objective To investigate whether the variation of the pathogenicity of enterovirus71( EV71 ) was related to the variation of the nucleotide sequencing of the virus of the full length genomes of EV71 strains isolated from pediatric patients with hand.foot and mouth disease ( HFMD ) presenting different symptoms in Beijing from 2008 to 2009.Methods Five EV71 strains in 2008 and 4 EV71 strains in 2009 isolated from Laboratory of Virology of Capital Institute of Pediatrics were chosen, of which 4 EV71 strains were isolated from severe HFMD children with high fever, continuous convulsion and loss of consciousness.Five EV71 strains were isolated from mild HFMD children.The full length genomes from 9 EV71 strains isolated from children with various clinical presentations were sequenced by amplification with RT-PCR and sequencing of 10 overlapped gene fragments covering full length of the genomes.Their nucleotide and amino acid sequences were aligned and phylogenetic relations of the sequences were analyzed by using DNAStar and MEGA software ( version 4.1 ).Results The full length of 9 EV71 strains isolated from children in Beijing from 2008 to 2009 was 7 405 bp or 7 406 bp ( Poly A tail not included ) with a deletion in the 5' UTR for 6 out of these 9 strains.The overall nucleotide sequence identities of these 9 EV71 isolates were in the range of 96.3％99.4％ ,while amino acid sequence identities were 98.2％ - 99.6％.The homology of the nucleotide sequences of VPI of these 9 EV71 isolates was 96.9％ - 99.9％ , while the homology of the amino acid sequences was 98.3％ - 100.0％.The majority of nucleotide changes were.located at the third codon positions which caused silent mutations.thus the deduced amino acid sequence changes among those 9 EV71 isolates were scanty.The residue 144 in the VP2 protein and the residues 140 and 263 in the 3D polymerase ( 3Dpol) which were T.R and I were substituted hy S, K and V, respectively in 3 out of 4 neurovirulent strains.Two bases.G and A at the nuc:leotides 208 and 254 in the 5'UTR were substituted respectively by A and G in the same 3 of those 4 neurovirulent strains.The nucleotide sequences of 9 EV71 isolates in the 5'UTR.P1, P2.P3 and 3'UTR regions exhibited the highest homology to the sub-genotype C4 of EV71.However, when compared with CV-A16 prototype strain G10.the nucleotide identities within the 3D and 3'UTR regions were higher than those of known genotypes A, B, C1 - C3 and C5 of EV71.These 9 EV71 isolates formed a hranch with other EV71 isolates circulating in mainland China and Taiwan previously identified as genotype C4 on the two phylogenetic trees.Conclusions The data from this study suggests that mutations at the residue 144 in the VP2 protein ( T→S ), the residues 140 and 263 in the 3Dpol( R→K and Ⅰ→Ⅴ ) and the nucleotide 254 in the 5'UTR ( A→G ) may be associated with the severity of clinical presentations.Based on the entire VPI gene sequences, the EV71 viruses circulating in Beijing from 2008 to 2009 should be classified as the sub-genotype C4.The non-structural protein encoding genomic regions may play a role in the evolution of EV71.%目的 了解2008至2009年从北京地区手足口病(HFMD)患儿分离到的肠道病毒71型(EV71)全基因组序列特点(未包括多聚腺苷尾),以探讨基因序列的改变是否与病毒的致病性有关.方法 选取首都儿科研究所病毒研究室2008年分离到的5株EV71毒株和2009年分离到的4株EV71毒株,其中4株来源于重症HFMD患儿(伴高热、持续抽搐及意识丧失等中枢神经系统症状), 5株来源于轻症HFMD患儿.设计覆盖病毒全基因组的10对特异性引物,对9株EV71毒株进行RT-PCR扩增、全基因组序列测定和分析.结果 9株EV71毒株的全基因组长度为7 406 bp或7 405 bp,部分毒株在5′UTR存在1处1个碱基的缺失.9株EV71毒株的全基因组核苷酸和氨基酸同源性分别为96.3%～99.4%和98.2%～99.6%,在VP1区核苷酸和氨基酸同源性分别为96.9%～99.9%和98.3%～100.0%.重症HFMD来源的4株毒株中有3株在VP2蛋白第144位及3D聚合酶(3Dpol)第140和263位同时出现相同的氨基酸变异(T144S、 R140K和I263V),并且在5′UTR区第208和254位同时出现相同的碱基变异(G208A和A254G).9株EV71毒株的全基因组与C4亚型毒株具有最高的核苷酸同源性,在VP1区为94.3%～95.5%;在3D及3′UTR区与CV-A16/G10的同源性(84.3%～85.0%和89.0%～91.5%)高于与EV71-B型、A型及C型(C1～3、C5)的同源性.VP1和3D基因的遗传进化分析显示,9株EV71毒株与C4亚型毒株属同一分支.结论 VP2蛋白第144位氨基酸突变(T→S)、3Dpol第140和263位氨基酸突变(R→K和I→V)及5′UTR区第254位碱基突变(A→G)可能与EV71感染后引起的不同临床症状有关.根据VP1核苷酸序列,2008至2009年北京地区流行的EV71属于C4亚型;非结构蛋白基因在EV71进化中可能有一定的作用.
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