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We screened phage display libraries of porcine reproductive and respiratory syndrome virus (PRRSV) protein fragments with sera from experimentally infected pigs to identify linear B-cell epitopes that are commonly recognized during infection in vivo. We identified 10 linear epitope sites (ES) 11 to 53 amino acids in length. In the replicase polyprotein, a total of eight ES were identified, six of which localized to the Nsp2 replicase polyprotein processing end product. In the structural proteins, a total of two ES were identified, in the ORF3 and ORF4 minor envelope glycoproteins. The ORF4 ES was previously identified by monoclonal antibody mapping (J. J. M. Meulenberg, A. P. van Nieuwstadt, A. van Essen-Zandenbergen, and J. P. M. Langeveld, J. Virol. 71:6061-6067, 1997), but its immunogenicity had not been examined in pigs. We found that six experimentally PRRSV-infected pigs consistently had very high antibody titers against the ORF4 ES. In some animals, sera diluted 1:62,500 still gave weak positive enzyme immunoassay reactivity against the ORF4 ES. This hitherto unrecognized immunodominance likely caused phages displaying the ORF4 ES to outcompete phages displaying other ES during library screening with porcine sera and accounted for our failure to identify more than two ES in the structural genes of PRRSV. Genetic analysis showed that variable ES were also the most immunogenic in vivo. Serological analysis indicated differences in the immunoglobulin A responses between short-term and longer-term viremic pigs towards some ES. The implications of these findings for PRRSV diagnostics and immunopathogenesis are discussed.Porcine reproductive and respiratory syndrome virus (PRRSV) is a recently emerged pathogen of domesticated swine. The virus, which belongs to the Arteriviridae family, has a 15-kb positive-sense, single-stranded RNA genome. PRRSV encodes an approximately 4,000-amino-acid large replicase polyprotein (open reading frame [ORF] 1a and 1b) and six structural proteins of 130 to 265 amino acids (ORFs 2 to 7) (reviewed in references 6, 36, and 45). The replicase polyprotein is processed by autoproteolytic cleavage into nonstructural protein fragments (Nsps). The replicase polyprotein processing cascade has recently been reviewed by Ziebuhr et al. (45), and the Nsp and protease domain nomenclature suggested by Ziebuhr et al. is used throughout this article. Two main PRRSV genotypes exist, the American (US) and European (EU) types, which are only approximately 60% identical at the nucleotide level. For reasons currently not understood, these two distantly related PRRSV types emerged virtually simultaneously on their respective continents in the late 1980s. Since then, intermingling of the genotypes has occurred through the use of a live, US-type PRRSV vaccine in Europe (2, 18).PRRSV infection poses a challenge to current serodiagnostic and vaccination strategies. Although live PRRSV vaccines provide protection against homologous challenge, the genetic diversity of field PRRSV isolates is very high, and v...

The nucleocapsid protein of the European genotype of porcine reproductive and respiratory syndrome virus (type 1, PRRSV-1) exhibited extensive size polymorphism (124-130 amino acids), correlating with phylogenetic grouping of ORF7 as well as ORF5 nucleotide sequences, thereby validating ORF7 size as an independent PRRSV-1 subtype marker. Based on new sequence information from the Russian Federation, we propose division of European genotype PRRSV-1 into 3 subtypes: a pan-European subtype 1 and East European subtypes 2 and 3, with nucleocapsid protein sizes of 128, 125 and 124 amino acids, respectively. The genetic differences between European genotype PRRSV subtypes affected diagnostic RT-PCR primer binding sites. Using Escherichia coli-expressed ORF7 protein, we confirmed that even the relatively closely related PRRSV subtypes 2 and 3 were antigenically different. Finally, the isoelectric point (pI) correlated with the nucleocapsid protein size for European genotype PRRSV subtypes, suggesting subtype-specific compensatory structural changes associated with subtype-specific ORF7 sizes. Thus, the new ORF7-based subtype division of PRRSV-1 proposed here is biologically meaningful and practically relevant.

Foot-and-mouth disease (FMD) is a highly contagious

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