北京婴幼儿腹泻中G1、G3型轮状病毒VP7基因分子进化动态分析
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摘要
北京儿童腹泻中G1和G3型轮状病毒(Rotavirus,RV)的流行随时间动态变化。鉴于G1和G3型RV在疫苗研发设计中的重要地位,本研究依据前期对2007～2017北京腹泻就诊患儿中RV流行的监测结果,分别选取G1(15例)和G3(18例)型RV标本,应用DNAStar、MEGA 6.0和BEAST软件包及Datamonkey在线工具,分析和预测11年来其主要中和抗原VP7基因的分子进化动态。结果显示G1和G3型RV同近年世界各地的各自主要流行株之间的进化距离近,相比同一进化树分支内参考株的VP7基因并没有表现出特有的氨基酸变异,提示并未有特别的基因变异直接改变北京G1和G3型RV的流行。本研究中北京G1和G3型RV最近共同祖先株形成时间分别约为2005年和1996年,其VP7基因平均进化速率分别为2.29×10~(-3)和5.11×10~(-4)氨基酸替换·位点~(-1)·年~(-1)。尽管G1型的变异速率大于G3型,但有意思的是它们均无正向选择变异位点。群体流行历史(Bayesian skyline plot,BSP)分析显示人群中G1和G3型RV的感染水平呈现缓慢下降,并均在2010年前后出现了跳跃式下降,随后有所上升并维持在较平稳的感染水平,基本符合北京腹泻儿童中RV流行趋势监测,据此推测群体免疫压力可能是影响G1和G3型RV的流行分布随时间动态变化的原因。分子分析结果还显示本研究中G1和G3型RV和相应型别疫苗株之间的进化距离远,且均在主要抗原区存在变异,这些是否对疫苗应用效果产生影响仍需要对RV的流行进行持续监测。
The distribution of rotavirus G-types in children appears to be changing over time in Beijing,China,especially at the present time. G9 has been re-emergent and absolutely predominant according to data from continuous surveillance,whereas G3 and G1(which used to be common G-types of rotaviruses)have declined.In view of the importance of G1 and G3 rotaviruses for the design and application of vaccines,the mechanisms underlying the epidemic changes of G1 and G3 rotaviruses must be investigated further. We wished to understand the genetic and evolutionary characteristics of the main outer-capsid glycoprotein VP7 that induces neutralizing antibodies. Hence,the VP7 genes of G1(15)and G3(18)from rotavirus-positive stool samples collected during 2007～2017 in Beijing,China were amplified by reverse transcription-polymerase chain reaction(RT-PCR) and sequenced. Multiple-sequence alignments and phylogenetic analyses were conducted using DNAStar and MEGA 6.0,respectively. The time to formulation,the most recent common ancestor(tMRCA)and evolutionary rate as well as the Bayesian skyline plot(BSP;to estimate the epidemic history in the population) were evaluated and constructed. Meanwhile,the positive sites under selective pressure were predicated by Datamonkey ?. We found that the VP7 genes of G1 and G3 rotaviruses were closely related to those of the corresponding major epidemic reference strains around the world in recent years. Particular aminoacid substitutions compared with the reference strains within the same evolutionary tree lineage were not observed,which suggested that drift or shift of individual genes might not have been the direct cause of the epidemic changes of genotypes G1 and G3 in Beijing,China. Evolutionary rates of Beijing G1 and G3 rotaviruses in our study were estimated to be 2.29×10~-3 and 5.11×10~-4 substitutions per site per year,and their tMRCA originated from 2005 and 1996,respectively. Although G1 rotaviruses exhibited a higher rate of accumulation of amino-acid substitutions than that of G3 rotaviruses,neither had positive selection sites. In addition,BSPs showed that infection levels in populations for G1 and G3 genotypes declined slowly with time and dropped abruptly down around 2010,then increased and maintained relatively stable infection levels.Interestingly,the infection changes in the population were in accordance with the epidemic trend of G1 and G3 rotaviruses circulating in children in Beijing,China monitored by our research team. Therefore,we speculated that a herd immune pressure might have affected the epidemic changes of G1 and G3 rotaviruses over time.Notably,the VP7 genes from G1 and G3 rotaviruses in the present study were distant from corresponding vaccine strains contained in currently licensed rotavirus vaccines,and possessed some amino-acid substitutions in major neutralizing antigenic epitope regions by deduced amino-acid alignments. Whether these antigenic changes have an impact on vaccine efficacy in the long term merit close investigation.
The distribution of rotavirus G-types in children appears to be changing over time in Beijing,China,especially at the present time. G9 has been re-emergent and absolutely predominant according to data from continuous surveillance,whereas G3 and G1(which used to be common G-types of rotaviruses)have declined.In view of the importance of G1 and G3 rotaviruses for the design and application of vaccines,the mechanisms underlying the epidemic changes of G1 and G3 rotaviruses must be investigated further. We wished to understand the genetic and evolutionary characteristics of the main outer-capsid glycoprotein VP7 that induces neutralizing antibodies. Hence,the VP7 genes of G1(15)and G3(18)from rotavirus-positive stool samples collected during 2007～2017 in Beijing,China were amplified by reverse transcription-polymerase chain reaction(RT-PCR) and sequenced. Multiple-sequence alignments and phylogenetic analyses were conducted using DNAStar and MEGA 6.0,respectively. The time to formulation,the most recent common ancestor(tMRCA)and evolutionary rate as well as the Bayesian skyline plot(BSP;to estimate the epidemic history in the population) were evaluated and constructed. Meanwhile,the positive sites under selective pressure were predicated by Datamonkey ?. We found that the VP7 genes of G1 and G3 rotaviruses were closely related to those of the corresponding major epidemic reference strains around the world in recent years. Particular aminoacid substitutions compared with the reference strains within the same evolutionary tree lineage were not observed,which suggested that drift or shift of individual genes might not have been the direct cause of the epidemic changes of genotypes G1 and G3 in Beijing,China. Evolutionary rates of Beijing G1 and G3 rotaviruses in our study were estimated to be 2.29×10~-3 and 5.11×10~-4 substitutions per site per year,and their tMRCA originated from 2005 and 1996,respectively. Although G1 rotaviruses exhibited a higher rate of accumulation of amino-acid substitutions than that of G3 rotaviruses,neither had positive selection sites. In addition,BSPs showed that infection levels in populations for G1 and G3 genotypes declined slowly with time and dropped abruptly down around 2010,then increased and maintained relatively stable infection levels.Interestingly,the infection changes in the population were in accordance with the epidemic trend of G1 and G3 rotaviruses circulating in children in Beijing,China monitored by our research team. Therefore,we speculated that a herd immune pressure might have affected the epidemic changes of G1 and G3 rotaviruses over time.Notably,the VP7 genes from G1 and G3 rotaviruses in the present study were distant from corresponding vaccine strains contained in currently licensed rotavirus vaccines,and possessed some amino-acid substitutions in major neutralizing antigenic epitope regions by deduced amino-acid alignments. Whether these antigenic changes have an impact on vaccine efficacy in the long term merit close investigation.
引文
[1]GBD Diarrhoeal Diseases Collaborators.Estimates of global,regional,and national morbidity,mortality,and aetiologies of diarrhoeal diseases:a systematic analysis for the Global Burden of Disease Study 2015[J].Lancet Infect Dis,2017,17(9):909-948.
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[5]Liu N,Xu Z,Li D,Zhang Q,Wang H,Duan Z J.Update on the disease burden and circulating strains of rotavirus in China:a systematic review and metaanalysis[J].Vaccine,2014,32(35):4369-4375.
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[7]董慧瑾,孙宇,赵林清,邓莉,朱汝南,陈冬梅,贾立平,刘立颖,张又,钱渊.北京腹泻儿童中G9-VI轮状病毒再次出现并呈优势流行[J].病毒学报,2016,32(4):436-444.DOI:10.13242/j.cnki.bingduxuebao.002983
[8]Xu C,Fu J,Ai J,Zhang J,Liu C,Huo X,Bao C,Zhu Y.Phylogenetic analysis of human G9P[8]rotavirus strains circulating in Jiangsu,China between2010 and 2016[J].J Med Virol,2018,90(9):1461-1470.
[9]Glass R I,Keith J,Nakagomi O,Nakagomi T,Askaa J,Kapikian A Z,Chanock R M,Flores J.Nucleotide sequence of the structural glycoprotein VP7 gene of Nebraska calf diarrhea virus rotavirus:comparison with homologous genes from four strains of human and animal rotaviruses[J].Virology,1985,141,292-298.
[10]Green K Y,Midthun K,Gorziglia M,Hoshino Y,Kapikian A Z,Chanock R M,Flores J.Cornparison of the amino acid sequences of the major neutralization protein of four human rotavirus serotypes[J].Virology,1987,161,153-159.
[11]董慧瑾,钱渊,张又,邓莉,赵林清,朱汝南,陈冬梅,刘立颖,贾立平.2009~2010年北京儿童腹泻中轮状病毒新VP4基因亚型的研究[J].病毒学报,2011,27(6):565-570.DOI:10.13242/j.cnki.bingduxuebao.002223
[12]Gouvea V,Glass R I,Woods P,Taniguchi K,Clark HF,Forrester B,Fang Z Y.Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens[J].J Clin Microbiol,1990,28(2):276-282.
[13]Suchard M A,Lemey P,Baele G,Ayres D L,Drummond A J,Rambaut A.Bayesian phylogenetic and phylodynamic data integration using BEAST 1.10[J].Virus Evol,2018,4(1):vey016.
[14]Rambaut A,Drummond A J,Xie D,Baele G,Suchard M A.Posterior Summarization in Bayesian Phylogenetics Using Tracer 1.7[J].Syst Biol,2018,67(5):901-904.
[15]李国华,钱渊,熊朝晖,靖宇.北京地区G1-G4型人轮状病毒地方株VP7编码基因的序列分析[J].病毒学报,1998,14(2):126-132.DOI:10.13242/j.cnki.bingduxuebao.001094
[16]Martella V,Ba′nyai K,Matthijnssens J,Buonavoglia C,Ciarlet M.Zoonotic aspects of rotaviruses[J].Vet Microbiol,2010,140(3-4):246-255.
[17]董慧瑾,钱渊,农艺,张又,莫兆军,李荣成.一株与儿童腹泻相关的跨多种属的基因重配G3P[3]型轮状病毒[J].病毒学报,2014,32(2):129-140.DOI:10.13242/j.cnki.bingduxuebao.002897
[18]Tian Y,Chughtai A A,Gao Z,Yan H,Chen Y,Liu B,Huo D,Jia L,Wang Q,MacIntyre C R.Prevalence and genotypes of group A rotavirus among outpatient children under five years old with diarrhea in Beijing,China,2011-2016[J].BMC Infect Dis,2018,18(1):497.
[19]Zhang S,Yin J,Yang J,Tian L,Li D,Zhang Q,Chen J,Xu W,Zhou X.Epidemiology and genetic diversity of group A rotavirus in acute diarrhea patients in pre-vaccination era in southwest China[J].J Med Virol,2017,89(1):71-78.
[2]Estes M K,Greenberg H B(2013)Rotaviruses.In:Knipe D M,Howley P M(eds)Fields virology,6th edn[M].Lippincott Williams and Wilkins,Philadelphia,pp 1347-1401.
[3]Riddle M S,Chen W H,Kirkwood C D,MacLennan CA.Update on vaccines for enteric pathogens[J].Clin Microbiol Infect,2018,24(10):1039-1045.
[4]Wu D,Yen C,Yin Z D,Li Y X,Liu N,Liu Y M,Wang H Q,Cui F Q,Gregory C J,Tate J E,Parashar U D,Yin D P,Li L.The public health burden of rotavirus disease in children younger than five years and considerations for rotavirus vaccine introduction in China[J/OL].Pediatr Infect Dis J,2016,35(12):e392-e398.
[5]Liu N,Xu Z,Li D,Zhang Q,Wang H,Duan Z J.Update on the disease burden and circulating strains of rotavirus in China:a systematic review and metaanalysis[J].Vaccine,2014,32(35):4369-4375.
[6]Li Y,Wang S M,Zhen S S,Chen Y,Deng W,Kilgore P E,Wang X Y.Diversity of rotavirus strains causing diarrhea in<5 years old Chinese children:a systematic review[J/OL].PLoS One,2014,9(1):e84699.
[7]董慧瑾,孙宇,赵林清,邓莉,朱汝南,陈冬梅,贾立平,刘立颖,张又,钱渊.北京腹泻儿童中G9-VI轮状病毒再次出现并呈优势流行[J].病毒学报,2016,32(4):436-444.DOI:10.13242/j.cnki.bingduxuebao.002983
[8]Xu C,Fu J,Ai J,Zhang J,Liu C,Huo X,Bao C,Zhu Y.Phylogenetic analysis of human G9P[8]rotavirus strains circulating in Jiangsu,China between2010 and 2016[J].J Med Virol,2018,90(9):1461-1470.
[9]Glass R I,Keith J,Nakagomi O,Nakagomi T,Askaa J,Kapikian A Z,Chanock R M,Flores J.Nucleotide sequence of the structural glycoprotein VP7 gene of Nebraska calf diarrhea virus rotavirus:comparison with homologous genes from four strains of human and animal rotaviruses[J].Virology,1985,141,292-298.
[10]Green K Y,Midthun K,Gorziglia M,Hoshino Y,Kapikian A Z,Chanock R M,Flores J.Cornparison of the amino acid sequences of the major neutralization protein of four human rotavirus serotypes[J].Virology,1987,161,153-159.
[11]董慧瑾,钱渊,张又,邓莉,赵林清,朱汝南,陈冬梅,刘立颖,贾立平.2009~2010年北京儿童腹泻中轮状病毒新VP4基因亚型的研究[J].病毒学报,2011,27(6):565-570.DOI:10.13242/j.cnki.bingduxuebao.002223
[12]Gouvea V,Glass R I,Woods P,Taniguchi K,Clark HF,Forrester B,Fang Z Y.Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens[J].J Clin Microbiol,1990,28(2):276-282.
[13]Suchard M A,Lemey P,Baele G,Ayres D L,Drummond A J,Rambaut A.Bayesian phylogenetic and phylodynamic data integration using BEAST 1.10[J].Virus Evol,2018,4(1):vey016.
[14]Rambaut A,Drummond A J,Xie D,Baele G,Suchard M A.Posterior Summarization in Bayesian Phylogenetics Using Tracer 1.7[J].Syst Biol,2018,67(5):901-904.
[15]李国华,钱渊,熊朝晖,靖宇.北京地区G1-G4型人轮状病毒地方株VP7编码基因的序列分析[J].病毒学报,1998,14(2):126-132.DOI:10.13242/j.cnki.bingduxuebao.001094
[16]Martella V,Ba′nyai K,Matthijnssens J,Buonavoglia C,Ciarlet M.Zoonotic aspects of rotaviruses[J].Vet Microbiol,2010,140(3-4):246-255.
[17]董慧瑾,钱渊,农艺,张又,莫兆军,李荣成.一株与儿童腹泻相关的跨多种属的基因重配G3P[3]型轮状病毒[J].病毒学报,2014,32(2):129-140.DOI:10.13242/j.cnki.bingduxuebao.002897
[18]Tian Y,Chughtai A A,Gao Z,Yan H,Chen Y,Liu B,Huo D,Jia L,Wang Q,MacIntyre C R.Prevalence and genotypes of group A rotavirus among outpatient children under five years old with diarrhea in Beijing,China,2011-2016[J].BMC Infect Dis,2018,18(1):497.
[19]Zhang S,Yin J,Yang J,Tian L,Li D,Zhang Q,Chen J,Xu W,Zhou X.Epidemiology and genetic diversity of group A rotavirus in acute diarrhea patients in pre-vaccination era in southwest China[J].J Med Virol,2017,89(1):71-78.