一株鼠冠状病毒的全基因组序列特征分析

Characterizationof complete genome sequence of a rat coronavirus

目的确定一株新型鼠冠状病毒全基因组序列特征及系统进化特征。方法采用高通量测序在内蒙地区采集的鼠肠道组织发现与已知冠状病毒差异较大的冠状病毒,拼接获得部分基因组序列,采用PCR验证扩增病毒基因组并采用RACE方法扩增基因组末端序列。采用MEGA软件进行多序列比对并构建系统发育树。结果本研究中发现的鼠冠状病毒经扩增获得其全基因组长度为27674 bp,命名为NMR-13,包括两端非编码区和8个开放阅读框,依次为5′UTR-ORF1ab-S-ORF3-E-M-ORF6-N-ORF8-3′UTR,其中ORF8仅在部分鼠类Alpha冠状病毒存在,功能尚不清楚,并且缺乏许多鼠类Alpha冠状病毒存在的NS2基因。同源性分析结果显示该病毒株属于Alpha冠状病毒属成员,与美国发现的FiCoV/UMN2020病毒株具有最高同源性,核苷酸同源性为91.3%,其次与我国浙江省发现的Lucheng/Lijiang-170,Lucheng/Ruian-83和Lucheng/Lijiang-71病毒株核苷酸同源性较高,分别为79.5%,80.6%和81.0%。系统发育分析也显示NMR-13与FiCoV/UMN2020具有最近的亲缘关系。重组分析未发现重组现象。结论本研究发现了一株鼠冠状病毒,丰富了鼠类中Alpha冠状病毒的多样性,为了解国内鼠冠状病毒的分子遗传特征及分子进化提供了参考依据。

This study determined the whole genome sequence and phylogenetic characteristics of a rat coronavirus.Nucleic acids were extracted from rat intestinal tissues collected in Inner Mongolia,and high-throughput sequencing was performed.A novel alphacoronavirus was present in the samples.The complete genome was amplified with PCR and RACE.Multiple sequence alignment and a phylogenetic tree were constructed in MEGA.The whole genome of the rat coronavirus,denoted NMR-13,was 27674 bp and included two non-coding regions and eight open reading frames,successively 5′UTR-ORF1ab-S-ORF3-E-M-ORF6-N-ORF8-3′UTR.Sequence identity analysis indicated that NMR-13 was most closely related to alphacoronavirus,which shared 91.3%nt identity with strain FiCoV/UMN2020.NMR-13h shared the next highest-sequence identitywiththe strains Lucheng/Lijiang-170,Lucheng/Ruian-83 and Lucheng/Lijiang-71 found in Zhejiang Province,China(79.49%,80.6%and 81.0%,respectively).Phylogeneticanalysis indicated that NMR-13 clustered with FiCoV/UMN2020.Recombination analysis indicated no recombination phenomenon.A rat coronavirus was isolated in this study,thus enriching the diversity of known alphacoronaviruses,and providing a reference for understanding the molecular genetic characteristics and molecular evolution of mouse coronaviruses in China.