致蛋鸡血管瘤J亚群禽白血病病毒cDNA全序列分析

Full-length cDNA sequence analysis of avian leukosis viruses subgroup J isolated from chickens with clinical hemangioma

【目的】了解近年来我国商品蛋鸡群中以血管瘤为主要表型的J亚群禽白血病病毒（Avian Leukosis Virus subgroup J,ALV-J）的分子生物学特性,为控制ALV-J在鸡群中流行提供基础资料。【方法】采用PCR扩增和序列分析技术,对分离自血管瘤或者血管瘤与髓样细胞瘤（Myeloid Leukosis,ML）并存的3株蛋鸡ALV-J毒株前病毒DNA的全序列及3株商品蛋鸡血管瘤型分离毒和1株商品蛋鸡ML型分离毒的致瘤关键性序列进行研究。【结果】来自血管瘤或者血管瘤与ML并存的商品蛋鸡分离毒株与来自肉鸡分离毒株的全序列差异明显,在遗传进化树上分属两个大的分支;研究发现商品蛋鸡血管瘤及ML混合病例分离毒JS09GY3与JS09GY6株的引物结合位点（Primer Binding Site,PBS）-Leader中出现极为罕见的连续19bp的插入突变,其与劳斯相关病毒1（Rous Associated Virustype1,RAV-1）、劳斯相关病毒2（Rous Associated Virustype2,RAV-2）及劳斯肉瘤病毒施密特-鲁宾二氏[Rous sarcoma virus（strain Schmidt-RuppinB）,RSV-SRB]毒株序列相同;通过对U3区调控元件的分析,发现血管瘤商品蛋鸡病例分离毒NHH与JS09GY5的U3区各发生1处连续序列缺失,出现了极为独特的c-Est-1、TCF11及C/EBP结合位点,这些调控元件可能与病毒的致肿瘤特性相关;所测的5株血管瘤商品蛋鸡分离毒均保留完整E元件,而所有肉鸡分离毒的E元件均发生了几乎相同的大部分序列缺失;首次发现血管瘤商品蛋鸡分离毒JS09GY3的E元件中有11bp的连续插入序列。【结论】商品蛋鸡血管瘤型ALV-J与肉鸡分离毒在全序列上差异明显,U3、DR1和E元件等区域有一部分特殊的突变与毒株的宿主类型和肿瘤表型有一定关系,其功能尚需进一步研究。而血管瘤型、髓细胞瘤型ALV-J可能是ALV-J与其它反转录病毒的重组毒。

[Objective]To understand the molecular characteristics of subgroup J Avian Leukosis Viruses（ALV-J） isolated from chickens with clinical hemangioma,as well as to get more information for controlling the spread of ALV-J in layer chickens flocks.[Method]We amplified the full-length viral cDNA sequences of three layers isolates associated with simple hemangioma or coexisting of hemangioma and myeloid leukosis（ML） by PCR.We also obtained some partial sequences of three layer isolates related to hemangioma and one layer isolate from ML case.Then we analyzed and compared the sequences by using DNAstar software.[Results]The phylogentic analysis showed the significant differences in the complete sequences between isolates from layer hemangioma cases and from broilers,which were grouped to two branches in the phylogenetic tree.We noted a special 19bp insertion mutation in Primer Binding Site（PBS）-Leader sequences in both JS09GY3 and JS09GY6 isolates from layer chickens with hemangioma and ML,which sequence was same to Rous Associated Virus type 1（RAV-1）,Rous Associated Virus type 2（RAV-2） and Rous sarcoma virus（strain Schmidt-Ruppin B）（RSV-SRB）.In addition,different continuous sequences deletions were found in the U3 regions of NHH and JS09GY5.By motif analysis,we found some distinct motifs including c-Est-1,TCF11 and C /EBP only in the isolates from layers with hemangioma.The five isolates associated with layer hemangioma exhibited intact E element sequences but almost identical substantial deletion was found in all Chinese broiler isolates.An 11bp continuous nucleotide insertion in the E element of JS09GY3 was found.[Conclusion]Isolates from layer showing hemangioma and broilers exhibited evident difference.We found some special mutation sites in U3,DR1 and the E element showed some potential relationship with the host breeds and the tumor phenotype,which function needs to be investigated in future.The isolates from layer cases with coexisting of hemangioma and ML were the recommbinants of ALV-J and other retroviruses.