长沙市诺如病毒暴发疫情中GⅡ.2[P16]型全基因组分子进化特征分析

目的 对长沙市2020年急性胃肠炎暴发疫情中诺如病毒GⅡ.2[P16]型进行全基因组序列测定以及遗传进化特征分析。方法 采集2020年诺如病毒暴发疫情标本,采用逆转录聚合酶链反应（RT-PCR）方法扩增开放阅读框（ORF）1和ORF 2连接区进行分型鉴定。GⅡ.2[P16]型阳性标本在二代测序Miseq平台进行测序。测序结果进行序列比对及进化分析。结果 2020年本市共发生诺如病毒暴发疫情29起,共采集标本277份,阳性检出率为44.04%（122/277）。分型鉴定发现诺如病毒GⅡ.2[P16]型占比45.08%（55/122）。从疫情分布月份和机构来看,9—12月为暴发高峰,疫情发生在幼儿园和学校占比89.66%（26/29）。对53份GⅡ.2[P16]型ORF1和ORF2连接区序列进行进化树分析发现毒株处于多个分支,序列之间的同源性为98.5%~100%。对4株GⅡ.2[P16]型全基因组序列进行同源性比较发现毒株之间的同源性为98.5%~99.5%,与GⅡ.2[P16]2016-2017（KY771081）毒株同源性为97.9%~98.2%,进化树处于不同亚分支。RdRp区域氨基酸位点发生T396A和T464A突变,HBGA结合位点保守未发生突变。结论 长沙地区诺如病毒暴发疫情中以GⅡ.2[P16]型突变株传播为主,毒株由GⅡ.2[P16]2016-2017持续传播进化而来,并进化为另一个分支。本地区暴发的诺如病毒疫情主要发生在学校以及幼托机构,应持续加强对诺如病毒的监测。

Genome-wide molecular evolutionary characterization of GⅡ.2[P16] type in norovirus outbreaks in Changsha

Objective To investigate the genetic evolutionary characteristics of norovirus GⅡ.2[P16] type from acute gastroenteritis outbreaks in Changsha city in 2020 by whole genome sequencing. Methods Norovirus outbreak samples were collected in 2020 year. The open reading frame (ORF) 1 and ORF 2 junction regions were amplified by reverse transcription polymerase chain reaction (RT-PCR) for further norovirus genotyping. The full-genome of GⅡ.2[P16] positive samples were obtained from Miseq platform (Illumina). Sequence alignment and evolutionary analysis were performed. Results There were 29 outbreaks of norovirus in Changsha city in 2020. 277 samples were collected, and the positive rate was 44.04% (122/277). Genotype identification showed that GⅡ.2[P16] norovirus was accounted for 45.08% (55/122). The peak of norovirus pandemic appeared from September to December. Kindergarten and school of the outbreak places were accounted for 89.66% (26/29). Based on ORF1 and ORF2 junction nucleotide sequence, phylogenetic tree analysis revealed that 53 norovirus strains distributed in different branches. Those strains shared 98.5%-100% similarity with each other. The 4 strains of full-genome GⅡ.2[P16] sequences showed 98.5%-99.5% similarity with each other, and shared 97.9%-98.2% similarity with GⅡ.2[P16]2016-2017(KY771081) strain. Phylogenetic tree showed the 4 strains in this research belong to different sub-branches. The amino acid sites in RdRp region contain T396A and T464A mutations. And the HBGA binding sites in GⅡ.2[P16] viruses were conserved. Conclusions The spread of GⅡ.2[P16] type mutant strain is predominant in the norovirus outbreak in Changsha city, which evolved from GⅡ.2[P16]2016-2017 and have evolved into a different branch. Outbreaks are mainly reported in kindergarten and school. Our findings have improved the general understanding of the evolution, genetic diversity, and wide distribution of noroviruses.