一株鸭源传染性支气管炎病毒的分离鉴定及结构蛋白基因和血清型分析
|
摘要
【目的】对从广西某鸭场发生呼吸道感染的11天龄樱桃谷肉鸭分离到的病毒株进行鉴定,并探索此鸭源病毒分离株的遗传变异情况。【方法】通过血凝试验、鸡胚接种实验、3?端非编码区(3'UTR)基因扩增与序列测定对分离株进行鉴定,并对该分离株的结构基因S1、E、M和N分别进行序列测定以及相似性、系统进化树分析和血清型鉴定。【结果】血凝试验为阴性,接种鸡胚盲传5代后出现侏儒胚,3?UTR基因测序结果表明为传染性支气管炎病毒(IBV)序列。该分离株S蛋白的裂解位点为RRSRR,S1、E、M和N基因与IBV毒株H120、4/91、LTD3核苷酸相似性分别为:78.6%–99.7%、85.4%–100.0%、91.6%–93.2%、86.7%–91.7%。除N基因存在点突变外,S1、E和M基因均存在氨基酸的突变、插入和(或)缺失。系统进化树分析显示,其S1基因属于4/91型,E、M和N基因均为LDT3型。血清型分析表明,该分离株的血清型不同于疫苗株H120和4/91。【结论】此鸭源病毒分离株为IBV,且该分离株的基因型与血清型均发生了变异。本研究结果暗示禽类传染性支气管炎的防控面临着更严峻的挑战。
[Objective] It is to identify and explore the genetic variation of an infectious bronchitis virus isolated from 11-day-old cherry valley ducks that developed suspected respiratory infection in Guangxi province. [Methods] The virus was identified by hemagglutinin test, chicken embryo inoculation and gene sequence analysis of the 3'untranslated region(UTR). The S1, E, M, and N genes of the virus were amplified by reverse transcriptase polymerase chain reaction(RT-PCR), cloned, sequenced, similarity comparison, phylogenetic tree and serotype analyais was conducted. [Results] The hemagglutination test was negative and dwarf embryo appeared after 5 passages of inoculation of chicken embryos. The sequencing result of 3?UTR gene indicated the virus was IBV. The sequence of cleavage site within S protein of the virus was RRSRR. The nucleotide similarities of S1, E, M, and N genes with those of H120, 4/91 and LTD3 reference strains were 78.6%–99.7%, 85.4%–100.0%, 91.6%–93.2% and 86.7%–91.7%, respectively. Compared with the reference strains, there is no insertion or deletion in N gene of the virus except mutations. However, substitutions, insertions and(or) deletions were found in S1, E, M genes of the isolated strain. According to the phylogenetic tree analysis, it was clustered into 4/91-type based on S1 gene and LDT3-type based on E, M and N genes. The serotype analysis showed that the serotype of the isolated strain was different from vaccine strains H120 and 4/91. [Conclusion] The strain isolated from ducks was IBV and the isolated strain existed variation both in structural genes and serotype. The present study suggests that the prevention and control of IB in poultry is facing more severe challenges.
[Objective] It is to identify and explore the genetic variation of an infectious bronchitis virus isolated from 11-day-old cherry valley ducks that developed suspected respiratory infection in Guangxi province. [Methods] The virus was identified by hemagglutinin test, chicken embryo inoculation and gene sequence analysis of the 3'untranslated region(UTR). The S1, E, M, and N genes of the virus were amplified by reverse transcriptase polymerase chain reaction(RT-PCR), cloned, sequenced, similarity comparison, phylogenetic tree and serotype analyais was conducted. [Results] The hemagglutination test was negative and dwarf embryo appeared after 5 passages of inoculation of chicken embryos. The sequencing result of 3?UTR gene indicated the virus was IBV. The sequence of cleavage site within S protein of the virus was RRSRR. The nucleotide similarities of S1, E, M, and N genes with those of H120, 4/91 and LTD3 reference strains were 78.6%–99.7%, 85.4%–100.0%, 91.6%–93.2% and 86.7%–91.7%, respectively. Compared with the reference strains, there is no insertion or deletion in N gene of the virus except mutations. However, substitutions, insertions and(or) deletions were found in S1, E, M genes of the isolated strain. According to the phylogenetic tree analysis, it was clustered into 4/91-type based on S1 gene and LDT3-type based on E, M and N genes. The serotype analysis showed that the serotype of the isolated strain was different from vaccine strains H120 and 4/91. [Conclusion] The strain isolated from ducks was IBV and the isolated strain existed variation both in structural genes and serotype. The present study suggests that the prevention and control of IB in poultry is facing more severe challenges.
引文
[1]Masters PS.The molecular biology of coronaviruses.Advances in Virus Research,2006,66:193-292.
[2]Han ZX,Sun CY,Yan BL,Zhang XN,Wang Y,Li CR,Zhang QX,Ma YZ,Shao YH,Liu QR,Kong XG,Liu SW.A 15-year analysis of molecular epidemiology of avian infectious bronchitis coronavirus in China.Infection,Genetics and Evolution,2011,11(1):190-200.
[3]Cavanagh D.Coronavirus avian infectious bronchitis virus.Veterinary Research,2007,38(2):281-297.
[4]Chen YQ,Jiang L,Zhao WJ,Liu LL,Zhao Y,Shao YH,Li HX,Han ZX,Liu SW.Identification and molecular characterization of a novel serotype infectious bronchitis virus(GI-28)in China.Veterinary Microbiology,2017,198:108-115.
[5]Thor SW,Hilt DA,Kissinger JC,Paterson AH,Jackwood MW.Recombination in avian gamma-coronavirus infectious bronchitis virus.Viruses,2011,3(9):1777-1799.
[6]Jiang L,Han ZX,Chen YQ,Zhao WJ,Sun JF,Zhao Y,Liu SW.Characterization of the complete genome,antigenicity,pathogenicity,tissue tropism,and shedding of a recombinant avian infectious bronchitis virus with a ck/CH/LJL/140901-like backbone and an S2 fragment from a4/91-like virus.Virus Research,2018,244:99-109.
[7]Cavanagh D,Davis PJ,Cook JKA,Li D,Kant A,Koch G.Location of the amino acid differences in the S1 spike glycoprotein subunit of closely related serotypes of infectious bronchitis virus.Avian Pathology,1992,21(1):33-43.
[8]Fan WS,Wang HY,Tang N,Dong ZH,Wei TC,Mo ML,Wei P.Establishment of identification method for infectious bronchitis virus vaccine strains and Guangxi field isolates.Progress in Veterinary Medicine,2018,39(5):26-30.(in Chinese)范文胜,王海勇,唐宁,董志华,韦天超,磨美兰,韦平.鸡传染性支气管炎疫苗株与广西流行野毒株鉴别方法的建立.动物医学进展,2018,39(5):26-30.
[9]Mo ML,Li M,Huang BC,Fan WS,Wei P,Wei TC,Cheng QY,Wei ZJ,Lang YH.Molecular characterization of major structural protein genes of avian coronavirus infectious bronchitis virus isolates in southern China.Viruses,2013,5(12):3007-3020.
[10]Valastro V,Holmes EC,Britton P,Fusaro A,Jackwood MW,Cattoli G,Monne I.S1 gene-based phylogeny of infectious bronchitis virus:An attempt to harmonize virus classification.Infection,Genetics and Evolution,2016,39:349-364.
[11]Ignjatovic J,Galli U.Immune responses to structural proteins of avian infectious bronchitis virus.Avian Pathology,1995,24(2):313-332.
[12]Muradrasoli S,Bálint A,Wahlgren J,Waldenstr?m J,Belák S,Blomberg J,Olsen B.Prevalence and phylogeny of coronaviruses in wild birds from the Bering Strait area(Beringia).PLoS ONE,2010,5(10):e13640.
[13]Jackwood MW.Review of infectious bronchitis virus around the world.Avian Diseases,2012,56(4):634-641.
[14]de Wit JJS,Cook JKA,van der Heijden HMJF.Infectious bronchitis virus variants:a review of the history,current situation and control measures.Avian Pathology,2011,40(3):223-235.
[15]Li M,Wang XY,Wei P,Chen QY,Wei ZJ,Mo ML.Serotype and genotype diversity of infectious bronchitis viruses isolated during 1985-2008 in Guangxi,China.Archives of Virology,2012,157(3):467-474.
[16]Muradrasoli S,Mohamed N,Hornyák A,Fohlman J,Olsen B,Belák S,Blomberg J.Broadly targeted multiprobe QPCR for detection of coronaviruses:Coronavirus is common among mallard ducks(Anas platyrhynchos).Journal of Virological Methods,2009,159(2):277-287.
[17]Chen GQ,Zhuang QY,Wang KC,Liu S,Shao JZ,Jiang WM,Hou GY,Li JP,Yu JM,Li YP,Chen JM.Identification and survey of a novel avian coronavirus in ducks.PLoS ONE,2013,8(8):e72918.
[18]Yao SX,Ou CB,Liu XY,Wang XW,Yao ZH,Liu JJ.Isolation of a novel serotype strain of infectious bronchitis virus ZZ2004 from ducks in China.Virus Genes,2016,52(5):660-670.
[19]Wu XP,Pan SL,Zhou WD,Wu YJ,Huang YF,Wu BC.The isolation and identification of infectious bronchitis virus PTFY strain in muscovy ducks.Chinese Journal of Virology,2016,32(2):203-209.(in Chinese)吴晓平,潘书磊,周五朵,吴异健,黄一帆,吴宝成.番鸭源传染性支气管炎病毒PTFY株的分离鉴定.病毒学报,2016,32(2):203-209.
[20]Cavanagh D.Coronaviruses in poultry and other birds.Avian Pathology,2005,34(6):439-448.
[21]Woo PCY,Lau SKP,Lam CSF,Tsang AKL,Hui SW,Fan RYY,Martelli P,Yuen KY.Discovery of a novel bottlenose dolphin coronavirus reveals a distinct species of marine mammal coronavirus in Gammacoronavirus.Journal of Virology,2014,88(2):1318-1331.
[22]Mo ML,Hong SM,Kwon HJ,Kim IH,Song CS,Kim JH.Genetic diversity of spike,3a,3b and e genes of infectious bronchitis viruses and emergence of new recombinants in Korea.Viruses,2013,5(2):550-567.
[23]Archetti I,Horsfall FL.Persistent antigenic variation of influenza A viruses after incomplete neutralization in ovo with heterologous immune serum.The Journal of Experimental Medicine,1950,92(5):441-462.
[24]Han ZX,Jiang L,Zhao WJ,Chen YQ,Xu LW,Sun JF,Zhao Y,Liu SW.Isolation and characteristics of the Arkansas-type infectious bronchitis virus in China.Avian Diseases,2018,62(1):18-27.
[25]Jiang L,Zhao WJ,Han ZX,Chen YQ,Zhao Y,Sun JF,Li HX,Shao YH,Liu LL,Liu SW.Genome characterization,antigenicity and pathogenicity of a novel infectious bronchitis virus type isolated from south China.Infection,Genetics and Evolution,2017,54:437-446.
[26]Wu CL,He YN,Li HM,Sun XK,Tan YC,Wei TC,Mo ML,Wei P.Isolation and analysis of structural protein genes variation and serotype of a recombinant strain of chicken infectious bronchitis virus.Acta Veterinaria Et Zootechnica Sinica,2015,46(5):815-823.(in Chinese)吴翠兰,何怡宁,李和鸣,孙新宽,谭渝才,韦天超,磨美兰,韦平.一株重组鸡传染性支气管炎病毒的分离及结构蛋白基因变异和血清型分析.畜牧兽医学报.2015,46(5):815-823.
[2]Han ZX,Sun CY,Yan BL,Zhang XN,Wang Y,Li CR,Zhang QX,Ma YZ,Shao YH,Liu QR,Kong XG,Liu SW.A 15-year analysis of molecular epidemiology of avian infectious bronchitis coronavirus in China.Infection,Genetics and Evolution,2011,11(1):190-200.
[3]Cavanagh D.Coronavirus avian infectious bronchitis virus.Veterinary Research,2007,38(2):281-297.
[4]Chen YQ,Jiang L,Zhao WJ,Liu LL,Zhao Y,Shao YH,Li HX,Han ZX,Liu SW.Identification and molecular characterization of a novel serotype infectious bronchitis virus(GI-28)in China.Veterinary Microbiology,2017,198:108-115.
[5]Thor SW,Hilt DA,Kissinger JC,Paterson AH,Jackwood MW.Recombination in avian gamma-coronavirus infectious bronchitis virus.Viruses,2011,3(9):1777-1799.
[6]Jiang L,Han ZX,Chen YQ,Zhao WJ,Sun JF,Zhao Y,Liu SW.Characterization of the complete genome,antigenicity,pathogenicity,tissue tropism,and shedding of a recombinant avian infectious bronchitis virus with a ck/CH/LJL/140901-like backbone and an S2 fragment from a4/91-like virus.Virus Research,2018,244:99-109.
[7]Cavanagh D,Davis PJ,Cook JKA,Li D,Kant A,Koch G.Location of the amino acid differences in the S1 spike glycoprotein subunit of closely related serotypes of infectious bronchitis virus.Avian Pathology,1992,21(1):33-43.
[8]Fan WS,Wang HY,Tang N,Dong ZH,Wei TC,Mo ML,Wei P.Establishment of identification method for infectious bronchitis virus vaccine strains and Guangxi field isolates.Progress in Veterinary Medicine,2018,39(5):26-30.(in Chinese)范文胜,王海勇,唐宁,董志华,韦天超,磨美兰,韦平.鸡传染性支气管炎疫苗株与广西流行野毒株鉴别方法的建立.动物医学进展,2018,39(5):26-30.
[9]Mo ML,Li M,Huang BC,Fan WS,Wei P,Wei TC,Cheng QY,Wei ZJ,Lang YH.Molecular characterization of major structural protein genes of avian coronavirus infectious bronchitis virus isolates in southern China.Viruses,2013,5(12):3007-3020.
[10]Valastro V,Holmes EC,Britton P,Fusaro A,Jackwood MW,Cattoli G,Monne I.S1 gene-based phylogeny of infectious bronchitis virus:An attempt to harmonize virus classification.Infection,Genetics and Evolution,2016,39:349-364.
[11]Ignjatovic J,Galli U.Immune responses to structural proteins of avian infectious bronchitis virus.Avian Pathology,1995,24(2):313-332.
[12]Muradrasoli S,Bálint A,Wahlgren J,Waldenstr?m J,Belák S,Blomberg J,Olsen B.Prevalence and phylogeny of coronaviruses in wild birds from the Bering Strait area(Beringia).PLoS ONE,2010,5(10):e13640.
[13]Jackwood MW.Review of infectious bronchitis virus around the world.Avian Diseases,2012,56(4):634-641.
[14]de Wit JJS,Cook JKA,van der Heijden HMJF.Infectious bronchitis virus variants:a review of the history,current situation and control measures.Avian Pathology,2011,40(3):223-235.
[15]Li M,Wang XY,Wei P,Chen QY,Wei ZJ,Mo ML.Serotype and genotype diversity of infectious bronchitis viruses isolated during 1985-2008 in Guangxi,China.Archives of Virology,2012,157(3):467-474.
[16]Muradrasoli S,Mohamed N,Hornyák A,Fohlman J,Olsen B,Belák S,Blomberg J.Broadly targeted multiprobe QPCR for detection of coronaviruses:Coronavirus is common among mallard ducks(Anas platyrhynchos).Journal of Virological Methods,2009,159(2):277-287.
[17]Chen GQ,Zhuang QY,Wang KC,Liu S,Shao JZ,Jiang WM,Hou GY,Li JP,Yu JM,Li YP,Chen JM.Identification and survey of a novel avian coronavirus in ducks.PLoS ONE,2013,8(8):e72918.
[18]Yao SX,Ou CB,Liu XY,Wang XW,Yao ZH,Liu JJ.Isolation of a novel serotype strain of infectious bronchitis virus ZZ2004 from ducks in China.Virus Genes,2016,52(5):660-670.
[19]Wu XP,Pan SL,Zhou WD,Wu YJ,Huang YF,Wu BC.The isolation and identification of infectious bronchitis virus PTFY strain in muscovy ducks.Chinese Journal of Virology,2016,32(2):203-209.(in Chinese)吴晓平,潘书磊,周五朵,吴异健,黄一帆,吴宝成.番鸭源传染性支气管炎病毒PTFY株的分离鉴定.病毒学报,2016,32(2):203-209.
[20]Cavanagh D.Coronaviruses in poultry and other birds.Avian Pathology,2005,34(6):439-448.
[21]Woo PCY,Lau SKP,Lam CSF,Tsang AKL,Hui SW,Fan RYY,Martelli P,Yuen KY.Discovery of a novel bottlenose dolphin coronavirus reveals a distinct species of marine mammal coronavirus in Gammacoronavirus.Journal of Virology,2014,88(2):1318-1331.
[22]Mo ML,Hong SM,Kwon HJ,Kim IH,Song CS,Kim JH.Genetic diversity of spike,3a,3b and e genes of infectious bronchitis viruses and emergence of new recombinants in Korea.Viruses,2013,5(2):550-567.
[23]Archetti I,Horsfall FL.Persistent antigenic variation of influenza A viruses after incomplete neutralization in ovo with heterologous immune serum.The Journal of Experimental Medicine,1950,92(5):441-462.
[24]Han ZX,Jiang L,Zhao WJ,Chen YQ,Xu LW,Sun JF,Zhao Y,Liu SW.Isolation and characteristics of the Arkansas-type infectious bronchitis virus in China.Avian Diseases,2018,62(1):18-27.
[25]Jiang L,Zhao WJ,Han ZX,Chen YQ,Zhao Y,Sun JF,Li HX,Shao YH,Liu LL,Liu SW.Genome characterization,antigenicity and pathogenicity of a novel infectious bronchitis virus type isolated from south China.Infection,Genetics and Evolution,2017,54:437-446.
[26]Wu CL,He YN,Li HM,Sun XK,Tan YC,Wei TC,Mo ML,Wei P.Isolation and analysis of structural protein genes variation and serotype of a recombinant strain of chicken infectious bronchitis virus.Acta Veterinaria Et Zootechnica Sinica,2015,46(5):815-823.(in Chinese)吴翠兰,何怡宁,李和鸣,孙新宽,谭渝才,韦天超,磨美兰,韦平.一株重组鸡传染性支气管炎病毒的分离及结构蛋白基因变异和血清型分析.畜牧兽医学报.2015,46(5):815-823.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |