东莞地区诺如病毒ORF2基因克隆与进化树分析

Phylogenetic analysis of ORF2gene clone of human norovirus in Dongguan

目的探讨东莞地区婴幼儿感染的诺如病毒ORF2基因序列特性,分析东莞地区诺如病毒的基因型。方法收集2004年和2009年东莞地区婴幼儿腹泻标本66份,参考诺如病毒Farmington Hills株(AY502023)基因组序列,自行设计了引物两段扩增ORF2基因的引物,短片段作为检测标本的片段,分段扩增病毒ORF2全长基因,PCR产物克隆于T载体上,序列测定,用ClustalW/X和MEGA5.0等软件进行序列特性分析和基因型分析。结果获得了3株ORF2全长基因,全长为1623bp,ORF2编码主要结构蛋白衣壳蛋白(VP1),VP1蛋白有两个主要区域:P区(protruding)和S区(shell);将病毒株NVdgsl0902、NVdgsl0910的P2区与诺如病毒GⅡ-4基因型中a、b、c、d、e、f 6个亚型的P2区氨苷酸序列进行同源性比较,同源性为86.0%～91.0%,而与2006b新变株为97.0%,病毒株NVdgsl0412的P2区与诺如病毒GⅡ-4基因型中a、b、c、d、f5个亚型的P2区氨苷酸序列进行同源性比较,同源性为88.0%～93.0%,与e亚型的同源性为98.1%。结论 NVdgsl0412属于GⅡ-4e型,NVdgsl0902、NVdgsl0910属于GⅡ-4f亚型的2006b新变异株,2004年与2009年东莞地区的诺如病毒流行株发生了变异,目前是以2006b新变异株作为主要的流行株。

OBJECTIVE To explore the characteristics of ORF2 gene in human norovirus causing infections in the infants in Dongguan of China so as to analyze the genotypes of norovirus in Dongguan.METHODS A total of 66 stool samples were collected from patients with gastroenteritis from seven hospitals in 2004 and 2009.Primers were designed and amplified ORF2 gene by referring the sequence of genotypes of Farmington Hills（AY502023）.The PCR-products were cloned to the Tvector and sequenced,the nucleotide sequences were analyzed with the programs ClustalW/X and MEGA5.0.RESULTS The ORF2 gene of 3 strains Norovirus were cloned,ORF2 encoded the major structural protein VP1,VP1 folded into two major domains designated S for the shell domain and P for the protruding domain.The P2 domain amino acid sequences of NVdgsl0902 and NVdgsl0910 strain were compared with Norovirus GⅡ-4a,b,c,d,e,and f subtypes,revealing that the homology with GⅡ-4 subtypes ranged between 86.0% and 91.0%,the homology with GⅡ-4 variants 2006b virus was 97%;the P2 domain amino acid sequences of NVdgsl0412 strain was compared with Norovirus GⅡ-4a,b,c,d,and f subtypes,revealing that the homology with GⅡ-4 subtypes ranged between 88.0% and 93.0%,the homology with GⅡ-4e subtypes was 98%.CONCLUSION The sequence and phylogenetic analysis of the NVdgsl0902 and NVdgsl0910 strains reveals that these isolates belongs to the GⅡ-4f novel variant 2006b virus,NVdgsl0412 strain belongs to GⅡ-4e subtype.The Norovirus in Dongguan between 2004 and 2009 have been varied,the novel variants 2006b virus are at present the predominant strains.