云南新分离2株基因Ⅰ型流行性乙型脑炎病毒的全基因组序列分析

Full-genome sequencing analysis of two stains of Japanese encephalitis virus isolated from Yunnan,China

目的对云南省新分离的2株流行性乙型脑炎(乙脑)病毒进行全基因组序列测定和分析,了解当地乙脑病毒基因组结构及分型特征。方法设计乙脑病毒全基因组扩增引物,RT-PCR扩增片段,PCR产物直接测序,拼接后获得全基因组序列。通过Clustal X(1.8)、DNASTAR、GENEDOC(3.2)等生物学软件进行核苷酸序列及氨基酸序列分析和病毒的系统进化分析。结果从2007年云南省捕获的三带喙库蚊中分离到2株乙脑病毒(TC07018、TC07172),并对病毒株的全基因组序列进行了分子生物学研究,结果提示TC07018、TC07172株与JEVMie40株的核苷酸同源性最高,为98.4%和98.7%,与JKT6468株核苷酸同源性最低,为83.6%和83.5%。2个分离株与JEVMie40、SH17M-07和HEN0701株氨基酸同源性最高,均为99.6%,与JKT6468株氨基酸同源性最低,为95.5%和95.4%。2个新分离株与我国现在最常用的疫苗株SA14-14-2相比较,核苷酸同源性为88.7%和88.6%,氨基酸同源性为97.1%。基于全基因序列,从基因库中选择了36株乙脑病毒与分离株进行进化分析,2个新分离株与KV1899和HEN0701的进化关系最接近,都属于基因Ⅰ型。结论新分离的2株乙脑病毒属于基因Ⅰ型,此为滇西地区首次分离该型病毒。这2株病毒与国内外基因Ⅰ型乙脑病毒流行株间存在一定差异,但根据抗原和毒力位点分析表明,乙脑SA14-14-2疫苗株能保护该型病毒。

Objective Characterization and structure analysis of the complete nucleotide sequence of the two strains of Japanese encephalitis virus（JEV） isolated in 2007 from Yunnan,China.Methods Overlapping primers were designed according to the full-genome reference sequence of JEV obtained from GenBank.JEV genome fragments were amplified using RT-PCR.PCR products were directly sequenced and all fragment sequences were aligned to the reference sequence to acquire the complete nucleotide sequence of the two JEV strains.Nucleotide sequence analysis,amino acid deduction,and phylogenetic construction were done by using softwares Clustal X（1.8）,DNASTAR and GENEDOC（3.2）.Results Two JEV strains（TC07018 and TC07172） were isolated from Culex tritaeniorhynchus in Yunnan,China;the complete nucleotide sequences of the two strains were obtained.Comparison analyses of these two strains with that of 14 fully-sequenced reference JEV strains showed that nucleotide homology ranged from 98.7%（JEV Mie40） to 83.5%（JKT6468） and deducted amino acid homology from 99.6%（JEV Mie40） to 95.4%（JKT6468）.These two JEV strains share the highest homology of both nucleotide and amino acid sequences with JEV strain mie40.In comparison with the strain SA14-14-2 which is currently used for live vaccine in China,strains TC07018 and TC07172 showed relatively low homology in nucleotide sequence（88.7% and 88.6%,respectively） although the homo-logy of deducted amino acid sequence was considerably high（97.1%）.Phylogenetic analysis of these two strains with 36 JEV strains obtained from GenBank indicated that both TC07018 and TC07172 strains were closely related to JEV strains KV1899 and HEN0701,which were grouped into genotype I.Conclusions The full genome of the two JEV strains isolated from Yunnan,China were sequenced and compared with currently available reference JEV sequences.Phylogenetic construction grouped these two JEV strains into genotype I and these two strains are closely-related to the Chinese isolate HEN0701 and the Korean isolate KV1899.