摘要
为了解信阳地方土鸡中J亚群禽白血病病毒(ALV-J)的遗传进化情况,对疑似感染ALV的鸡进行了核酸检测、病理组织学观察,并采集肝脏组织接种鸡胚成纤维细胞DF-1进行病毒分离;对分离到的ALV-J全基因组进行PCR扩增并测序;使用MegAlign软件对病毒全序列进行核苷酸相似性比对,同时对gp85、3′UTR和U3区进行序列分析,并使用MEGA 11.0软件构建遗传进化树。结果:PCR扩增出ALV-J特异性条带;病理组织学观察结果显示,病鸡肝脏、脾脏组织结构紊乱,可见大量髓样瘤细胞增生;肝脏组织处理液接种DF-1细胞后,细胞上清液经ELISA检测P27抗原呈阳性,将分离株命名为HN24XY03。全基因组序列分析表明,HN24XY03株全长7609 bp,符合典型禽反转录病毒基因组结构特点,与25株ALV-J参考毒株全基因组核苷酸序列的同源性为94.3%~95.8%,其中与广西分离株GX14NN01和GX16YL02株同源性最高,亲缘关系最近;gp85基因及推导氨基酸序列分析显示,HN24XY03株与ALV-J广西分离株GX14NN01、GX17NN05亲缘关系较近,处于同一分支;与ALV-J原型株HPRS103相比,HN24XY03 gp85蛋白存在34个氨基酸变异位点和1个位点(D61-)缺失,其中9个位于hr1区,9个位于hr2区;3′UTR序列分析结果显示,分离株HN24XY03为UTR-Δr-TM毒株;利用SoftBerry NSITE在线分析程序分析发现,与英国分离株HPRS103相比,HN24XY03 U3区转录调控元件相对保守。本研究结果丰富了信阳地方土鸡ALV-J的分子流行病学资料,为进一步研究ALV-J在我国地方品种鸡群中的遗传进化和致病性奠定了基础。
In order to understand the genetic evolution of avian leukosis virus subgroup J(ALV-J)in local chickens in the Xinyang area,histopathological observation and PCR detection were carried out in chickens suspected of avian leukosis.Liver tissue samples were collected from the chickens and were inoculated with DF-1 cells for virus isolation.Then,the whole genome of ALV-J was amplified and sequenced by polymerase chain reaction(PCR),the nucleotide similarity was compared by the MegAlign software,and the gp85,3′UTR and U3 regions were analyzed.Based on the whole genome,the gp85 gene and its deduced amino acid sequence,the genetic evolution tree was constructed using the MEGA11.0 software.The results were as follows:The PCR test showed that the positive sample amplified the ALV-J-specific band.The histopathological results showed that the liver and spleen tissues of the diseased chickens were structurally disordered,and a large number of medullary tumor cells were proliferated.After DF-1 cells were inoculated with liver tissue treatment solution,the cell supernatant was positive for P27 antigen by ELISA.The isolate was named HN24XY03.The whole genome sequence analysis showed that HN24XY03 was 7609 bp in length,which was consistent with the genomic structure of typical retroviruses,and the homology of the whole genome nucleotide with 25 ALV-J reference strains was 94.3%-95.8%.Among them,it had the highest homology with the Guangxi isolates GX14NN01 and GX16YL02,and they had the closest genetic relationship.The analysis of the gp85 gene and the deduced amino acid sequence showed that HN24XY03 was closely related to the ALV-J Guangxi isolates GX14NN01 and GX17NN05,and they was in the same branch.Compared with HPRS103,there were 34 amino acid variant sites and 1 locus(D61-)deletion of the HN24XY03 gp85 protein,of which 9 were located in the HR1 region and 9 were located in the HR2 region.The analysis of the 3′UTR sequence showed that HN24XY03 was a UTR-Δr-TM strain.The SoftBerry NSITE online analysis program showed that,compared with the British isolate HPRS103,the transcriptional regulatory elements in the U3 region of HN24XY03 were relatively conserved.The results of this study provided data for the molecular epidemiological study of ALV-J in local chickens in the Xinyang area,and laid a foundation for further research on the genetic evolution and pathogenicity of ALV-J.
作者
焦凤超
赵瑜
董建国
何书海
曲哲会
李迎晓
陈敏
JIAO Fengchao;ZHAO Yu;DONG Jianguo;HE Shuhai;QU Zhehui;LI Yingxiao;CHEN Min(College of Animal Science and Technology,Xinyang Agricultural and Forestry University,Xinyang 464000,China)
出处
《畜牧与兽医》
北大核心
2025年第7期103-114,共12页
Animal Husbandry & Veterinary Medicine
基金
信阳农林学院科技服务团队项目(2022FWTD)
信阳农林学院青年教师科研基金项目(QN2023019)
信阳农林学院科技创新团队项目(KJCXTD-201901)。
