1株基因Ⅶ型新城疫病毒的生物学特性研究及全基因组序列分析
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摘要
为研究当前流行的新城疫病毒(Newcastle disease virus,NDV)基因型、致病性及其与传统新城疫病毒疫苗株(La Sota等)的核苷酸差异,试验从某发病鸡场病死鸡体内分离到1株疑似NDV毒株,经红细胞凝集试验(HA)和红细胞凝集抑制试验(HI)初步确定为鸡源NDV。参照GenBank公布的NDV F基因部分片段(登录号:JF950510.1)设计1对引物,通过RT-PCR技术扩增分离株的F基因并克隆、测序,测序结果与NCBI中NDVF基因序列进行比对,构建系统进化树并分析其基因型;通过测定鸡胚平均致死时间(MDT)、1日龄鸡脑内致病指数(ICPI)和6周龄鸡静脉致病指数(IVPI)判断病毒致病性;参照GenBank公布的NDV全基因组序列(登录号:JF950510.1)设计9对引物对分离株进行全基因组序列测定,并分析其基因组结构。结果表明,RT-PCR扩增得到F基因长约500bp,基于F基因构建的系统进化树显示分离株为基因Ⅶ型NDV;MDT、ICPI和IVPI分别为52.8h、1.675和2.46,表明分离株属于强毒株。全基因组序列分析显示,分离株全基因组全长15 192bp,与传统La Sota株基因组相比,序列多出6个碱基,核苷酸序列同源性82.8%。本研究成功分离到1株基因Ⅶ型NDV强毒株,且与传统疫苗毒株La Sota的核苷酸序列同源性差异较大。
In order to study the genotype,pathogenicity and nucleotide difference between Newcastle disease virus(NDV)isolates and the traditional NDV vaccine strain(La Sota),a suspected NDV virus was isolated from a chicken farm.The isolate was preliminarily determined by HA and HI tests.A pair of primers was designed based on the partial fragment of NDVFgene published at GenBank(accession No.:JF950510.1).Fgene was amplified by RT-PCR method,and cloned and sequenced,the sequencing result was compared with the Fgene sequences published at GenBank,and the evolutionary tree was constracted to analyze the genotypes.The pathogenicity of the virus was determined by the mean death time(MDT)of chicken embryos,intracerebral pathogenicity index(ICPI)in one-day-old chickens and intravenous pathogenicity index(IVPI)in sixweek-old chickens,respectively.Based on the NDV genome sequence published in GenBank(accession No.:JF950510.1),nine pairs of primers were designed to amplify the genome sequence of the isolate strain,and its structure was analyzed.The results showed that the length of Fgene wasabout 500 bp,a NDV strain of genotypeⅦ was isolated.The MDT,ICPI and IVPI were 52.8 h,1.675 and 2.46,respectively.The whole genome sequence analysis results showed that the full genome length of the isolated strain was 15 192 bp,and had 6 nucleotides more than the strain of La Sota,and the homology between the two strains was 82.8%.Therefore,a virulent NDV strain of genotypeⅦ was isolated,and contained a large difference in the nucleotide sequence compared with the La Sota strain.
In order to study the genotype,pathogenicity and nucleotide difference between Newcastle disease virus(NDV)isolates and the traditional NDV vaccine strain(La Sota),a suspected NDV virus was isolated from a chicken farm.The isolate was preliminarily determined by HA and HI tests.A pair of primers was designed based on the partial fragment of NDVFgene published at GenBank(accession No.:JF950510.1).Fgene was amplified by RT-PCR method,and cloned and sequenced,the sequencing result was compared with the Fgene sequences published at GenBank,and the evolutionary tree was constracted to analyze the genotypes.The pathogenicity of the virus was determined by the mean death time(MDT)of chicken embryos,intracerebral pathogenicity index(ICPI)in one-day-old chickens and intravenous pathogenicity index(IVPI)in sixweek-old chickens,respectively.Based on the NDV genome sequence published in GenBank(accession No.:JF950510.1),nine pairs of primers were designed to amplify the genome sequence of the isolate strain,and its structure was analyzed.The results showed that the length of Fgene wasabout 500 bp,a NDV strain of genotypeⅦ was isolated.The MDT,ICPI and IVPI were 52.8 h,1.675 and 2.46,respectively.The whole genome sequence analysis results showed that the full genome length of the isolated strain was 15 192 bp,and had 6 nucleotides more than the strain of La Sota,and the homology between the two strains was 82.8%.Therefore,a virulent NDV strain of genotypeⅦ was isolated,and contained a large difference in the nucleotide sequence compared with the La Sota strain.
引文
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[2] ALDERS R G.Making Newcastle disease vaccines available at village level[J].The Veterinary Record,2014,174(20):502-503.
[3]陈娟娟.胶体金免疫层析试纸条检测新城疫病毒方法的建立及初步应用[D].扬州:扬州大学,2009.CHEN J J.Establishiment of colloidal gold immunochromatographic assay for NDV detection and its preliminary application[D].Yangzhou:Yangzhou University,2009.(in Chinese)
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[7] PANDA A,HUANG Z,ELANKUMARAN S,et al.Role of fusion protein cleavage site in the virulence of Newcastle disease virus[J].Microbial Pathogenesis,2004,36(1):1-10.
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[11]段博芳,王静静,赵云玲,等.2011—2016年云南新城疫病毒感染状况调查[J].畜牧兽医学报,2017,48(9):1711-1717.DUAN B F,WANG J J,ZHAO Y L,et al.Epidemiological survey of Newcastle disease in Yunnan province during 2011-2016[J].Acta Veterinaria et Zootechnica Sinica,2017,48(9):1711-1717.(in Chinese)
[12] SNOECK C J,OWOADE A A,COUACY-HYMANN E,et al.High genetic diversity of Newcastle disease virus in poultry in West and Central Africa:Cocirculation of genotypeand newly defined genotypesⅩⅦandⅩⅧ[J].Journal of Clinical Microbiology,2013,51(7):2250-2260.
[13] QIN Z M,TAN L T,XU H Y,et al.Pathotypical characterization and molecular epidemiology of Newcastle disease virus isolates from different hosts in China from 1996to 2005[J].Journal of Clinical Microbiology,2008,46(2):601-611.
[14]黄勇.鹅源新城疫病毒ZJ1株全基因组序列的测定及含该毒株全基因组cDNA的转录载体的构建[D].扬州:扬州大学,2003.HUANG Y.The full length genome sequence of a Newcastle disease virus(ZJ1)of goose origin and the construction of a transcription vector containing the cDNA of its entire genome[D].Yangzhou:Yangzhou University,2003.(in Chinese)
[15] CATTOLI G,FUSARO A,MONNE I,et al.Emergence of a new genetic lineage of Newcastle disease virus in West and Central Africa-implications for diagnosis and control[J].Veterinary Microbiology,2010,142(3):168-176.
[16] KIM L M,KING D J,CURRY P E,et al.Phylogenetic diversity among low-virulence Newcastle disease viruses from waterfowl and shorebirds and comparison of genotype distributions to those of poultry-origin isolates[J].Journal of Virology,2007,81(22):12641-12653.
[17] PEROZO F,MARCANO R,AFONSO C L.Biological and phylogenetic characterization of a genotypeⅦNewcastle disease virus from Venezuela:Efficacy of field vaccination[J].Journal of Clinical Microbiology,2012,50(4):1204-1208.
[18] XIAO S,NAYAK B,SAMUEL A,et al.Generation by reverse genetics of an effective,stable,live-attenuated Newcastle disease virus vaccine based on a currently circulating,highly virulent Indonesian strain[J].PLoS One,2012,7(12):e52751.
[19]王建忠.基于CMV启动子NDV基因Ⅶ型NA-1株活载体构建及免疫特性评价[D].长春:吉林大学,2015.WANG J Z.Generation and immunological characteristics evaluation of a recombinant genotypeⅦNDV strain NA-1as a viral vector based on CMV promoter[D].Changchun:Jilin University,2015.(in Chinese)
[20] TAKEUCHI K,TAKEDA M,MIYAJIMA N,et al.Recombinant wild-type and edmonston strain measles viruses bearing heterologous H proteins:Role of H protein in cell fusion and host cell specificity[J].Journal of Virology,2002,76(10):4891-4900.
[21] OHGIMOTO S,OHQIMOTO K,NIEWIESK S,et al.The haemagglutinin protein is an important determinant of measles virus tropism for dendritic cells in vitro[J].The Journal of General Virology,2001,82(8):1835-1844.
[22]李晓文,刘佑明,梁国智,等.4株NDV流行株的分离鉴定、生物学特性及遗传学特性的研究[J].中国畜牧兽医,2017,44(7):1925-1933.LI X W,LIU Y M,LIANG G Z,et al.Isolation,identification of four Newcastle disease virus strains and study on their biological characteristics and genetic characteristics[J].China Animal Husbandry&Veterinary Medicine,2017,44(7):1925-1933.(in Chinese)
[23] ROMER-OBERDORFER A,WERNER O,VEITIS J,et al.Contribution of the length of the HN protein and the sequence of the F protein cleavage site to Newcastle disease virus pathogenicity[J].The Journal of General Virology,2003,84(11):3121-3129.
[24] KUMAR S,NAYAK B,COLLINS P L,et al.Evaluation of the Newcastle disease virus F and HN proteins in protective immunity by using a recombinant avian paramyxovirus type 3vector in chickens[J].Journal of Virology,2011,85(13):6521-6534.
[2] ALDERS R G.Making Newcastle disease vaccines available at village level[J].The Veterinary Record,2014,174(20):502-503.
[3]陈娟娟.胶体金免疫层析试纸条检测新城疫病毒方法的建立及初步应用[D].扬州:扬州大学,2009.CHEN J J.Establishiment of colloidal gold immunochromatographic assay for NDV detection and its preliminary application[D].Yangzhou:Yangzhou University,2009.(in Chinese)
[4] AMARASINGHE G K,BAO Y,BASLER C F,et al.Taxonomy of the order Mononegavirales:Update2017[J].Archives of Virology,2017,162(8):2493-2504.
[5]殷震,刘景华.动物病毒学[M].第2版.北京:科学技术出版社,1997.YIN Z,LIU J H.Animal Toxicology[M].2nd Edition.Beijing:Science Press,1997.(in Chinese)
[6] ALDOUS E W,ALEXANDER D J.Detection and differentiation of Newcastle disease virus(avian paramyxovirus type 1)[J].Avian Pathology,2001,30(2):117-128.
[7] PANDA A,HUANG Z,ELANKUMARAN S,et al.Role of fusion protein cleavage site in the virulence of Newcastle disease virus[J].Microbial Pathogenesis,2004,36(1):1-10.
[8] YANG C Y,SHIEH H K,LIN Y L,et al.Newcastle disease virus isolated from recent outbreaks in Taiwan phylogenetically related to viruses(genotypeⅦ)from recent outbreaks in Western Europe[J].Avian Diseases,1999,43(1):125-130.
[9] PEETERS B P,DE LEEUW O S,KOCH G,et al.Rescue of Newcastle disease virus from cloned cDNA:Evidence that cleavability of the fusion protein is a major determinant for virulence[J].Journal of Virology,1999,73(6):5001-5009.
[10] PEDERSEN J C,SENNE D A,WOOLCOCK P R,et al.Phylogenetic relationships among virulent Newcastle disease virus isolates from the 2002-2003outbreak in California and other recent outbreaks in North America[J].Journal of Clinical Microbiology,2004,42(5):2329-2334.
[11]段博芳,王静静,赵云玲,等.2011—2016年云南新城疫病毒感染状况调查[J].畜牧兽医学报,2017,48(9):1711-1717.DUAN B F,WANG J J,ZHAO Y L,et al.Epidemiological survey of Newcastle disease in Yunnan province during 2011-2016[J].Acta Veterinaria et Zootechnica Sinica,2017,48(9):1711-1717.(in Chinese)
[12] SNOECK C J,OWOADE A A,COUACY-HYMANN E,et al.High genetic diversity of Newcastle disease virus in poultry in West and Central Africa:Cocirculation of genotypeand newly defined genotypesⅩⅦandⅩⅧ[J].Journal of Clinical Microbiology,2013,51(7):2250-2260.
[13] QIN Z M,TAN L T,XU H Y,et al.Pathotypical characterization and molecular epidemiology of Newcastle disease virus isolates from different hosts in China from 1996to 2005[J].Journal of Clinical Microbiology,2008,46(2):601-611.
[14]黄勇.鹅源新城疫病毒ZJ1株全基因组序列的测定及含该毒株全基因组cDNA的转录载体的构建[D].扬州:扬州大学,2003.HUANG Y.The full length genome sequence of a Newcastle disease virus(ZJ1)of goose origin and the construction of a transcription vector containing the cDNA of its entire genome[D].Yangzhou:Yangzhou University,2003.(in Chinese)
[15] CATTOLI G,FUSARO A,MONNE I,et al.Emergence of a new genetic lineage of Newcastle disease virus in West and Central Africa-implications for diagnosis and control[J].Veterinary Microbiology,2010,142(3):168-176.
[16] KIM L M,KING D J,CURRY P E,et al.Phylogenetic diversity among low-virulence Newcastle disease viruses from waterfowl and shorebirds and comparison of genotype distributions to those of poultry-origin isolates[J].Journal of Virology,2007,81(22):12641-12653.
[17] PEROZO F,MARCANO R,AFONSO C L.Biological and phylogenetic characterization of a genotypeⅦNewcastle disease virus from Venezuela:Efficacy of field vaccination[J].Journal of Clinical Microbiology,2012,50(4):1204-1208.
[18] XIAO S,NAYAK B,SAMUEL A,et al.Generation by reverse genetics of an effective,stable,live-attenuated Newcastle disease virus vaccine based on a currently circulating,highly virulent Indonesian strain[J].PLoS One,2012,7(12):e52751.
[19]王建忠.基于CMV启动子NDV基因Ⅶ型NA-1株活载体构建及免疫特性评价[D].长春:吉林大学,2015.WANG J Z.Generation and immunological characteristics evaluation of a recombinant genotypeⅦNDV strain NA-1as a viral vector based on CMV promoter[D].Changchun:Jilin University,2015.(in Chinese)
[20] TAKEUCHI K,TAKEDA M,MIYAJIMA N,et al.Recombinant wild-type and edmonston strain measles viruses bearing heterologous H proteins:Role of H protein in cell fusion and host cell specificity[J].Journal of Virology,2002,76(10):4891-4900.
[21] OHGIMOTO S,OHQIMOTO K,NIEWIESK S,et al.The haemagglutinin protein is an important determinant of measles virus tropism for dendritic cells in vitro[J].The Journal of General Virology,2001,82(8):1835-1844.
[22]李晓文,刘佑明,梁国智,等.4株NDV流行株的分离鉴定、生物学特性及遗传学特性的研究[J].中国畜牧兽医,2017,44(7):1925-1933.LI X W,LIU Y M,LIANG G Z,et al.Isolation,identification of four Newcastle disease virus strains and study on their biological characteristics and genetic characteristics[J].China Animal Husbandry&Veterinary Medicine,2017,44(7):1925-1933.(in Chinese)
[23] ROMER-OBERDORFER A,WERNER O,VEITIS J,et al.Contribution of the length of the HN protein and the sequence of the F protein cleavage site to Newcastle disease virus pathogenicity[J].The Journal of General Virology,2003,84(11):3121-3129.
[24] KUMAR S,NAYAK B,COLLINS P L,et al.Evaluation of the Newcastle disease virus F and HN proteins in protective immunity by using a recombinant avian paramyxovirus type 3vector in chickens[J].Journal of Virology,2011,85(13):6521-6534.