潍坊市9例重症与轻症手足口病患者肠道病毒71型5′非编码区、VP1基因特征分析

目的 探讨重症手足口病肠道病毒71型(enterovirus 71,EV71)5′非编码区(untranslated region, UTR)、VP1区是否存在潜在的毒力位点。 方法 分离培养不同临床症状手足口病患者粪便标本中EV71,提取病毒总RNA,逆转录PCR扩增5′-UTR、VP1区后测定基因序列,对两个区域核苷酸序列及VP1区编码氨基酸序列进行比对和同源性分析,构建VP1区及不同临床症状EV71病毒5′- UTR基因进化树。 结果 本实验共获得9株EV71毒株(5株来源于重症病例,4株来源于轻症),5′-UTR、VP1区核苷酸同源性均为94.5%~ 99.7%,VP1区氨基酸同源性为97.3%~99.9%。9株EV71毒株5′-UTR共有67个核苷酸突变位点,与4株轻症EV71毒株相比,5株重症EV71毒株VP1区编码氨基酸有2处发生替换(S283T、A289T),5′-UTR共有37个核苷酸位点发生突变。VP1区基因进化树显示9个EV71毒株均属于C4a基因亚型,并且两组不同临床症状毒株处于同一较小分支中。5′-UTR核苷酸序列的系统进化树显示临床表现不同的EV71株呈交错分布,相同临床表现不单独聚类。 结论 9株EV71毒株均属于C4a基因亚型,5′-UTR核苷酸突变和VP1区氨基酸替换可能影响病毒毒力,对EV71病毒的全基因组序列特征分析及与宿主之间的相互作用机制进行研究非常必要。

Gene characteristics of 5’-UTR and VP1 of enterovirus 71 isolated from 9 patients with severe or mild hand,foot and mouth disease in Weifang City

Objective To explore whether the 5’-untranslated region(5’-UTR) and VP1 region of enterovirus 71(EV71) isolated from severe hand, foot and mouth disease(HFMD) contain potential virulence sites. Methods EV71 strains isolated from fecal specimens collected from HFMD patients with different clinical symptoms were cultured by RD cells. The viral total RNA was extracted. The 5’-UTR and VP1 in genomes were amplified by RT-PCR and then sequenced. The nucleotide sequences of 5’-UTR and VP1 and the amino acid sequences of VP1 were compared, and the homology analysis was performed. The phylogenetic tree was constructed based on the VP1 gene sequences and the 5’-UTR of EV71 with different clinical symptoms. Results A total of 9 EV71 strains(5 strains from severe cases and 4 strains from mild cases) were obtained in this study. The nucleotide homologies of 5’-UTR and VP1 regions were both 94.5%-99.7%, and the amino acid homology of VP1 region was 97.3%-99.9%. There were 67 mutation sites in 5’-UTR of the 9 EV71 strains. Compared with the 4 EV71 strains isolated from mild cases, 2 amino acids(S283 T, A289 T) of VP1 from the 5 EV71 strains isolated from severe cases were substituted, and a total of 37 nucleotide positions were mutated in 5’-UTR sequences. The phylogenetic tree based on VP1 sequences showed that the 9 EV71 strains belonged to sub-genogroup C4 a, and the strains isolated from different clinical manifestation cases of two groups were clustered in the same branch. Phylogenetic tree based on the nucleotide sequences of 5’-UTR showed that EV71 strains from different clinical manifestation cases exhibited a staggered distribution, and the strains from same clinical manifestation cases did not cluster separately. Conclusion The nine EV71 strains belonged to the sub-genogroup C4 a. Nucleotide mutations at 5’-UTR and amino acid substitutions at VP1 may affect the virulence of EV71. Accordingly, further study on the whole genome characterization of the EV71 strains and the interaction mechanism between the EV71 strains and the host protein factors are necessary.