肠道病毒ECHO_(13)中国分离株的基因特征

The Genetic Characterization of Echovirus 13 Isolated in China

为研究ECHO13病毒中国分离株的分子特征及其与世界其它分离株之间的基因关系,对1998年、2000年从中国福建省分离到2株ECHO13病毒进行基因序列对比分析。2株病毒分别命名为Fujian98-1和Fujian00-1,用逆转录-聚合酶链反应(RT-PCR)扩增出VP1蛋白编码基因全长861个核苷酸片段并进行序列测定,将2株E CHO13病毒的VP1序列与所有已发表的ECHO13病毒VP1基因全长进行同源性比较。结果显示,福建分离株之间核苷酸同源性为79 6%,氨基酸同源性为93 4%;与遗传距离最近的法国CF1089-91(AJ537604)毒株的核苷酸同源性分别为80%和88%,与代表株DelCarmen的核苷酸同源性分别为75 8%和77 9%。通过VP1基因分析,福建2株病毒均属于ECHO13病毒,与血清中和试验鉴定结果一致。下载所有已发表的ECHO13病毒VP1序列并构建进化树,发现福建2株病毒分属不同的分枝,提示这2株病毒来自不同的病毒传播链。进一步分析发现,整个E CHO13病毒可划分为3个不同的基因型:A、B和C基因型。福建Fujian98-1和Fujian00-1分别被划分在基因型B和C中,各基因型之间的核苷酸差异均大于20%。为验证该分型方法,将26株来自不同国家和时间的ECHO13病毒和2株福建分离ECHO13病毒部分VP1基因序列进行对比分析,建立进化树。结果显示,所有ECHO13病毒被分在A。

2 strains of enterovirus were isolated from Fujian Province in China in 1998 and 2000.The 2 isolates,Fujian98-1 and Fujian00-1,were Echovirus 13 identified by WHO panel of enterovirus sera using neutralization assay.The complete VP1 sequences of the 2 isolates were successfully amplified by RT-PCR with two enterovirus general primer pairs.The whole coding region of VP1 of the Fujian98-1 and Fujian00-1 were sequenced and analyzed.861 nucleotides of VP1 coding gene of the 2 Fujian isolates were compared with other Echovirus 13 isolates available from GenBank.The highest nucleotide homogeneity of Fujian98-1 and Fujian00-1 were 80.0% and 88.0% with French strain CF1083-91,and 75.8%,77.9% with Echovirus 13 reference strain(Del Carmen)respectively.The two strains from Fujian Province were thus clearly identified as Echovius 13 that agreed with neutralization result.The homogeneity analysis showed that the 2 Fujian isolates shared 79.6% similarity in nucleotide level and 93.4% in amino acid level.Phylogenetic tree based on complete VP1 sequence assigned the two isolates to different branches,which indicated there were at least two transmitting chains of Echovirus 13 prevailed in Fujian Province.We also found there were 3 genotypes(A,B and C)of Echovirus 13,the diversity of complete VP1 sequence among them was more than 20%.In order to confirm the genotyping,a phylogenetic tree based on the 5′ terminus of VP1 gene was constructed,including 26 strains of Echovirus 13 from different countries and different times.As the result,the Fujian strains,Fujian98-1 and Fujian00-1,still belonged to genotype B and genotype C respectively.With one exception,the diversity of genotype cluster from each other was more than 15%.So far,this is the first report about sequencing data and analysis of VP1 gene of Echovirus 13 isolated in China.