2000-2008中国H_1基因型麻疹野病毒血凝素和血溶素基因特征分析
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摘要
麻疹是由麻疹病毒(Measles virus,MV)引起的急性呼吸道传染病,在中国目前规定为乙类传染病。虽然有效的疫苗在我国广泛使用,但部分地区麻疹发病率仍居高不下,2005年中国所在的世界卫生组织(WHO)西太平洋区所有国家承诺于2012年消除麻疹。为达到这一目标,除了要加强常规免疫,提高二次接种效率,还要对现用疫苗的保护效率做科学的评估,为免疫策略的有效实施提供科学依据。本课题从MV两个重要蛋白质的基因水平分析近年来在中国流行的MV的变异特点,探讨近年来的流行毒株与疫苗株之间的基因差异,评价现用疫苗的保护效率。
目的研究2000—2008年中国流行的H1基因型麻疹野病毒代表株血溶素(Fusion,F)、血凝素(Haemagglutinin,H)基因特征和变异规律。评估中国现用疫苗的保护效率,分析蛋白质重要氨基酸位点的变异情况。方法从2000—2008年中国大陆各省送检的麻疹野病毒中依据N基因C末端基因亲缘性关系树选取差异较大的30株代表株,用RT-PCR方法从代表株中扩增出病毒F、H基因,对上述PCR扩增产物进行核苷酸序列测定和分析,构建基因亲缘性关系树,进行核苷酸(Nucleotide,nt)、氨基酸(Amino acid,aa)同源性分析。结果中国疫苗株与30株所测毒株H基因nt同源性在94.3%～95.5%(83—106nt差异),30株间nt同源性97.1%～99.6%(7-53nt差异),中国疫苗株与30株所测毒株aa同源性在94.8%～95.7%(26—32aa差异),30株间aa同源性96.4%～100%(0-22aa差异)。中国疫苗株与30株所测毒株F基因nt同源性在98.1%～98.6%(23～31nt差异),30株间nt同源性99%～100%(0-16nt差异),中国疫苗株与30株所测毒株F基因aa同源性在96.7%～97.8%(12～18aa差异),30株间aa同源性98.1%～100%(0-10aa差异)。经序列比对分析,H1a基因型中H蛋白第238～240糖基化位点丢失,H1b仍保留这个糖基化位点。F蛋白3个糖基化位点未发生变异,其他重要aa位点未发生变异,H基因368～396、195和200位氨基酸是神经功能区域,无变异。与Edmonston株比,所有毒株的HA蛋白546氨基酸(Gly→Ser)发生改变。结论:2000—2008年间30株所测毒株H、F基因nt/aa差异不明显,但与疫苗株相比仍有一定差异,H基因有随时间推移差异增大趋势,重要aa位点无明显变异(除238～240位糖基化位点缺失,546位aa变异外),F基因相对较保守,重要aa无变异,现用疫苗仍能保护机体免受感染以及病毒仍能感染细胞导致细胞病变效应(CPE),据此推测近年我国流行的MV HA蛋白和F蛋白在功能和结构上无较大变异,从序列比对结果看,两基因无明显变异规律,无突变积累现象,属随机漂移。
Measles is an acute respiratory infectious disease caused by Measle viruses,belongs to "C" class infectious disease in China.Now measles still remains a substantial health risk despite extensive vaccination efforts,and the incidence rate hold the high level in some areas in China.The goal of measles elimination before 2012 has been made in China.Therefore,strengthening the routine immunization, improving second-dose vaccination rate and evaluating vaccine-preverntable efficiency are crucial for the elimination goal.In this study,we focus on measles viruses circulating in China from 2000 to 2008.Variation characristic of the significance protein of measles virus,the gene diversity between epidemic measles virus strains and the vaccine strains,and vaccine-preverntable efficiency were studied.
Objective Study the genetic characterization and variation principle of Fusion(F)、haemagglutinin(HA) protein of H1 genotype of wild-type measles virus circulated in China from 2000 to 2008,estimate vaccine-preverntable efficiency,analysis the variation of key amino acid site of F and HA protein.
Methods 30 isolates of H1 genotype sent by provincial Measles Laboratories measles viruses were selected based on N-COOH phylogenetic tree.RNA from measles isolates were extracted,Then complete coding regions of fusion(1662nt) and hemagglutinin(1 854nt) were amplified by RT-PCR and sequenced.Phylogenetic tree were constructed,and nucleotide and amino acid diversity were further analysised.
Results Among 30 representative strains circulated in China during 2000 to 2008, the homology of nucleotide acid and amino acid of hemagglutinin were 97.1%～99.6% and 96.4%～100%respectively,and the homology were 94.3%～95.5%and 94.8%～95.7%when compared with Chinese vaccine strains;Among 30 representative strains circulated in China during 2000 to 2008,the homology of nucleotide acid and amino acid of fusion were 99%～100%and 98.1%～100%respectively,and were 98.1%～98.6%and 98.1%～100%respectively compared with Chinese vaccine strains; Though amino acid aliment,we found one N-glycosylation site in 240~(th) amino acids disappeared among H_(1a) sub-genotype strains,but not for H_(1b) sub-genotype strains.No any change was found in three N-glycosylation sites in Fusion protein.The 546~(th) amino acid of HA protein in all 30 strains changed from glycine to serine when compared with Edmonston strains.
Conclusion The nucleotide and amino acid variation of fusion and hemagglutinin is unapparent,all of the key amino acid is unvariated except the 240~(th) and 546~(th) amino acids in hemagglutinin.Because the measles vaccine in China can prevent measles infection and the measles virus can cause the classic CPE on sensitivity cell,so we suppose that structure and fuction of Fusion and hemagglutinin is stable.No obvious variation regular and mutation accumulation were found based on sequence alignment results,and we believe these variations belong to random shift.
目的研究2000—2008年中国流行的H1基因型麻疹野病毒代表株血溶素(Fusion,F)、血凝素(Haemagglutinin,H)基因特征和变异规律。评估中国现用疫苗的保护效率,分析蛋白质重要氨基酸位点的变异情况。方法从2000—2008年中国大陆各省送检的麻疹野病毒中依据N基因C末端基因亲缘性关系树选取差异较大的30株代表株,用RT-PCR方法从代表株中扩增出病毒F、H基因,对上述PCR扩增产物进行核苷酸序列测定和分析,构建基因亲缘性关系树,进行核苷酸(Nucleotide,nt)、氨基酸(Amino acid,aa)同源性分析。结果中国疫苗株与30株所测毒株H基因nt同源性在94.3%～95.5%(83—106nt差异),30株间nt同源性97.1%～99.6%(7-53nt差异),中国疫苗株与30株所测毒株aa同源性在94.8%～95.7%(26—32aa差异),30株间aa同源性96.4%～100%(0-22aa差异)。中国疫苗株与30株所测毒株F基因nt同源性在98.1%～98.6%(23～31nt差异),30株间nt同源性99%～100%(0-16nt差异),中国疫苗株与30株所测毒株F基因aa同源性在96.7%～97.8%(12～18aa差异),30株间aa同源性98.1%～100%(0-10aa差异)。经序列比对分析,H1a基因型中H蛋白第238～240糖基化位点丢失,H1b仍保留这个糖基化位点。F蛋白3个糖基化位点未发生变异,其他重要aa位点未发生变异,H基因368～396、195和200位氨基酸是神经功能区域,无变异。与Edmonston株比,所有毒株的HA蛋白546氨基酸(Gly→Ser)发生改变。结论:2000—2008年间30株所测毒株H、F基因nt/aa差异不明显,但与疫苗株相比仍有一定差异,H基因有随时间推移差异增大趋势,重要aa位点无明显变异(除238～240位糖基化位点缺失,546位aa变异外),F基因相对较保守,重要aa无变异,现用疫苗仍能保护机体免受感染以及病毒仍能感染细胞导致细胞病变效应(CPE),据此推测近年我国流行的MV HA蛋白和F蛋白在功能和结构上无较大变异,从序列比对结果看,两基因无明显变异规律,无突变积累现象,属随机漂移。
Measles is an acute respiratory infectious disease caused by Measle viruses,belongs to "C" class infectious disease in China.Now measles still remains a substantial health risk despite extensive vaccination efforts,and the incidence rate hold the high level in some areas in China.The goal of measles elimination before 2012 has been made in China.Therefore,strengthening the routine immunization, improving second-dose vaccination rate and evaluating vaccine-preverntable efficiency are crucial for the elimination goal.In this study,we focus on measles viruses circulating in China from 2000 to 2008.Variation characristic of the significance protein of measles virus,the gene diversity between epidemic measles virus strains and the vaccine strains,and vaccine-preverntable efficiency were studied.
Objective Study the genetic characterization and variation principle of Fusion(F)、haemagglutinin(HA) protein of H1 genotype of wild-type measles virus circulated in China from 2000 to 2008,estimate vaccine-preverntable efficiency,analysis the variation of key amino acid site of F and HA protein.
Methods 30 isolates of H1 genotype sent by provincial Measles Laboratories measles viruses were selected based on N-COOH phylogenetic tree.RNA from measles isolates were extracted,Then complete coding regions of fusion(1662nt) and hemagglutinin(1 854nt) were amplified by RT-PCR and sequenced.Phylogenetic tree were constructed,and nucleotide and amino acid diversity were further analysised.
Results Among 30 representative strains circulated in China during 2000 to 2008, the homology of nucleotide acid and amino acid of hemagglutinin were 97.1%～99.6% and 96.4%～100%respectively,and the homology were 94.3%～95.5%and 94.8%～95.7%when compared with Chinese vaccine strains;Among 30 representative strains circulated in China during 2000 to 2008,the homology of nucleotide acid and amino acid of fusion were 99%～100%and 98.1%～100%respectively,and were 98.1%～98.6%and 98.1%～100%respectively compared with Chinese vaccine strains; Though amino acid aliment,we found one N-glycosylation site in 240~(th) amino acids disappeared among H_(1a) sub-genotype strains,but not for H_(1b) sub-genotype strains.No any change was found in three N-glycosylation sites in Fusion protein.The 546~(th) amino acid of HA protein in all 30 strains changed from glycine to serine when compared with Edmonston strains.
Conclusion The nucleotide and amino acid variation of fusion and hemagglutinin is unapparent,all of the key amino acid is unvariated except the 240~(th) and 546~(th) amino acids in hemagglutinin.Because the measles vaccine in China can prevent measles infection and the measles virus can cause the classic CPE on sensitivity cell,so we suppose that structure and fuction of Fusion and hemagglutinin is stable.No obvious variation regular and mutation accumulation were found based on sequence alignment results,and we believe these variations belong to random shift.
引文
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54Liesegang TJ.Varicella-Zoster Virus eye disease:Cornea 1999;18(5):511-31