肠道病毒71型武汉分离株(EV71-WH029)全基因组cDNA克隆及序列分析

Constructing a full-length genomic cDNA clone and sequencing of a Wuhan isolate of enterovirus 71(EV71-WH029)

目的对肠道病毒71型武汉分离株（EV71-WH029）进行全基因组克隆、序列测定及分析,了解其基因组结构和遗传进化特征,并初步探索EV71毒力相关位点。方法采用RT-PCR扩增覆盖基因组全长的cDNA片段,利用Over-lap extension PCR得到WH029全基因组cDNA克隆;cDNA片段测序后利用BioEdit 4.8.8和MEGA 5.2.2等软件进行序列分析和遗传进化分析;采用Fisher＇s exact test进行编码区氨基酸变异位点的统计分析;采用RNAfold服务器进行非编码区二级结构分析。结果 WH029分离株基因组全长7 406nt（未包含多聚腺苷酸尾）,5′-和3′-UTR分别为743nt和81nt,中间为一个全长6 582nt开放阅读框（ORF）,编码一个长度为2 194aa的多聚蛋白,编码区无核苷酸的缺失或插入;该毒株与安徽阜阳株EU703813及上海株JQ736684的核苷酸及氨基酸序列同源性均最高（分别为97.53%-100%和97.41%-99.73%）,系统进化分析也表明该毒株与以上两株的亲缘关系最近;编码区氨基酸序列分析发现有7个位点变异。非编码区的二级结构预测提示,该区域的核苷酸位点变异与毒力无直接相关性,而与地域有相关性。结论 WH029分离株全基因组结构符合EV71病毒特征,经序列比对及进化分析证实WH029分离株属于EV71C4a基因亚型,并推断该株可能来源于2008年安徽HFMD疫情。对不同临床症状的EV71毒株序列的比较分析提示,EV71毒力与编码区单一位点的突变及非编码区二级结构的变异无直接相关性,可能与多位点突变共同作用及患者的个体差异相关。

Objective To construct a full-length genomic cDNA clone and sequence a Wuhan isolate（EV71-WH029）of human enterovirus 71（EV71）in order to characterize the genomic structure of WH029 and obtain phylogenetic information on that isolate so as to further examine sites related to the virulence of EV71. Methods cDNA fragments spanning the entire genome of WH029 were amplified with RT-PCR using viral RNA as a template,and full-length genomic cDNA was obtained using multiple rounds of overlap extension PCR.Genome sequence analyses and phylogenetic analyses of the entire genomic sequence,coding sequences,and UTR regions were performed using the software BioEdit 4.8.8and MEGA5.2.2.Amino acid changes in coding regions were tallied and analyzed statistically using Fisher＇s exact test.The secondary structures of non-coding regions were predicted using the RNAfold Web server. Results The entire genome of the EV71-WH029 strain was 7 406nucleotides（nts）in length（excluding apoly（A）tail）.The 5＇-untranslated region（5′-UTR）was 743 nt in length and the 3′-UTR was 81 nt in length.The entire open reading frame（ORF）contained 6,582 nt,encoding apolyprotein of 2 194 amino acids（aa）.The ORF did not include any deletion or insertion of nt.The sequence of the entire genome of WH029 had characteristics like the sequences of typical EV71 strains.Sequencing of individual regions（P1,P2,P3,5′-UTR,and 3′-UTR）in different EV71 subtypes indicated that the WH029 strain was most similar to the Fuyang strain EU703813 and the Shanghai strain JQ736684（（97.53%-100% similarity to EU703813 and 97.41%-99.73%similarity to JQ736684）.Phylogenetic analyses also suggested that WH029 strain was most closely related to these two EV71 strains.Sequencing of the coding regions in different EV71 subtypes revealed seven aa mutations.Analysis of the secondary structures of non-coding regions did not reveal a direct relationship between structure and virulence.However,EV71 strains isolated fromthe same region have very similar sequences and secondary structures. Conclusion The genomic structure of WH029 strain is consistent with that of a typical EV71 strain.Phylogenetic analyses suggested that the WH029 strain belongs to the C4asub-genotype of EV71.The WH029 strain might have originated from an outbreak of hand,foot,and mouth disease associated with EV71 that occurred in Anhui Province in 2008.Sequencing of EV71 strains from patients with different clinical symptoms suggested that point mutations in coding regions and changes in the secondary structure of non-coding regions are not the primary determinants of virulence.Different clinical symptoms observed in patients infected with EV71 strains may be the result of numerous factors,such as multiple mutations or individual differences in patients.