贵州地区威宁鸡禽白血病病毒分离鉴定及env基因遗传进化分析

Isolation and identification of Avian leukosis virus from Weining chickens in Guizhou region and genetic evolution analysis of env gene

为了对贵州省纳雍县某养殖场发病威宁鸡种鸡进行禽白血病病毒(Avian leukosis virus,ALV)的分离鉴定及env基因遗传进化分析,试验对送检的病死鸡进行临床症状和剖检病变观察,以及采集发病鸡肝脏进行细菌分离、ALV分离、ALV p27抗原的ELISA检测和病毒PCR鉴定,进一步对分离株的env基因进行克隆、测序,与GenBank收录的其他ALV参考株进行序列比对分析,并构建遗传进化树。结果表明:剖检可见病死鸡腹腔中及肝脏上有肿瘤结节;取病死鸡的肝脏、心脏接种于含5%新生牛血清的TSA培养基和麦康凯琼脂培养基培养24 h,未见有细菌生长;取病死鸡肝脏组织,处理后接种鸡胚成纤维细胞(DF-1)细胞,用ALV ELISA抗原检测试剂盒检测发现ALV p27抗原呈阳性;用ALV-J亚群env基因引物进行PCR检测,结果呈阳性,PCR产物大小约为924 bp;将分离毒株命名为ALV-GZ株,该毒株与国内外分离的ALV-J亚群的核苷酸相似性为97.0%~99.4%,与国内外分离的其他亚群的核苷酸相似性为50.8%~53.0%,与AHFX/16916株(GenBank登录号为MF614031.1)处于同一遗传进化分支,亲缘关系最近。说明贵州省纳雍县某养殖场发病威宁鸡种鸡感染了ALV-J亚群,且ALV-J亚群毒株在缓慢的变异演化中向地方品系鸡蔓延传播。

In order to carry out the isolation and identification of Avian leukosis virus from sick Weining chickens in a breeding farm in Nayong County of Guizhou Province and genetic evolution analysis of env gene,in the experiment,the dead chickens were observed for clinical symptoms and autopsy lesions;the livers of the sick chickens were collected for bacterial isolation,ALV isolation,AlV p27 antigen ELISA detection and viral PCR identification.The env gene of the isolates was further cloned and sequenced;sequence alignment analysis was performed with other ALV reference strains included in GenBank,and a genetic evolutionary tree was constructed.The results showed that there were tumor nodules in the abdominal cavities and livers of the dead chickens during autopsy.The livers and hearts of the dead chickens were taken and inoculated with 5%calf serum TSA medium and McConkay medium for 24 h,and no bacterial growth was seen.Dead chicken liver tissue was taken and inoculated on chicken embryo fibroblast(DF-1)cells after treatment,and ALV p27 antigen was found to be positive by using ALV ELISA antigen detection kit.PCR detection with ALV-J subgroup env gene primers was positive,and the PCR product size was approximately 924 bp.The isolated strain was named ALV-GA strain.The nucleotide similarity between the isolated ALV-J strain and the ALV-J subgroup isolated at home and abroad was 97.0%-99.4%,and the nucleotide similarity with other subgroup isolated at home and abroad was 50.8%-53.0%,which was closely related to AHFX/16916 strain(GenBank accession number MF614031.1)and was in the same genetic evolutionary branch.The results indicated that the sick Weining chicken breeders were infected with ALV-J subgroup in a farm in Nayong County of Guizhou Province,and the ALV-J subgroup spread to the local breeds in a slow mutation evolution.