12个新城疫病毒分离株的F基因和HN基因序列分析
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摘要
自1997年-2016年从广东佛山、肇庆、云浮等地疑似新城疫的家禽病料分离鉴定获得12株新城疫病毒,采用RT-PCR方法对所有分离株的F基因和HN基因进行扩增,产物经克隆并测序后与GenBank数据库中的参考毒株做遗传进化分析。12个分离株的F基因遗传进化分析结果显示,9株属于基因Ⅶ型,2株属于基因Ⅸ型,1株属于基因Ⅵ型。F基因同源性分析结果显示,12个毒株与目前疫苗株La Sota、B1、Mukteswar和Clone30的同源性在81.6%~91.5%之间,12个毒株间同源性的差异较大,9个Ⅶ型分离株之间的同源性在84.8%~99%之间;HN基因同源性分析结果显示,12个毒株与目前疫苗株La Sota、B1、Mukteswar和Clone30的同源性在80.9%~91.9%之间,12个毒株间同源性的差异较大,9个Ⅶ型分离株之间的同源性在84%~98.5%之间。氨基酸序列分析表明,12个分离株与NDV强毒株的氨基酸特征相符,有10个分离株的HN基因第514氨基酸残基出现Ⅰ→Ⅴ的变异,有9个分离株的F基因和HN基因的中和抗原位点出现变异。试验提示,当前广东部分地区的分离株与传统疫苗株之间存在抗原性差异,故应加强广东地区新城疫病毒的分子遗传监控。
In this research,samples were collected from different poultry in Foshan,Zhaoqing,Yunfu,etc,Guangdong province from 1997 to 2016.The F genes and HN genes of 12 NDV isolates were amplified through RT-PCR.PCR products were cloned into 18-T vector and sequenced.The sequence results were analyzed by comparing with NDV reference strains published in GenBank.The genetic evolution analysis of F gene showed that 9 of 12 NDV isolates belong to genotypeⅦ,2 of 12 NDV isolates belong to genotypeⅨ,1 of 12 NDV isolates belongs to genotypeⅥ.The aligment and analysis results showed that the F gene has similarities from 81.6% to 91.5% by comparing with those reference strains,such as La Sota,B1,Mukteswar and Clone 30.The difference of similarity among 12 strains is larger.The similarities among 9 strains of typeⅦisolates are between 84.8%to 99%.The aligment and analysis results showed the HN gene has similarities from 80.9% to 91.9% by comparing with those reference strains,such as La Sota,B1,Mukteswar and Clone 30.The difference of similarity among 12 strains is larger.The similarities among 9 strains of type Ⅶ isolates are between 84%to 98.5%.The analysis of sequences of amino acids showed that 12 isolates were virulent strains of NDV.The 514 th amino acid residues in the HN gene of 10 isolates showedⅠtoⅤ variation.And there are 9 isolates having the variation of the neutralizing antigen loci of the F and HN genes.The results suggest that there are antigenic differences between isolates and traditional vaccine strains in some areas of Guangdong.Therefore,molecular genetic monitoring of Newcastle disease virus strains in Guangdong area should be strengthened.
In this research,samples were collected from different poultry in Foshan,Zhaoqing,Yunfu,etc,Guangdong province from 1997 to 2016.The F genes and HN genes of 12 NDV isolates were amplified through RT-PCR.PCR products were cloned into 18-T vector and sequenced.The sequence results were analyzed by comparing with NDV reference strains published in GenBank.The genetic evolution analysis of F gene showed that 9 of 12 NDV isolates belong to genotypeⅦ,2 of 12 NDV isolates belong to genotypeⅨ,1 of 12 NDV isolates belongs to genotypeⅥ.The aligment and analysis results showed that the F gene has similarities from 81.6% to 91.5% by comparing with those reference strains,such as La Sota,B1,Mukteswar and Clone 30.The difference of similarity among 12 strains is larger.The similarities among 9 strains of typeⅦisolates are between 84.8%to 99%.The aligment and analysis results showed the HN gene has similarities from 80.9% to 91.9% by comparing with those reference strains,such as La Sota,B1,Mukteswar and Clone 30.The difference of similarity among 12 strains is larger.The similarities among 9 strains of type Ⅶ isolates are between 84%to 98.5%.The analysis of sequences of amino acids showed that 12 isolates were virulent strains of NDV.The 514 th amino acid residues in the HN gene of 10 isolates showedⅠtoⅤ variation.And there are 9 isolates having the variation of the neutralizing antigen loci of the F and HN genes.The results suggest that there are antigenic differences between isolates and traditional vaccine strains in some areas of Guangdong.Therefore,molecular genetic monitoring of Newcastle disease virus strains in Guangdong area should be strengthened.
引文
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[13]陈亚波.新城疫病毒HN蛋白与F蛋白结构域共表达对细胞融合的影响[D].浙江杭州:浙江大学,2006.
[14]McGinnes L,Sergel T,Reitter J,et al.Carbohydrate modifications of the NDV fusion protein heptad repeat domains influence maturation and fusion activity[J].Virology,2001,283(2):332-342.
[15]Rui Z,Juan P,Jingliang S,et al.Phylogenetic characterization of Newcastle disease virus isolated in the mainland of China during2001-2009[J].Vet Microbiol,2010,141(3-4):246-257.
[16]Umali D V,Ito H,Shirota K,et al.Characterization of complete genome sequence of genotypeⅥandⅦvelogenic Newcastle disease virus from Japan[J].Virus Genes,2014,49(1):89-99.
[2]de Leeuw O S,Koch G,Hartog L,et al.Virulence of Newcastle disease virus is determined by the cleavage site of the fusion protein and by both the stem region and globular head of the haemagglutinin-neuraminidase protein[J].J Gen Virol,2005,86(6):1759-69.
[3]Jin J,Zhao J,Ren Y,et al.Contribution of HN protein length diversity to Newcastle disease virus virulence,replication and biological activities[J].Sci Rep,2016,11(6):36890.
[4]卡尔尼克.禽病学[M].北京:中国农业出版社,1999.
[5]Umali D V,Ito H,Katoh H,et al.Surveillance of avian paramyxovirus in migratory waterfowls in the sanin region of Watern Japan from 2006to 2012[J].Vet Med Sci,2014,76(3):423-430.
[6]Miller P J,Decanini E L,Afonso C L.Newcastle disease:Evolution of genotypes and the related diagnostic challenges[J].Infect Genet Evol,2010,10(1):26-35.
[7]刘华雷,王永坤,周继宏,等.鹅副粘病毒毒力特性的研究[J].扬州大学学报:农业与生命科学版,2000,21(2):15-20.
[8]丁壮,伊仁福,薛聪,等.新城疫流行病学新特点及鹅新城疫防控策略[J].中国兽医学报,2015,35(1):159-168.
[9]Liu H,Wang Z,Wu Y,et al.Molecular characterization and phylogenetic analysis of new newcastle disease virus isolates from the mainland of China[J].Res Vet Sci,2008,85(3):612-616.
[10]贺文美,韦平.感染水禽的新城疫病毒特性及其预防控制[J].广西畜牧兽医,2011,27(4):252-256.
[11]刘秀梵,胡顺林.新城疫病毒的进化及其新型疫苗的研制[J].中国兽医杂志,2010,44(1):12-18.
[12]Liu X F,Wan H Q,Ni X X,et al.Pathotypical and genotypical characterization of strains of Newcastle disease virus isolated from outbreaks in chicken and goose flocks in some regions of China during 1985-2001[J].Arch Virol,2003,148(7):1387-1403.
[13]陈亚波.新城疫病毒HN蛋白与F蛋白结构域共表达对细胞融合的影响[D].浙江杭州:浙江大学,2006.
[14]McGinnes L,Sergel T,Reitter J,et al.Carbohydrate modifications of the NDV fusion protein heptad repeat domains influence maturation and fusion activity[J].Virology,2001,283(2):332-342.
[15]Rui Z,Juan P,Jingliang S,et al.Phylogenetic characterization of Newcastle disease virus isolated in the mainland of China during2001-2009[J].Vet Microbiol,2010,141(3-4):246-257.
[16]Umali D V,Ito H,Shirota K,et al.Characterization of complete genome sequence of genotypeⅥandⅦvelogenic Newcastle disease virus from Japan[J].Virus Genes,2014,49(1):89-99.