摘要
目的对2002年分离自广州登革热患者的1型登革病毒GZ2002株进行全基因组序列测定及分析,为探讨其来源和基因组特征提供依据。方法利用C6/36细胞增殖GZ2002株,出现典型细胞病变后收集感染细胞及病毒液提取R NA。根据5株登革病毒1型标准株AY145123、FJ176780、FJ176779、DVU88536、EF025110全序列设计6对相互覆盖的引物。采用R T-PCR法扩增覆盖GZ2002株基因组全长的6个cDNA片段,并将其分别克隆到pMD-18T载体上,通过序列测定和重叠序列拼接获得全基因组序列。结果 GZ2002株基因组全长10 735 nt,单一阅读框长10 176 nt,推测编码3 392个氨基酸,5'及3'-UTR分别为94 nt和462 nt,无显著的密码子偏嗜性。GenBank登录号:JN205310。系统进化分析显示GZ2002株属于DENV-1型基因IV型。比较不同致病性的DENV-1型毒株发现,随DENV毒株致病力的增强,编码区氨基酸突变位点明显增多。GZ2002株、泰国16007株、柬埔寨株及印度尼西亚98901530株的5'-UTR二级结构高度保守,3'-UTR分析显示强毒力泰国16007株与柬埔寨株均存在高突变区。结论 GZ2002株属于DENV-1型基因IV型,可能与1998年印度尼西亚DENV-1流行株相关,编码区株特有氨基酸变异位点及3'-UTR高突变区的生物学意义值得进一步探讨。
【Objective】 To analyze the possible origin and the genomic characteristics,the whole genome of a Dengue virus serotype 1（DENV-1） strain GZ2002 isolated from the serum of a dengue fever patient in Guangzhou province in 2002 was sequenced.【Methods】 The virus suspension of GZ2002 infected C6/36 cells was harvested and used for RNA extraction.Six primer pairs were designed according to the genome sequences of five DENV-1 reference strains： AY145123,FJ176780,FJ176779,DVU88536,and EF025110.Six overlapping fragments of GZ2002 genome were amplified by reverse transcription polymerase chain reaction,and then cloned into pMD-18T vector.The sequence determination and assembly of positive clones were performed to obtain GZ2002 full-length genomic sequence.【Results】 The complete genome of GZ2002 is composed of 10735 nucleotides（nt） with a single long open reading frame of 10176 nt which encodes a polyprotein of 3392 amino acids.The 5′-and 3′-untranslated regions（UTR） contain 94 nt and 462 nt,respectively.The GenBank accession number is JN205310.The analysis of evolutionary tree showed that GZ2002 be longes to DENV-1 genotype IV.Comparison of the genomic sequences of different pathogenic strains（16007,Cambodia,98901530） revealed that the number of amino acid mutational sites in coding region is associated with the pathogenicity of virus strain.Furthermore,the 5′-untranslated regions are highly conserved,and the 3′-untranslated regions of 16007 and Cambodia strains have a hypervariable region.【Conclusion】 GZ2002 belongs to DENV-1 genotype IV and maybe closely related to the DENV-1 strain isolated in Indonesia in 1998.The biological significances of strain specific amino acid mutational sites in coding region and the hypervariable regions in 3′-untranslated region are worth to further study.
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
2012年第14期25-33,共9页
China Journal of Modern Medicine
基金
国家自然科学基金(No:31070811)
重庆市自然科学基金(No:CSTC
2009BB5015)
第三军医大学科研基金(No:2009XYY04
2009XHG03)
国家重大新药创制(2012ZX09103301-038)
关键词
登革病毒1型
全基因组测序
序列分析
Dengue virus serotype 1
genome sequencing
sequence analysis
