全基因组分析揭示A组肠道病毒型间重组的复杂模式
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摘要
A组肠道病毒(Enterovirus A,EV-A)是手足口病的主要病原体。基因组流行病学将基因组技术与生物信息学和流行病学有机整合,可快速对致病病原体进行鉴定和溯源。基因重组是肠道病毒进化的驱动力之一。本研究以EV-A的基因组为研究对象,对其进行重组特点分析,为基于EV-A基因组的手足口病病原体溯源技术体系提供基础资料。本研究以GenBank中全部1 180条EV-A的全长基因组序列为研究对象,为降低序列的冗余性和减少计算强度,对序列进行整理,剔除核苷酸相似性>98%的序列,构建包含346条EV-A代表性全基因组序列的数据库,对EV-A不同血清型全基因组的重组形式和重组热点进行了研究。利用T-RECs软件,对上述346条EV-A全基因组序列进行基因重组分析,并应用Simplot软件进行验证。结果显示,EV-A衣壳编码区未发生基因重组,而在整个P2区和P3区均发生了高频率的重组事件,尤其P2区的2A区是重组热点。尽管EV-A中有21个血清型,但只有少数血清型作为频繁的重组供体发挥着核心作用,其中肠道病毒A组71型和柯萨奇病毒A组6型是EV-A的最主要的重组供体。本研究凸显了全基因组序列分析在EV-A溯源中的重要作用,随着基因组学技术、生物信息学以及大数据管理技术的迅速发展,可对海量全基因组序列数据进行加工整理,准确进行病毒溯源,洞悉病原体进化过程,发现其毒力和致病因子、耐药基因,从而为病毒病"精准防控"提供理论依据。
Enterovirus A(EV-A)is the main pathogen of hand,foot and mouth disease(HFMD). Genomic epidemiology integrates genome technology with bioinformatics and epidemiology,which can quickly identify and trace pathogens. Recombination is one of the driving forces of virus evolution. In the present study,the genome of EV-A was taken as the research object,and the recombination characteristics of EV-A genome were analyzed to provide basic data for trace ability technology system of pathogens of HFMD based on EV-A genomes. 1,180 full-length genomes of EV-A in GenBank were selected as the research objects. To reduce redundancy and the computational intensity,the genome was cleaned up. Genomic sequences with nucleotide similarity > 98% were deleted. Finally,a database of 346 representative full-length genome sequences of EV-A was obtained. The intertypic recombination forms and recombination hotspots between the serotypes of EV-A were studied. The genome recombination events of 346 EV-A genome sequences were analyzed by using T-RECs software,and the results were verified by Simplot software. We found no recombination in the coding region of the EV-A capsid region,but high-frequency recombination events occurred in the P2 and the P3 coding regions. In particular,the 2A region of the P2 coding region was a recombinaiton hotspot. Although there are 21 serotypes in EV-A,only a few serotypes have central roles as frequent recombinant donors.Enterovirus A71 and coxsackievirus A6 are the main recombinant donors of EV-A. Our study highlighted the important role of analyses of the full-length genome sequences in EV-A. With rapid development of genomics technology,bioinformatics,and technology to manage large amount of data,many full-length genome sequences can be processed. This strategy can be used to trace the origin of viruses accurately,understand pathogen evolution,and discover virulence,pathogenic factors,and drug-resistant genes. In this way,a theoretical basis for precise control of viral diseases can be formulated.
Enterovirus A(EV-A)is the main pathogen of hand,foot and mouth disease(HFMD). Genomic epidemiology integrates genome technology with bioinformatics and epidemiology,which can quickly identify and trace pathogens. Recombination is one of the driving forces of virus evolution. In the present study,the genome of EV-A was taken as the research object,and the recombination characteristics of EV-A genome were analyzed to provide basic data for trace ability technology system of pathogens of HFMD based on EV-A genomes. 1,180 full-length genomes of EV-A in GenBank were selected as the research objects. To reduce redundancy and the computational intensity,the genome was cleaned up. Genomic sequences with nucleotide similarity > 98% were deleted. Finally,a database of 346 representative full-length genome sequences of EV-A was obtained. The intertypic recombination forms and recombination hotspots between the serotypes of EV-A were studied. The genome recombination events of 346 EV-A genome sequences were analyzed by using T-RECs software,and the results were verified by Simplot software. We found no recombination in the coding region of the EV-A capsid region,but high-frequency recombination events occurred in the P2 and the P3 coding regions. In particular,the 2A region of the P2 coding region was a recombinaiton hotspot. Although there are 21 serotypes in EV-A,only a few serotypes have central roles as frequent recombinant donors.Enterovirus A71 and coxsackievirus A6 are the main recombinant donors of EV-A. Our study highlighted the important role of analyses of the full-length genome sequences in EV-A. With rapid development of genomics technology,bioinformatics,and technology to manage large amount of data,many full-length genome sequences can be processed. This strategy can be used to trace the origin of viruses accurately,understand pathogen evolution,and discover virulence,pathogenic factors,and drug-resistant genes. In this way,a theoretical basis for precise control of viral diseases can be formulated.
引文
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[2]Zhang Y,Tan X J,Wang H Y,Yan D M,Zhu S L,Wang D Y,Ji F,Wang X J,Gao Y J,Chen L,An HQ,Li D X,Wang S W,Xu A Q,Wang Z J,Xu W B.An outbreak of hand,foot,and mouth disease associated with subgenotype C4 of human enterovirus 71 in Shan-dong,China[J].J Clin Virol,2009,44(4):262-267.
[3]Zhang Y,Xu W B.Molecular epidemiology of enterovi-ruses associated with hand,foot,and mouth disease in the mainland of China[J].Biomed Environ Sci,2013;26(11):875-876.
[4]Zhang Y,Wang,D Y,Yan D M,Zhu S L,Liu J F,Wang H Y,Zhao S C,Yu D S,Nan L J,An J J,Chen L,An H Q,Xu A Q,Xu W B.Molecular evidence of persistent epidemic and evolution of subgenotype B1 cox-sackievirus A16-associated hand,foot,and mouth dis-ease in China[J].J Clin Microbiol,2010,48(2):619-622.
[5]Song Y,Zhang Y,Ji T J,Gu X R,Yang Q,Zhu S L,Xu W,Xu Y,Shi Y,Huang X Y,Li Q,Deng H,Wang X J,Yan D M,Yu W,Wang S,Yu D S,Xu WB.Persistent circulation of coxsackievirus A6 of geno-type D3 in mainland of China between 2008 and 2015[J].Sci Rep,2017,7(1):5491.
[6]Muslin C,Joffret M L,Pelletier I,Blondel B,Delpey-roux F.Evolution and emergence of enteroviruses through intra-and inter-species recombination:plasticity and phenotypic impact of modular genetic exchanges in the 5′untranslated region[J/OL].PLoS Pathog,2015,11(11):e1005266.
[7]Kyriakopoulou Z,Pliaka V,Amoutzias G D,Markoula-tos P.Recombination among human non-polio enterovi-ruses:implications for epidemiology and evolution[J].Virus Genes,2015,50(2):177-188.
[8]Lukashev A N.Role of recombination in evolution of en-teroviruses[J].Rev Med Virol,2005,15(3):157-167.
[9]Woodman A,Arnold J J,Cameron C E,Evans D J.Biochemical and genetic analysis of the role of the viral polymerase in enterovirus recombination[J].Nucleic Ac-ids Res,2016,44(14):6883-6895.
[10]Lukashev A N,Shumilina E Y,Belalov I S,Ivanova OE,Eremeeva T P,Reznik V I,Trotsenko O E,Drex-ler J F,Drosten C.Recombination strategies and evolu-tionary dynamics of the human enterovirus A global gene pool[J].J Gen Virol,2014,95(Pt 4):868-873.
[11]Simmonds P,Welch J.Frequency and dynamics of re-combination within different species of human enterovi-ruses[J].J Virol,2006,80(1):483-493.
[12]Kew O M,Wright P F,Agol V I,Delpeyroux F,Shi-mizu H,Nathanson N,Pallansch M A.Circulating vac-cine-derived polioviruses:current state of knowledge[J].Bull World Health Organ,2004,82(1):16-23.
[13]Kew O,Morris-Glasgow V,Landaverde M,Burns C,Shaw J,Garib Z,Andre J,Blackman E,Freeman C J,Jorba J,Sutter R,Tambini G,Venczel L,Pedreira C,Laender F,Shimizu H,Yoneyama T,Miyamura T,van Der Avoort H,Oberste M S,Kilpatrick D,Cochi S,Pallansch M,de Quadros C.Outbreak of poliomyeli-tis in Hispaniola associated with circulating type 1 vaccine-derived poliovirus[J].Science,2002,296(5566):356-359.
[14]Edgar R C.Search and clustering orders of magnitude faster than BLAST[J].Bioinformatics,2010,26(19):2460-2461.
[15]Tsimpidis M,Bachoumis G,Mimouli K,Kyriakopou-lou Z,Robertson D L,Markoulatos P,Amoutzias G D.T-RECs:rapid and large-scale detection of recombina-tion events among different evolutionary lineages of viral genomes[J].BMC Bioinformatics,2017,18(1):13.
[16]Fan Q,Zhang Y,Hu L,Sun Q,Cui H,Yan D M,Si-kandaner H,Tang H S,Wang D Y,Zhu Z,Zhu S L,Xu W B.A novel recombinant enterovirus type EV-A89with low epidemic strength in Xinjiang,China[J].Sci Rep,2015,5:18558.
[17]Huang K Q,Zhang Y,Song Y,Cui H,Yan D M,Zhu S L,Sun Q,Tang H S,Wang D Y,Xu W B.Antigen-ic characteristics and genomic analysis of novel EV-A90enteroviruses isolated in Xinjiang,China[J].Sci Rep,2018,8(1):10247.
[18]Oberste,M S,Penaranda S,Pallansch M A.RNA re-combination plays a major role in genomic change during circulation of coxsackie B viruses[J].J Virol,2004,78(6):2948-2955.
[19]McWilliam Leitch E C,Cabrerizo M,Cardosa J,Harva-la H,Ivanova O E,Koike S,Kroes A C,Lukashev A,Perera D,Roivainen M,Susi P,Trallero G,Evans DJ,Simmonds P.The association of recombination events in the founding and emergence of subgenogroup evolu-tionary lineages of human enterovirus 71[J].J Virol,2012,86(5):2676-2685.
[20]Wang J,Ke YH,Zhang Y,Huang K Q,Wang L,Shen X X,Dong X P,Xu W B,Ma X J.Rapid and ac-curate sequencing of enterovirus genomes using MinIONnanopore sequencer[J].Biomed Environ Sci,2017,30(10):718-726.
[21]Beato M S,Marcacci M,Schiavon E,Bertocchi L,Di Domenico M,Peserico A,Mion M,Zaccaria G,Cavic-chio L,Mangone I,Soranzo E,Patavino C,Camma C,Lorusso A.Identification and genetic characterization of bovine enterovirus by combination of two next genera-tion sequencing platforms[J].J Virol Methods,2018,260:21-25.
[22]Zhao K,Han X,Wang G J,Hu W,Zhang W Y,Yu XF.Circulating coxsackievirus A16 identified as recombi-nant type A human enterovirus,China[J].Emerg Infect Dis,2011,17(8):1537-1540.
[23]Noisumdaeng P,Sangsiriwut K,Prasertsopon J,Klinm-alai C,Payungporn S,Mungaomklang A,Chokephaib-ulkit K,Buathong R,Thitithanyanont A,Puthavathana P.Complete genome analysis demonstrates multiple in-troductions of enterovirus 71 and coxsackievirus A16 re-combinant strains into Thailand during the past decade[J].Emerg Microbes Infect,2018,7:214.
[24]Song Y,Zhang Y,Fan Q,Cui H,Yan D M,Zhu S L,Tang H S,Sun Q,Wang D Y,Xu W B.Phylogenetic characterizations of highly mutated EV-B106 recombi-nants showing extensive genetic exchanges with other EV-B in Xinjiang,China[J].Sci Rep,2017,7:43080.
[2]Zhang Y,Tan X J,Wang H Y,Yan D M,Zhu S L,Wang D Y,Ji F,Wang X J,Gao Y J,Chen L,An HQ,Li D X,Wang S W,Xu A Q,Wang Z J,Xu W B.An outbreak of hand,foot,and mouth disease associated with subgenotype C4 of human enterovirus 71 in Shan-dong,China[J].J Clin Virol,2009,44(4):262-267.
[3]Zhang Y,Xu W B.Molecular epidemiology of enterovi-ruses associated with hand,foot,and mouth disease in the mainland of China[J].Biomed Environ Sci,2013;26(11):875-876.
[4]Zhang Y,Wang,D Y,Yan D M,Zhu S L,Liu J F,Wang H Y,Zhao S C,Yu D S,Nan L J,An J J,Chen L,An H Q,Xu A Q,Xu W B.Molecular evidence of persistent epidemic and evolution of subgenotype B1 cox-sackievirus A16-associated hand,foot,and mouth dis-ease in China[J].J Clin Microbiol,2010,48(2):619-622.
[5]Song Y,Zhang Y,Ji T J,Gu X R,Yang Q,Zhu S L,Xu W,Xu Y,Shi Y,Huang X Y,Li Q,Deng H,Wang X J,Yan D M,Yu W,Wang S,Yu D S,Xu WB.Persistent circulation of coxsackievirus A6 of geno-type D3 in mainland of China between 2008 and 2015[J].Sci Rep,2017,7(1):5491.
[6]Muslin C,Joffret M L,Pelletier I,Blondel B,Delpey-roux F.Evolution and emergence of enteroviruses through intra-and inter-species recombination:plasticity and phenotypic impact of modular genetic exchanges in the 5′untranslated region[J/OL].PLoS Pathog,2015,11(11):e1005266.
[7]Kyriakopoulou Z,Pliaka V,Amoutzias G D,Markoula-tos P.Recombination among human non-polio enterovi-ruses:implications for epidemiology and evolution[J].Virus Genes,2015,50(2):177-188.
[8]Lukashev A N.Role of recombination in evolution of en-teroviruses[J].Rev Med Virol,2005,15(3):157-167.
[9]Woodman A,Arnold J J,Cameron C E,Evans D J.Biochemical and genetic analysis of the role of the viral polymerase in enterovirus recombination[J].Nucleic Ac-ids Res,2016,44(14):6883-6895.
[10]Lukashev A N,Shumilina E Y,Belalov I S,Ivanova OE,Eremeeva T P,Reznik V I,Trotsenko O E,Drex-ler J F,Drosten C.Recombination strategies and evolu-tionary dynamics of the human enterovirus A global gene pool[J].J Gen Virol,2014,95(Pt 4):868-873.
[11]Simmonds P,Welch J.Frequency and dynamics of re-combination within different species of human enterovi-ruses[J].J Virol,2006,80(1):483-493.
[12]Kew O M,Wright P F,Agol V I,Delpeyroux F,Shi-mizu H,Nathanson N,Pallansch M A.Circulating vac-cine-derived polioviruses:current state of knowledge[J].Bull World Health Organ,2004,82(1):16-23.
[13]Kew O,Morris-Glasgow V,Landaverde M,Burns C,Shaw J,Garib Z,Andre J,Blackman E,Freeman C J,Jorba J,Sutter R,Tambini G,Venczel L,Pedreira C,Laender F,Shimizu H,Yoneyama T,Miyamura T,van Der Avoort H,Oberste M S,Kilpatrick D,Cochi S,Pallansch M,de Quadros C.Outbreak of poliomyeli-tis in Hispaniola associated with circulating type 1 vaccine-derived poliovirus[J].Science,2002,296(5566):356-359.
[14]Edgar R C.Search and clustering orders of magnitude faster than BLAST[J].Bioinformatics,2010,26(19):2460-2461.
[15]Tsimpidis M,Bachoumis G,Mimouli K,Kyriakopou-lou Z,Robertson D L,Markoulatos P,Amoutzias G D.T-RECs:rapid and large-scale detection of recombina-tion events among different evolutionary lineages of viral genomes[J].BMC Bioinformatics,2017,18(1):13.
[16]Fan Q,Zhang Y,Hu L,Sun Q,Cui H,Yan D M,Si-kandaner H,Tang H S,Wang D Y,Zhu Z,Zhu S L,Xu W B.A novel recombinant enterovirus type EV-A89with low epidemic strength in Xinjiang,China[J].Sci Rep,2015,5:18558.
[17]Huang K Q,Zhang Y,Song Y,Cui H,Yan D M,Zhu S L,Sun Q,Tang H S,Wang D Y,Xu W B.Antigen-ic characteristics and genomic analysis of novel EV-A90enteroviruses isolated in Xinjiang,China[J].Sci Rep,2018,8(1):10247.
[18]Oberste,M S,Penaranda S,Pallansch M A.RNA re-combination plays a major role in genomic change during circulation of coxsackie B viruses[J].J Virol,2004,78(6):2948-2955.
[19]McWilliam Leitch E C,Cabrerizo M,Cardosa J,Harva-la H,Ivanova O E,Koike S,Kroes A C,Lukashev A,Perera D,Roivainen M,Susi P,Trallero G,Evans DJ,Simmonds P.The association of recombination events in the founding and emergence of subgenogroup evolu-tionary lineages of human enterovirus 71[J].J Virol,2012,86(5):2676-2685.
[20]Wang J,Ke YH,Zhang Y,Huang K Q,Wang L,Shen X X,Dong X P,Xu W B,Ma X J.Rapid and ac-curate sequencing of enterovirus genomes using MinIONnanopore sequencer[J].Biomed Environ Sci,2017,30(10):718-726.
[21]Beato M S,Marcacci M,Schiavon E,Bertocchi L,Di Domenico M,Peserico A,Mion M,Zaccaria G,Cavic-chio L,Mangone I,Soranzo E,Patavino C,Camma C,Lorusso A.Identification and genetic characterization of bovine enterovirus by combination of two next genera-tion sequencing platforms[J].J Virol Methods,2018,260:21-25.
[22]Zhao K,Han X,Wang G J,Hu W,Zhang W Y,Yu XF.Circulating coxsackievirus A16 identified as recombi-nant type A human enterovirus,China[J].Emerg Infect Dis,2011,17(8):1537-1540.
[23]Noisumdaeng P,Sangsiriwut K,Prasertsopon J,Klinm-alai C,Payungporn S,Mungaomklang A,Chokephaib-ulkit K,Buathong R,Thitithanyanont A,Puthavathana P.Complete genome analysis demonstrates multiple in-troductions of enterovirus 71 and coxsackievirus A16 re-combinant strains into Thailand during the past decade[J].Emerg Microbes Infect,2018,7:214.
[24]Song Y,Zhang Y,Fan Q,Cui H,Yan D M,Zhu S L,Tang H S,Sun Q,Wang D Y,Xu W B.Phylogenetic characterizations of highly mutated EV-B106 recombi-nants showing extensive genetic exchanges with other EV-B in Xinjiang,China[J].Sci Rep,2017,7:43080.