广西猕猴中发现GⅡ.17型诺如病毒及其全基因组序列分析

摘要

目的 了解广西省龙虎山猕猴粪便标本中的GⅡ.17型诺如病毒的流行情况、基因结构和进化特征.方法 2015年3月～8月收集400份野生猕猴粪便标本,利用建立好的HISEQ高通量测序技术在粪便标本中发现了GⅡ.17型诺如病毒,命名为GX213.采用反转录-聚合酶链反应(Reverse transcription-polymerase chain reaction,RT-PCR)技术对其进行了序列鉴定、全长扩增和检测研究,并对三个开放阅读框(Open reading frames,ORFs)进行了序列和遗传进化分析.结果 筛查结果显示阳性率为0.5％ (2/400).诺如病毒GX213毒株基因组全长为7 565 bp(包括PloyA尾),其基因组分为三个ORFs:ORF1(10～5112nt)、ORF2(5093 ～ 6715 nt)和ORF3(6715 ～ 7494nt),其中ORF1与ORF2之间重叠20 bp,ORF2和ORF3之间重叠1 bp.GX213病毒株全基因组序列分析显示,其与引起2014年至2015年亚洲部分地区暴发的新型人GⅡ.17型诺如病毒变异株CUHK-NS-613(GenBank ID:KU561248)同源性最高(同源性最高为99.5％).ORF1和ORF3区核苷酸和氨基酸序列都与CUHK-NS-613同源性最高(分别为99.5％,99.4％;99.5％,99.2％).ORF2区分析序列发现其与CUHK-NS-491毒株(GenBank ID:KP698928)同源性最高,核苷酸和氨基酸同源性分别为99.4％和99.8％,仅P1.1区发现了一个氨基酸的突变aa245P→S.另外,RdRp核苷酸和VP1氨基酸序列进化树分析显示该病毒株与人GⅡ.17型诺如病毒CUHK-NS-613和CUHK-NS-491的进化关系最近,而与首次被发现的猴GⅡ.17型诺如病毒KM1509(GenBank ID:KX356908)次之.结论 本研究发现的GX213属于2014年至2015年在亚洲地区引起暴发的新型人GⅡ.17型诺如病毒变异株,提示GⅡ.17型诺如病毒为人猕猴共患病毒.%Objective To analyze the prevalence,genetic structure and evolutionary characteristics of G Ⅱ.17 norovirus isolated from the fecal samples of rhesus monkeys in Longhu Mountain of Guangxi Zhuang Autonomous Region.Methods A total of 400 stool specimens were collected from wild rhesus monkeys from March to August of 2015.The G Ⅱ.17 norovirus named as GX213 was identified in fecal samples by high-throughput sequencing technology.Reverse transcription-polymerase chain reaction was used to confirm and screen GX213,as well as amplify its complete gene sequence.Then the sequence and phylogenetic analysis of three ORFs of GX213 were constructed by software MEGA 6.0.Results Two out of 400 fecal samples were positive.The full-length genome of GX213 was 7 565 bp (containing PloyA tail),which was composed of three open reading frames (ORFs):ORF1 (10-5112 nt),ORF2(5093-6715 nt) and ORF3(6715-7494 nt),with 20 bp overlapping between ORF1 and ORF2,and 1 bp overlapping between ORF2 and ORF3.Analysis of the complete sequence of GX213 showed that it shared the highest homology with the strain of human G Ⅱ.17 norovirus C UHK-NS-613 (GenBank ID:KU561248) (99.5％ identity),and ORF1 and ORF3 also shared the highest homology with the strain CUHK-NS-613 [99.5％ and 99.4％ in nucleotide (nt);99.5％ and 99.2％ in amino acid (aa),respectively],which was the main cause of human norovirus outbreaks in some regions of Asia from 2014 to 2015.ORF2 sequence analysis showed that it displayed the highest identity (99.4％ in nt and 99.8％ in aa) to the strain CUHK-NS-491 (GenBank ID:KP698928),only one aa mutation aa245P→S (P1.1 region) was observed in the GX213 VP1 protein.Furthermore,the phylogenetic analysis showed that GX213 was more related to CUHK-NS-613 and CUHK-NS-491 than the strain KM1509 (GenBank ID:KX356908) of G Ⅱ.17 norovirus recently identified in rhesus monkeys.Conclusions GX213 belongs to the human G Ⅱ.17 norovirus variant causing the norovirus outbreaks from 2014 to 2015.Our research suggests that G Ⅱ.17 norovirus can infect not only humans but also rhesus monkeys.