VIRAL IMMUNOLOGY Volume 19, Number 3, 2006 Mary Ann Liebert, Inc. Pp.

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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) protein from 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; N2F7 corresponded 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 5158 represent an immunodominant region of the N protein. Sequence alignment revealed that these epitopes are well conserved among North American and European genotypes 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

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 most economically 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 and effective vaccine has been hampered by a variety of factors, including the high degree of variation of this virus (35), 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 2040% 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 final 11 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, Peoples Republic of China.

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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 diagnostic method 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 recombinant protein (HisN, glutathione S-transferase [GST]M, GSTGP5, HisGP4, GSTGP3, and GSTGP2) was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE).

Generation of mAbs
PRRSV was cultivated in Marc-145 cells and then semipurified by ultracentrifugation through a sucrose cushion as described by Yang et al. (16). BALB/c mice were immunized with PRRSV to produce hybridomas according to the method of Yang et al. (16) and Zhang et al. (17). Hybridoma culture supernatants were screened, using PRRSV whole virus-coated plates and an enzymelinked immunosorbent assay (ELISA) kit (IDEXX Laboratories, Westbrook, ME). This screen resulted in the selection of a total of 16 mAbs for further study. To assess specificity, six His- or GST-tagged recombinant proteins (HisN, GSTM, GSTGP5, HisGP4, GSTGP3, and GSTGP2) were expressed in E. coli and tested for reactivity against each monoclonal antibody by ELISA. Briefly, ELISA plates were coated overnight with cell lysate (5 g/well), containing approximately 1 g of recombinant protein, and blocked for 2 h with 4% nonfat milk at 37C, followed by three washes with phosphatebuffered salineTween 20 (PBST). Plates were then incubated with hybridoma supernatant at 37C for 1 h. Bound mAbs were detected with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Sigma, St. Louis, MO), and developed with o-phenylenediamine dihydrochloride (OPD; Sigma). Each sample was analyzed in triplicate.

MATERIALS AND METHODS Virus and sera

PRRSV CH-1a strain (GenBank accession no. AY032626) was maintained in our laboratory. PRRSV CH-1a-positive pig sera were kindly provided by the PRRSV research team of the Harbin Veterinary Research Institute (Chinese Academy of Agricultural Sciences, Harbin, Peoples Republic of China).

Construction of expression vectors

On the basis of the analysis of signal peptides, transmembrane regions, and domains with high hydrophilicity or antigenicity among all the structural proteins of PRRSV CH-1a, four truncated genes, tORF2, tORF3, tORF5, and tORF6 (lacking the signal sequence and highly hydrophobic domain), were prepared by reverse transcription-polymerase chain reaction (RT-PCR) and inserted into the expression vector pGEX-6p-1 (Amersham Biosciences, Uppsala, Sweden). Two full-length genes, ORF4 and ORF7, were cloned into the expression vector pET-30a (Novagen, Darmstadt, Germany). All the resulting recombinant plasmids were validated by restriction analysis and nucleotide sequencing. For recombinant protein expression, plasmids were transformed into Escherichia coli DH5 (for the pGEX-6p-1 vector)
TABLE 1. RT-PCR PRIMERS

Broad epitope mapping with overlapping N protein fragments

Using the analysis software DNAStar (DNASTAR, Madison, WI), the ORF7 gene was divided into four overORF7 FRAGMENTS Position

FOR

AMPLIFICATION

OF

Name ORF7-N1 ORF7-N2 ORF7-N3 ORF7-N4 5 5 5 5 5 5 5 5

Sequences of PCR primers -TTGGAATTCATGCCAAATAACAACG-3 -GTTGTCGACTCAGATGATCTTACC-3 -GGCGAATTCATGAATCAGCTGTGC-3 -CAAGTCGACTCAACTAGGGGTGAAG-3 -AATGAATTCATGAGCCCGGAGAAG-3 -CCCGTCGACTCACAGGGTACAAGT-3 -TTGGAATTCATGTCGATCCAGACT-3 -TACCGTCGACTCATGCTGAGGGTG-3

In genome 1488114968 1493515087 1502215154 1510915248

In N protein 130 2070 4991 78123

EPITOPE CLUSTER IN N PROTEIN OF PRRSV

TABLE 2. Name NEP1 NEP2 NEP3 NEP3-1 NEP3-2 NEP3-3 NEP3-4 NEP3-5 NEP3-6 NEP3-7 NEP3-8 NEP3-9 AMINO ACID SEQUENCE Sequences of peptide SPEKPHFPLATEDDVRHHFTPS TEDDVRHHFTPS SPEKPHFPLA PEKPHFPLA EKPHFPLA SPEKPHFPL SPEKPHFP KPHFPLA KPHFPL PHFPLA HFPLA EKPHFP
AND

385
POSITION
OF

FUSION PEPTIDES USED

IN

STUDY Size (amino acids) 22 12 10 9 8 9 8 7 6 6 5 6

Positions of amino acids 4970 5870 4948 5058 5158 4957 4956 5258 5257 5358 5458 5156

lapping fragments for amplification by PCR (Table 1). These proteins were then used to coat the wells of ELISA plates at a concentration of 5 g/well, and then probed with anti-N mAbs as described above.

Intermediate-level epitope mapping with polypeptides

On the basis of the epitope analysis using larger fragments of the N protein described above, the overlapping region of fragments ORF7-N2 and ORF7-N3 (residues 4970) was divided into three additional fragments (NEP1, NEP2, and NEP3), described in Table 2. To express these polypeptides, complementary oligonucleotide pairs encoding each peptide were synthesized, annealed, and cloned into the BamHI (TaKaRa, Dalian, China) and XhoI (TaKaRa) sites of pGEX-6p-1, resulting in three recombinant plasmids. GST fusion proteins were expressed and used to screen the mAbs by ELISA as described above.

GSTN2, GSTN3, GSTN4, GSTNEP3, and GST were resolved by SDSPAGE and electrophoretically transferred to nitrocellulose membrane. After blocking with 5% nonfat milk in PBS overnight at 4C, the membrane was incubated with PRRSV-positive pig serum (diluted 1:100 in PBS) at 37C for 1 h, washed three times with PBST, and probed with a 1:5000 dilution of HRPconjugated rabbit anti-pig IgG (Sigma) at 37C for 1 h. Reactivity was visualized with the substrate diaminobenzidine (DAB; Sigma).

RESULTS Development of mAbs

Sixteen mAbs were generated by screening hybridoma supernatants against PRRSV antigen by ELISA. Crossreactivity with other structural proteins (GP2GP5 and M) was assessed. Results showed that the mAbs were all directed against the N protein, and that none of them could react with GP2GP5 or M protein. In addition, the mAbs were demonstrated by immunofluorescence assay (IFA) to be well recognized by Marc-145 cells infected with PRRSV CH-1a (data not shown).

Precise location of the epitopes

To finely map the minimal epitopes of the N protein, the reactive polypeptide identified above was truncated by deleting amino acids one by one from both ends (Table 2). Construction of recombinant vectors, fusion expression of shorter peptides, and ELISAs were performed as described above. On the basis of the ELISA results, the reactive peptides were retruncated by deleting amino acids one by one (Table 2) and then subjected to ELISA. The procedure was repeated until the smallest functional unit recognized by each mAb was identified.

Expression and identification of epitopecontaining N protein fragments

Four overlapping N protein gene fragments, ORF7-N1, ORF7-N2, ORF7-N3, and ORF7-N4, were prepared by RT-PCR and cloned into a GST fusion protein expression vector. After validation by restriction analysis and nucleotide sequencing, recombinant proteins encoded by each of these constructs were expressed in E. coli. The resulting recombinant proteins were used in an ELISA to identify the reactivity of each of the 16 anti-N mAbs. Seven of the mAbs (N1A2, N1G4, N1E3, N2E5, N1H11,

Reactivity with immune serum

To investigate whether antibodies against these recombinant N protein fragments can be found in PRRSVpositive pig serum, GST fusion proteins GSTN1,

386 N2F7, and N2H7) were found to react with both ORF7N2 and ORF7-N3 (data not shown), whereas other mAbs reacted only with complete N protein, and none show cross-reactivity with GST.

ZHOU ET AL. (Fig. 3). This suggests that the epitope cluster defined in the present study is immunodominant in PRRSV-infected pigs.

Mapping of minimal epitopes

The overlapping region (residues 4970) of fragments ORF7-N2 and ORF7-N3 was then further divided into three fragments (NEP1NEP3). When the panel of mAbs was probed with the NEP1NEP3 recombinant polypeptides, six were found to react with NEP3 (residues 4958). In contrast, mAb N1H11 reacted only weakly with NEP-1, a fragment consisting of N protein residues 4970, but strongly with intact N protein, suggesting that this region contains at least part of the epitope recognized by N1H11 (Fig. 1). To more precisely define the minimal epitopes recognized by these mAbs, the NEP3 polypeptide was further truncated from both ends (NEP31NEP-9) (Table 2). The minimal antigenic determinants recognized by mAbs N2H7 and N2F7 were found to be H54FPLA58 and K52PHFPLA58, respectively. Monoclonal antibodies N1A2, N1E3, N1G4, and N2E5 reacted against a third distinct epitope comprising E51KPHFP56 (Fig. 2).

Epitope sequence is well conserved among diverse isolates

Sequence alignment of the region encompassing the three antigenic epitopes demonstrated that this sequence is well conserved among 53 isolates of PRRSV (including both North American and European genotypes), with the notable exception of a single amino acid substitution in the Canadian isolate PA-8 (P56 for CH-1a and L56 for PA-8) (Fig. 4).

DISCUSSION
Monoclonal antibodies have been employed to precisely map the antigenic epitopes of a variety of infectious agents, including PRRSV. Previously, epitopes were discovered in several European and North American type isolates of this virus. The work of Yang et al. identified 8 epitopes recognized by 36 mAbs directed against the N protein from ISU-P or KY35 strains. Five of these epitopes were found to be linear (EPORF7-A through EPORF7-E), whereas others were conformational epitopes (EPORF7-Fd, EPORF7-Gd, and EPORF7-Hd); however, the positions of these epitopes within the N protein sequence were not described (16,18). Another group, Wootton et al., using mAbs against the N protein, identified four linear epitopes in the PA-8 strain, which were located at residues

Epitope cluster is recognized by PRRSV-positive serum

Western blot analysis indicated that fragments ORF7N2, ORF7-N3, and NEP3 were recognized by PRRS-positive pig sera, whereas ORF7-N1 and ORF7-N4 were not

1.4 1.2 1 0D490 nm 0.8 0.6 0.4 0.2 0 NEP1 NEP2 NEP3 GST N N1A2 N1E3 N1G4 N1H11 N2E5 N2F7 N2H7

FIG. 1. Reactivity of mAbs against NEP1NEP3 fragments by ELISA. Reactivity of seven mAbs to NEP1NEP3 peptides was tested by an indirect ELISA. Briefly, ELISA plate wells were coated with GST-conjugated NEP1NEP3, full-length N protein, or GST, followed by blocking with nonfat milk, and then incubated with hybridoma supernatant. Bound mAbs were detected with HRP-conjugated goat anti-mouse IgG. Each sample was analyzed in triplicate. All seven mAbs shown reacted with NEP1 and NEP3, as well as with the complete N protein; however, none reacted with NEP2. As anticipated, each of the mAbs reacted with full-length N protein (N), whereas none showed cross-reactivity to GST.

EPITOPE CLUSTER IN N PROTEIN OF PRRSV

1.8 1.6 1.4 1.2 0D490 nm 1 0.8 0.6 0.4 0.2 NEP3-1 NEP3-2 NEP3-3 NEP3-4 NEP3-5 NEP3-6 NEP3-7 NEP3-8 NEP3-9 NEP3 GST 0 N1A2 N1E3 N1G4 N2E5 N2F7 N2H7

387

FIG. 2. Reactivity of mAbs to peptides NEP3-1NEP3-9 by ELISA. Overlapping peptides NEP3-1NEP3-9, generated by serial amino acid deletion from both termini, were coated onto ELISA plates and probed with a panel of six mAbs, followed by addition of HRP-conjugated goat anti-mouse IgG, and then developed with OPD solution. Each sample was analyzed in triplicate. mAb N2H7 reacted with peptides NEP3-1, NEP3-2, NEP3-5, NEP3-7, and NEP3-8; N2F7 reacted with peptides NEP3-1, NEP3-2, and NEP3-5; whereas each of the other four mAbs N1A2, N1E3, N1G4, and N2E5 reacted with peptides NEP3-1NEP34, and with NEP3-9.

3052, 3752, 69123, and 112123, as well as one conformational epitope composed of amino acids 5269 and 112123 (13). Meulenberg et al. contributed an additional three linear epitopes (residues 212, 2530, and 4046) and one conformational epitope utilizing residues 5167 and 8090, from European isolates. Of the Meulenberg epitopes, residues 2530 are highly conserved in all European and North American isolates, whereas the residue 212 and 4046 epitopes are conserved in European, but not North American, types. The conformational epitope, composed of residues 5167 and 8090, is not conserved in either the European or American isolates (19). In the present study, we immunized mice with purified PRRS virion of the CH-1a strain, which belongs to the North American type (5,20,21). We found that all 16 mAbs generated in this study were directly against the N protein. Initially we used four overlapping recombinant fragments to broadly map the epitope-containing regions of the N protein. Seven of the 16 mAbs reacted with 2 of these recombinant fragments, ORF7-N2 and ORF7N3, but other antibodies recognized only full-length N protein by ELISA, suggesting that these mAbs may recognize conformational rather than linear epitopes. When the N protein fragments were reacted with PRRSV-positive pig serum by Western blot, we similarly observed that immune serum was recognized only by ORF7-N2 and ORF7-N3 fragments, along with full-length N pro-

tein. To more finely map the antigenic determinants in the region represented by the overlap of the ORF7-N2 and ORF7-N3 proteins (residues 4970), this region was expressed contiguously (NEP1) or subdivided into two smaller recombinant fragments (NEP2 and NEP3). Although the proteins used to coat the well were not purified, the ELISA was performed under strict conditions with a GST cell lysate control in parallel to each monoclonal antibody sample for the purpose of cross-reaction.

0RF7-N1

0RF7-N2

0RF7-N3

0RF7-N4

NEP3

FIG. 3. Reactivity of PRRS-positive pig serum by Western blotting. ORF7-N1ORF7-N4 and NEP3 GST fusion proteins were transferred from SDSpolyacrylamide gels to nitrocellulose membrane. After being blocked with 4% nonfat milk, the membrane was reacted with PRRSV-positive pig serum, followed by incubation with HRP-conjugated rabbit anti-pig IgG. Reactivity was visualized with the substrate DAB. ORF7-N2, ORF7-N3, and NEP3 proteins produced a positive reaction with pig serum. In contrast, the serum did not recognize ORF7-N1, ORF7-N2, or GST alone.

GST

388
30 40 50 60 70 80 90

ZHOU ET AL.

CH-1a CQMLGKIIAQQNQSRGKGPG-KKNKKKSPEKPHFPLATEDDVRHHFTPSERQLCLSSIQTAFNQGAGTCTLG 93 93 BJ-4 93 BJ-Ye S N RS E S 93 HB-2 (sh) I 93 HN1 93 S1 93 VR-2332 93 MLV RespPRRS R 93 I AF-Klop 93 15308 93 8981 93 16244B 93 25544 93 Carolina 93 DK3506-12 93 DK5163-17 93 DK5163-23 R N 93 IAF93-653 93 IAF93-2616 R 93 IAF94-287 S R 93 IAF94-3182 93 IAF-BAJ 93 ISU-22 93 ISU-55 SG 93 ISU-1894 I 93 ISU-3927 I N 93 JA142 H N 93 Kitasato 93-1 S I N 93 NVSL97-7895 I 93 NVSL97-7985IA1 R DRK 93 ONT-TS A I S 93 P129 G V L 93 PA-8 F R 93 Spain 2000 G 93 SP Y 93 Quebec 807-94 93 pig32-84bo I A AS L QT S Q L AM KS R QPR QA K 98 LV I A AS L QT S Q L AM KS R QPR QA K 98 EuroPRRSV I A AS L QT S Q L AM KS R QPR QA K 98 DV A AS L QT S Q L AM KS R QPR QA K 98 ABV 32-13 I A AL L QT S Q L AM KS R P QA K 98 AGS-96 I A AS L QT S Q L AM KS R QPR QA K 98 Denmark 49 I A AS L QT S Q L AM KS R QSR QA K 98 DK111-92 I A AAS 98 L QT S Q L AM KS R QPR QA K Nethelandsr4 1 I A AS L QT S Q L AM KS R QPR QA K 98 Nethelandr60 I A AS L QT S Q L AM KS R QPR QA K 98 Netherlands3 2 I A AS L QT S Q L AM KS R QPR QA K 98 Olot-91-G2540A I A AS L QT S Q L AM KS R QPR QA K 98 France 50-18 I A AS L QT S Q L AM KS R QPR QA K 98 SD-01-07 I A AS L QT S Q L AM KS R QPR QA K 98 SD-01-08 I A AS L QT S Q L AM KS R QPR QA K 98 SD-02-10

FIG. 4. Sequence alignment of PRRSV N protein (amino acids 23 to 93) among North American and European type isolates. Amino acid sequences of residues 2393 of PRRSV N protein from a total of 53 strains, including 38 North American and 15 European isolates, were analyzed with CLUSTAL W (23). The sequence of the epitope cluster described in this study is highly conserved among nearly all isolates, with the exception of a single amino acid substitution (56P 56L) in the PA-8 strain.

The ELISA results indicated that none of mAbs react to the GST or cell lysate. In accord with the results of Rodriguez et al. (22), who described an epitope at amino acids 5066, six of the seven mAbs reacted with NEP3 (residues 4958). The other, mAb N1H11, reacted only weakly with NEP1, suggesting that amino acids 4970 may form only a part of the epitope recognized by this antibody. To precisely identify the minimal epitopes of PRRSV N protein, the sequence of NEP3 (residues 4958) was further serially truncated from both ends, one residue at a time. In the process of serial truncation, we found that

different truncated peptides reacted with different monoclonal antibodies. Reactivity against these short peptides resolved the six mAbs into three distinct groups. Group 1, of which N2H7 is the only member, did not tolerate deletion of any amino acids from the C terminus, but permitted the removal of five residues from the N terminus, indicating that the minimal epitope unit is composed of residues 5458 (H54FPLA58). Group 2, represented by mAb N2F7, required a similar but slightly larger region, residues 5258 (K52PHFPLA58). The remaining four mAbs, (N1A2, N1E3, N1G4, and N2E5) form group 3, which reacted with amino acids 5156 (E51KPHFP56).

EPITOPE CLUSTER IN N PROTEIN OF PRRSV It was notable that three different epitopes were clustered in a region only eight amino acids in size, E51KPHFPLA58. Importantly, this sequence is highly conserved among all European and North American type isolates, as determined by sequence alignment. Taken together, these data are consistent with a previous study positing that residues 5268 form a conserved antigenic region. This eight-amino acid sequence is found in the most hydrophilic area of the N protein, and is included in the nuclear localization signal sequence (amino acids 4172) (15). On the basis of secondary structure and solubility analysis, Rodriguez et al. predicted residues 5060 to be highly accessible on the surface of the N protein, perhaps accounting for the antigenicity of this region (22). In the study described here, NEP3 (residues 4958) was strongly recognized by PRRSV CH-1a-positive pig serum, and three short peptides within the region were reactive to different mAbs specific for N protein of PRRSV, suggesting that this region contains a linear epitope cluster. Furthermore, this epitope is distinct from the previously described conformational epitopes from strains PA-8 (residues 5269 and 112123) (13) and LV (residues 5167 and 8090) (18). Although the three epitopes (residues 5156, 5258, and 5458) we have identified overlap with part of the two conformational epitopes, we have in fact shown them to be independent linear epitopes. The results of this study contribute to our understanding of the antigenic structure of PRRSV and perhaps may form the basis for an improved diagnostic method. Although the region of the epitope cluster is small, only 10 amino acids in length, it reacted strongly with pig serum, which is helpful in constructing an antigen based on multiple conserved epitopes for diagnostic purposes.

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American isolates of porcine reproductive and respiratory syndrome virus (PRRSV) are encoded by the carboxy-terminal portion of viral open reading frame 3. Vet. Microbiol. 1995;44:6576. 4. Meng XJ, Paul PS, Halbur PG, and Lum MA: Phylogenetic analysis of the putative M (ORF6) and N (ORF7) genes of porcine reproductive and respiratory syndrome virus (PRRSV): Implication for the existence of two genotypes of PRRSV in the US and Europe. Arch. Virol. 1995; 140:745755. 5. Qiu HJ, Guo BQ, Tong GZ, Liu WX, and Yu L: Genotyping of porcine reproductive and respiratory syndrome virus strain CH-1a isolated in China by RT-PCR. Chin. J. Vet. Sci. 1998;18:118121. 6. Albina E, Madec E, Cariolet R, and Torrison J: Immune response and persistence of the porcine reproductive and respiratory syndrome virus in infected pigs and farm units. Vet. Rec. 1994;134:567573. 7. Yoon KJ, Wu LL, Zimmerman JJ, Hill HT, and Platt KB: Antibody-dependent enhancement (ADE) of porcine reproductive and respiratory syndrome virus (PRRSV) infection in pigs. Viral Immunol. 1996;9:5163. 8. Voicu IL, Silim A, Morin M, and Elazhary MA: Interaction of porcine reproductive and respiratory syndrome virus with swine monocytes. Vet. Rec. 1994;134:422423. 9. Wensvoort G, Terpstra C, Pol JM, ter Laak EA, Bloemraad M, de Kluyver EP, Kragten C, van Buiten L, den Besten A, Wagenaar F, et al.: Mystery swine disease in the Netherlands: The isolation of Lelystad virus. Vet. Q. 1991; 13:121130. 10. Mardassi H, Mounir S, and Dea S: Identification of major differences in the nucleocapsid protein genes of a Quebec strain and European strains of porcine reproductive and respiratory syndrome virus. J. Gen. Virol. 1994;75:681 685. 11. Murtaugh MP, Elam MR, and Kakach LT: Comparison of the structural protein coding sequences of the VR-2332 and Lelystad virus strains of the PRRS virus. Arch.Virol. 1995;140:14511460. 12. Wootton S, Koljesar G, Yang L, Yoon KJ, and Yoo D: Antigenic importance of the carboxy-terminal -strand of the porcine reproductive and respiratory syndrome virus nucleocapsid protein. Clin. Diagn. Lab. Immunol. 2001;8: 598603. 13. Wootton SK, Nelson EA, and Yoo D: Antigenic structure of the nucleocapsid protein of porcine reproductive and respiratory syndrome virus. Clin. Diagn. Lab. Immunol. 1998; 5:773779. 14. Rowland RR, Kervin R, Kuckleburg C, Sperlich A, and Benfield DA: The localization of porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus of infected cells and identification of a potential nucleolar localization signal sequence. Virus Res. 1999;64: 112.

ACKNOWLEDGMENTS
The study was supported by grants from the National Basic Research Program (973) of China (no. 2005CB523200) and the National Natural Science Foundation of China (no. 30470072).

REFERENCES
1. Keffaber KK: Reproductive failure of unknown etiology. Am. Assoc. Swine Practitioners Newsl. 1989;1:110. 2. Guo BQ, Chen ZS, Liu WX, and Cui YZ: Isolation and identification of porcine reproductive and respiratory syndrome (PRRS) virus from aborted fetuses suspected of PRRS. Chin. J. Infect. Dis. Anim. Poult. 1996;18:15. 3. Katz JB, Shafer AL, Eernisse KA, Landgraf JG, and Nelson EA: Antigenic differences between European and

390
15. Rowland RR, and Yoo D: Nucleolarcytoplasmic shuttling of PRRSV nucleocapsid protein: A simple case of molecular mimicry or the complex regulation by nuclear import, nucleolar localization and nuclear export signal sequences. Virus Res. 2003;95:2333. 16. Yang L, Yoon KJ, Li Y, Lee JH, Zimmerman JJ, Frey ML, Harmon KM, and Platt KB: Antigenic and genetic variations of the 15 kD nucleocapsid protein of porcine reproductive and respiratory syndrome virus isolates. Arch. Virol. 1999;144:525546. 17. Zhang GS, and Rong BP (Eds.). Application of Monoclonal Antibody in Medicine. Shanghai Science and Technology Publishing House, Shanghai, China, 1987, pp. 299300. 18. Yang L, Frey ML, Yoon KJ, Zimmerman JJ, and Platt KB: Categorization of North American porcine reproductive and respiratory syndrome viruses: Epitopic profiles of the N, M, GP5 and GP3 proteins and susceptibility to neutralization. Arch. Virol. 2000;145:15991619. 19. Meulenberg JJ, van Nieuwstadt AP, van Essen-Zandbergen A, Bos-de Ruijter JN, Langeveld JP, and Meloen RH: Localization and fine mapping of antigenic sites on the nucleocapsid protein N of porcine reproductive and respiratory syndrome virus with monoclonal antibodies.Virology 1998;252:106114. 20. Tong GZ, Qiu HJ, Zhou YJ, Guo BQ, Zhang SJ, Wang L, Cai XH, and Liu B: Characterize and sequence analysis of the structural proteins of porcine reproductive and respira-

ZHOU ET AL.
tory syndrome virus CH-1a strain. Prog. Natural Sci. 2000;10:147153. 21. Xue Q, Zhou YJ, Liu GQ, Qiu HJ, and Tong GZ: Construction of a full length genomic cDNA clone of porcine reproductive and respiratory syndrome virus. Chin. J. Prev. Vet. Med. 2003;25:401407. 22. Rodriguez MJ, Sarraseca J, Garcia J, Sanz A, Plana-Duran J, and Ignacio Casal J: Epitope mapping of the nucleocapsid protein of European and North American isolates of porcine reproductive and respiratory syndrome virus. J. Gen. Virol. 1997;78:22692278. 23. Thompson JD, Higgins DG, and Gibson TJ: CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994;22:46734680.

Address reprint requests to: Dr. Guang-Zhi Tong National Key Laboratory of Veterinary Biotechnology Harbin Veterinary Research Institute, CAAS 427 Maduan Street Harbin 150001, Peoples Republic of China E-mail: gztong@hvri.ac.cn Received November 29, 2005; accepted April 18, 2006.