Porcine reproductive and respiratory syndrome virus (PRRSV) has been one of the most economically damaging pathogens for the swine industry world-wide. Since it first emerged in 1996, the virus has spread widely throughout pig-producing provinces of China, imposing a considerable economic burden on the swine industry, especially after the outbreak of highly pathogenic PRRS in 2006 [14, 15]. Studies have been performed on genetic variability of these isolates, revealing extensive sequence variation among the Chinese PRRSV strains [17, 18]. Nevertheless, the molecular characterization of the ORF7 gene among PRRSVs circulating in China has not been documented. Here, we determined the partial Nsp2 sequences and the complete ORF7 sequences of 7 PRRSV strains isolated in different swine herds during 2007-2010 and compared them with the published sequences of 91 Chinese strains and 2 North American strains (VR-2332 and its attenuated vaccine virus RespPRRS MLV).
Pair wise comparisons showed that 6 isolates characterized in this study (07N, 128, PC, TS, XIN, and XB) and 64 Chinese reference isolates contained a discontinuous deletion of 30 amino acids in the Nsp2 gene. The deletion is considered a gene marker for highly pathogenic PRRSV; however, it is not related to virulence . Further analysis of the complete ORF7 sequences revealed that all the 98 Chinese PRRSV isolates analyzed exhibited a very high degree of genetic diversity, and clustered into 5 subgroups, suggesting the coexistence of related non-identical PRRS viral variants evolving independently. Subgroup I isolates shared a high identity with the MLV vaccine and its parent virus VR-2332. Subgroup II isolates were highly homologous to the CH-1R vaccine and its parent virus CH-1a. Isolates in subgroups IV and V were all highly pathogenic PRRSVs, and distinct from the MLV or CH-1R vaccine. These highly pathogenic subgroups IV and V PRRSVs are the dominant strains circulating in China, and so should be the focus when formulating preventive and control measures against PRRSV.
No apparent relationship between geographic and genetic distance was found for the isolates based on the N protein in the study, especially for the highly pathogenic strains, since these atypical viruses existed throughout the mainland of China. A correlation between temporal and genetic distance was also not found, as the highly pathogenic PRRS outbreak occurred in several pig farms in the summer of 2006 and rapidly spread to almost all pig-producing provinces of China. Our data indicate that the disease is still circulating in China.
The nucleocapsid protein (N) encoded by ORF7 is highly immunogenic, and several antigenic domains have been mapped onto N in both the European and the North American PRRSV. A common linear epitope conserved among different isolates of European and North American origin was located in the amino acid segment 50-66 . Another linear epitope, conserved in European and North American isolates, was identified in amino acids 25-30 . Wootton et al. found three additional linear epitopes (residues 30-52, 37-52, and 69-123) and one discontinuous epitope utilizing residues 52-69 and 112-123 . In addition, four other linear epitopes at 23-33, 30-48, 30-50, and 43-56 were observed in VR2332 . For the North American isolate CH-1a, the first Chinese isolate, epitopes were reported in amino acid segments 51-58 and 79-87 [32, 33].
Extensive substitutions were observed in the 123-residue nucleocapsid protein on the basis of the alignment. Substitutions K46R and V117A occurred in all the highly pathogenic PRRSVs, which might impede the recognition of the epitopes encompassing or flanking the two substitutions by anti-N mAbs. A previous study confirmed that the 11 C-terminal residues 112-123 were essential for the generation of discontinuous epitopes . Single amino acid substitutions introduced into the C-terminal domain show that the requirement of the C terminus for conformation-dependent mAb binding correlates with the proper formation of the predicted beta-strand formed by amino acids 111-117 . Therefore, it is likely that the mutation V117A observed in highly pathogenic PRRSVs could exert great influence on the structure and antigenicity of N protein.
The substitution K46R might also alter the function of the nuclear localization signal (NLS) motif (41-47) and the nucleolar localization signal (NoLS) motif (41-72) . Previous studies have demonstrated that mutations at 43 and 44 within the NLS attenuated viral replication . Whether the mutation at amino acid position 46 has the similar or opposite impact on viral pathogenicity remains to be determined.
Two other substitutions, R11K and D15N, occurred in subgroups II, III, IV, and V, although there are a large number of Lys (K) and Asn (N) residues in the N-terminal half of the 123-residue nucleocapsid protein. The accumulation of these residues in the N terminus might function in the interaction with genomic viral RNA .
Some conserved sites were also observed from our alignment analysis. For example, three cysteine residues at amino acid positions 23, 75, and 90 were highly conserved in all isolates. Covalent interactions were formed through disulfide linkages between conserved cysteines at position 23 in North American strains, while the domain 30-37, which was also conserved in all isolates in this study, was shown to be essential for non-covalent interactions . The C75 S mutant induced cytopathic effects and produced infectious strains with plaque morphology indistinguishable from the wild type clone. In contrast, the C23 S and C90 S mutations completely abolished viral infectivity, indicating that C23 and C90 play critical roles in PRRSV infection .
Conserved regions were also found by variability analysis at positions 16-45, 55-90, and 92-108. These highly conserved amino acid segments are probably associated with nucleocapsid structure and/or function. Meanwhile, non-synonymous mutations did not occur more frequently than synonymous mutations among the Chinese isolates, and the main variable sites and non-synonymous mutations (residues 11, 15, 46, 91, 109, and 117) were distributed in the hydrophilic regions prone to immune pressure.
The N protein contains important immunogenic epitopes, and the majority of antibodies produced during PRRSV infection are specific for it [20, 40]. Thus, the N protein has been targeted as a suitable candidate for the detection and diagnosis of PRRS. Numerous serological diagnostic tests have been developed based on the N protein [21, 22, 41–43]. Additionally, PCR is another widely used method for detecting the viruses, and ORF7 has been regarded as a promising target gene [25, 44, 45] due to its sequence stability relative to other structural genes . However, the high genetic variability among the ORF7 sequences of the Chinese PRRSV isolates observed in this study should be taken into consideration when designing serological or molecular detection methods for PRRSV diagnosis and epidemiological surveillance.
In conclusion, all the ORF7 sequences of PRRSV isolates from 1996 to 2010 in China belonged to the North American type. Chinese strains were categorized into five subgroups. The highly pathogenic PRRSVs have become the dominant strains in China. Our study provides the first genetic analysis of the Chinese PRRSV N protein. These results could lead to a better understanding of the molecular variation of PRRSV in China and to the development of more effective vaccines and reliable diagnostic methods.