鸡传染性法氏囊病一个自然病例的病原分离及其分子特征分析  

Isolation and Identification of Infectious Bursal Disease Virus from a Clinical Flock and Molecular Characterization Analysis

作　　者：何秀苗[1] 官丁明[1] 韦平[2] 秦爱建 

机构地区：[1]广西民族大学,广西南宁530006 [2]广西大学,广西南宁530004 [3]扬州大学,江苏扬州225009

出　　处：《中国家禽》2009年第14期18-22,共5页China Poultry

基　　金：广西自然科学基金项目(桂科自0728058);广西科技攻关项目(2007A04024;0537008-3A);广西民族大学引进人才启动基金项目(0409025);扬州大学江苏省动物预防医学重点实验室开放课题(K06012)

摘　　要：设计针对传染性法氏囊病病毒(IBDV)VP2基因高变区(vVP2)的引物对湖南永州送检的一群疑似传染性法氏囊病病例的法氏囊组织通过逆转录-聚合酶链式反应(RT-PCR)技术进行检测,应用鸡胚绒毛尿囊膜(CAM)接种的方法对阳性样品进行病毒分离,同时对IBDV分离株中的vVP2进行限制性内切酶分析和核苷酸序列测定,分析关键位点的氨基酸特征和同源性,绘制遗传进化树。结果成功分离出了4个IBDV毒株,其中HuN0801、HuN0802和HuN0803的特征性位点的氨基酸为222A、249Q、254G、256I、279D、284A、294I和299S,属于vvIDBV的特征,并与vvIBDV参考株具有较高的同源性;HuN0804的则为222P、249Q、254G、256V、279N、284T、294I和299N,符合弱毒株的特征,与弱毒参考株的同源性较高;4个分离株与常用疫苗株的同源性普遍较低。遗传进化分析表明,3个vvIBDV分离株与vvIBDV参考株和中等偏强毒力株YSLMB同处于一个大的分支,并与欧洲株DV86、湖南株HuN亲缘关系最近;而HuN0804与弱毒株、经典毒株和常用疫苗株同处于一个大分支。研究表明,本病例中存在不同致病型IBDV毒株的混合感染。RT-PCR based on a set of primers infectious bursal disease virus（IBDV） was used to flock in Yongzhou region of Hunan province. Viral embryo inoculation via ehorio-allantoic membrane vVP2 of the 4 isolates were analyzed by restriction designed to amplify the hypervariable region of VP2 gene （vVP2） of detect the tissue samples of Fabficus＇ bursa collected from a clinical isolation was performed on the RT-PCR positive samples by chicken （CAM） and 4 isolates of IBDV were obtained. Amplified samples of enzyme analysis （REA）and sequenced analysis respectively,and their phylogenetic tree was made based on the nucleotide sequences. The results indicated that the isolate HUN0801 ,HUN0802 and HUN0803 were classified to be very virulent IBDV （vvIBDV）with the specific amino acids at critical sites as 222A, 249Q,254G,2561,279D,284A,2941 and 299S and shared the highest homologies with other vvIBDV strains,while isolate HUN0804 was identified as an attenuated strain with the specific amino acids at critical sites as 222P,249Q,254G, 256V,279N,284T,294I and 299N,shared the highest homology with other attenuated strains,and the homologies between the isolates and the commonly used vaccine strains were low. The vvIBDV isolates were belong to a large group with vvIBDV reference strains and intermediate-plus virulent strain YSLMB,and have a close relationship to Europe strain DV86 and Hunan strain HuN,while HUN0804 was belonged to another group with attenuated strains and other commonly used vaccine strains in the phylogenetic tree. The results of the study demonstrated that there were various pathotypes of IBDV co-infection in the flock.

关 键 词：传染性法氏囊病 超强毒株 弱毒株 RT-PCR VP2基因高变区 分子特征 

分 类 号：S858.315.3[农业科学—临床兽医学] S858.31[农业科学—兽医学]