found that the mAb of the first competition group reacted with linear peptides whose core sequenc es consisted of amino acids 2-12 (site A), the mAbs of the second group rea cted with peptides whose core sequences consisted of amino acids 25-30 (sit e B), and the mAb of the third group reacted with peptides whose core seque nces consisted of amino acids 40-46 (site C). However, the fourth group of mAbs binding to an antigenic region, provisionally designated as domain D, reacted very weakly or did not react at all with solid-phase dodecapeptides . To further characterize the structure of the epitopes in domain D, we pro duced chimeric constructs composed of the N protein sequences of Lelystad v irus and another arterivirus lactate dehydrogenase-elevating virus, which w as used because its N protein has similarity in amino acid sequence and hyd ropathicity profile but does not react with our mAbs. When the mAbs specifi c to domain D were tested for binding to the chimeric N proteins expressed by semliki Forest virus, we found that the regions between amino acids 51-6 7 and amino acids 80-90 are involved in the formation or are part of the ep itopes in domain D. Therefore, we conclude that the N protein contains four distinct antigenic regions. The epitopes mapped to sites A-C are linear, w hereas the epitopes mapped to domain D are more conformation dependent or d iscontinuous. Sites A and C contain epitopes that are conserved in European but not in North American isolates; site B contains epitopes that are cons erved in European and North American isolates; and site D contains epitopes that are either conserved or not conserved in European and North American isolates. The antigenic regions identified here might be important for the development of diagnostic test for PRRSV in particular tests that discrimin ate between different antigenic types of PRRSV. (C) 1998 Academic Press.