广东地方品种鸡ALV-K分子流行病学及gag基因12 bp缺失对病毒体外复制能力影响的研究

doi:10.11843/j.issn.0366-6964.2022.11.021

畜牧兽医学报 ›› 2022, Vol. 53 ›› Issue (11): 3956-3966.doi: 10.11843/j.issn.0366-6964.2022.11.021

广东地方品种鸡ALV-K分子流行病学及gag基因12 bp缺失对病毒体外复制能力影响的研究

郭妍妍, 梁灿新, 李锦群, 董欣怡, 廖明, 曹伟胜*

华南农业大学兽医学院, 人兽共患病防控制剂国家地方联合工程实验室, 农业部兽用疫苗创制重点实验室, 广东省动物源性人兽共患病预防与控制重点实验室, 广州 510642

收稿日期:2022-03-31 出版日期:2022-11-23 发布日期:2022-11-25
通讯作者: 曹伟胜,主要从事家禽重要传染病防控和净化研究,E-mail:caoweish@scau.edu.cn
作者简介:郭妍妍(1996-),女,安徽安庆人,硕士,主要从事动物传染病研究,E-mail:15397915301@163.com
基金资助:
广东省重点领域研发计划项目（2020B020222001）；广东省家禽产业技术体系（2022KJ128）；国家肉鸡产业技术体系（CARS-41-G10）

Study on Molecular Epidemiology of ALV-K in Guangdong Local Breed Chickens and the Effect of 12 bp Deletion of gag Gene on Virus Replication Ability in vitro

GUO Yanyan, LIANG Canxin, LI Jinqun, DONG Xinyi, LIAO Ming, CAO Weisheng*

National and Local Joint Engineering Laboratory for Zoonoses Prevention and Control Agents;Key Laboratory of Veterinary Vaccine Creation of Ministry of Agriculture;Key Laboratory of Prevention and Control of Zoonotic Diseases of Guangdong Province, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China

Received:2022-03-31 Online:2022-11-23 Published:2022-11-25

摘要/Abstract

摘要： 为了解目前广东地方品种鸡K亚群禽白血病病毒（avian leukosis virus，ALV-K）的分子特征，于2020-2021年，从广东7家（A～G）正在进行外源性ALV净化的、外观健康的规模化种禽场核心群采集抗凝血样品共8 042份。通过血浆接种DF-1细胞、ELISA检测p27抗原，共检出357份阳性，并对ELISA结果S/P值0.2～0.6的150份阳性样品进行PCR鉴定和env基因测序，共分离到32株ALV-K，进一步选取16株进行全基因组扩增及测序分析。结果显示，这16株ALV-K全长为7 481～7 496 bp，基因组符合5'-LTR-UTR-gag-pol-env-UTR-LTR-3'典型的反转录病毒结构，不含已知致癌基因；其gp85基因与ALV-K参考株位于同一进化分支上且相似性最高（>94.0％）；其pol、gp37基因均比较保守，与ALV各亚群相似性均较高（>94.0％）；其LTR与内源性ALV及大多数ALV-K参考株相似性最高（91.4％～99.5％），且LTR U3区与内源性ALV LTR有相同的转录调控元件；此外，观察到31.3％（5/16）的分离株在gag基因373-384位核苷酸存在12 bp的缺失，进一步以缺失株GD20 JM10为亲本，通过PCR分段扩增和同源重组的方法分别构建了缺失株（rGD20 JM10）和回补株（rGD20 JM10 A12）的cDNA克隆，将其转染DF-1细胞，ELISA和IFA结果表明，病毒拯救成功；体外复制动力学结果显示，两者差异不显著（P>0.05）。本研究所调查的广东7个种禽场的ALV-K流行株均携带内源性LTR，且分子特征较为稳定，故采用更敏感的检测技术在开展ALV净化时尤为重要，此外，gag基因373—384位核苷酸的规律性缺失对病毒的体外复制能力无显著影响。

关键词: K亚群禽白血病病毒, 地方品种鸡, 分子流行病学, gag基因, 感染性克隆

Abstract: To investigate the molecular characteristics of ALV-K isolates from local breed chickens in Guangdong, 8 042 anticoagulant samples were collected from core flocks of healthy appearance in seven large-scale breeding poultry farms (A-G) in Guangdong that are undergoing eradication of exogenous ALV during 2020-2021. Among them, 357 positive samples were identified by DF-1 cell culture and p27 antigen ELISA test. PCR identification and env gene sequencing were performed on 150 positive samples with S/P values of 0.2 to 0.6 by ELISA, and 32 ALV-K strains were isolated. Sixteen isolates were further selected for whole-genome sequencing analysis. The results showed that the total genome length of the 16 ALV-K isolates ranged from 7 481 to 7 496 bp, and the genome conformed to the typical retrovirus structure of 5'-LTR-UTR-gag-pol-env-UTR-LTR-3', without known oncogenes. The gp85 gene of the isolates and ALV-K reference strains were located in the same evolutionary branch and had the highest similarity (>94.0%). The pol and gp37 genes were relatively conserved, with the similarity more than 94.0% to other ALV subgroups. The LTR showed highest similarity with endogenous ALV and most ALV-K reference strains (91.4%-99.5%), and the LTR U3 region had the same transcriptional regulatory elements as the endogenous ALV LTR. Additionally, 31.3% (5/16) of isolates were observed to have a 12 bp deletion at nucleotides 373-384 of the gag gene. Further, the full-length cDNA clones of the deletion strain (rGD20 JM10) and the complementary strain (rGD20 JM10 A12) were respectively constructed by PCR segmental amplification and homologous recombination based on the deletion strain GD20 JM10, and the infectious clones were transfected into DF-1 cells. The ELISA and IFA results showed the two viruses were successfully rescued, and the in vitro replication curves showed no significant difference between the two viruses. The epidemic ALV-K strains from seven breeding poultry farms in Guangdong investigated in this study all carried endogenous LTR, so it is particularly important to use a more sensitive detection technology when carrying out exogenous ALV eradication. Additionally, the growth curves showed that such gag gene with the 12 bp deletion had no evident effect on the virus replication in vitro.

Key words: ALV-K, local breed chickens, molecular epidemiology, gag gene, infectious clone

中图分类号:

S852.659.3

郭妍妍, 梁灿新, 李锦群, 董欣怡, 廖明, 曹伟胜. 广东地方品种鸡ALV-K分子流行病学及gag基因12 bp缺失对病毒体外复制能力影响的研究[J]. 畜牧兽医学报, 2022, 53(11): 3956-3966.

GUO Yanyan, LIANG Canxin, LI Jinqun, DONG Xinyi, LIAO Ming, CAO Weisheng. Study on Molecular Epidemiology of ALV-K in Guangdong Local Breed Chickens and the Effect of 12 bp Deletion of gag Gene on Virus Replication Ability in vitro[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(11): 3956-3966.

参考文献 24

[1]	CHENG Z Q, LIU J Z, CUI Z Z, et al.Tumors associated with avian leukosis virus subgroup J in layer hens during 2007 to 2009 in China[J].J Vet Med Sci, 2010, 72(8):1027-1033.
[2]	PAYNE L N, NAIR V.The long view:40 years of avian leukosis research[J].Avian Pathol, 2012, 41(1):11-19.
[3]	LI H J, WANG P K, LIN L L, et al.The emergence of the infection of subgroup J avian leucosis virus escalated the tumour incidence in commercial yellow chickens in southern China in recent years[J].Transbound Emerg Dis, 2019, 66(1):312-316.
[4]	王鑫, 赵鹏, 崔治中.我国地方品种鸡分离到的一个禽白血病病毒新亚群的鉴定[J].病毒学报, 2012, 28(6):609-614.WANG X, ZHAO P, CUI Z Z.Identification of a new subgroup of avian leukosis virus isolated from Chinese indigenous chicken breeds[J].Chinese Journal of Virology, 2012, 28(6):609-614.(in Chinese)
[5]	CUI N, SU S, CHEN Z M, et al.Genomic sequence analysis and biological characteristics of a rescued clone of avian leukosis virus strain JS11C1, isolated from indigenous chickens[J].J Gen Virol, 2014, 95(11):2512-2522.
[6]	DONG X, ZHAO P, XU B, et al.Avian leukosis virus in indigenous chicken breeds, China[J].Emerg Microbes Infect, 2015, 4(12):e76.
[7]	SHAO H X, WANG L, SANG J J, et al.Novel avian leukosis viruses from domestic chicken breeds in mainland China[J].Arch Virol, 2017, 162(7):2073-2076.
[8]	ZHAO Z J, RAO M Z, LIAO M, et al.Phylogenetic analysis and pathogenicity assessment of the emerging recombinant subgroup K of avian leukosis virus in south China[J].Viruses, 2018, 10(4):194.
[9]	SU Q, LI Y, LI W H, et al.Molecular characteristics of avian leukosis viruses isolated from indigenous chicken breeds in China[J].Poult Sci, 2018, 97(8):2917-2925.
[10]	SU Q, LI Y, CUI Z Z, et al.The emerging novel avian leukosis virus with mutations in the pol gene shows competitive replication advantages both in vivo and in vitro[J].Emerg Microbes Infect, 2018, 7(1):1-11.
[11]	SUN T, WANG X M, HAN W, et al.Complete genome sequence of a novel recombinant avian leukosis virus isolated from a three-yellow chicken[J].Arch Virol, 2020, 165(11):2615-2618.
[12]	LI X Y, YU Y, MA M G, et al.Molecular characteristic and pathogenicity analysis of a novel multiple recombinant ALV-K strain[J].Vet Microbiol, 2021, 260:109184.
[13]	CHEN J, ZHAO Z J, CHEN Y Y J, et al.Development and application of a SYBR green real-time PCR for detection of the emerging avian leukosis virus subgroup K[J].Poult Sci, 2018, 97(7):2568-2574.
[14]	戚永娜.山东省某地方品种鸡ALV-K的分离鉴定及其全基因组序列分析[D].泰安:山东农业大学, 2019.QI Y N.Isolation, identification and evolution analysis of ALV-K isolated from local Chinese chickens in Shandong province[D].Tai'an:Shandong Agricultural University, 2019.(in Chinese)
[15]	SWANSTROM R, GRAHAM W D, ZHOU S T.Sequencing the biology of entry:the retroviral env gene[M]//HUNTER E, BISTER K.Viruses, Genes, and Cancer.Cham:Springer, 2017:65-82.
[16]	WANG P K, LI M, LI H J, et al.Full-length cDNA sequence analysis of 85 avian leukosis virus subgroup J strains isolated from chickens in China during the years 1988-2018:coexistence of 2 extremely different clusters that are highly dependent upon either the host genetic background or the geographic location[J].Poult Sci, 2020, 99(7):3469-3480.
[17]	赵子君, 饶明章, 陈建, 等.K亚群禽白血病病毒5'LTR序列及启动活性分析[J].畜牧兽医学报, 2018, 49(4):754-760.ZHAO Z J, RAO M Z, CHEN J, et al.The research of 5'LTR promoter activity of avian leukosis virus subgroup K[J].Acta Veterinaria et Zootechnica Sinica, 2018, 49(4):754-760.(in Chinese)
[18]	ROBINSON H L, BLAIS B M, TSICHLIS P N, et al.At least two regions of the viral genome determine the oncogenic potential of avian leukosis viruses[J].Proc Natl Acad Sci U S A, 1982, 79(4):1225-1229.
[19]	KLINGLER J, ANTON H, RÉAL E, et al.How HIV-1 gag manipulates its host cell proteins:a focus on interactors of the nucleocapsid domain[J].Viruses, 2020, 12(8):888.
[20]	王静.鸡Viperin蛋白与ALV-J相互作用的研究[D].泰安:山东农业大学, 2021.WANG J.Study on the interaction between chicken Viperin protein and ALV-J[D].Tai'an:Shandong Agricultural University, 2021.(in Chinese)
[21]	SU Q, CUI Z Y, ZHANG Z H, et al.Whole-genome analysis of an emerging recombinant avian leukosis virus in yellow chickens, south China[J].Transbound Emerg Dis, 2020, 67(5):2254-2258.
[22]	WANG P K, SHI M Y, HE C W, et al.A novel recombinant avian leukosis virus isolated from gamecocks induced pathogenicity in Three-Yellow chickens:a potential infection source of avian leukosis virus to the commercial chickens[J].Poult Sci, 2019, 98(12):6497-6504.
[23]	WANG P K, NIU J R, XUE C, et al.Two novel recombinant avian leukosis virus isolates from Luxi gamecock chickens[J].Arch Virol, 2020, 165(12):2877-2881.
[24]	LI J, LIU L L, NIU X X, et al.Research note:a novel recombinant subgroup E isolate of the avian leukosis virus with a subgroup B-like Gp85 region in China[J].Poult Sci, 2021, 100(7):101137.

广东地方品种鸡ALV-K分子流行病学及gag基因12 bp缺失对病毒体外复制能力影响的研究

Study on Molecular Epidemiology of ALV-K in Guangdong Local Breed Chickens and the Effect of 12 bp Deletion of gag Gene on Virus Replication Ability in vitro

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献 24

相关文章 15

编辑推荐

Metrics

本文评价

[1]	常益铭, 汤承, 岳华. 牦牛源牛呼吸道合胞体病毒的分子流行病学调查[J]. 畜牧兽医学报, 2023, 54(5): 2030-2041.
[2]	王梦娇, 蒋倩, 马学军, 夏瑞阳, 郭雪萍, 孙磊, 钟旗, 马雪连, 姚刚. 新疆地区牛冠状病毒的分子流行病学调查[J]. 畜牧兽医学报, 2023, 54(12): 5125-5133.
[3]	周群, 杨苑, 宋鑫, 何欣怡, 曹慧, 张斌. 2017—2021年成都市猫细小病毒的检测及其VP2基因的变异分析[J]. 畜牧兽医学报, 2022, 53(4): 1303-1309.
[4]	蔡伟龙, 李娜, 冯耀宇, 肖立华. 牛十二指肠贾第虫的分子流行病学研究进展[J]. 畜牧兽医学报, 2021, 52(2): 300-310.
[5]	赫秀甜, 向阳, 袁东波, 阳爱国, 范小虎, 谭雄, 钟叶青, 郝力力. 四川省松潘县牦牛体表蜱、高原鼠兔携带巴尔通体和无形体的PCR检测与进化分析[J]. 畜牧兽医学报, 2020, 51(6): 1438-1446.
[6]	陈新诺, 肖敏, 阮文强, 覃思楠, 岳华, 汤承, 张斌. 川藏地区牦牛牛病毒性腹泻病毒分子流行病学调查及分离鉴定[J]. 畜牧兽医学报, 2018, 49(3): 606-613.
[7]	郭振华, 陈鑫鑫, 李睿, 乔松林, 郭军庆, 张改平. 中国猪繁殖与呼吸综合征病毒流行历史及现状[J]. 畜牧兽医学报, 2018, 49(1): 1-9.
[8]	丁轲，余祖华，彭春平，赵战勤，何雷，贾艳艳，张春杰，程相朝，夏咸柱. 2011—2013年河南省犬细小病毒分子流行病学调查与分析[J]. 畜牧兽医学报, 2014, 45(10): 1671-1678.
[9]	林艳，夏静，邹年莉，郭明萍，王富妍，赵扬，文心田，曹三杰，黄勇. 1株髓细胞瘤型J亚群禽白血病病毒感染性克隆的构建与病毒拯救[J]. 畜牧兽医学报, 2013, 44(5): 754-759.
[10]	王蓓，刘芳，王汉清，李宁，魏建忠，刘光清，王桂军. 鸡J亚群禽白血病病毒AH-J11株的全基因组感染性克隆构建[J]. 畜牧兽医学报, 2013, 44(12): 1976-1981.
[11]	郝飞，汤德元，李春燕，罗险峰，张华，马萍，曾智勇，洪尼宁，刘霞. 猪博卡病毒流行病学研究[J]. 畜牧兽医学报, 2012, 43(10): 1609-1617.
[12]	吕健，韦祖樟，高飞，郑海红，童光志，袁世山. 猪繁殖与呼吸综合征病毒强弱毒株嵌合感染性克隆的构建及鉴定[J]. 畜牧兽医学报, 2012, 43(10): 1598-1602.
[13]	李文洁;李文涛;严伟东;陈焕春;何启盖. 中国部分地区猪圆环病毒2型的基因型分析[J]. 畜牧兽医学报, 2009, 40(9): 1358-1362.
[14]	张国庆;朱书;盖新娜;郭鑫;陈艳红;查振林;杨汉春. 猪脑心肌炎病毒分离株BJC3全长感染性克隆的构建与拯救病毒鉴定[J]. 畜牧兽医学报, 2009, 40(9): 1350-1357.
[15]	范运峰;赵启祖;赵耘;邹兴启;徐璐;张仲秋;王琴;宁宜宝. 猪瘟病毒低温诱变疫苗Thiverval株感染性克隆的构建及病毒拯救[J]. 畜牧兽医学报, 2009, 40(9): 1420-1425.