摘要
新型冠状病毒(SARS-CoV-2)作为引起全球新型冠状病毒肺炎疫情蔓延的病原体而受到广泛关注。安徽省自2020年1月起陆续出现990例新型冠状病毒肺炎确诊病例。为探索SARS-CoV-2分离培养和二代测序方法构建,了解该病毒在安徽省传播的变异情况,为药物筛选和疫苗研发等工作提供基础和依据,本研究选取1例新型冠状病毒肺炎确诊病例咽拭子样本接种Vero细胞进行病毒分离培养,并逐日观察细胞病变,经荧光定量PCR检测确认病毒分离培养结果;采用SARS-CoV-2全基因组捕获技术及二代测序对毒株进行全基因组测序,并通过DNASTAR Lasergene MegAlign v7.1.0和PhyloSuite v1.2.1进行序列比对和进化分析;将全基因组序列上传国家基因组科学数据中心鉴定变异位点。结果显示,样本接种Vero细胞第4d出现细胞病变,经荧光定量PCR检测确认为SARS-CoV-2毒株。该毒株基因组序列全长29 860bp,与SARS-CoV-2参比序列相似度均在99%以上,共有9个位点发生突变。与蝙蝠来源SARS样冠状病毒Bat Yunnan RaTG13相似度最高,为96.8%。本研究通过Vero细胞成功分离培养了新型冠状病毒,并分析了安徽省首个SARS-CoV-2毒株全基因组序列,为了解病毒生物学特性提供了重要的参考信息。
SARS-CoV-2 was worldwide concerned as the pathogen of coronavirus disease 2019(COVID-19).Since January 2020,there were 990 confirmed cases reported in Anhui Province,China. We wish to explore the isolation and next-generation sequencing(NGS)of SARS-CoV-2. In the way,we wanted to understand the variation of SARS-CoV-2 in Anhui Province,China and provide a basis for the research and development of a vaccine. Vero cells were infected with throat swab samples. Cells were monitored daily for the cytopathic effect(CPE). Nucleic acids of SARS-CoV-2 in cell supernatants were detected by reverse transcription-polymerase chain reaction(RT-PCR). The whole genome sequence of SARS-CoV-2 was generated by genome capture and NGS. Sequence alignment was done by Lasergene MegAlign v7.1.0. Phylogenetic trees were constructed by PhyloSuite v1.2.1. The sequence was uploaded to the National Genomic Data Center to identify variations.SARS-CoV-2 isolation showed that a CPE appeared on the fourth day of incubation,and the result was confirmed by RT-PCR. The whole length of the virus genome was 29,860 bp,which shared more than 99%sequence identity with other SARS-CoV-2 strains and varied in nine positions. The most similar SARS-like coronavirus in bats was the Yunnan RaTG13 strain(96.8% sequence identity). These data showed that we isolated SARS-CoV-2 with Vero cells and analyzed(for the first time)the whole genome sequence of SARSCoV-2 in Anhui Province,China. Our results will aid understanding the characteristics of SARS-CoV-2.
作者
栗薇薇
孙永
袁媛
俞俊岭
陈晴晴
葛盈露
张竹慧
何军
LI Weiwei;SUN Yong;YUAN Yuan;YU Junling;CHEN Qingqing;GE Yinglu;ZHANG Zhuhui;HE Jun(A nhui Provincial Center fo r Disease Control and Prevention,Hefei 230601,China)
出处
《病毒学报》
CAS
CSCD
北大核心
2020年第5期751-757,共7页
Chinese Journal of Virology
基金
安徽省新型冠状病毒肺炎疫情应急科研攻关(项目号:202004a07020004),题目:新型冠状病毒基因分子变异与进化研究
安徽省新型冠状病毒肺炎疫情应急科研攻关(项目号:202004a07020002),题目:新冠病毒肺炎流行病学及临床特征研究。
