我国部分省区鸡传染性法氏囊病病毒的分子流行病学研究

Molecular Epidemiology of Infectious Bursal Disease Viruses Isolated from Some Provinces of China

应用RT-PCR技术,对2000-2006年间收集于广西、江苏、浙江、安徽、海南的临床疑似传染性法氏囊病病鸡的法氏囊、脾脏和骨髓样品进行检测,对检测呈阳性的病料采用9～11日龄的鸡胚通过绒毛尿囊膜或鸡胚成纤维细胞接种的方法进行鸡传染性法氏囊病病毒(IBDV)的分离和传代,结果成功分离到23株IBDV;设计针对IB-DVVP2基因高变区(用vVP2表示)的引物对这些分离株及5株常用的中等毒力商品疫苗株进行RT-PCR扩增并对其进行酶切分析和核苷酸序列测定,分析比较23个分离株与参考毒株的vVP2的序列并绘制遗传系谱树。结果表明BH09、BH11、JS1、JS7、YY1、YY6、YL051、050222、TSC-2(9)、TZ(3)、HN0602共11个分离株属于超强毒株,与已发表的vvIBDV其它中国分离株、日本分离株OKYM、欧洲分离株DV86和UK661的亲源关系均较近;040124、YL052、020180、YLZF2、040131、BH15、YY2、050045、050057、050258、TSC-1(3)、A038共12株属于经典毒株,其中040124、YL052 2株属于中等偏强毒力疫苗株,其余10株则属于弱毒疫苗株。本研究的结果表明,近6年来在我国5个省区养鸡业中流行的IBDV主要为vvIBDV毒株,而且所有分离株VP2基因高变区均缺乏明显的时间和地域的遗传特征。

Tissue samples of Fabricius＇ bursal, spleen and bone marrow collected from the provinces of Gangxi, Anhui, Jiangshu, Zhejiang and Hainan during the years of 2000-2006, were detected by a developed reverse transcriptase polymerase chain reaction （RT-PCR） technique for IBDV. Viral isolations were performed on the positive samples by chicken embryo inoculation via chorio-allantoic membrane （CAM） or cell culture with chicken embryo fibroblast （CEF） and 23 isolates of IBDV were obtained. A set of primer was designed to amplify the vVP2 of 23 isolates and 5 commercial vaccine strains with intermediate and intermediate-plus virulence by RT-PCR. The PCR products were analyzed by restriction enzyme analysis （REA） and sequenced respectively. The sequences of all the isolates and reference viruses were analyzed and compared, and their phylogenetic tree was made based on the nucleotide sequences. The results indicated that isolate BH09, BH11, JS1, JST, YY1, YY6, YL051, 050222, TSC-2（9） ,TZ（3） and HN0602 were classified tO be very virulent IBDV （vvlBDV）, and have close phylogenetic relationship with other Chinese isolates, Japanese isolate OKYM and European isolate DV86 and UK66, while isolate 040124, YL052, 020180, YLZF2, 040131, BH15, YY2, 050045, 050057, 050258, TSC-1（3） and A038 were classified to be classical IBDV （clBDV）. Isolate 040124 and YL052 were further identified as intermediate and intermediate-plus virulent vaccine strains, and the rest 10 isolates were identified as attenuated vaccine strains. The results of the study demonstrated that the viruses prevailing in chickens in these 5 provinces during 2000-2006 are vvIBDV and there is no significant evident divergence in vVP2 of all the isolates on both time and geology.