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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 investigate the whole genome sequence of dengue virus type 1 Dengue virus strain GZ2002 isolated from dengue fever in Guangzhou in 2002 and provide evidence for its origin and genomic characteristics. Methods C6 / 36 cells were used to proliferate GZ2002 strain. After typical cytopathic effect, the infected cells and virus liquid were collected to extract R NA. Six pairs of primers covering each other were designed according to the complete sequences of five strains of Dengue virus type 1 AY145123, FJ176780, FJ176779, DVU88536 and EF025110. Six cDNA fragments covering the full-length genome of GZ2002 strain were amplified by RT-PCR and cloned into pMD-18T vector respectively. The complete genome sequence was obtained by sequencing and overlapping sequence. Results The genome of GZ2002 strain was 10 735 nt in length with a single reading frame of 10 176 nt and deduced 3 392 amino acids. The 5 ’and 3’ UTRs of GZ2002 were 94 nt and 462 nt, respectively. There was no significant codon bias. GenBank accession number: JN205310. Phylogenetic analysis showed that strain GZ2002 belonged to DENV-1 genotype IV. Compared with different pathogenic DENV-1 strains, it was found that with the increase of pathogenicity of DENV strains, the amino acid mutation sites in the coding region were significantly increased. The secondary structure of 5’-UTR of GZ2002 strain, Thailand 16007 strain, Cambodia strain and Indonesia 98901530 strain was highly conserved. The 3’-UTR analysis showed high virulence. Conclusion The GZ2002 strain belongs to the type IV of DENV-1 gene, which may be related to the epidemic strain of DENV-1 in Indonesia in 1998. The biological significance of the specific amino acid variation sites and 3’-UTR hyper mutated regions of the coding region strain should be further explored.