诺如病毒河北卢龙株NHBGR59全基因组序列分析

Complete sequence analysis of norovirus strain NHBGR59 of Hebei Lulong

目的 分析河北卢龙县腹泻儿童粪便标本中存在的诺如病毒株NHBGR59的全基因组序列，了解其基因结构特点和进化特征。方法根据454高通量测序获得的两个重叠群（contig）和诺如病毒参考株序列（GenBank登录号KM198534）设计NHBGR59特异性引物，用重叠反转录聚合酶链反应（ReversetranscriptionPCR，RT—PCR）和cDNA末端快速扩增的技术（rapid—amplificationofcDNAends，RACE）的方法扩增其全长基因，经过分子克隆、测序获得全基因组序列，然后进行三个开放阅读框（OpenReadingFrames，ORFs）序列和系统进化树的分析。结果诺如病毒河北卢龙珠HBGR59病毒全基因组全长为7563bp（除了PolyA尾），病毒基因组分为三个ORFs：ORF1（5～5098nt）、ORF2（5079—6731nt）和ORF3（6731～7510nt），其中ORFl和ORF2之间有20bp重叠，ORF2和ORF3之间有lbp重叠。经过全基因组序列分析发现其和基因组II基因型6（genogroupIIGenotypeVI，GII．6）诺如病毒株30443同源性最高（同源性为98．O％）。ORF2以及ORF3核苷酸和氨基酸序列与株30443同源性最高（分别为97．0％，98．0％；99．0％，98．0％）。ORFI的基因和氨基酸序列与台湾暴发的急性胃肠炎GII．6株11-FJ-5（GenBank号KM461694）同源性最高（99．0％，99．3％），只在高突变P2区发生一个氨基酸突变位点：aa308D—N；其中衣壳区的氨基酸序列与其他GII-6型诺如病毒相似度为73．0％～99．3％。进化树分析发现河北卢龙株HBGR59与GII．6型株30443和11-FJ-5亲缘关系最近。结论河北卢龙株HBGR59为诺如病毒GII．6型，进化关系上与已报道的GII.6型株30443和11-FJ-5最近。

Objective To study the gene and evolution of the complete sequence of NHBGR59 strain isolated from children with diarrhea in Hebei Lulong. Methods The full-length genome was amplified based on the two contigs and reference sequence of strain of 30443 （KM198534） by methods of reverse transcription PCR and rapid-amplification of cDNA ends. The PCR products was cloned and sequenced. HBGR59 was performed on sequence analysis and phylogenetie tree. Results The complete sequence of NHBGR59 was 7 563 bp （excluding PolyA tail） , including three open reading frames （ORFs） ： ORF1 （5 - 5 098 nt） , ORF2 （5 079 -6 731 nt） and ORF3（6 731 -7 510 nt） , with 20 nueleotide （nt） overlapping in ORFI and ORF2, and 1 nt overlapping in ORF2 and ORF3. By sequence analysis, NHBGR59 showed the highest homology in complete sequence with norovirus strain 30443 （98% identity）, and the ORF2 and ORF3 also showed the highest identity to strain 30443 （97.0% and 99.0% in nt; 98.0% and 98.0% in amino acid （aa）） , while the ORF2 displayed the highest homology （99.0% in nt, 99.3% in aa） with the strain 11-FJ-5 （KM461694）, which was related to GII. 6 norovirus gastroenteritis outbreaks in Taiwan in 2014, and only one amino acid was different （aa308 D-N in P2 region） between them. The phylogenetic analysis showed NHBGR59 clustered tightly with strains of 30443 and ll-FJ-5 in norovirus genogroup II genotype 6 （GII. 6）. Conclusions NHBGR59 belongs to norovirus GII. 6, which is the mostly close to the recently reported strain 30443 and 11-FJ-5.