Phylogenetic analysis and molecular characteristics of seven variant Chinese field isolates of PRRSV
© Wang et al; licensee BioMed Central Ltd. 2010
Received: 28 February 2010
Accepted: 20 May 2010
Published: 20 May 2010
Porcine reproductive and respiratory syndrome (PRRS) has now been widely recognized as an economically important disease. The objective of this study was to compare the molecular and biological characteristics of porcine reproductive and respiratory syndrome virus (PRRSV) field isolates in China to those of the modified live virus (MLV) PRRS vaccine and its parent strain (ATCC VR2332).
Five genes (GP2, GP3, GP4, GP5 and NSP2) of seven isolates of PRRSV from China, designated LS-4, HM-1, HQ-5, HQ-6, GC-2, GCH-3 and ST-7/2008, were sequenced and analyzed. Phylogenetic analyses based on the nucleotide sequence of the ORF2-5 and NSP2 showed that the seven Chinese isolates belonged to the same genetic subgroup and were related to the North American PRRSV genotype. Comparative analysis with the relevant sequences of another Chinese isolate (BJ-4) and North American (VR2332 and MLV) viruses revealed that these isolates have 80.8-92.9% homology with VR-2332, and 81.3-98.8% identity with MLV and 80.7-92.9% with BJ-4. All Nsp2 nonstructural protein of these seven isolates exhibited variations (a 29 amino acids deletion) in comparison with other North American PRRSV isolates. Therefore, these isolates were novel strain with unique amino acid composition. However, they all share more than 97% identity with other highly pathogenic Chinese PRRSV strains. Additionally, there are extensive amino acid (aa) mutations in the GP5 protein and the Nsp2 protein when compared with the previous isolates.
These results might be useful to study the genetic diversity of PRRSV in China and to track the infection sources as well as for vaccines development.
Porcine reproductive and respiratory syndrome virus (PRRSV) is recognized as one of the major infective agents in the pig industry worldwide since its appearance in the 1980s. It was first diagnosed in the USA in 1987 , immediately found in Europe, soon disseminated to the rest of the world . The disease is characterized by reproductive failure in pregnant sows and respiratory distress particularly in suckling piglets, thereupon getting its name. PRRSV is a single-stranded positive RNA virus and a member of the family Arteriviridae in the order of Nidovirales . Based on phylogenetic analyses of different virus isolates around the world, PRRSV can be differentiated into two genotypes: Type I, represented by the European prototype Lelystad strain LV, and Type II, the prototype being the Northern American ATCC strain VR2332. Chinese isolates were assigned as members of the genotype II . Extensive molecular studies show that PRRSV is highly variable in antigenicity, virulence and sequence diversity [5, 6].
PRRSV is a small, enveloped, single positive-stranded RNA virus including a genome of about 15 kb, encoding nine ORFs [2, 7, 8]. The PRRSV genome is comprised of two polymerase genes, ORF1a and 1b, and seven structural genes, ORF2a, 2b, 3, 4, 5, 6, and 7 . ORF1a and ORF1b constitutes approximately 75% of the viral genome, and are characterized by a process of ribosomal frame shifting translated into a large polyprotein; which by self-cleavage gives rise to the non-structural proteins (NSPs) including the RNA-dependent RNA polymerase . Open reading frames 2a, 3, 4 and 5 all encode glycosylated proteins, designated GP2a, GP3, GP4, and GP5, respectively [7, 11]. The newly defined ORF2b encodes the smallest protein of the virus particle designated GP2b [8, 12]. ORF7 encodes the non-glycosylated nucleocapsid protein (N), constituting 20-40% of the protein content of the virion [8, 13, 14]. ORF6 encodes the likewise non-glycosylated matrix protein (M) [8, 12]. Heterodimers constituted by GP5 and M have been found in the endoplasmic reticulum of infected cells , and have been suggested to be involved in virus-host cell receptor interaction . A rapid genetic divergence of PRRSV was revealed by an experiment of serial in vivo passage of a PRRSV strain  and by an analysis of naturally infected pigs. The presence of genetically divergent viruses in a swine population may complicate the disease control by vaccination, because the PRRSV vaccine efficacy is reduced when the challenge virus is a virus of a different genotype  or of a different phylogenetic cluster within the same genotype .
In China the first outbreak of PRRS was recorded in 1995 which encountered almost all provinces (include Hong Kong). Due to its economic impact in China, the disease has been recognized as one of the most severe viral diseases for pig farms. The first Chinese strain of PRRSV was isolated in 1996, and the complete genome sequence of the Chinese PRRSV isolate BJ-4 was first reported in 2001 . Highly pathogenic PRRSV is the causative agent of porcine high fever syndrome and characterized by high fever and high death rates in pigs of all ages. Since May 2006, the highly pathogenic PRRSV has emerged in China. Recently, the genomic characteristics of two other Chinese isolates of PRRSV were described with comparisons to some American and European isolates . It has been documented that PRRSV strains differ in virulence [20–23] and vary genetically [24, 25]. Concerns that vaccine strains or derivatives of the vaccine strains may induce disease continue to be discussed [26–28]. The objective of this research was to compare the genetic and molecular characteristics of seven Chinese PRRSV field isolates to that of a known high-virulence PRRSV isolate (BJ-4), the Ingelvac PRRS MLV vaccine, and the parent strain of the vaccine (ATCC VR2332). The results inferred from this study might be useful for infection tracking as well as for vaccines development.
Results and discussion
For a long time, outbreaks of highly pathogenic (acute, atypical) PRRS in many Chinese territories have been attributed to the highly virulent Chinese-type PRRSV (H-PRRSV) strains. From January to July 2007, 39455 morbid pigs died among 143,221 infected pigs according to the administrative files . New types of PRRSV variants with high pathogenicity were identified in China was responsible for severe impact on pig industry as well as food safety . Concurrently, this Chinese variant of PRRSV was detected in Vietnam where it caused a serious epidemic .
Glycoprotein 5 (gp5) is one of the major structural proteins encoded by PRRSV and forms disulfide-linked heterodimers with M protein in the viral envelope . The ORF5 of PRRSV encodes a 24.5-26 kDa envelope protein with a characteristic hydropathy profile and putative glycosylation sites [11, 14, 36]. Amplicons of ORF5 genes derived from the 7 tested isolates had the same size of 603 bp (deduced amino acids are 201). The sequence alignments indicated that they had an identity of 99-100% at the nucleotide level and 98-100% at the amino acid level between MLV and BJ-4. However, the deduced amino acid sequence comparison indicated that those isolates show an higher evolutionary divergence of 2.372-2.429 with VR-2332 and MLV,3.314-3.471 with BJ-4 (Additional file 1), and displayed considerable genetic variation.
The Nsp2 protein has been shown to be highly variable among arteriviruses, with similarities observed only in the amino- and carboxy-terminal domains whereas the central region of the protein varies in both length and amino acid composition . Interestingly, the Nsp2 protein was found to contain the highest frequency of immunogenic epitopes including positions 27-42, 37-52, 483-497, 503-517,823-837 and 833-847, when compared to reference virus strains examined in this study (Figure 6). In addition, these immuno-dominant B-cell epitopes were scattered along the protein sequence, and most of them were localized within predicted hydrophilic regions of the protein by predicting hydropathy Kyte-Doolittle method (Additional file 9). These results were not unexpected since hydrophilic amino acid sequences are likely to be exposed on the surface of the protein and thus may be more easily recognized by B-lymphocytes. A previous report has also demonstrated the occurrence of a cluster of B-cell epitopes in Nsp2 of an EUtype PRRSV isolate and a north America PRRSV isolate, NVSL 97-7895 strain [33, 48].
In conclusion, this study presented detailed molecular and phylogenetic analyses for seven field isolates of PRRSV from China. The collected results revealed that the highly pathogenic PRRSV variants with the 30-aa deletion in Nsp2 were still the dominating viruses in China. The genetic diversity of PRRSV strain existed in the field in China. These results might be useful for the origin and genetic diversity of PRRSV Chinese isolates and the development of vaccine candidates in the future.
Cell culture and viruses
Swine Alveolar Macrophages (SAM) were obtained from about 4 week-old pigs as previously described . The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and antibiotics (25 U/ml penicillin, 25 μg/ml streptomycin, 40 μg/ml gentamicin, 25 μg/ml neomycin and 300 U/ml polymyxin). Monkey kidney cell line, MARC-145 , was cultured in Eagle's minimum essential medium supplemented with 5% fetal bovine serum. Infectious PRRSV, LS-4, HM-1, HQ-5, GCH-3, GC-2, HQ-6 and ST-7 strains from Shijiazhuang of Hebei province (Additional file 10), were isolated in our laboratory at National Center of Wildlife Born Diseases, by inoculation of the sera or the tissue homogenates into SAM or MARC-145 cells.
RNA extraction, reverse transcriptase PCR (RT-PCR) and nucleotide sequencing
Primers used for PCR amplification of ORF2--ORF5 and NSP2 from PRRSV
Construction of phylogenetic trees
Nucleotide BLASTn analysis http://www.ncbi.nlm.nih.gov/BLAST was used to identify related genes of the viruses, and the reference sequences were obtained from GenBank. Pair-wise sequence alignments were also performed with the MEGA4.0 program http://www.megasoftware.net/ to determine nucleotide sequence similarities. Alignments of each virus sequence were generated using program ClustalW http://clustalw.genome.ad.jp/. Phylogenetic analyses of the aligned sequences for 5 gene segments (ORF2-5 and NSP2) were performed by the neighbor-joining method with 1000 bootstraps and Maximum-Likelihood with 100 bootstraps by using PHYLIP version 3.67 http://evolution.gs.washington.edu/phylip.html. All gene accession number of the isolates and other references virus were shown as Additional file 11.
Comparison and analysis of amino acid sequences in gp2, gp3, gp4, gp5 and nsp2
Amino acid sequences of Chinese isolate virus (BJ-4), VR2332 and MLV gp2, gp3, gp4 and gp5 proteins were retrieved from the public domain database Entrez Protein, and compared each of them with all the 7 isolate virus proteins using the software ClustalW .
This work was supported by grants from: Hi-Tech Research and development program of China (863, 2007AA100606), National Science and Technology Ministry(ID:2009BAI83B01) and The National Key Basic Research and Development Program of China (973, 2007BC109103).
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