Veterinaria

VIRAL IMMUNOLOGY Volume 19, Number 3, 2006 © Mary Ann Liebert, Inc. Pp. 383–390

Identification of a Conserved Epitope Cluster in the N Protein of Porcine Reproductive and Respiratory Syndrome Virus
YAN-JUN ZHOU, TONG-QING AN, JIN-XIA LIU, HUA-JI QIU, YUN-FENG WANG, and GUANG-ZHI TONG

ABSTRACT In this study, 4 overlapping fragments and 12 overlapping peptides of the nucleocapsid (N) proteinfrom porcine reproductive and respiratory syndrome virus (PRRSV) were expressed as glutathione S-transferase (GST) fusion proteins and used to probe a panel of 16 anti-N protein monoclonal antibodies (mAbs) by ELISA. The minimal epitope sequence of the following seven mAbs was determined by sequential deletion of terminal amino acid residues from each peptide: N2H7 corresponded to H54FPLA58; N2F7corresponded to K52PHFPLA58; and N1A2, N1E3, N1G4, and N2E5 were reactive against E51KPHFP56. Furthermore, a polypeptide containing this epitope cluster was recognized by PRRSV-immune pig serum by Western blot, suggesting that residues 51–58 represent an immunodominant region of the N protein. Sequence alignment revealed that these epitopes are well conserved among North American and Europeangenotypes of PRRSV. These findings enhance our knowledge of the antigenic structure of N protein and may facilitate the development of better diagnostic methods for PRRSV.

INTRODUCTION

P

ORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME

(PRRS), characterized by reproductive failure of pregnant sows and respiratory distress of piglets and juvenile pigs, continues to be one of the mosteconomically important diseases in the pig-farming industry worldwide since its emergence in North America in 1987 (1). Eight years later, in 1995, the PRRSV CH-1a strain became the first isolate from China (2). On the basis of nucleotide sequence similarity and antigenicity, CH-1a was assigned to the North American type. While this virus continues to be an economic burden, development of a safe andeffective vaccine has been hampered by a variety of factors, including the high degree of variation of this virus (3–5), persistent viral infection (6), effects of antibody-

dependent enhancement (ADE) (7), and the fact that the primary permissive cells for infection are of the monocytic lineage (8,9). PRRSV nucleocapsid (N) protein, encoded by ORF7, is the most abundant protein in the virion,accounting for 20–40% of the total protein content (10). N protein is a 15-kDa, nonglycosylated protein composed of 123 or 128 amino acids in the North American and European type isolates, respectively. The N terminus is presumed to play a role in the interaction with genomic viral RNA because of the high composition of basic amino acids, such as lysine and arginine (4,11). At the C terminus, the final11 residues have been shown to be necessary for maintaining proper tertiary structure of this protein (12,13). During the process of viral replication of both the North American and European isolates, N protein is lo-

National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People’s Republic of China.

383384 cated in the cytoplasm and nucleolus of porcine alveolar macrophages (PAMs) and Marc-145 cells (14,15). Because antibodies generated against N protein develop rapidly and persist for a long time, it is a preferred antigen for diagnosis. Defining epitopes of the N protein will prove useful not only for understanding its antigenic structure, but also to further the establishment a diagnosticmethod based on multiple epitopes. In the present study, we have identified a cluster of epitopes within the PRRSV nucleocapsid protein, using a panel of monoclonal antibodies (mAbs) and overlapping peptide series.

ZHOU ET AL. or BL21(DE3) (for the pET-30a vector) cells, followed by the addition of 1 mM isopropyl-D-thiogalactopyranoside (IPTG; Pharmacia Biosciences) for induction. Each...