一株肉鸡呼肠孤病毒变异株的分离鉴定
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摘要
旨在研究肉鸡呼肠孤病毒分离株致病与基因组变异情况。2017年从山东潍坊地区跗关节肿胀、出血严重商品肉鸡中收集病料,进行病毒分离,通过RT-PCR检测、电镜观察对病毒进行鉴定;将分离到的病毒回归商品肉鸡;设计18对引物对分离株全基因组扩增测序,并进行了遗传进化分析;将分离毒株与经典毒株S1133进行血清交叉中和试验。结果表明分离到一株禽呼肠孤病毒(命名为WF17),回归商品肉鸡能完全复制出临床症状,并能从试验鸡中重新分离到该病毒。该分离株基因组完全符合禽呼肠孤病毒基因组结构特点,主要抗原σC蛋白基因与台湾918株最接近,相似性为92.7%,与S1133株的相似性只有55.9%。WF17株与S1133株的抗原相关指数(R值)只有0.19。目前以S1133株为主的商品化疫苗无法对禽呼肠孤病毒变异株产生有效保护。
This experiment was conducted to study the pathogenicity and genome variation of broiler reovirus isolates in Shandong Province. In 2017, a virus was isolated from commercial broilers with tarsal joint swelling and severe bleeding in Weifang, Shandong, and identified by RT-PCR and electron microscopy. And the virus was returned to commercial broilers. The whole genome of the isolated strain was amplified by using18 pairs of primers, then sequenced, and its genetic evolution was analyzed. Serum cross neutralization test was carried out between the isolated strain and the classical strain S1133. Our results showed that a strain of avian reovirus(named WF17) was isolated. The clinical symptoms could be completely replicated in commercial broilers and the virus could be re-isolated from experimental chickens. The genome of the isolate fully conformed to the genomic structure of avian reovirus. And the main antigen(σC protein) gene was closest to 918 strain from Taiwan Province, with 92.7% homology, and 55.9% homology with S1133 strain. The antigen-related index(R value) between WF17 strain and S1133 strain was only 0.19. At present, commercialized vaccine based on S1133 strain can not effectively protect variant avian reovirus strains.
This experiment was conducted to study the pathogenicity and genome variation of broiler reovirus isolates in Shandong Province. In 2017, a virus was isolated from commercial broilers with tarsal joint swelling and severe bleeding in Weifang, Shandong, and identified by RT-PCR and electron microscopy. And the virus was returned to commercial broilers. The whole genome of the isolated strain was amplified by using18 pairs of primers, then sequenced, and its genetic evolution was analyzed. Serum cross neutralization test was carried out between the isolated strain and the classical strain S1133. Our results showed that a strain of avian reovirus(named WF17) was isolated. The clinical symptoms could be completely replicated in commercial broilers and the virus could be re-isolated from experimental chickens. The genome of the isolate fully conformed to the genomic structure of avian reovirus. And the main antigen(σC protein) gene was closest to 918 strain from Taiwan Province, with 92.7% homology, and 55.9% homology with S1133 strain. The antigen-related index(R value) between WF17 strain and S1133 strain was only 0.19. At present, commercialized vaccine based on S1133 strain can not effectively protect variant avian reovirus strains.
引文
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[17] PALOMINO-TAPIA V,MITEVSKI D,INGLIS T,et al.Molecular characterization of emerging avian reovirus variants isolated from viral arthritis cases in Western Canada 2012-2017 based on partial sigma (σ)C gene[J].Virology,2018,522:138-146.
[18] 黄莉,谢芝勋,蓝元喜,等.禽呼肠孤病毒感染对SPF鸡外周血淋巴细胞中天然免疫相关基因转录的影响[J].畜牧兽医学报,2017,48(1):116-123.HUANG L,XIE Z X,LAN Y X,et al.Effects of avian reovirus infection on transcription of innate immune genes in peripheral blood lymphocytes of SPF chickens[J].Acta Veterinaria et Zootechnica Sinica,2017,48(1):116-123.(in Chinese)
[19] 吴海洋.禽呼肠孤病毒p10蛋白诱导宿主细胞凋亡及自身降解的分子机理[D].北京:中国农业大学,2016.WU H Y.The molecular mechanism underlying ARV p10-induced apoptosis and its degradation in host cells[D].Beijing:China Agricultural University,2016.(in Chinese)
[2] SAIF Y M.禽病学[M].苏敬良,高福,索勋,译.12版.北京:中国农业出版社,2012:355-365.SAIF Y M.Diseases of poultry[M].SU J L,GAO F,SUO X,trans.12th ed.Beijing:China Agriculture Press,2012:355-365.(in Chinese)
[3] 赵圆圆,黄晓慧,王蓓,等.安徽省禽呼肠孤病毒感染的血清学调查[J].中国微生态学杂志,2011,23(9):813-815.ZHAO Y Y,HUANG X H,WANG B,et al.Serological investigation of Avian reovirus infection in Anhui province[J].Chinese Journal of Microecology,2011,23(9):813-815.(in Chinese)
[4] 黄莉,谢芝勋,谢丽基,等.禽呼肠孤病毒强毒株和弱毒疫苗株感染Vero细胞的蛋白质组学研究[J].畜牧兽医学报,2016,47(2):331-339.HUANG L,XIE Z X,XIE L J,et al.Proteomic analysis of Vero cells infected by avian reovirus virulent strain and attenuated vaccine strain[J].Acta Veterinaria et Zootechnica Sinica,2016,47(2):331-339.(in Chinese)
[5] 王兴和.鸡病毒性关节炎的研究进展[J].中国动物保健,2016,18(11):81-84.WANG X H.Research progress of avian viral arthritis in chicken[J].China Animal Health,2016,18(11):81-84.(in Chinese)
[6] 张志强.禽呼肠孤病毒Sigma-C蛋白引起宿主细胞凋亡的分子机理研究[D].北京:中国农业大学,2015.ZHANG Z Q.The molecular mechanism underlying ARV Sigma-C-induced apoptosis in host cells[D].Beijing:China Agricultural University,2015.(in Chinese)
[7] TANG Y,LU H G.Genomic characterization of a broiler reovirus field strain detected in Pennsylvania[J].Infect Genet Evol,2015,31:177-182.
[8] 殷震,刘景华.动物病毒学[M].2版.北京:科学出版社,1997.YIN Z,LIU J H.Animal virology[M].2nd ed.Beijing:Science Press,1997.(in Chinese)
[9] 孙美玉.禽呼肠孤病毒检测方法的建立及病毒分离鉴定[D].北京:中国农业科学院,2012.SUN M Y.Establishment of detection methods and isolation and identification of avian reovirus[D].Beijing:Chinese Academy of Agricultural Sciences,2012.(in Chinese)
[10] LUBLIN A,GOLDENBERG D,ROSENBLUTH E,et al.Wide-range protection against avianreovirus conferred by vaccination with representatives of four defined genotypes[J].Vaccine,2011,29(47):8683-8688.
[11] TROXLER S,RIGOMIER P,BILIC I,et al.Identification of a newreovirus causing substantial losses in broiler production in France,despite routine vaccination of breeders[J].Vet Rec,2013,172(21):556.
[12] 姜晓宁.鸡源呼肠孤病毒的分离鉴定及对肉鸡的致病性研究[D].泰安:山东农业大学,2018.JIANG X N.Isolation and identification of chicken-derived avian reovirus and its pathogenicity to broilers[D].Tai’an:Shandong Agricultural University,2018.(in Chinese)
[13] 钟丽.禽呼肠孤病毒流行株的进化及致病性研究[D].北京:中国农业科学院,2017.ZHONG L.Evolution and pathogenicity of avian reovirus isolates[D].Beijing:Chinese Academy of Agricultural Sciences,2017.(in Chinese)
[14] 池晓娟,邵文涵,陈吉龙,等.禽源呼肠孤病毒诱导宿主免疫应答研究进展[J].畜牧兽医学报,2017,48(12):2249-2257.CHI X J,SHAO W H,CHEN J L,et al.The research advance of host immune response induced by avian-origin reovirus[J].Acta Veterinaria et Zootechnica Sinica,2017,48(12):2249-2257.(in Chinese)
[15] LIU H J,HUANG P H.Sequence and phylogenetic analysis of the σA-encoding gene of avian reovirus[J].J Virol Mehtods,2001,98(2):99-107.
[16] 康亚男,毛雅元,王寿山,等.鸡病毒性关节炎灭活疫苗与同类产品比较研究[J].黑龙江畜牧兽医,2014(4):97-98.KANG Y N,MAO Y Y,WANG S S,et al.Comparative study on inactivated vaccine against chicken viral arthritis and similar products[J].Heilongjiang Animal Science and Veterinary Medicine,2014(4):97-98.(in Chinese)
[17] PALOMINO-TAPIA V,MITEVSKI D,INGLIS T,et al.Molecular characterization of emerging avian reovirus variants isolated from viral arthritis cases in Western Canada 2012-2017 based on partial sigma (σ)C gene[J].Virology,2018,522:138-146.
[18] 黄莉,谢芝勋,蓝元喜,等.禽呼肠孤病毒感染对SPF鸡外周血淋巴细胞中天然免疫相关基因转录的影响[J].畜牧兽医学报,2017,48(1):116-123.HUANG L,XIE Z X,LAN Y X,et al.Effects of avian reovirus infection on transcription of innate immune genes in peripheral blood lymphocytes of SPF chickens[J].Acta Veterinaria et Zootechnica Sinica,2017,48(1):116-123.(in Chinese)
[19] 吴海洋.禽呼肠孤病毒p10蛋白诱导宿主细胞凋亡及自身降解的分子机理[D].北京:中国农业大学,2016.WU H Y.The molecular mechanism underlying ARV p10-induced apoptosis and its degradation in host cells[D].Beijing:China Agricultural University,2016.(in Chinese)