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).
- Albina E: Epidemiology of porcine reproductive and Respiratory syndrome (PRRS): an overview. Vet Microbiol. 1997, 55: 309-316. 10.1016/S0378-1135(96)01322-3.View ArticlePubMedGoogle Scholar
- Wensvoort G, Terpstra C, Pol JM, Ter Laak EA, Bloemraad M, De Kluyver EP: Mystery swine disease in The Netherlands: the isolation of Lelystad virus. Vet Q. 1991, 13 (3): 121-130.View ArticlePubMedGoogle Scholar
- Cavanagh D: Nidovirales: a new order comprising Coronaviridae and Arteriviridae. Arch Virol. 1997, 142 (3): 629-633.PubMedGoogle Scholar
- Gao ZQ, Guo X, Yang HC: Genomic characterization of two Chinese isolates of porcine respiratory and reproductive syndrome virus. Arch Virol. 2004, 149: 1341-1351. 10.1007/s00705-004-0292-0.View ArticlePubMedGoogle Scholar
- Stadejek T, Oleksiewicz MB, Potapchuk D, Podgorska K: Porcine reproductive and respiratory syndrome virus strains of exceptional diversity in eastern europe support the definition of new genetic subtypes. J Gen Virol. 2006, 87: 1835-1841. 10.1099/vir.0.81782-0.View ArticlePubMedGoogle Scholar
- An TQ, Zhou YJ, Liu GQ, Tian ZJ, Li J, Qiu HJ, Tong GZ: Genetic diversity and phylogenetic analysis of glycoprotein 5 of PRRSV isolates in Mainland China from 1996 to 2006: coexistence of two NA-subgenotypes with great diversity. Vet Microbiol. 2007, 123: 43-52. 10.1016/j.vetmic.2007.02.025.View ArticlePubMedGoogle Scholar
- Dea S, Gagnon CA, Mardassi H, Pirzadeh B, Rogan D: Current knowledge on the structural proteins of porcine reproductive and respiratory syndrome (PRRS) virus: comparison of the North American and European isolates. Arch Virol. 2000, 145 (4): 659-688. 10.1007/s007050050662.View ArticlePubMedGoogle Scholar
- Wu WH, Fang Y, Farwell R, Steffen-Bien M, Rowland RR, Christopher-Hennings J, Nelson EA: A 10-kDa structural protein of porcine reproductive and respiratory syndrome virus encoded by ORF2b. Virology. 2001, 287 (1): 183-191. 10.1006/viro.2001.1034.View ArticlePubMedGoogle Scholar
- Labarque G, Van Reeth K, Nauwynck H, Drexler C, Van Gucht S, Pensaert M: Impact of genetic diversity of European-type porcine reproductive and respiratory syndrome virus strains on vaccine efficacy. Vaccine. 2004, 22 (31-32): 4183-4190. 10.1016/j.vaccine.2004.05.008.View ArticlePubMedGoogle Scholar
- Meulenberg JJ: PRRSV, the virus. Vet Res. 2000, 31 (1): 11-21.PubMedGoogle Scholar
- Meulenberg JJ, Petersen-den BA, De Kluyver EP, Moormann RJ, Schaaper WM, Wensvoort G: Characterization of proteins encoded by ORFs 2 to 7 of Lelystad virus. Virology. 1995, 206 (1): 155-163. 10.1016/S0042-6822(95)80030-1.View ArticlePubMedGoogle Scholar
- Snijder EJ, van Tol H, Pedersen KW, Raamsman MJ, de Vries AA: Identification of a novel structural protein of arteriviruses. J Virol. 1999, 73 (8): 6335-6345.PubMed CentralPubMedGoogle Scholar
- Nelson EA, Christopher-Hennings J, Drew T, Wensvoort G, Collins JE, Benfield DA: Differentiation of U.S. and European isolates of porcine reproductive and respiratory syndrome virus by monoclonal antibodies. J Clin Microbiol. 1993, 31 (12): 3184-3189.PubMed CentralPubMedGoogle Scholar
- Mardassi H, Massie B, Dea S: Intracellular synthesis, processing, and transport of proteins encoded by ORFs 5 to 7 of porcine reproductive and respiratory syndrome virus. Virology. 1996, 221 (1): 98-112. 10.1006/viro.1996.0356.View ArticlePubMedGoogle Scholar
- Delputte PL, Vanderheijden N, Nauwynck HJ, Pensaert MB: Involvement of the matrix protein in attachment of porcine reproductive and respiratory syndrome virus to a heparin-like receptor on porcine alveolar macrophages. J Virol. 2002, 76 (9): 4312-4320. 10.1128/JVI.76.9.4312-4320.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Chang CC, Yoon KJ, Zimmerman JJ, Harmon KM, Dixon PM, Dvorak CM, Murtaugh MP: Evolution of porcine reproductive and respiratory syndrome virus during sequential passages in pigs. J Virol. 2002, 76 (10): 4750-4763. 10.1128/JVI.76.10.4750-4763.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Goldberg TL, Lowe JF, Milburn SM, Firkins LD: Quasispecies variation of porcine reproductive and respiratory syndrome virus during natural infection. Virology. 2003, 317 (2): 197-207. 10.1016/j.virol.2003.07.009.View ArticlePubMedGoogle Scholar
- VanWoensel PA, Liefkens K, Demaret S: Effect on viraemia of an American and a European serotype PRRSV vaccine after challenge with European wild-type strains of the virus. Vet Rec. 1998, 142 (9): 510-512.View ArticleGoogle Scholar
- Yang HC, Huang FF, Guo X, Gao Y, Li H, Chen S: Sequencing of genome of porcine reproductive and respiratory syndrome virus isolate BJ-4. J Agric Biotechnol. 2001, 9 (3): 212-218.Google Scholar
- Halbur PG, Paul PS, Frey ML, Landgraf J, Eernisse K, Meng XJ, Lum MA, Andrews JJ, Rathje JA: Comparison of the pathogenicity of two U.S. porcine reproductive and respiratory syndrome virus isolates with that of the Lelystad virus. Vet Pathol. 1995, 34: 648-660. 10.1177/030098589503200606.View ArticleGoogle Scholar
- Halbur PG, Paul PS, Meng XJ, Lum MA, Andrews JJ, Rathje JA: Comparative pathogenicity of nine U.S. porcine reproductive and respiratory syndrome virus (PRRSV) isolates in a 5-week-old cesareanderived-colostrum-deprived pig model. J Vet Diagn Investig. 1996, 8: 11-20.View ArticleGoogle Scholar
- Halbur PG, Paul PS, Frey ML, Landgraf J, Eernisse K, Meng XJ, Andrews JJ, Lum MA, Rathje JA: Comparison of the antigen distribution of two U.S. porcine reproductive and respiratory syndrome virus isolates with that of the Lelystad virus. Vet Pathol. 1996, 33: 159-170. 10.1177/030098589603300205.View ArticlePubMedGoogle Scholar
- Mengeling WL, Lager KM, Vorwald AC: Clinical consequences of exposing pregnant gilts to strains of porcine reproductive and respiratory syndrome (PRRS) virus isolated from field cases of "atypical" PRRS. Am J Vet Res. 1998, 59: 1540-1544.PubMedGoogle Scholar
- Meng XJ, Paul PS, Halbur PG: Molecular cloning and nucleotide sequencing of the 3'-terminal genomic RNA of porcine reproductive and respiratory syndrome virus. J Gen Virol. 1994, 75: 1795-1801. 10.1099/0022-1317-75-7-1795.View ArticlePubMedGoogle Scholar
- Meng XJ, Paul PS, Morozov I, Halbur PG: A nested set of six or seven subgenomic mRNAs is formed in cells infected with different isolates of porcine reproductive and respiratory syndrome virus. J Gen Virol. 1996, 77: 1265-1270. 10.1099/0022-1317-77-6-1265.View ArticlePubMedGoogle Scholar
- Key KF, Haqshenas G, Guenette DK, Swenson SL, Toth TE, Meng XJ: Genetic variation and phylogenetic analyses of the ORF5 gene of acute porcine reproductive and respiratory syndrome virus isolates. Vet Microbiol. 2001, 83: 249-263. 10.1016/S0378-1135(01)00427-8.View ArticlePubMedGoogle Scholar
- Meng XJ: Heterogeneity of porcine reproductive and respiratory syndrome virus: implications for current vaccine efficacy and future vaccine development. Vet Microbiol. 2000, 74: 309-329. 10.1016/S0378-1135(00)00196-6.View ArticlePubMedGoogle Scholar
- Torrison JL, Knoll M, Wiseman B: Evidence of pig-to-pig transmission of a modified live vaccine. Proceedings of the 27th Annual Meeting of the American Association of Swine Practitioners, Nashville, Tenn. American Society of Swine Veterinarians, Perry, Iowa. 1996, 89-91.Google Scholar
- Zhou L, Chen SX, Zhang JL, Zeng JW, Guo X, Ge XN, Zhang DB, Yang HC: Molecular variation analysis of porcine reproductive and respiratory syndrome virus in China. Virus Res. 2009, 145 (1): 97-105. 10.1016/j.virusres.2009.06.014.View ArticlePubMedGoogle Scholar
- Tian KG, Yu XL, Zhao TZ, Feng YJ, Cao Z1, Wang CB, Hu Y, Chen XZ, Hu DM, Tian XS, Liu D, Zhang S, Deng XY, Ding YQ, Yang L, Zhang YX, Xiao HX, Qiao MM, Wang B, Hou LL, Wang XY, Yang XY, Kang LP, Sun M, Jin P, Wang SJ, Kitamura Y, Yan JH, Gao GF: Emergence of fatal PRRSV variants: unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hallmark. PLoS ONE. 2007, 2: e526-10.1371/journal.pone.0000526.PubMed CentralView ArticlePubMedGoogle Scholar
- Feng YJ, Zhao TZ, Nguyen T, Inui K, Ma Y, Nguyen TH, Nguyen VC, Liu D, Bui QA, Thanh TL, Wang CB, Tian KG, Gao GF: Porcine respiratory and reproductive syndrome virus variants, Vietnam and China, 2007. Emerg Infect Dis. 2008, 14: 1774-1776. 10.3201/eid1411.071676.PubMed CentralView ArticlePubMedGoogle Scholar
- Snijder EJ, Meulenberg JM: Arteriviruses in Fields Virology. Edited by: Kniper D, et al. 2001, LippincottWilliams and Wilkins, Philadelphia, 1: 1205-1220. 4Google Scholar
- Marcelo de L, Asit KP, Eduardo FF, Fernando AO: Serologic marker candidates identified among B-cell linear epitopes of Nsp2 and structural proteins of a North American strain of porcine reproductive and respiratory syndrome virus. Virology. 2006, 353: 410-421. 10.1016/j.virol.2006.05.036.View ArticleGoogle Scholar
- Zhou YJ, An TQ, He YX, Liu JX, Qiu HJ, Wang YF, Tong G: Antigenic structure analysis of glycosylated protein 3 of porcine reproductive and respiratory syndrome virus. Virus Res. 2006, 118: 98-104. 10.1016/j.virusres.2005.11.019.View ArticlePubMedGoogle Scholar
- Meulenberg JJ, van Nieuwstadt AP, van Essen-Zandbergen A, Langeveld JP: Posttranslational processing and identification of a neutralization domain of the GP4 protein encoded by ORF4 of Lelystad virus. J Virol. 1997, 71 (8): 6061-6067.PubMed CentralPubMedGoogle Scholar
- Mardassi H, Mounir S, Dea S: Molecular analysis of the ORF3-7 of porcine reproductive and respiratory syndrome virus, Quebec reference strain. Arch Virol. 1995, 140: 1405-1418. 10.1007/BF01322667.View ArticlePubMedGoogle Scholar
- Israrul HA, Byungjoon K, Fernando AO, Asit KP: Influence of N-Linked Glycosylation of Porcine Reproductive and Respiratory Syndrome Virus GP5 on Virus Infectivity, Antigenicity, and Ability To Induce Neutralizing Antibodies. J Virol. 2006, 80 (8): 3994-4004. 10.1128/JVI.80.8.3994-4004.2006.View ArticleGoogle Scholar
- Plagemann PGW, Rowland RRR, Faaberg KS: The primary neutralization epitope of porcine respiratory and reproductive syndrome virus strain VR-2332 is located in the middle of the GP5 ectodomain. Arch Virol. 2002, 147: 2337-2347. 10.1007/s00705-002-0887-2.View ArticleGoogle Scholar
- Wissink EH, Kroese MV, Maneschijn-Bonsing JG, Meulenberg JJ, van Rijn PA, Rijsewijk FA, Rottier PJ: Significance of the oligosaccharides of the porcine reproductive and respiratory syndrome virus glycoproteins GP2a and GP5 for infectious virus production. J Gen Virol. 2004, 85: 3715-3723. 10.1099/vir.0.80402-0.View ArticlePubMedGoogle Scholar
- Oleksiewicz MB, Botner A, Normann P: Porcine B-cells recognize epitopes that are conserved between the structural proteins of American- and European-type porcine reproductive and respiratory syndrome virus. J Gen Virol. 2002, 83 (6): 1407-1418.View ArticlePubMedGoogle Scholar
- Zhou YJ, Yu H, Tian ZJ, Liu JX, An TQ, Peng JM: Monoclonal antibodies and conserved antigenic epitopes in the C terminus of GP5 protein of the North American type porcine reproductive and respiratory syndrome virus. Vet Microbiol. 2009, 138 (1-2): 1-10. 10.1016/j.vetmic.2009.01.041.View ArticlePubMedGoogle Scholar
- Li Y, Wang X, Bo K, Tang B, Yang B, Jiang W, Jiang P: Emergence of a highly pathogenic porcine reproductive and respiratory syndrome virus in the Mid-Eastern region of China. Vet J. 2007, 174: 577-584. 10.1016/j.tvjl.2007.07.032.View ArticlePubMedGoogle Scholar
- Hu HX, Li XL, Zhang ZF, Shuai JB, Chen N, Liu GQ, Fang WH: Porcine reproductive and respiratory syndrome viruses predominant in southeastern China from 2004 to 2007were from a common source and underwent further divergence. Arch Virol. 2009, 154: 391-398. 10.1007/s00705-009-0316-x.View ArticlePubMedGoogle Scholar
- Lv J, Zhan JW, Sun Z, Liu WQ, Yuan SS: An infectious cDNA clone of a highly pathogenic porcine reproductive and respiratory syndrome virus variant associated with porcine high fever syndrome. J Gen Virol. 2008, 89: 2075-2079. 10.1099/vir.0.2008/001529-0.View ArticlePubMedGoogle Scholar
- Zhou YJ, Hao XF, Tian ZJ, Tong GZ, Yoo D, An TQ: Highly virulent porcine reproductive and respiratory syndrome virus emerged in China. Trans Emerg Dis. 2008, 55: 152-164. 10.1111/j.1865-1682.2008.01020.x.View ArticleGoogle Scholar
- Zhou L, Zhang J, Zeng J, Yin S, Li Y, Zheng L: The 30-amino-acid deletion in the Nsp2 of highly pathogenic porcine reproductive and respiratory syndrome virus emerging in China is not related to its virulence. J Virol. 2009, 83: 5156-5167. 10.1128/JVI.02678-08.PubMed CentralView ArticlePubMedGoogle Scholar
- Allende R, Lewis TL, Lu Z, Rock DL, Kutish GF, Ali A, Doster AR, Osorio FA: North American and European porcine reproductive and respiratory syndrome viruses differ in non-structural protein coding regions. J Gen Virol. 1999, 80 (2): 307-315.View ArticlePubMedGoogle Scholar
- Oleksiewicz MB, Botner A, Toft P, Normann P, Storgaard T: Epitope mapping porcine reproductive and respiratory syndrome virus by phage display: the nsp2 fragment of the replicase polyprotein contains a cluster of B-cell epitopes. J Virol. 2001, 75 (7): 3277-3290. 10.1128/JVI.75.7.3277-3290.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Mengeling WL, Lager KM, Vorwald AC: Diagnosis of porcine reproductive and respiratory syndrome. J Vet Diagn Invest. 1995, 7 (1): 3-16.View ArticlePubMedGoogle Scholar
- Kim HS, Kwang J, Yoon IJ, Joo HS, Frey ML: Enhanced replication of porcine reproductive and respiratory syndrome (PRRS) virus in a homogeneous subpopulation of MA-104 cell line. Arch Virol. 1993, 133 (3-4): 477-483. 10.1007/BF01313785.View ArticlePubMedGoogle Scholar
- Kumar S, Tamura K, Jakobsen IB: MEGA2: Molecular evolutionary genetics analysis software. Bioinformatics. 2001, 17: 1244-1245. 10.1093/bioinformatics/17.12.1244.View ArticlePubMedGoogle Scholar
- Thompson JD, Higgins DG, 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: 4673-4680. 10.1093/nar/22.22.4673.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.