Epitope Mapping Porcine Reproductive and Respiratory Syndrome Virus by Phage Display: the nsp2 Fragment of the Replicase Polyprotein Contains a Cluster of B-Cell Epitopes

Abstract

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...

C-Terminal nsP1a Protein of Human Astrovirus Colocalizes with the Endoplasmic Reticulum and Viral RNA

“…Most of the proteins from other viral families that showed sequence similarity to HAstV nsP1a/4 belonged to singlestranded RNA viral families and were related to the formation of the viral replication complex, to the regulation of viral replication, and/or to transcription (8,9,10,26,30,31,42,43,45,46,49,50,51,52,54,55,57,58,61). This observation provides additional support to the idea that nsP1a/4 protein may be involved in RNA replication.…”

“…This observation provides additional support to the idea that nsP1a/4 protein may be involved in RNA replication. Of note, added to sequence similarity, HAstV nsP1a/4 protein shared some other features with all these proteins, such as, for example, a high degree of tolerance to insertions and deletions (51,54), the occurrence of phosphorylation sites (30,46,52,55) and immunogenic sequences (42), and intracellular colocalization with viral replication sites (31,49).…”

Identification of Neutralizing and Nonneutralizing Epitopes in the Porcine Reproductive and Respiratory Syndrome Virus GP5 Ectodomain

“…In contrast, swine HI sera did not react with the mimotope recognized by MAb ISU25-C1 but instead reacted with the native sequence deduced from PRRSV GP5. Differences in recognition by mouse and pig antibodies have been reported before (25). It is not surprising since swine antibodies are limited to the usage of the products of only one family of variable heavy genes which are homologous to the 7183 VH family of mouse antibodies (4) whereas MAb ISU25-C1 uses the most distant J558 VH family of antibodies (unpublished results), confirming that epitopes can only be defined in terms of their complementary paratopes (36).…”

“…period (16). Immunodominant epitopes in PRRSV structural and nonstructural proteins have been characterized (20,25,26,31). However, to date, there has been no molecular characterization of PRRSV neutralizing epitopes present in GP5.…”

Characterization of Emerging European-Like Porcine Reproductive and Respiratory Syndrome Virus Isolates in the United States

“…NSP2 has been reported to be the most variable part of the genome (11,61), and the ORF 1a sequence of EuroPRRSV was consistent with that notion, displaying a 17-aa deletion within the NSP2 protein, the only deletion or insertion observed in relation to the LV genomic sequence. Although NSP2 has been shown to be variable in type 2 viruses, this is the first instance of variation in this region for type 1 viruses and suggests that this PRRSV protein is uniformly unstable, perhaps due to immunologic pressure, as NSP2 was previously shown to contain a cluster of B-cell epitopes (47). The protein is well conserved at residues thought to be important in proteolytic cleavage events but differs in downstream amino acids.…”