B亚型禽偏肺病毒的分离鉴定及致病性研究

doi:10.11843/j.issn.0366-6964.2022.10.026

畜牧兽医学报 ›› 2022, Vol. 53 ›› Issue (10): 3540-3549.doi: 10.11843/j.issn.0366-6964.2022.10.026

B亚型禽偏肺病毒的分离鉴定及致病性研究

于蒙蒙¹, 包媛玲¹, 王素艳¹, 辛子琪², 刘鹏¹, 冯笑艳¹, 孟令宅¹, 郭茹¹, 张艳萍¹, 刘长军¹, 祁小乐¹, 李俊平², 王笑梅^1,3, 高玉龙^1*

1. 中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室禽免疫抑制病创新团队, 哈尔滨 150069;
2. 中国兽医药品监察所, 北京 100081;
3. 扬州大学江苏省动物重要疫病与人畜共患病协同创新中心, 扬州 225009

收稿日期:2022-02-21 出版日期:2022-10-23 发布日期:2022-10-26
通讯作者: 高玉龙,主要从事家禽免疫抑制病诊断与防治研究,E-mail:gaoyulong@caas.cn
作者简介:于蒙蒙(1993-),女,河南通许人,博士生,主要从事禽免疫抑制病研究,E-mail:yumengmeng1029@126.com
基金资助:
国家肉鸡产业技术体系（CARS-41-G15）；黑龙江省自然科学基金（TD2019C003）

Isolation, Identification and Pathogenicity of Subtype B Avian Metapneumovirus

YU Mengmeng¹, BAO Yuanling¹, WANG Suyan¹, XIN Ziqi², LIU Peng¹, FENG Xiaoyan¹, MENG Lingzhai¹, GUO Ru¹, ZHANG Yanping¹, LIU Changjun¹, QI Xiaole¹, LI Junping², WANG Xiaomei^1,3, GAO Yulong^1*

1. Avian Immunosuppressive Diseases Division, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China;
2. China Institute of Veterinary Drug Control, Beijing 100081, China;
3. Jiangsu Co-innovation Center for Prevention and Control of Important Animal InfectiousDiseases and Zoonoses, Yangzhou University, Yangzhou 225009, China

Received:2022-02-21 Online:2022-10-23 Published:2022-10-26

摘要/Abstract

摘要： 旨在确定我国部分养鸡场的鸡出现甩头、精神萎靡和肿头综合征是否由禽偏肺病毒（aMPV）引起，本研究从山东、福建、黑龙江等地的发病蛋鸡和肉鸡场采集鼻甲骨、气管和肺等样品，首先利用aMPV特异性的RT-PCR方法对临床样品进行初步检测，将RT-PCR检测阳性样品接种Vero细胞进行病毒分离，然后利用G/F基因序列分析及间接免疫荧光试验（IFA）等鉴定病毒的亚型，最后将分离株感染SPF鸡进行致病性分析。结果显示：在采集的220份样品中，RT-PCR检测结果显示，有3份鼻甲骨样品在228 bp左右出现特异性条带，将阳性病料接种Vero细胞盲传5代后，细胞出现变圆、聚集和融合等aMPV特征性细胞病变（CPE），表明成功分离到3株aMPV，将其分别命名为SD2001、SD2002和HLJ2101。G和F基因同源性分析显示，来自蛋鸡的SD2001、SD2002和来自肉鸡的HLJ2101分离株的G和F基因与其他B亚型aMPV毒株的核苷酸和氨基酸序列相似性均较高，核苷酸的相似性分别为93.4%～98.6%和95.6%～100.0%，氨基酸的相似性分别为88.7%～97.8%和97.6%～100.0%；而与A、C和D亚型的G和F基因同源性较低，核苷酸的相似性分别为27.1%～61.8%和66.8%～74.8%，氨基酸的相似性分别为16.1%～36.7%和72.5%～86.5%，这些结果表明，SD2001、SD2002和HLJ2101分离株属于B亚型aMPV。进一步利用B亚型aMPV特异性的阳性血清进行IFA检测，接种SD2001、SD2002和HLJ2101的Vero细胞均可以观察到特异性的绿色荧光信号，进一步证实3个分离株属于B亚型aMPV。选择SD2001感染3周龄SPF鸡进行了致病性研究，结果发现SPF鸡感染后3～6 d出现精神萎靡、甩头和流鼻涕等症状，鼻甲骨、气管和肺也出现病理性损伤，其发病率为90%（18/20）。3株B亚型aMPV的分离不仅有助于明确我国部分养鸡场出现肿头综合征的发病原因，同时也证实B亚型aMPV流行毒株对鸡有明显的致病性，这些结果为我国家禽疫病的诊断和有效防控提供了重要理论依据。

关键词: B亚型禽偏肺病毒, 分离, 鉴定, 致病性

Abstract: The purpose of this study was to identify the avian metapneumovirus (aMPV) as the pathogen of head shaking, mental depression and swollen head syndrome in some poultry farms in China. In this study, nasal turbinate and lung samples were collected from chicken farms in Shandong, Fujian and Heilongjiang. Firstly, the clinical samples were detected by aMPV specific RT-PCR method, and then the positive samples were inoculated into Vero cells for virus isolation. Secondly, the subtypes of the viruses were identified by G and F gene sequence analysis and indirect immunofluorescence assay (IFA). Finally, the pathogenicity of the isolate to SPF chickens was analyzed. Among the 220 samples, the RT-PCR results indicated that three nasal turbinate samples had specific bands at about 228 bp. After the positive samples were inoculated into Vero cells for 5 consecutive generations, the cells showed aMPV characteristic cytopathy (CPE) such as rounding, aggregation and fusion. These results showed that three isolates of aMPV were successfully isolated and named SD2001, SD2002 and HLJ2101, respectively. Phylogenetic analysis showed that the G and F genes of layer-originated SD2001, SD2002 and broiler-originated HLJ2101 isolates had high nucleotide and amino acid sequence homologies with other subtype B aMPV strains, and the nucleotide similarities were 93.4%-98.6% and 95.6%-100.0%, respectively, and the amino acid similarities were 88.7%-97.8% and 97.6%-100.0%, respectively. However, the homologies with the G and F genes of subtypes A, C and D aMPV strains were low, and the nucleotide similarities were 27.1%-61.8% and 66.8%-74.8%, respectively, and the amino acids similarities were 16.1%-36.7% and 72.5%-86.5%, respectively. These results indicated that SD2001, SD2002 and HLJ2101 isolates belonged to subtype B aMPV. Further, the specific positive serum of subtype B aMPV was used for IFA test. The specific green fluorescence signal could be observed in Vero cells inoculated with SD2001, SD2002 and HLJ2101, respectively, which furtherly confirmed that the three isolates belonged to subtype B aMPV. Next, the pathogenicity of the isolate to SPF chickens was tested. SPF chickens infected with SD2001 isolate showed clinical symptoms such as depression, head shaking and sneezing 3-6th day after infection. Histopathological analysis showed that there were pathological lesions in the turbinate, trachea and lung of infected SPF chickens. The incidence rate was 90%(18/20). In this study, three strains of subtype B aMPV were successfully isolated. Our results not only identified the pathogen of swollen head syndrome in some poultry farms in China, but also confirmed that the epidemic strain of subtype B aMPV had obvious pathogenicity to chickens. These results provided an important theoretical basis for the diagnosis and effective prevention of aMPV infection in China.

Key words: subtype B avian metapneumovirus, isolation, identify, pathogenicity

中图分类号:

S852.659.5

于蒙蒙, 包媛玲, 王素艳, 辛子琪, 刘鹏, 冯笑艳, 孟令宅, 郭茹, 张艳萍, 刘长军, 祁小乐, 李俊平, 王笑梅, 高玉龙. B亚型禽偏肺病毒的分离鉴定及致病性研究[J]. 畜牧兽医学报, 2022, 53(10): 3540-3549.

YU Mengmeng, BAO Yuanling, WANG Suyan, XIN Ziqi, LIU Peng, FENG Xiaoyan, MENG Lingzhai, GUO Ru, ZHANG Yanping, LIU Changjun, QI Xiaole, LI Junping, WANG Xiaomei, GAO Yulong. Isolation, Identification and Pathogenicity of Subtype B Avian Metapneumovirus[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(10): 3540-3549.

参考文献 27

[1]	BÄYON-AUBOYER M H, ARNAULD C, TOQUIN D, et al. Nucleotide sequences of the F, L and G protein genes of two non-A/non-B avian pneumoviruses (APV) reveal a novel APV subgroup[J]. J Gen Virol, 2000, 81(Pt 11):2723-2733.
[2]	GUIONIE O, TOQUIN D, SELLAL E, et al. Laboratory evaluation of a quantitative real-time reverse transcription PCR assay for the detection and identification of the four subgroups of avian metapneumovirus[J]. J Virol Methods, 2007, 139(2):150-158.
[3]	EASTON A J, DOMACHOWSKE J B, ROSENBERG H F. Animal pneumoviruses:molecular genetics and pathogenesis[J]. Clin Microbiol Rev, 2004, 17(2):390-412.
[4]	BUYS S B, DU PREEZ J H, ELS H J. The isolation and attenuation of a virus causing rhinotracheitis in turkeys in South Africa[J]. Onderstepoort J Vet Res, 1989, 56(2):87-89.
[5]	BENNETT R S, LARUE R, SHAW D, et al. A wild goose metapneumovirus containing a large attachment glycoprotein is avirulent but immunoprotective in domestic turkeys[J]. J Virol, 2005, 79(23):14834-14842.
[6]	BANET-NOACH C, SIMANOV L, LAHAM-KARAM N, et al. Longitudinal survey of avian metapneumoviruses in poultry in Israel:infiltration of field strains into vaccinated flocks[J]. Avian Dis, 2009, 53(2):184-189.
[7]	CHACÓN J L, MIZUMA M, VEJARANO M P, et al. Avian metapneumovirus subtypes circulating in Brazilian vaccinated and nonvaccinated chicken and turkey farms[J]. Avian Dis, 2011, 55(1):82-89.
[8]	TANAKA M, TAKUMA H, KOKUMAI N, et al. Turkey rhinotracheitis virus isolated from broiler chicken with swollen head syndrome in Japan[J]. J Vet Med Sci, 1995, 57(5):939-941.
[9]	FRANZO G, LEGNARDI M, MESCOLINI G, et al. Avian metapneumovirus subtype B around Europe:a phylodynamic reconstruction[J]. Vet Res, 2020, 51(1):88.
[10]	CHABOKI P M, GHALYANCHILANGEROUDI A, KARIMI V, et al. Prevalence of avian metapneumovirus subtype B in live bird market in Gilan Province, Iran[J]. Vet Res Forum, 2018, 9(1):93-97.
[11]	沈瑞忠, 曲立新, 于康震, 等. 禽肺病毒的分离鉴定[J]. 中国预防兽医学报, 1999, 21(1):76-77.SHEN R Z, QU L X, YU K Z, et al. Isolation and characterization of an avian pneumovirus form chickens in China[J]. Chinese Journal of Preventive Veterinary Medicine, 1999, 21(1):76-77. (in Chinese)
[12]	郭龙宗, 曲立新. 种鸡禽肺病毒感染的血清学调查[J]. 中国畜牧兽医, 2009, 36(4):149-150.GUO L Z, QU L X. Serological investigation of avian pneumoviruses infection in breeders[J]. China Animal Husbandry & Veterinary Medicine, 2009, 36(4):149-150. (in Chinese)
[13]	贠炳岭, 刘在斯, 吴关, 等. 我国部分地区种鸡禽肺病毒感染的血清学调查[J]. 中国家禽, 2012, 34(12):64-65.YUN B L, LIU Z S, WU G, et al. Serological investigation of avian pneumoviruses infection in breeding chickens in some areas of China[J]. China Poultry, 2012, 34(12):64-65. (in Chinese)
[14]	朱艳梅, 宫晓, 郭伟伟, 等. 2012年-2015年我国部分地区禽偏肺病毒的分子流行病学分析[J]. 动物医学进展, 2016, 37(10):30-34.ZHU Y M, GONG X, GUO W W, et al. Molecular epidemiology analysis of aMPV in some regions in China during 2012 to 2015[J]. Progress in Veterinary Medicine, 2016, 37(10):30-34. (in Chinese)
[15]	YU M M, XING L X, CHANG F F, et al. Genomic sequence and pathogenicity of the first avian metapneumovirus subtype B isolated from chicken in China[J]. Vet Microbiol, 2019, 228:32-38.
[16]	包媛玲, 何锡栋, 于蒙蒙, 等. B亚型禽偏肺病毒对蛋鸡的致病性研究[J]. 中国家禽, 2021, 43(6):25-30.BAO Y L, HE X D, YU M M, et al. Pathogenicity of avian metapneumovirus subtype B on layer chicken[J]. China Poultry, 2021, 43(6):25-30. (in Chinese)
[17]	张丹俊, 戴银, 赵瑞宏, 等. 安徽省部分地区鸡群禽偏肺病毒感染的血清学调查[J]. 动物医学进展, 2017, 38(2):126-129.ZHANG D J, DAI Y, ZHAO R H, et al. Serological survey on avian metapneumovirus infection in chicken flocks in Anhui Province[J]. Progress in Veterinary Medicine, 2017, 38(2):126-129. (in Chinese)
[18]	梅晨, 李淑芳, 闫旭, 等. 北京油鸡混合感染副鸡禽杆菌和禽偏肺病毒的诊治[J]. 中国兽医杂志, 2017, 53(6):56-58.MEI C, LI S F, YAN X, et al. Diagnosis and treatment of mixed infection of paraavian bacilli and avian metapneumovirus in Beijing oil chickens[J]. Chinese Journal of Veterinary Medicine, 2017, 53(6):56-58. (in Chinese)
[19]	付岳林, 崔明仙, 白晓, 等. 规模化肉鸡场鼻气管鸟杆菌、禽呼肠孤病毒和禽偏肺病毒感染状况的血清学调查[J]. 今日畜牧兽医, 2019, 35(2):3-4.FU Y L, CUI M X, BAI X, et al. Serological investigation on the infection of avian bacilli, avian reovirus and avian metapneumovirus in nasotracheal of large-scale broiler farms[J]. Today Animal Husbandry and Veterinary Medicine, 2019, 35(2):3-4. (in Chinese)
[20]	葛菲菲, 齐新永, 李鑫, 等. 雉鸡禽偏肺病毒、鸡毒支原体及细菌混合感染的诊断[J]. 中国家禽, 2017, 39(10):65-66.GE F F, QI X Y, LI X, et al. Diagnosis of mixed infection of avian metapneumovirus, mycoplasma gallisepticum and bacteria in pheasants[J]. China Poultry, 2017, 39(10):65-66. (in Chinese)
[21]	RICKETTS C, PICKLER L, MAURER J, et al. Identification of strain-specific sequences that distinguish a Mycoplasma gallisepticum vaccine strain from field isolates[J]. J Clin Microbiol, 2017, 55(1):244-252.
[22]	CHANG P C, LEE Y L, SHIEN J H, et al. Rapid differentiation of vaccine strains and field isolates of infectious laryngotracheitis virus by restriction fragment length polymorphism of PCR products[J]. J Virol Methods, 1997, 66(2):179-186.
[23]	XIE Z X, XIE L J, CHEN A L, et al. Complete genome sequence analysis of a newcastle disease virus isolated from a wild egret[J]. J Virol, 2012, 86(24):13854-13855.
[24]	DE WIT J J, KOCH G, KANT A, et al. Detection by immunofluorescent assay of serotype-specific and group-specific antigens of infectious bronchitis virus in tracheas of broilers with respiratory problems[J]. Avian Pathol, 1995, 24(3):465-474.
[25]	陈秀琴, 郑敏, 黄梅清, 等. 福建省规模化鸡场禽偏肺病毒感染情况的血清学调查[J]. 动物医学进展, 2021, 42(1):130-133.CHEN X Q, ZHENG M, HUANG M Q, et al. Serosurvey of avian metapneumovirus in scaled chicken farms of Fujian[J]. Progress in Veterinary Medicine, 2021, 42(2):130-133. (in Chinese)
[26]	NAYLOR C J, LUPINI C, BROWN P A. Charged amino acids in the AMPV fusion protein have more influence on induced protection than deletion of the SH or G genes[J]. Vaccine, 2010, 28(41):6800-6807.
[27]	HTUT AUNG Y, LIMAN M, NEUMANN U, et al. Reproducibility of swollen sinuses in broilers by experimental infection with avian metapneumovirus subtypes A and B of turkey origin and their comparative pathogenesis[J]. Avian Pathol, 2008, 37(1):65-74.

B亚型禽偏肺病毒的分离鉴定及致病性研究

Isolation, Identification and Pathogenicity of Subtype B Avian Metapneumovirus

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献 27

相关文章 15

编辑推荐

Metrics

本文评价

[1]	陈玥彤, 刘晓涵, 王芷洋, 赵宇馨, 周铁忠, 胡增金, 朱悦, 王少辉, 田明星, 丁思羽, 祁晶晶, 于圣青. 广东规模化鸡场死鸡胚中鸡毒支原体的分离鉴定、致病性及药物敏感性[J]. 畜牧兽医学报, 2024, 55(1): 290-299.
[2]	于秀菊, 张敏爱, 胡燕姣, 朱芷葳, 王海东, 杨丽华, 范阔海. 枯草芽孢杆菌细菌素的分离、表达及稳定性分析[J]. 畜牧兽医学报, 2024, 55(1): 323-333.
[3]	杨恒, 李占鸿, 宋子昂, 高林, 李卓然, 廖德芳, 肖雷, 李华春. 帕利亚姆病毒实时荧光定量RT-PCR检测方法的建立与应用[J]. 畜牧兽医学报, 2024, 55(1): 395-400.
[4]	余洲, 杨柏高, 张航, 徐茜, 张培培, 冯肖艺, 曹建华, 牛一凡, 杜卫华, 郝海生, 朱化彬, 阿布力孜·吾斯曼, 赵学明. 奶水牛发情标记物的研究进展[J]. 畜牧兽医学报, 2023, 54(9): 3623-3630.
[5]	杨悦, 王锐, 甘源, 郝飞, 谢星, 张磊, 邵国青, 孟庆国, 陈蓉, 冯志新. 基于串联亲和层析法纯化并鉴定猪初乳中SIgA[J]. 畜牧兽医学报, 2023, 54(9): 3884-3894.
[6]	刘鑫欢, 恽佳蕾, 毛立, 李基棕, 郝飞, 何苗锋, 杨蕾蕾, 张纹纹, 程子龙, 孙敏, 刘茂军, 王少辉, 白娟, 李文良. 羊腹泻样本中大肠杆菌的分离、毒力基因与耐药性分析[J]. 畜牧兽医学报, 2023, 54(8): 3445-3454.
[7]	彭硕, 陶亮, 查文文, 陈成鹏, 辜丽川, 朱诚, 焦俊. 基于稀疏分量分析的生猪音频欠定盲源分离研究[J]. 畜牧兽医学报, 2023, 54(7): 2794-2809.
[8]	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.
[9]	程相宁, 张韵, 何佳蔚, 陈政锟, 金美兰, 丁红雷, 王庆华. 鸽源白色念珠菌的分离鉴定及药物敏感性分析[J]. 畜牧兽医学报, 2023, 54(7): 3002-3011.
[10]	赵菲菲, 李杰, 韩宁, 谢仕廷, 曾振灵. 分离自屠宰场的肺炎克雷伯菌的耐药性分析[J]. 畜牧兽医学报, 2023, 54(7): 3044-3053.
[11]	刘弘毅, 罗霆宇, 李昌文, 于海波, 路小野, 陈洪岩, 夏长友, 高彩霞. 实验用荣昌猪SLA-1等位基因鉴定及分子遗传特征分析[J]. 畜牧兽医学报, 2023, 54(7): 3064-3077.
[12]	胥辉豪, 冯雪倩, 朴雪玲, 慎晓军, 郑小波, 杨恒, 林珈好, 金艺鹏, 林德贵. 猫角膜缘干细胞不同采集、分离与培养鉴定方法的对比研究[J]. 畜牧兽医学报, 2023, 54(7): 3091-3101.
[13]	袁生, 李安琪, 吕文珂, 羊露露, 周峰, 黄良宗, 白挨泉, 温峰, 黄淑坚, 郭锦玥. 一株猪伪狂犬病病毒的主要毒力相关基因的变异分析及其对家兔的致病性[J]. 畜牧兽医学报, 2023, 54(5): 2195-2199.
[14]	库旭钢, 于学祥, 孙琪, 李盼盼, 张梦佳, 罗锐, 钱平, 何启盖. 猪圆环病毒3型的分离及致病性分析[J]. 畜牧兽医学报, 2023, 54(4): 1568-1578.
[15]	刘建奎, 徐叶, 刘辰, 于慧, 杨圆, 何乐, 李佳睿, 但惠娟, 戴爱玲, 杨小燕, 魏春华. 基于全基因组分析2017—2021年福建省猪繁殖与呼吸综合征病毒基因组特征[J]. 畜牧兽医学报, 2023, 54(4): 1579-1589.