不同毒力EV71济南分离株全基因组序列比较分析

目的研究EV71济南分离株的基因特点，寻找潜存的EV7l毒力决定位点。方法将6株EV71济南分离株（JN200803、JN200804、Jinanl002、Jinanl004、Jinan1005和Jinan1006株）分别接种1d龄BALB／c小鼠，确定其对小鼠的致病性程度。对6株EV71全基因组核苷酸、氨基酸序列进行比较，对非编码区进行RNA二级结构的预测和分析，对VPl区进行遗传进化分析。结果通过动物实验可将6株EV71济南分离株分为强、中、弱毒力株。小鼠感染强毒株（JN200804、Jinan1002、Jinanl004、Jinanl005株）的主要症状为后肢麻痹并死亡，感染弱毒株（Jinanl006株）的主要症状为后肢麻痹但能自愈，感染无毒株（JN200803株）未引起明显症状。不同毒力EV71分离株全基因组序列比对发现，共有40个核苷酸位点的差异，导致编码区8个氨基酸突变，其中第937位氨基酸（位于2A片段）在强毒株、弱毒株和无毒株间发生了连续突变（937aa：S→C→G）。非编码区RNA二级结构预测表明，5’UTR第115位核苷酸突变对内部核糖体进入位点（IRES）的结构有较大影响。遗传进化分析显示，与同内2003年以后的流行毒株相同，6株EV71济南分离株均属C4亚型的C4a进化分支。结论全基因组序列比较分析发现不同毒力EV71济南分离株之间存在差异位点，为采用反向遗传学方法鉴定EV71毒力决定位点奠定了基础。

Comparative analysis of the complete genome sequences of enterovirus 71 strains with differing virulence isolated in Jinan

Objectives To investigate the genetic characteristics of EV71 strains prevalent in Jinan and to explore poten- tial determinants of EV71 virulence. Methods One-day-old BALB/c mice were inoculated with 6 EV71 strains. Com- plete genome sequences of the 6 EV71 strains were analyzed using bioinformatics. Results The 6 EV71 strains from Ji-- nan can be divided into 3 types： highly virulent strains （JN200804, Jinanl002, Jinanl004, and Jinanl005）, a slightly vir- ulent strain （Jinanl006）, and an avirulent strain （JN200803）. These types are based on symptoms of EVT1 infection in mice, such as hind limb paralysis, spontaneous recovery, and death. Sequence analysis indicated that the 3 types of EV71 strains with differing virulence have 40 nucleotide mutations and 8 amino acid mutations. Of note was a unique difference in amino acids （Ser937--＂Cys937--~Gly937） that distinguished highly virulent strains from the slightly virulent strain and the avirulent strain. A mutation in ntll5 led to obvious changes in the RNA secondary structure at the internal ribozyme entry site （IRES）. Phylogenetic analysis based on VP1 nucleotide sequences revealed that all 6 EVTI strains fell under the C4a cluster of subgenotype C4. Conclusion The potential determinants of the virulence of EV71 strains from Jinan were found by complete genome sequence analysis of 6 EV71 strains from Jinan with differing virulence. These results also pro- vide a basis to investigate the determinants of EVT1 virulence through a reverse genetics approach.