3株鸭乙型肝炎病毒全基因组克隆与序列分析
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摘要
对河南南阳、安徽亳州、湖北潜江三地鸭血清样本用PCR进行鸭乙型肝炎病毒(DHBV)检测,并从三地经检测为DHBV阳性的样本中各挑选1份进行DHBV全基因的扩增、克隆、测序及序列分析。结果显示,河南南阳、安徽亳州、湖北潜江三地的鸭乙型肝炎自然感染率分别为17.6%、13.6%、16.1%,差异不显著(P>0.05)。河南南阳、安徽亳州、湖北潜江3株DHBV基因组全长分别为3 024、3 027、3 024 bp,均含有编码P、S和C蛋白的3个开放阅读框。通过各开放阅读框氨基酸同源性比较,发现编码P蛋白的区域差异较大。全基因序列分析显示,3株DHBV核苷酸同源性为95.1%～97.4%,与选取的25株参考株同源性为90.3%～96.2%。遗传进化分析及P蛋白关键位点分析结果表明,3株均为类中国基因型,湖北潜江的DHBV毒株P蛋白3位-345位关键氨基酸残基位点与中国毒株基因型相同,在367位-771位关键氨基酸残基位点与西方毒株基因型相同,推测其产生了重组。结果表明,成功克隆了华中地区的3株鸭DHBV全基因序列,为DHBV的分子流行病学调查提供参考。
To investigated the infections and genetic variation of duck hepatitis B virus(DHBV),serum samples of ducks from Nanyang of Henan province,Bozhou of Anhui province and Qianjiang of Hubei province were detected by PCR.One of the positive samples from each province was selected for cloning,sequencing and sequence analysis of the whole genomes of DHBV.The results showed that the positive rates of DHBV were 17.6%,13.6% and 16.1% in Nanyang,Bozhou and Qianjiang,respectively.The total genomes of these DHBV strains were 3 024 bp,3 027 bp,3 024 bp,respectively,and all contained 3 open reading frames(ORF) that encoding P,S and C proteins.The amino acid homology of the ORFs were compared,which was found that the most major differences sited in the coding region of P protein.Sequence analysis showed that nucleotides of these DHBVs were homologous from 95.1% to 97.4%.The homology between the 3 strains and the 25 reference strains was from 90.3% to 96.2%.Genetic evolution analysis and key sites analysis of P protein showed that all 3 strains were Chinese-like genotypes.In this study, three DHBV strains were successfully cloned and sequence-analyzed,which would provide foundation for the molecular epidemiological investigation of the virus.
To investigated the infections and genetic variation of duck hepatitis B virus(DHBV),serum samples of ducks from Nanyang of Henan province,Bozhou of Anhui province and Qianjiang of Hubei province were detected by PCR.One of the positive samples from each province was selected for cloning,sequencing and sequence analysis of the whole genomes of DHBV.The results showed that the positive rates of DHBV were 17.6%,13.6% and 16.1% in Nanyang,Bozhou and Qianjiang,respectively.The total genomes of these DHBV strains were 3 024 bp,3 027 bp,3 024 bp,respectively,and all contained 3 open reading frames(ORF) that encoding P,S and C proteins.The amino acid homology of the ORFs were compared,which was found that the most major differences sited in the coding region of P protein.Sequence analysis showed that nucleotides of these DHBVs were homologous from 95.1% to 97.4%.The homology between the 3 strains and the 25 reference strains was from 90.3% to 96.2%.Genetic evolution analysis and key sites analysis of P protein showed that all 3 strains were Chinese-like genotypes.In this study, three DHBV strains were successfully cloned and sequence-analyzed,which would provide foundation for the molecular epidemiological investigation of the virus.
引文
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[2] Liu Q,Jia R,Wang M,et al.Cloning,expression and purification of duck hepatitis B virus (DHBV) core protein and its use in the development of an indirect ELISA for serologic detection of DHBV infection[J].Arch Virol,2014,159(5):897-904.
[3] Jilbert A,Kotlarski I.Immune responses to duck hepatitis B virus infection[J].Dev Comp Immunol,2000,24(2):285-302.
[4] 苏何玲,黄日东,何松青,等.桂林地区麻鸭DHBV基因全长克隆和序列分析以及DHBV定量检测方法的建立[J].病毒学报,2013,29(2):180-184.
[5] 刘强,贾仁勇.鸭乙型肝炎病毒基因组结构及其编码蛋白的研究进展[J].病毒学报,2012(6):681-688.
[6] Zhou F,Xu H,Chen M,et al.X gene/core promoter deletion mutation:a novel mechanism leading to hepatitis B 'e' antigennegative chronic hepatitis B[J].Mol Med Rep,2014,10(2):799-803.
[7] Datta S,Chatterjee S,Veer V,et al.Molecular biology of the hepatitis B virus for clinicians[J].J Clin Exp Hepatol,2012,2(4):353-365.
[8] Günther S,Li B C,Miska S,et al.A novel method for efficient amplification of whole hepatitis B virus genomes permits rapid functional analysis and reveals deletion mutants in immunosuppressed patients[J].J Virol,1995,69(9):5437-5444.
[9] Tamura K,Peterson D,Peterson N,et al.MEGA5:molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J].Mol Biol Evol,2011,28(10):2731-2739.
[10] Guo W N,Zhu B,Ai L,et al.Animal models for the study of hepatitis B virus infection[J].Zool Res,2018,39(1):25-31.
[11] 沈永恕,刘扬科,陈陆,等.河南省樱桃谷鸭乙型肝炎病毒全基因组克隆与序列分析[J].中国兽医学报,2013,33(9):1394-1398.
[12] Li Q,Jia R,Liu S,et al.Complete genome sequence of the novel duck hepatitis B virus strain SCP01 from Sichuan Cherry Valley duck[J].Springerplus,2016,5(1):1353-1358.
[13] Su H L,Huang R D,He S Q,et al.Cloning and sequence analysis of the DHBV genome of the brown ducks in Guilin region and establishment of the quantitative method for detecting DHBV[J].China J Virol,2013,29(2):180-184.
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