1株鸡传染性支气管炎病毒的分离鉴定及生物学特性研究
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摘要
为跟踪传染性支气管炎病流行情况,2015年我们从在江苏某养鸡场疑似传染性支气管炎发病鸡群采集分离到1株疑似传染性支气管炎病毒,对其进行了鉴定和生物学特性研究。结果在鸡胚上连传6代出现鸡胚典型病变,死亡鸡胚全身出血明显,未死鸡胚发育受阻,呈明显的侏儒胚、蜷缩。分离株S1基因RT-PCR扩增可见到清晰的1. 7 kb左右大小的条带,与引物设计的预期结果相符,与Gen Bank中的IBV序列进行比对和分析,从进化树看,分离株属于IBV当前主要流行的QX型。电镜下观察可见直径为80～120 nm,有囊膜和纤突的冠状病毒粒子;经卵磷脂酶C处理后能凝集鸡红细胞,并能特异性地被该分离株的高免血清所抑制;对热、酸碱、胰酶有一定耐受性,对脂溶剂敏感,符合鸡传染性支气管关病毒(IBV)有囊膜的特性;该分离株的最小致病量为102. 0EID50。该毒株灭活苗0. 25 m L/只免疫即可达到良好的免疫效果。以上结果表明,该分离株符合鸡传染性支气管炎病毒的特性。
In order to invesgate the epidemics of infectious bronchitis,we isolated an avian infectious bronchitis virus from chickens,suspected of infectious bronchitis,in a chicken farm in Jiangsu province,2015. We identified and characterized this virus.We obtained an avian infectious bronchitis virus through a continuous passageof six generations in embryos,which resulted in typical embryo lesions and martality. The embryo lesions included hemorrhage,stunting and curling of the embryo. The RT-PCR amplification of the S1 gene of the isolate showed a clear band of about 1. 7 kb,which was in consistent with the expected target of the primer design. We compared the sequence with the IBV sequence in Genbank,and the phylogenetic tree analysis showed that the isolate belonged to the popular QX type. Biological characteristics showed that diameter of this virus particle was approximately 80 ～ 120 nm,and the particle had capsules and dendritic coronavirus particles. The virus agglutinated chicken red blood cells after treating with lecithinase C,and virus-neutralizing test showed that its growth was inhibited by hyper-immune serum. The virus was resistant to heat,trypsin,acid and alkali,but sensitive to lipid solvent,which were consistent with the capsule characteristics. The minimum pathogenic dose was 102. 0 EID50. 0. 25 m L immunity dose of this isolate protected vaccinated chickens against challenge. The results indicated that the strain was an avian infectious bronchitis virus
In order to invesgate the epidemics of infectious bronchitis,we isolated an avian infectious bronchitis virus from chickens,suspected of infectious bronchitis,in a chicken farm in Jiangsu province,2015. We identified and characterized this virus.We obtained an avian infectious bronchitis virus through a continuous passageof six generations in embryos,which resulted in typical embryo lesions and martality. The embryo lesions included hemorrhage,stunting and curling of the embryo. The RT-PCR amplification of the S1 gene of the isolate showed a clear band of about 1. 7 kb,which was in consistent with the expected target of the primer design. We compared the sequence with the IBV sequence in Genbank,and the phylogenetic tree analysis showed that the isolate belonged to the popular QX type. Biological characteristics showed that diameter of this virus particle was approximately 80 ～ 120 nm,and the particle had capsules and dendritic coronavirus particles. The virus agglutinated chicken red blood cells after treating with lecithinase C,and virus-neutralizing test showed that its growth was inhibited by hyper-immune serum. The virus was resistant to heat,trypsin,acid and alkali,but sensitive to lipid solvent,which were consistent with the capsule characteristics. The minimum pathogenic dose was 102. 0 EID50. 0. 25 m L immunity dose of this isolate protected vaccinated chickens against challenge. The results indicated that the strain was an avian infectious bronchitis virus
引文
[1]刘兴友.传染性支气管炎病毒S1基因载体构建和荧光抗体检测的研究[D].北京:中国农业大学,2000.
[2]秦丽莉,李孟,何坤.广西2009~2011年鸡传染性支气管炎病毒分离株的基因分型及血清分型[J].病毒学报,2014,30(2):162-169.
[3]黄向阳,刘振生,郭永立.中华人民共和国兽药典[M].北京:中国农业出版社. 2005.
[4] Han Z,Sun C,Yan B,et al. A 15-year analysis of molecular epidemiology of avian infectious bronchitis coronavirus in China[J].Infect Genet Evol,2011,11(1):190-200.
[5] Ji J,Xie J,Chen F,Shu D,et al. Phylogenetic distribution and predominant genotype of the avian infectious bronchitis virus in China during 2008—2009[J]. Virology journal,2011,8:184.
[6] Li M,Wang XY,Wei P,et al. Serotype and genotype diversity of infectious bronchitis viruses isolated during 1985-2008 in Guangxi,China[J]. Archives of virology,2012,157(3):467-474.
[7] Liu X,Ma H,Xu Q,et al. Characterization of a recombinant coronavirus infectious bronchitis virus with distinct S1 subunits of spike and nucleocapsid genes and a 3'untranslated region[J].Veterinary micro-biology,2013,162(2-4):429-436.
[8] Luo H,Qin J,Chen F,et al. Phylogenetic analysis of the S1 glycoprotein gene of infectious bronchitis viruses isolated in China during 2009-2010[J]. Virus genes,2012,44(1):19-23.
[9]王玉东,王永玲,张子春,等.鸡腺胃型传染性支气管炎病毒(QX IBV)的分离和鉴定[J].中国动物检疫,1998,15(1):3-5.
[10] Liu S,Kong X. A new genotype of nephropathogenic infectious bronchitis virus circulating in vaccinated and non-vaccinated flocks in China[J]. Avian Pathol,2004,33(3):321-327.
[11] Terregino C,Toffan A,Beato M S,et al. Pathogenicity of a QX strain of infectious bronchitis virus in specific pathogen free and commercial broiler chickens,and evaluation of protection induced by a vaccination programme based on the Ma5 and 4/91 serotypes[J]. Avian Pathol,2008,37(5):487-493.
[12]张小荣. 2009—2011年国内传染性支气管炎病毒分子流行病学研究及以马立克病毒为载体的基因工程疫苗研制[D].扬州:扬州大学,2012.
[13]刘胜旺.我国鸡传染性支气管炎流行现状及原因分析[J].中国家禽,2010,32(16):5-9.
[14]贺秀媛,刘胜旺,孔宪刚,等.我国鸡传染性支气管炎病毒地方分离株生物学特性的研究[J].中国预防兽医学报,2002,24(5):349-353.
[2]秦丽莉,李孟,何坤.广西2009~2011年鸡传染性支气管炎病毒分离株的基因分型及血清分型[J].病毒学报,2014,30(2):162-169.
[3]黄向阳,刘振生,郭永立.中华人民共和国兽药典[M].北京:中国农业出版社. 2005.
[4] Han Z,Sun C,Yan B,et al. A 15-year analysis of molecular epidemiology of avian infectious bronchitis coronavirus in China[J].Infect Genet Evol,2011,11(1):190-200.
[5] Ji J,Xie J,Chen F,Shu D,et al. Phylogenetic distribution and predominant genotype of the avian infectious bronchitis virus in China during 2008—2009[J]. Virology journal,2011,8:184.
[6] Li M,Wang XY,Wei P,et al. Serotype and genotype diversity of infectious bronchitis viruses isolated during 1985-2008 in Guangxi,China[J]. Archives of virology,2012,157(3):467-474.
[7] Liu X,Ma H,Xu Q,et al. Characterization of a recombinant coronavirus infectious bronchitis virus with distinct S1 subunits of spike and nucleocapsid genes and a 3'untranslated region[J].Veterinary micro-biology,2013,162(2-4):429-436.
[8] Luo H,Qin J,Chen F,et al. Phylogenetic analysis of the S1 glycoprotein gene of infectious bronchitis viruses isolated in China during 2009-2010[J]. Virus genes,2012,44(1):19-23.
[9]王玉东,王永玲,张子春,等.鸡腺胃型传染性支气管炎病毒(QX IBV)的分离和鉴定[J].中国动物检疫,1998,15(1):3-5.
[10] Liu S,Kong X. A new genotype of nephropathogenic infectious bronchitis virus circulating in vaccinated and non-vaccinated flocks in China[J]. Avian Pathol,2004,33(3):321-327.
[11] Terregino C,Toffan A,Beato M S,et al. Pathogenicity of a QX strain of infectious bronchitis virus in specific pathogen free and commercial broiler chickens,and evaluation of protection induced by a vaccination programme based on the Ma5 and 4/91 serotypes[J]. Avian Pathol,2008,37(5):487-493.
[12]张小荣. 2009—2011年国内传染性支气管炎病毒分子流行病学研究及以马立克病毒为载体的基因工程疫苗研制[D].扬州:扬州大学,2012.
[13]刘胜旺.我国鸡传染性支气管炎流行现状及原因分析[J].中国家禽,2010,32(16):5-9.
[14]贺秀媛,刘胜旺,孔宪刚,等.我国鸡传染性支气管炎病毒地方分离株生物学特性的研究[J].中国预防兽医学报,2002,24(5):349-353.