摘要
II型猪圆环病毒(PCV2)是引起猪断奶多系统衰竭综合征(Postweaning Multisystemic Wasting Syndrome,PMWS)的主要病原。PMWS自1991年,加拿大首次暴发以后,继而迅速在美国、法国、英国等国家和地区也相继出现了与之相关的报道。并且在我国自2001年首次报道本病以来,有关该病的报道也是逐年增多。PCV2在我国大部分规模化养猪场里广泛存在,然而各地发病率及流行特点却有所不同。
本文对北京、河北、山东、山西四地规模化猪场或大型养殖户的共计213份送检样品进行PCR检测PCV,从流行病学调查结果中,选取强阳性或阳性病料进行病毒DNA的克隆、测序,分析各地PCV2流行毒株的ORF1、ORF2基因序列,比较各地毒株之间的同源性,再与国内外的流行毒株之间比较它们的亲缘关系,国内参考毒株为黑龙江(HLJ-AY122275)、北京(BJ-AY177626)、江苏(JS-AY686762)和广东(GD-AY65185) ;国外的参考毒株为美国株(AMA-AF264038)、加拿大株(CAN-AF109399)、德国株(GER-AF201311)、法国(FRA-AJ623306)、韩国株(KRO-AY672600)的ORF1、ORF2基因序列之间进行比对,进而通过序列分析这四地的毒株之间及与国内外流行毒株之间的内在联系,找出毒株遗传变异的特点,研究各毒株间的差异性,对于该类疾病的诊断、预防、治疗等可提供更有针对性的科学依据。
Porcine circovirus is a member of Circoviraceae Circoviridae, it was add in sixth Report of virus classification in International Committee on the Taxonomy of Viruses (ICTV), according to difference between antigenicity and genetic constitution it can be divided into two types, nonpathogenic PCV1 and pathogenic PCV2. research indicate that PCV1 is ubiquitous and nonpathogenic for pigs, whereas PCV2 is main initiation for postweaning multisystemic wasting syndrome (PMWS), it becomes including pig immunosuppression aspects disease to continue Porcine reproductive and respiratory syndrome. PMWS has made a huge economic loss for world's swine industry and aroused people's attention.
PCV confirmed by means of respiratory tract infection in pigs in different age groups, with 5 to 12-week-old early weaned piglets and adult pigs the most sensitive. It was also suggested that PCV transmitted to sow through to semen of boar, and then infected to piglets, discharges to the virus such as nasal fluid and waste, so the wide dissemination and it exists of PMWS, was a worldwide trend in Europe, the Americas, Asia countries in the region all have been reported. With in-depth study, PCV2 not only the main cause PMWS, but cause Porcine Dermatitis and Nephropathy Syndrome, Congenital Tremor type AII, Swine Respiratory disease syndrome, Sow Reproductive disorders at the same time and performance related to clinical symptoms and the corresponding changes in anatomical pathology. It can be said that after the farm infected PCV at once, it will be difficult to completely eliminate the virus, which has become a major problem for the pig farming industry.
Recently, it had established a number of PCV2 detection methods after a large number of researches and practices. Including viral isolation assay, Immunofluorescence assay, Enzyme-linked Immunosorbent assay, Polymerase Chain Reaction, In Situ nucleic acid Hybridization and so on, can be used to test DNA of virus or antibody levels in tissue sample and serum. According to the specific circumstances of each laboratory, these tests were used widely. The PCV genome studies have shown that Both PCV1 and PCV2 were known that they contain two major open reading frames (ORFs) that are ORF1 and ORF2 which were most important of all its. ORF1 (945bp) it major founction is encodes a viral replication protein (Rep protein). Rep protein of PCV1 shares about 85% identity with PCV2 through homology comparison, that is the most important reason of PCV1 and PCV2 have cross-reaction of antigenicity. ORF2 (702bp) it major founction is encodes a major structural protein of virus. They shares about 68% identity to contrast two types virus; however, they have no cross-reaction of antigenicity is observed between themslf. Therefore, the ORF2 protein is always used to distinguish PCV1 and PCV2, and have greater significance for porcine circovirus disease diagnosis and porcine circovirus vaccines.
Epidemiological investigation in this article part, through testing 76 samples from Beijing area, 44 samples of Hebei, 60 samples of Shandong region, 33 in Shanxi region, for a total of 213 tissue samples were detected by PCR, test results: positive for the virus in Beijing was 34.2%, Hebei 22.7%, Shanxi region 24.2%, Shandong region was 35% positive rate. In order to determine the various regions, PCV2 distribution of the prevalence in the large-scale pig farm for clinical medicine, and further rationalization of the recommendations provided to the pig farm, modify the immune comprehensive prevention and control procedures provide an important basis for measures. Then the focus of this study is the findings from the epidemiological, select positive sample to carry out DNA cloning, sequencing, analysis of the prevalence of PCV2 strain region ORF1, ORF2 gene sequences, compared to homology: PCV2 ORF1 shares about 97.8% ~ 99.4% range; PCV2 ORF2 shares about 91.4% ~ 99.7% of the range. Internal and abroad compared the prevalence of homology between the strains, internal reference strain as the strain of Heilongjiang (HLJ-AY122275), Beijing(BJ-AY177626), Jiangsu(JS-AY686762) and the Guangdong(GD-AY65185 ); abroad for the American strain (AMA-AF264038), the Canadian strain (CAN-AF109399), the German strain (GER-AF201311), France strain (FRA-AJ623306), the Korean strain (KRO-AY672600) of ORF1, ORF2 between gene sequence comparison, the results are: PCV2-ORF1 homology was 97.4% ~ 100%; PCV2 ORF2 homology was 91.3% ~ 100%. The results showed that a strain of PCV2 variation between ORF1 little difference between ORF2 and abroad mainly in between the strains, which results with the basic line. Through genetic analysis of phylogenetic trees found in different parts of the four PCV2-ORF2 sequences are closely related, indicating that these four areas of genetic variation may come from the same strain.
引文
[1] Allan G Meehan B,Todd D. Novel porcine circovirus from pigs with wasting disease syndrome[J] Vet Rec,1998,142:467-468.
[2] Al1an,G M.;McNeilly, E.;Kennedy,S.et al. PCV 2 PDNS in Northern Ireland in 1990.Veternary Record. 2000; 146(24):711-712.
[3] Hines,R and Lukert,P. Porcine circovirus as a cause of congential tremors in new born piglets.Proceedings of the American Association of Swine Practitioners. 1994; 344-345.
[4]曹胜波,陈涣春,猪圆环病毒2型的PCR检测方法的建立及应用.华中农业大学学报.2001(20)1:53-56.
[5]周绪斌,赵亚荣.猪圆环病毒病(PCVD)的流行动向与控制措施[J].今日养猪业. 2007(2):30-33.
[6] Ellis J,Hassard L,Clark E,et al.Isolation of circovirus from plesions of pigs with postweaning multisystemic wasting syndromejJ].Can Vet J,1998,39(1):44-51.
[7] Lukert PD, Boer GF, Dale JL, et al. The Circoviridae in Murpy FA, Fauquet CM, Bishop DH,Ghabrial SA, Jarvis AW, Martelli GP, Mayo MA, and MD Summer eds. Virus taxonomy 6th Report of the International Committee Taxonomy of Viruses. 1995,166-168.
[8] Allan G M,McNeilly F,Kennedy S,et a1.Isolation of porcine circovirus 2 like viruses from pigs with a wasting disease in the USA and Europe[J].J Vet Diagn Invest,1998,10(1):3-10.
[9] Hattermann K., Schmitt C., Soike D. et al. Cloning and sequencing of Duck circovirus (DuCV) [J]. Arch Virol, 2003, 148(12):2471-2480.
[10] Ritchie B, NiagroELukext P D, et al. Characterization of a new virus from cockatoos with psittacine beak and feather disease. Virology 1989; 171: 83-88.
[11] Bassami MR, Berrymani D, Wilcox D, et al. Psittacine beak and feather disease virus nucleotide sequence analysis and its relationship to porcine circovirus, plant circoviruses, and chicken anaemia virus. Virology 1998; 249:452459.
[12] Phenix K, Weston V, Ypelaar J H, et al. Nucleotide sequence analysis of anovel circovirus of canaries and its relationship to other members of the genus Circovirus of the family Circoviridae.[J] Gen Viro 2001;82:2805-2809.
[13] Todd D, Weston IH, Soike D, et al. Genome sequence determinations and analysis of novel circovirus from goose and pigeon. Virol 2001; 286: 354-362.
[14] Mankertz A, Hatermann Ehlers B, etal. Cloning and sequencing of columbid circovirus(CoCV), a new circovirus from pigeons. Arch Vrol 2000; 145:2469-2480.
[15] Woods LW, Latimer KS, Barr BC.et al. Crrcovirus-like infection in a pigeon.Vet Diagn Invest 1993; 5:609-612.
[16] Al1an,G M; McNeilly,E; Kennedy,S.et a1.Isolation of protein circovirus-like viruses from pigs with a wasting disease in the USA and Europe .Journal of Veterinary Diagnostic Investigation 1998;103-110.
[17]殷震,刘景华.动物病毒学(第2版) [M],北京:科学出版社, 1997, 1175~1183.
[18] Done, S.H., Cook, A., and A.C.J. Greshm. Porcine circovirus and post-weaning multisystemic wasting syndrome (PMWS); Factoids.The Pig Journal.2002.50:197-220.
[19] Hamel AL, Lin LL, and Nayar GP Nucleotide sequence of porcine circovirus associated withpostweaning multisystemic wasting syndrome in pigs.[J]Virol 1998; 72:5262-5267
[20] MeehanB M, McNeilly F ,Todd D ,et al. Characterization of novel circovirus DNAs. wasting syndromes in pigs[J].Journal of General Virology, 1998, 79:2171-2179.
[21] Steinfeldt T, Finsterbusch T, Mankertz A. Rep and Rep’protein of porcine circovirus type 1 bind to the origin of replication in vitro[J]. Virology, 2001, 91(1):152~160.
[22] Mankertz A ,MuellerB ,SteinfeldtT, et al .New reporter gene-based replication assay reveals exchange ability of replication factors of porcine circovirus types 1 and 2 [J].Journal of Virology ,2003,77(18):9885-9893.
[23] Andrew K.Cheung. The essential and nonessential transcription units for viral protein synthesis and DNA replication of porcine circovirus type 2[J]. Virology,2003, 291:152-160
[24] Truong C, Mahe D, Blanchard P, et al. Identification of an immunorelevant ORF2 epitope from porcine circovirus type2 as a serological marker for experimental and natural infection[J].Archive of Virology,2001,146(6):1197-1211.
[25] Liu Q, Tikoo S K, Babiuk L A. Nuclear localization of the ORF2protein encoded by porcine circovirus type2 [J]. Virology, 2001, 285(1):91-99.
[26] Allan GM, kennedy S, McNeilly F, Foster J C, Ellis J A, Krakowka S J, Meehan B M,Adair B M. Experimental reproduction of wasting disease and death by co-infectionof pigs with porcine circovirus and porcine parvovirus [J]. Comp Pathol.1999,121:1-11.
[27] Allan G M, McNeilly F, Kennedy S.et al. Pathogenesis of porcine circovirus-experimental infections of colostrum deprived piglets and examination of pig foetal.2003a, 44:49-64.
[28] Ellis JA, Konoby C, West KH, et al. Lack of antibodies to porcine circovirus type 2 virus in bees and dairy catle and horses in western Canada [J]. Can Vet J,2001,42((6):461-464.
[29] Nayar GP, Hamel AL, Lin L,.et a1.Evidence for circovirus in catle with respiratory disease and from aborted bovine fetuses [J].Can Vet J, 1999,40(4):277-278.
[30] Quintana J, Balasch M, Segales J,et al. Experimental inoculation of porcine circoviruses type 1(PCV1) and type 2 (PCV2) in rabbits and mice [J].Vet Res,2002,33(3):229-237.
[31] Tischer I,Mields W ,Wolf, D. Stydies on the pathogenicity of porcine circovirus[J]. Arch Viro l, 1986, 91, 271-276.
[32] Dulac G C, Afshar A. Porcine circovirus in Canadian pigs[J].Can J Vet Res, 1989, 53, 431-433.
[33] Tischer I, Bode L , Peters D. Distribution of antibodies to porcine circovirus in swine populations of different breeding farms [J]. Arch Virol,1995, 140, 737-743.
[34]郎洪武,张广川,吴发权等.断奶猪多系统衰弱综合征血清抗体检测[J].中国兽医科技,2000,30(3):3-5.
[35]曹胜波,陈焕春,肖少波等.猪环状病毒2型的PCR检测方法的建立和应用[J].华中农业大学学报,2001,(1):53-56.
[36]王忠田,杨汉春,郭鑫.规模化猪场猪圆环病毒2型感染的流行病学调查[J].中国兽医杂志,2002,38(10):3-6.
[37]孙泉云,沈悦,张苏华等.上海市规模化猪场猪圆环病毒病的血清学调查[J].上海畜牧兽医通讯,2003,(2):13.
[38] Staebler S., Sydler T, Buergi E. et a1. PMWS: an emerging disease identified in archived porcine tissues [J]. Vet J, 2005, 170(1):132-134.
[39] Krakowka S, Ellis J, McNeilly F, et a1. Features of porcine circovirus disease: correlations between lesions, amount and distribution of virus, and clinical outcome [J]. J Vet Diagn Invest, 2005, 17(3):213-222.
[40] Kim J, Jung K, Chae C, et a1. Prevalence of porcine circovirus type 2 in aborted fetuses and stillborn piglets [J]. Vet Rec, 2004, 155(16):489-492.
[41] Zhou J.Y, Chen Q.X, Ye J.X., et al. Serologic investigation and genomic characterization of PCV-2 isolates from different geographic regions of Zhejiang province in China[J]. Vet Res Commun, 2005, 30(2):205-220.
[42] Nawagitgul P, Harms P. A, Morozov I, et al. Modifide indirect porcine circovrus(PCV) type 2-based and recombinant capsid protein (ORF2) -based enzyme-linked immunosorbent assays for detection of antibodies to PCV [J]. Clin Diagn Lab Immanuel, 2002, 9(1):33-40.
[43] Walker I. W., Konoby C. A., Jewhurst V. A., et al. Development and application of a competitive enzyme-linked immunosorbent assay for the detection of serum antibodies to procine circovirus type 2[J]. J Vet Diagn Invest, 2000, 12(5):400-405.
[44] Larochelle R., Bielanki A., Muller R., et al. PCR detection and evidence of shedding of porcine circovirus type 2 in boar semen [J]. J Clin Microbiol, 2000, 38(12):4629-4632.
[45] Liu Qiang, Wang Li, Willson P. Quantitative competitive PCR analysis of porcine circovirus DNA in serum from pigs with postweaning multisystemic wasting syndrome[J]. Journal of Clinical Microbiology, 2000, 38:3474-3477.
[46] Junghyun Kim, Dong Un Han, Changsun Choi, et al. Differentiation of porcine circovirus PCV-1 and PCV-2 in boar semen using a multiplex nested polymerase chain reaction [J]. Journal of Virological Methods, 2001, 98:25-31.
[47] Celera VJ, Carasova P. First evidence of porcine circovirus type2 (PCV-2) infection of pigs in the Czech Republic by semi–nested PCR [J]. J Vet Med B Infect Dis Vet Public Health, 2002, 49(3):155-159.
[48] Kim J, Chae C. Multiplex nested PCR compared with in situ hybridization for the differentiation of porcine circoviruses and porcineparvovirus from pigs with postweaning multisystemic wasting syndrome[J]. Can J Vet Res, 2003, 67(2):133-137.
[49] Chung W.B, Chan W.H, Chaung H.C, et al. Real-time PCR for quantitation of porcine reproductive and respiratory syndromevirus and porcine circovirus type2 in naturally-infected and challenged pigs[J]. Virol Methods, 2005, 124:11-19.
[50]王新,郭万柱,徐志文等.应用PCR-RFLP方法对四川猪圆环病毒的检测与分型研究[J].黑龙江畜牧兽医, 2006, 6:7-9.
[51] Sirinarumitr T, Sorden S.D, Morozov I, et al. Double in situ hybridization for simultaneous detection of porcine reproductive and respiratory syndrome virus(PRRSV) and porcine circovirus (PCV) [J]. J Vet Diagn Invest, 2001, 13(1):68-71.
[52] Kim J., Chae C. Differentiation of porcine circovirus 1 and 2 in formalin-fixed, paraffin wax-embedded tissues from pigs with postweaning multisystemic wasting syndrome by in situ hybridization[J]. Res Vet Sci, 2001, 70(3):265-269.
[53] Tischar I,Mields W Wolf D.et al. Studies on epidemiology and pathogenity of porcine circovirus[J].Arch Viro1,1986, (91):271-276.
[54] Tischer I,Gelderblom H,Vettermann W et al..A very small porcine circovirus with circular single-standed DNA.[J].Nature, 1982,295(7):64-66.
[55]夏春香,肖啸,李志敏.猪圆环病毒病研究进展[J].动物医学进展, 2005, 26(1):35-38.
[56]丁淑燕,朱瑞良,高玲美.猪圆环病毒的研究进展[J].中国畜牧兽医, 2004, 31(3):41-43.
[57]金冬雁,黎孟枫.分子克隆实验指南[M].北京:科学出版社,1996,55-56.
[58]金冬雁,黎孟枫.分子克隆实验指南[M].北京:科学出版社,1992,19-22.