Amplification and Sequence Analysis of VP1 Gene Fragment of Local Epidemic IBDV Isolates

为了解鸡传染性法氏囊病病毒(IBDV)地方流行毒株在基因组B节段关键序列上的分子进化情况,通过反转录PCR(RT-PCR)技术对6个IBDV不同地方分离株的VP1基因中与病毒致病型相关的一个片段(1 518bp~1 997bp,共480bp)进行扩增,并对所获得的VP1 480bp片段进行核苷酸的序列测定,与IBDV参考株和常用疫苗株的相应序列进行比较分析和绘制遗传进化树。结果表明,6个分离株与参考株在VP1RNA聚合酶基序Ⅵ和基序Ⅶ上均保守,在特征性氨基酸位点上,3个广西分离株QX0604、WZ0704和NN0603与参考的vvIBDV相同,而分离株BH111(广西)、JS17(江苏)和HUN0804(湖南)则既具有vvIBDV的位点特征,又具有经典毒株特征,但不同毒株在不同位点上表现不同;在同源性分析上,JS17、BH111、QX0604、WZ0704和NN0603等5个毒株与参考用vvIBDV毒株的同源性较高,在核苷酸和氨基酸水平上分别为92.5%~100.0%和97.5%~100.0%,但与2个常用疫苗株和经典毒株的同源性仅分别为87.9%~90.0%和96.2%~96.9%,分离株HUN0804则与经典毒株和2个疫苗株更接近;在遗传进化树上,JS17和BH111等5个分离株与vvIBDV在一个分支上,亲缘关系更近,而HUN0804则与经典毒株和2个疫苗株在另一个大分支。表明不同分离株在VP1的功能基序上保持保守,但分离株在特征性氨基酸位点上则发生了变化,大多数分离株与常用疫苗株之间的同源性较低,亲缘关系较远。In order to look insight into the molecular evolution in B segment of different local epidemic infectious bursal disease virus（IBDV）isolates,the present study was carried out by amplification and sequencing of a fragment with 480bp（which is related to pathotype of IBDV）located in VP1gene（1 518bp-1 997bp）of IBDV by RT-PCR.The sequences of the epidemic IBDV isolates were compared with reference IBDV and commonly used vaccine strains,and the phylogenetic tree was established.The results showed that the motifⅥ and motifⅦ located in the VP1 480 bp region were highly conserved between the 6local epidemic IBDV isolates and reference IBDVs.The characteristic amino acid residues of isolates QX0604,WZ0704 and NN0603were conserved to vvIBDV,however,the other 3isolates showed mixed characteristic amino acid residues,some residues were conserved to vvIBDV,and some were conserved to classical IBDV.On the other hand,the five field isolates QX0604,WZ0704,NN0603,JS17 and BH111showed high homology to vvIBDV,with 92.5%-100.0% and 97.5%-100.0%in nucleotide and amino acid levels,respectively.They showed lower homology to the 2commonly used vaccine strains and reference classical IBDVs（87.9%-90.0%in nucleotide level,96.2%-96.9%in amino acid level）.HUN0804 showed high homology to the 2commonly used vaccine strains and reference classical IBDVs.In the phylogenetic tree,the five isolates were grouped with vvIBDV strain,HUN0804 grouped with vaccine strains and reference classical IBDVs.These results suggested that local epidemic IBDVs were conserved in the functional motif of VP1,however,some of the characteristic amino acid residues were changed and most of the isolates showed low homology and genetic relationship to commonly used vaccine strains.广西自然科学基金项目(2014GXNSFDA118018)； 广西高校科学技术研究项目(2013YB069,200809LX255)； 南宁市科技攻关项目(20142079