Monoclonal antibody and porcine antisera recognized B-cell epitopes of Nsp2 protein of a Chinese strain of porcine reproductive and respiratory syndrome virus

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important pathogens for swine industry. The non-structural protein 2 (Nsp2) is considered to be one of the immunogenic proteins of PRRSV. In this study, the B-cell epitopes of the Nsp2 protein of a North American type Chinese strain PRRSV BJ-4 were identified on a prokaryotic expressed Nsp2 fragment (73–567aa). A total of six monoclonal antibodies (mAbs) recognizing different epitopes on the expressed protein were prepared. All six mAbs exhibited immunoreactivity with the denatured Nsp2 protein in Western blotting and produced strong perinuclear staining in PRRSV infected MARC-145 cells in an immunofluorescence assay. Pepscan analysis revealed six distinct linear epitopes for the six mAbs, respectively, and of which four were identified to be novel linear Nsp2 B-cell epitopes: T⁷³LPERVRPPDDWAT⁸⁶, D³⁸⁵ELKDQMEED³⁹⁴, P⁴⁵²VPAPRRKVGSDCGS⁴⁶⁶, and P⁴⁶⁷VSLGGDVPNS⁴⁷⁷. All of the six mAb specific peptides could be recognized by porcine PRRSV antiserum, indicating that the epitopes involving these synthetic peptides were immunogenic and immunodominant during PRRSV infection in pigs. Our results provided valuable information for developing novel PRRSV vaccines using the Nsp2 epitopes as potential serological markers.

Introduction

Porcine reproductive and respiratory syndrome (PRRS) has become one of the most economically important infectious diseases for swine industry worldwide (Neumann et al., 2005, Pejsak et al., 1997) since its first recognition as a “mystery swine disease” in the United States in 1987 (Keffaber, 1989). Isolation and characterization of PRRS virus (PRRSV), the causative agent, was almost simultaneously reported in Europe and the United States (Christianson et al., 1992, Wensvoort et al., 1991). The disease is predominantly characterized by reproductive failure in breeding swine, pre-weaning mortality, and respiratory disorders in pigs of all ages, especially in suckling and nursery-age pigs, or influenza-like illness in growing and finishing pigs (Albina, 1997, Hopper et al., 1992, Pejsak et al., 1997, Rossow, 1998).

PRRSV, together with equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV), and simian hemorrhageic fever virus (SHFV), is a member of the family Arteriviridae in the order Nidovirales (Cavanagh, 1997). The genome of PRRSV is a single-stranded, non-segmented, positive-sense and polyadenylated RNA of approximately 15 kb in length that contains nine open reading frames (ORF1a, ORF1b, ORF2a, ORF2b and ORFs3 through 7) (Conzelmann et al., 1993, Meulenberg et al., 1993, Meulenberg et al., 1997, Murtaugh et al., 1995, Snijder and Meulenberg, 1998, van Nieuwstadt et al., 1996). Two large ORFs (ORF1a/ORF1b) comprise 80% of the genome size and encode a viral replicase polyprotein which is predicted to be autoproteolytically cleaved at 12 sites, producing 13 non-structural proteins ultimately (Allende et al., 1999, Bautista et al., 2002, den Boon et al., 1995, Snijder et al., 1995, Snijder et al., 1996, van der Meer et al., 1998, van Dinten et al., 1996). The other seven ORFs are located in the 3′-terminus of the genome and encode the viral structural proteins (GP2, E, GP3, GP4, GP5, M and N) (Mardassi et al., 1994, Meulenberg et al., 1995, Nelsen et al., 1999, Snijder and Meulenberg, 1998, Wu et al., 2005).

The non-structural protein 2 (Nsp2) of PRRSV possesses a chymotrypsin-like (or picornavirus 3C-like) cysteine protease domain (Dougherty and Semler, 1993). Many studies have been focused on the functions of this protein (Allende et al., 2000, Bautista et al., 2002, den Boon et al., 1995, Pedersen et al., 1999, Snijder et al., 1995, Snijder et al., 2001, van der Meer et al., 1998, Wassenaar et al., 1997, Welch et al., 2004, Ziebuhr et al., 2000). Previous data related to the genomic sequence and putative amino acid sequence of Nsp2-coding region indicated that Nsp2 sequence is highly variable (Allende et al., 1999, Fang et al., 2004, Gao et al., 2004, Ropp et al., 2004, Shen et al., 2000, Wootton et al., 2000). Meanwhile, interestingly, the Nsp2 protein was found to contain a large number of linear B-cell epitopes in both the European type and North American type PRRSV strains (de Lima et al., 2006, Oleksiewicz et al., 2001), indicating that Nsp2 protein of PRRSV is an immunogenic protein capable of eliciting specific antibody production during viral infections. Therefore, it is essential to further understand the epitopes and the functions of Nsp2 protein. Previous studies have demonstrated that Chinese PRRSV strains were related to the North American genotype (Gao et al., 2004, Yang et al., 2001) and the strain with unique deletions within Nsp2-coding region was found (Gao et al., 2004). However, data about the epitopes of Chinese PRRSV strains are scarce. It is unknown whether the Chinese and the North American PRRSV strains differ in Nsp2 epitopes. In this paper, we first expressed a variable domain fragment of a Chinese PRRSV Nsp2 protein, then prepared monoclonal antibodies (mAbs) against the protein fragment, and then analyzed by Pepscan approach the Nsp2 epitopes recognized by the mAbs and PRRSV antisera.