犬腺病毒2型E3基因自然缺失毒株的分离鉴定及其致病性

doi:10.11843/j.issn.0366-6964.2023.07.029

摘要/Abstract

摘要： 犬腺病毒2型（canine adenovirus type 2，CAV-2）是重要的犬呼吸道病原，本试验旨在调查CAV-2在川渝部分地区的流行情况及其分离毒株致病性。采用检测CAV-2的特异性PCR方法，对2021—2022年采集自川渝部分地区的121份患呼吸道疾病的犬鼻咽拭子进行检测，分离流行毒株并研究其基因组特征及致病性。结果显示：CAV-2的阳性率为29.8%，值得注意的是，69.4%的阳性样本在E3基因存在9个核苷酸连续缺失（1 035—1 043 nt），导致4个氨基酸缺失（345—348 aa）。成功分离出1株E3基因自然缺失毒株，噬斑纯化后的细胞半数感染值为10^-9.46TCID₅₀·0.1 mL^-1；该毒株经口鼻感染幼犬后，幼犬出现体温升高、打喷嚏、流鼻涕及咳嗽等典型呼吸道症状。该分离毒株基因组全长31 786 bp，与GenBank中CAV-2基因组的核苷酸相似性为98.8%~98.9%。与已知的CAV-2基因组相比，本研究分离毒株除了E3基因的独特突变外，还在Fiber、Hexon和E1A等蛋白有独特的氨基酸突变，使得该毒株在基因组进化树上区别于已知的CAV-2毒株。本试验首次获得了中国CAV-2毒株的基因组序列，发现了E3基因的自然缺失现象，有助于了解CAV-2的流行及遗传进化。

关键词: 犬腺病毒2型, E3基因自然缺失株, 分离鉴定, 基因组, 致病性

Abstract: Canine adenovirus type 2(CAV-2) is an important respiratory pathogen in Canine. The aim of this study was to investigate the prevalence and pathogenicity of CAV-2 in parts of Sichuan Province and Chongqing Municipality. The nasal swabs of 121 canines with respiratory tract diseases were collected from 2021 to 2022 for detecting CAV-2 by specific PCR primers, and the epidemic strain was isolated for further study, including its genome characteristics and pathogenicity. The results showed that the positive rate of CAV-2 in canine was 29.8%. Notably, 69.4% of the positive samples had identical 9 consecutive nucleotide deletions (1 035-1 043 nt) in the E3 genes, which resulting in 4 amino acid deletions in the E3 genes (345-348 aa). One strain with natural E3 deletion was successfully isolated, and the half infection value of MDCK cells was 10^-9.46TCID₅₀·0.1 mL^-1 after plaque purification. The puppies inoculated with the isolation showed typical respiratory symptoms, such as sneezing, runny nosing and coughing. The genome of the isolate was 31 786 bp, and shared the homology of 98.8%-98.9% in nt with the CAV-2 genomes in GenBank. Compared with the known CAV-2 genomes, besides the unique mutations of the E3 gene, the isolate in this study also had unique amino acid mutations in Fiber, Hexon, and E1A, which made this strain distinguish from the known CAV-2 strain in the genomic evolutionary tree. This study obtained the genome sequence of the Chinese CAV-2 strain for the first time, and found the strain with natural deletion in E3 genes, which is helpful to understand the prevalence and genetic evolution of CAV-2.

Key words: canine adenovirus type 2, E3 gene natural deletion strain, isolation and identification, genome, pathogenicity

中图分类号:

S852.659.1

王佳丽, 周宁, 陈曦, 岳华, 汤承. 犬腺病毒2型E3基因自然缺失毒株的分离鉴定及其致病性[J]. 畜牧兽医学报, 2023, 54(7): 2982-2990.

WANG Jiali, ZHOU Ning, CHEN Xi, YUE Hua, TANG Cheng. Isolation, Identification and Pathogenicity of Canine Adenovirus Type 2 Strain with Natural Deletion in E3 Gene[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 2982-2990.

参考文献 29

[1]	DECARO N, MARTELLA V, BUONAVOGLIA C. Canine adenoviruses and herpesvirus[J]. Vet Clin North Am Small Anim Pract, 2008, 38(4):799-814.
[2]	BENETKA V, WEISSENBÖCK H, KUDIELKA I, et al. Canine adenovirus type 2 infection in four puppies with neurological signs[J]. Vet Rec, 2006, 158(3):91-94.
[3]	MACARTNEY L, CAVANAGH H M A, SPIBEY N. Isolation of canine adenovirus-2 from the faeces of dogs with enteric disease and its unambiguous typing by restriction endonuclease mapping[J]. Res Vet Sci, 1988, 44(1):9-14.
[4]	DOWGIER G, LAHOREAU J, LANAVE G, et al. Sequential circulation of canine adenoviruses 1 and 2 in captive wild carnivores, France[J]. Vet Microbiol, 2018, 221:67-73.
[5]	DI FRANCESCO C E, GENTILE L, DI PIRRO V, et al. Serologic evidence for selected infectious diseases in Marsican brown bears (Ursus arctos marsicanus) in Italy (2004-09)[J]. J Wildl Dis, 2015, 51(1):209-213.
[6]	BALBONI A, VERIN R, MORANDI F, et al. Molecular epidemiology of canine adenovirus type 1 and type 2 in free-ranging red foxes (Vulpes vulpes) in Italy[J]. Vet Microbiol, 2013, 162(2-4):551-557.
[7]	MILLÁN J, LÓPEZ-BAO J V, GARCÍA E J, et al. Patterns of exposure of Iberian wolves (Canis lupus) to canine viruses in human-dominated landscapes[J]. EcoHealth, 2016, 13(1):123-134.
[8]	DITCHFIELD J, MACPHERSON L W, ZBITNEW A. Association of canine adenovirus (Toronto A 26/61) with an outbreak of laryngotracheitis ("Kennel Cough"):a preliminary report[J]. Can Vet J, 1962, 3(8):238-247.
[9]	张洋. 犬2型腺病毒分离、鉴定、特性分析及鼬獾血清腺病毒抗体调查[D]. 北京:中国人民解放军军事医学科学院, 2009.ZHANG Y. Isolation, identification and characterization of canine adenovirus type 2 and investigation of serum antibody to adenovirus in ferret badge[D]. Beijing:Academy of Military Medical Sciences, 2009. (in Chinese)
[10]	肖园, 吕艳丽, 孙艳争. 北京地区犬呼吸道冠状病毒感染状况的监测与分析[J]. 畜牧兽医学报, 2022, 53(10):3522-3529.XIAO Y, LV Y L, SUN Y Z. Surveillance and analysis of canine respiratory coronavirus infection in Beijing[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(10):3522-3529. (in Chinese)
[11]	YANG D K, KIM H H, LEE E J, et al. Recharacterization of the canine adenovirus type 1 vaccine strain based on the biological and molecular properties[J]. J Bacteriol Virol, 2019, 49(3):124-132.
[12]	WICKHAM T J, MATHIAS P, CHERESH D A, et al. Integrins αvβ3 and αvβ5 promote adenovirus internalization but not virus attachment[J]. Cell, 1993, 73(2):309-319.
[13]	LICHTENSTEIN D L, TOTH K, DORONIN K, et al. Functions and mechanisms of action of the adenovirus E3 proteins[J]. Int Rev Immunol, 2004, 23(1-2):75-111.
[14]	BURGERT H G, BLUSCH J H. Immunomodulatory functions encoded by the E3 transcription unit of adenoviruses[J]. Virus Genes, 2000, 21(1-2):13-25.
[15]	XIAO L, ZHANG M D, TIAN Z G, et al. Development of a recombinase polymerase amplification fluorescence assay for the detection of canine adenovirus 2[J]. Front Vet Sci, 2021, 8:596877.
[16]	薛林杰. 河南省部分地区犬腺病毒2型和犬细小病毒的流行病学研究[D]. 郑州:河南农业大学, 2022.XUE L J. Epidemiological study of canine parvovirus and canine adenovirus type 2 in some areas of Henan province[D]. Zhengzhou:Henan Agricultural University, 2022. (in Chinese)
[17]	张发明, 黄坚, 封海波, 等. 成都地区犬腺病毒2型分子流行病学调查及其纤突基因的生物信息学分析[J]. 黑龙江畜牧兽医, 2020(21):99-103.ZHANG F M, HUANG J, FENG H B, et al. Investigation of canine adenovirus type 2 Molecular epidemiology in Chengdu area and bioinformatics analysis of its spike gene[J]. Heilongjiang Animal Science and Veterinary Medicine, 2020(21):99-103. (in Chinese)
[18]	JI J, LI W Y, HU W, et al. Novel genotype definition and the first epidemiological investigation of canine adenovirus type 2 in dogs in central China[J]. Front Vet Sci, 2020, 7:534.
[19]	由欣月, 郝雲峰, 崔尚金, 等. 犬腺病毒SYBR Green Ⅰ荧光定量PCR检测方法的建立[J]. 中国动物传染病学报, 2019, 27(6):29-34.YOU X Y, HAO Y F, CUI S J, et al. Development of SYBR Green Ⅰ real-time PCR for the detection of canine adenovirus[J]. Chinese Journal of Animal Infectious Diseases, 2019, 27(6):29-34. (in Chinese)
[20]	蔡靖, 冯秀娟. 犬呼吸系统疾病研究进展[J]. 中国工作犬业, 2022(8):45-47.CAI J, FENG X J. Advances in canine respiratory diseases[J]. China Working Dog, 2022(8):45-47. (in Chinese)
[21]	王文佳, 王一斐, 常艳, 等. 犬新型病毒性呼吸道疾病的研究进展[J]. 中国兽医杂志, 2017, 53(1):70-72.WANG W J, WANG Y F, CHANG Y, et al. Research progress of novel viral respiratory diseases in dogs[J]. Chinese Journal of Veterinary Medicine, 2017, 53(1):70-72. (in Chinese)
[22]	RAJA P, SACHIN V, PARTHIBAN M, et al. Molecular characterization of canine adenovirus type 2 in dogs from India[J]. Virus Disease, 2021, 32(2):369-374.
[23]	RAMIDI A, GANJI V K, BUDDALA B, et al. E3 Gene-based genetic characterization of canine adenovirus-2 isolated from cases of canine gastroenteritis in India revealed a novel group of the virus[J]. Intervirology, 2019, 62(5-6):216-221.
[24]	BALBONI A, DONDI F, PROSPERI S, et al. Development of a SYBR Green real-time PCR assay with melting curve analysis for simultaneous detection and differentiation of canine adenovirus type 1 and type 2[J]. J Virol Methods, 2015, 222:34-40.
[25]	由欣月, 刘畅, 郝雲峰, 等. 犬腺病毒CAV-BJ02株的分离与鉴定[J]. 畜牧兽医学报, 2020, 51(8):2016-2021.YOU X Y, LIU C, HAO Y F, et al. Isolation and identification of canine adenovirus CAV-BJ02 strain[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(8):2016-2021. (in Chinese)
[26]	廖均乐. 犬腺病毒2型分离鉴定及感染模型建立与初步应用[D]. 郑州:河南农业大学, 2022.LIAO J L. Isolation and identification of canine adenovirus type 2 and establishment and preliminary application of infection model[D]. Zhengzhou:Henan Agricultural University, 2022. (in Chinese)
[27]	邱薇, 郑颖, 张富强, 等. 犬Ⅱ型腺病毒KM株的分离鉴定[C]//第17次全国犬业科技学术研讨会论文集. 太原:中国畜牧兽医学会, 2017:188-193.QIU W, ZHENG Y, ZHANG F Q, et al. Isolation and identification of canine adenovirus type II KM strain[C]//Proceedings of the 17th National Symposium on Canine Science and Technology. Taiyuan:Chinese Association of Animal Science and Veterinary Medicine, 2017:188-193. (in Chinese)
[28]	范泉水, 郑颖, 邱薇, 等. 犬2型腺病毒云南强毒株的分离鉴定[C]//第十三次全国养犬学术研讨会论文集. 泰州:中国畜牧兽医学会, 2009:124-132.FAN Q S, ZHENG Y, QIU W, et al. Isolation and identification of Yunnan virulent canine adenovirus type 2[C]//Proceedings of the 13th National Symposium on Dog Breeding. Taizhou:Chinese Association of Animal Science and Veterinary Medicine, 2009:124-132. (in Chinese)
[29]	张洋, 袁子国, 姜秋杰, 等. 犬2型腺病毒的分离鉴定及初步特性分析[J]. 军事医学科学院院刊, 2008, 32(6):541-544.ZHANG Y, YUAN Z G, JIANG Q J, et al. Isolation, identification and preliminary characteristic analysis of a CAV-2[J]. Bulletin of the Academy of Military Medical Sciences, 2008, 32(6):541-544. (in Chinese)