title: Localization and fine mapping of antigenic sites on the nucleocapsid protein N of porcine reproductive and respiratory syndrome virus with monoclonal antibodies.
abstract: The purpose of this study was to analyze the antigenic structure of the nucleocapsid protein N of the Lelystad virus isolate of porcine reproductive and 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 assays with other N-specific mAbs described previously (Drew et al., 1995; Nelson et al., 1993; van Nieuwstadt et al., 1996). Four different competition groups of mAbs were identified. Pepscan analysis with solid-phase dodecapeptides was used to identify specific antigenic regions in the N protein that were bound by the mAbs. In this pepscan analysis, we found that the mAb of the first competition group reacted with linear peptides whose core sequences consisted of amino acids 2-12 (site A), the mAbs of the second group reacted with peptides whose core sequences consisted of amino acids 25-30 (site B), and the mAb of the third group reacted with peptides whose core sequences 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 produced chimeric constructs composed of the N protein sequences of Lelystad virus and another arterivirus lactate dehydrogenase-elevating virus, which was used because its N protein has similarity in amino acid sequence and hydropathicity profile but does not react with our mAbs. When the mAbs specific 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 51-67 and amino acids 80-90 are involved in the formation or are part of the epitopes in domain D. Therefore, we conclude that the N protein contains four distinct antigenic regions. The epitopes mapped to sites A-C are linear, whereas the epitopes mapped to domain D are more conformation dependent or discontinuous. Sites A and C contain epitopes that are conserved in European but not in North American isolates; site B contains epitopes that are conserved 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 discriminate between different antigenic types of PRRSV.
authors.affiliation: Institute for Animal Science and Health, Lelystad, The Netherlands. J.J.M.Meulenberg@id.dlo.nl
authors.forename: J J
authors.initials: JJ
authors.lastname: Meulenberg
authors.forename: A P
authors.initials: AP
authors.lastname: van Nieuwstadt
authors.forename: A
authors.initials: A
authors.lastname: van Essen-Zandbergen
authors.forename: J N
authors.initials: JN
authors.lastname: Bos-de Ruijter
authors.forename: J P
authors.initials: JP
authors.lastname: Langeveld
authors.forename: R H
authors.initials: RH
authors.lastname: Meloen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D004279:DNA, Viral / Q000737:chemistry; D018604:Epitope Mapping / Q000379:methods; D051379:Mice; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D010449:Peptide Mapping / Q000379:methods; D019316:Porcine respiratory and reproductive syndrome virus / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D004279:DNA, Viral; D019590:Nucleocapsid Proteins; D011993:Recombinant Fusion Proteins