1株绵羊边界病病毒的分离鉴定与序列分析

doi:10.11843/j.issn.0366-6964.2014.03.013

畜牧兽医学报 ›› 2014, Vol. 45 ›› Issue (3): 434-442.doi: 10.11843/j.issn.0366-6964.2014.03.013

1株绵羊边界病病毒的分离鉴定与序列分析

刘霞^1,2，毛立²，李文良²，杨蕾蕾²，张纹纹²，魏建忠^{1 *}，江杰元^{2 *}

（1.安徽农业大学动物科技学院，合肥 230036；2.江苏省农业科学院兽医研究所，农业部兽用生物制品工程技术重点实验室，国家兽用生物制品工程技术研究中心，南京 210014)

收稿日期:2013-09-04 出版日期:2014-03-23 发布日期:2014-03-21
通讯作者: 魏建忠（1956-），男，教授，E-mail：wjz@ahau.edu.cn；江杰元（1957-），男，研究员， E-mail：jieyuanj57@gmail.com
作者简介:刘霞（1988-），女，安徽六安人，硕士生，主要从事动物疫病诊断研究,E-mail：ZHEDADA@126.com
基金资助:
江苏省农业科技自主创新资金项目（CX(12)3064）

Isolation and Sequence Analysis of a Border Disease Virus from Sheep

LIU Xia^1,2， MAO Li²， LI Wen-liang²， YANG Lei-lei²， ZHANG Wen-wen², WEI Jian-zhong^1*， JIANG Jie-yuan^2*

(1.College of Animal Science and Technology， Anhui Agricultural University，Hefei 230036， China;2.Key Laboratory of Veterinary Biological Engineering and Technology of Ministry of Agriculture，National Center for Engineering Research of Veterinary Bio-products，Institute of Veterinary Medicine，Jiangsu Academy of Agricultural Sciences，Nanjing 210014， China)

Received:2013-09-04 Online:2014-03-23 Published:2014-03-21

摘要/Abstract

摘要：

对华东地区某羊场采集定点监测的绵羊血液样品进行边界病的感染情况调查。通过RT-PCR方法检测瘟病毒5′-UTR基因，发现1只绵羊的血清样品在不同时间检测均为阳性，证实其为边界病病毒（BDV）持续性感染。将该阳性血清样品接种MDBK细胞，进行病毒分离鉴定。通过N^pro基因RT-PCR扩增、电镜观察、病毒全基因组序列分析，并对分离毒株5′-UTR和N^pro基因进行遗传进化分析。结果显示，该毒株在MDBK细胞上盲传至第8代，不发生细胞病变。RT-PCR扩增获得N^pro基因预期大小片段，电镜观察以及测序分析表明成功分离出1株BDV，将其命名为JSLS12-01。设计覆盖BDV全基因组的6对引物，RT-PCR扩增并测序，该分离株全基因组序列大小为12 227 bp（GenBank登录号为KC963426）。序列比对结果显示，该毒株基因组序列和氨基酸序列与已有BDV分离株之间相似性分别为72.3%~80.4%和80.1%~89.7%。5′-UTR和N^pro基因系统进化分析显示，该分离株与BDV 3参考毒株亲缘关系最近，相似性最高分别为87.7%和75.8%。结果证实从绵羊血液样品中鉴定并分离到1株ncp型BDV分离株，经序列分析表明该毒株属于BDV 3亚型。

关键词: 边界病病毒, 分离, 鉴定, 序列分析

Abstract:

In order to investigate the prevalence status of border disease，serum samples were collected from one sheep flock in east China and tested through RT-PCR method with the 5′-UTR generic primers of pestivirus.One sheep was detected as positive infection of border disease virus (BDV) at different sampling times.Positive serum sample was inoculated onto MDBK cells for viral isolation and identification.Then RT-PCR amplification of N^pro gene，electron microscopy detection of isolated virus，analysis of the complete genome sequences and phylogenetic analysis of the 5′-UTR and N^pro gene were performed.The results showed that no cytopathy effect (CPE) was observed until the eighth generation by serial passages.BDV was detected from cell cultures by RT-PCR using N^pro gene primers; and confirmed with sequence analysis as well as electron microscopy detection.The isolate was named as JSLS12-01.Amplification of complete genomic sequence was performed with six pairs of primers covering the near-full genome.The viral genome was about 12 227 nucleotides in size and had been submitted to the GenBank with registered number KC963426.Based on comparative sequence analysis of full genome and the deduced amino acids between this isolate and BDV reference strains，there were 72.3%-80.4% and 80.1%-89.7% identities，respectively.Phylogenetic analysis using partial 5′-UTR nucleotide sequence and N^pro sequence identified that the virus clustered within BDV 3 viruses，with the highest homology of 87.7% and 75.8%，respectively.Above all，we successfully isolated and identified one BDV 3 strain from sheep of the non-cytopathogenic biotype.

Key words: border disease virus (BDV), isolation, identification, sequence analysis

中图分类号:

S852.659.6

刘霞，毛立，李文良，杨蕾蕾，张纹纹，魏建忠，江杰元. 1株绵羊边界病病毒的分离鉴定与序列分析[J]. 畜牧兽医学报, 2014, 45(3): 434-442.

LIU Xia， MAO Li， LI Wen-liang， YANG Lei-lei， ZHANG Wen-wen, WEI Jian-zhong， JIANG Jie-yuan. Isolation and Sequence Analysis of a Border Disease Virus from Sheep[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(3): 434-442.

参考文献

［1］LIU L，XIA H，WAHLBERG N，et al.Phylogeny，classification and evolutionary insights into pestiviruses［J］.Virology，2009，385(2):351-357.
［2］BECHER P，SCHMEISER S，OGUZOGLU T C，et al.Complete genome sequence of a novel pestivirus from sheep［J］.J Virol，2012，86(20):11412.
［3］BECHER P，ORLICH M，THIEL H J.Complete genomic sequence of border disease virus，a pestivirus from sheep［J］.J Virol，1998，72(6):5165-5173.
［4］BECHER P，MEYERS G，SHANNON A D，et al.Cytopathogenicity of border disease virus is correlated with integration of cellular sequences into the viral genome ［J］.J Virol，1996，70(5):2992-2998.
［5］OGUZOGLU T C，TAN M T，TOPLU N，et al.Border disease virus (BDV) infections of small ruminants in Turkey: a new BDV subgroup［J］.Vet Microbiol，2009，135(3-4):374-379.
［6］CABEZÓN O，ROSELL R，VELARDE R，et al.Border disease virus shedding and detection in naturally infected Pyrenean chamois (Rupicapra pyrenaica)［J］.J Vet Diagn Invest，2010，22(5):744-747.
［7］CABEZÓN O，VELARDE R，MENTABERRE G，et al.Experimental infection with chamois border disease virus causes long-lasting viraemia and disease in Pyrenean chamois (Rupicapra pyrenaica)［J］. J Gen Virol，2011，92(Pt 11):2494-2501.
［8］DUBOIS E，RUSSO P，PRIGENT M，et al.Genetic characterization of ovine pestiviruses isolated in France,between 1985 and 2006［J］.Vet Microbiol，2008，130(1-2): 69-79.
［9］YAZICI Z，SERDAR M S，GUMUSOVA S O，et al.Molecular diagnosis and seroepidemiology of pestiviruses in sheep［J］.Vet Arhiv，2012，82:35-45.
［10］李文良，毛立，张纹纹，等.从山羊持续性腹泻病例中分离到边界病病毒（BDV）［J］.江苏农业学报，2013，29(1):222-224.
［11］VILCEK S，HERRING A J，HERRING J A，et al.Pestiviruses isolated from pigs，cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis［J］.Arch Virol，1994 ，136(3-4):309-323.
［12］VANTSIS J T，BARLOW R M，FRASER J，et al.Experiments in Border disease.Ⅷ.Propagation and properties of a cytopathic virus［J］.J Comp Pathol，1976，86(1):111-120.
［13］GIAMMARIOLI M，ROCCA S A L,STEINBACH F，et al.Genetic and antigenic typing of border disease virus (BDV) isolates from Italy reveals the existence of a novel BDV group［J］.Vet Microbiol，2011，147(3-4):231-236.
［14］THABTI F，LETELLIER C，HAMMAMI S，et al.Detection of a novel border ddisease virus subgroup in Tunisian sheep［J］.Arch Virol，2005，150(2):215-229.
［15］陆承平.兽医微生物学［M］.3版.北京：中国农业出版社，2001.
［16］LI W L，MAO L，ZHAO Y Q，et al.Detection of border disease virus(BDV) in goat herds suffering diarrhea in eastern China［J］.Virol J，2013，10:80-87.
［17］VALDAZO-GONZÁLEZ B，ALVAREZ M，SANDVIK T.Prevalence of border disease virus in Spanish lambs［J］.Vet Microbiol，2008，128 (3-4):269-278.
［18］FERNÁNDEZ-SIRERA L，CABEZÓN O，ALLEPUZ A，et al.Two different epidemiological scenarios of border disease in the populations of Pyrenean chamois (Rupicapra p.pyrenaica) after the first disease outbreaks［J］.PLoS ONE, 2012，7（12）：e51031.10.1371/journal.pone.0051031.
［19］DEPNER K R，HÜBSCHLE O J，LIESS B.Transplacental BVD virus transmission after experimental inoculation of goats in different pregnancy stages［J］.Dtsch tierarztl Wochenschr，1990，97(10):421-423.
［20］朱其太.边界病的研究进展［J］.甘肃畜牧兽医，1998(4):32-34.
［21］VALDAZO-GONZÁLEZ B，ALVAREZ-MARTÍNEZ M，GREISER-WILKE I.Genetic typing and prevalence of Border disease virus (BDV) in small ruminant flocks in Spain［J］. Vet Microbiol，2006，117(2-4):141-153.
［22］GARCÍA-PÉREZ A L，MINGUIJÓN E，ESTÉVEZ L，et al.Clinical and laboratorial findings in pregnant ewes and their progeny infected with Border disease virus (BDV-4 genotype)［J］.Res Vet Sci，2009，86(2):345-352.

[1]	康浩然, 刘重阳, 于勇, 张俊杰, 潘子豪, 姚火春. 2017-2018年我国部分地区牛支原体的分离鉴定及多位点序列分型[J]. 畜牧兽医学报, 2019, 50(9): 1857-1863.
[2]	李秀博, 刘存, 崔玉东, 梁琳, 张建伟, 崔尚金. 猪A型塞内卡病毒分离鉴定及其VP1基因变异分析[J]. 畜牧兽医学报, 2019, 50(9): 1945-1950.
[3]	马玉俊, 张凯, 王磊, 张康, 张宏, 王学智, 李杰, 梁子敬, 张景艳, 李建喜. 白蚁肠道纤维素降解菌的分离和鉴定[J]. 畜牧兽医学报, 2019, 50(9): 1951-1954.
[4]	邱静静, 丁轲, 余祖华, 贾艳艳, 张春杰, 汪洋, 赵战勤. 2014-2017年河南省副猪嗜血杆菌分离菌株的PCR鉴定及分型[J]. 畜牧兽医学报, 2019, 50(8): 1649-1657.
[5]	牛凯, 程汝佳, 许冠龙, 冯宇, 孙石静, 范学政, 彭小薇, 朱良全, 秦玉明, 丁家波, 常维山, 蒋卉. 一株鹿源牛种布鲁菌的鉴定及全基因组分析[J]. 畜牧兽医学报, 2019, 50(8): 1666-1675.
[6]	邱静静, 丁轲, 余祖华, 贾艳艳, 张春杰, 汪洋, 赵战勤. 2014-2017年河南省副猪嗜血杆菌分离菌株的PCR鉴定及分型[J]. 畜牧兽医学报, 2019, 50(8): 1649-1657.
[7]	牛凯, 程汝佳, 许冠龙, 冯宇, 孙石静, 范学政, 彭小薇, 朱良全, 秦玉明, 丁家波, 常维山, 蒋卉. 一株鹿源牛种布鲁菌的鉴定及全基因组分析[J]. 畜牧兽医学报, 2019, 50(8): 1666-1675.
[8]	申松玮, 王泽源, 范威峰, 祝常椿, 陆游, 石宝兰, 刘雷, 沈海峰, 焦库华, 秦涛, 陈素娟, 彭大新, 刘秀梵. 2017-2018年华东地区H9亚型禽流感病毒分离毒株的抗原差异分析[J]. 畜牧兽医学报, 2019, 50(6): 1230-1238.
[9]	申松玮, 王泽源, 范威峰, 祝常椿, 陆游, 石宝兰, 刘雷, 沈海峰, 焦库华, 秦涛, 陈素娟, 彭大新, 刘秀梵. 2017-2018年华东地区H9亚型禽流感病毒分离毒株的抗原差异分析[J]. 畜牧兽医学报, 2019, 50(6): 1230-1238.
[10]	刘琪, 史利军, 梁琳, 张玲玲, 袁维峰, 梁瑞英, 李金祥, 崔尚金. 一株猫泛白细胞减少症病毒的分离鉴定及其VP2和NS1基因的变异分析[J]. 畜牧兽医学报, 2019, 50(5): 1106-1112.
[11]	刘琪, 史利军, 梁琳, 张玲玲, 袁维峰, 梁瑞英, 李金祥, 崔尚金. 一株猫泛白细胞减少症病毒的分离鉴定及其VP2和NS1基因的变异分析[J]. 畜牧兽医学报, 2019, 50(5): 1106-1112.
[12]	吕天星, 李松建, 郝永清. 鉴别显色培养基在奶牛乳房炎病原菌快速鉴定中的应用[J]. 畜牧兽医学报, 2019, 50(4): 851-860.
[13]	吕天星, 李松建, 郝永清. 鉴别显色培养基在奶牛乳房炎病原菌快速鉴定中的应用[J]. 畜牧兽医学报, 2019, 50(4): 851-860.
[14]	李然, 汤承, 罗雪, 岳华. 一株牦牛源G6P[11]型牛轮状病毒的分离鉴定及部分基因的序列分析[J]. 畜牧兽医学报, 2019, 50(3): 592-601.
[15]	杨振兴, 孟锦昕, 肖雷, 朱建波, 廖德芳, 高林, 李占鸿, 杨恒, 李华春. 流行性出血病病毒(EHDV)血清7型毒株在中国的首次分离与鉴定[J]. 畜牧兽医学报, 2019, 50(3): 602-610.

1株绵羊边界病病毒的分离鉴定与序列分析

Isolation and Sequence Analysis of a Border Disease Virus from Sheep

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价