新型冠状病毒肺炎患者粪便中新型冠状病毒的分离鉴定  

Isolation and identification of a severe acute respiratory syndrome coronavirus 2 strain in feces from COVID-19 patients in Shanghai

作　　者：周艳秋 崔晓娴 牟嘉斌 房方皓 滕峥[1] 吴寰宇[2] 陈敏[1] 张曦[1] ZHOU Yanqiu;CUI Xiaoxian;MOU Jiabin;FANG Fanghao;TENG Zheng;WU Huanyu;CHEN Min;ZHANG Xi(Division of Microbiology,Shanghai Municipal Center for Disease Control and Prevention,Shanghai 200336,China;Shanghai Municipal Center for Disease Control and Prevention,Shanghai 200336,China)

机构地区：[1]上海市疾病预防控制中心病原生物检定所,上海200336 [2]上海市疾病预防控制中心,上海200336

出　　处：《上海预防医学》2022年第12期1173-1179,共7页Shanghai Journal of Preventive Medicine

基　　金：上海市科学技术委员会科研攻关项目(20411950100);上海市卫生计划生育委员会面上科研项目(201840033);上海市公共卫生体系建设三年行动计划(2020—2022年)学科人才建设计划(GWV-10.2-YQ03)。

摘　　要：【目的】分离鉴定新型冠状病毒肺炎(COVID-19)患者粪便标本中的新型冠状病毒(SARS-CoV-2),并进行生物学特性分析。【方法】利用Vero E6细胞对收集到的SARS-CoV-2核酸阳性粪便标本开展病毒分离,对产生细胞病变的培养物进行病毒核酸检测、免疫荧光抗原检测、病毒含量测定及全基因组测序分型。收集每代细胞培养物后进行半数组织培养感染剂量(TCID50)实验。【结果】8份粪便标本经处理后分别接种Vero E6细胞,培养48 h,1份标本在Vero E6细胞上发现明显细胞病变。8例COVID-19患者粪便病毒核酸阳性标本中分离到1株SARS-CoV-2,阳性分离率为12.5%。病毒能稳定传至第3代,P1代病毒TCID50为104.0/0.2 mL,P2代病毒TCID50为104.5/0.2 mL,P3代病毒TCID50为104.75/0.2 mL。8份粪便样本中仅1份有SARS-CoV-2活毒的复制扩增,传代培养的核酸检测Ct值约为10。该分离株的序列与Wuhan-Hu-1株序列(Gen Bank MN908947)的同源性>99.99%。【结论】从COVID-19患者粪便中分离到SARS-CoV-2活毒株,COVID-19患者中不仅存在粪便排毒的现象,且有活病毒存在。[Objective]To isolate and study the biological characteristics of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)from feces of coronavirus disease 2019(COVID-19)patients.[Methods]Vero E6 cells were used for virus isolation and the isolated strains were tested by nucleic acid test,immunofluorescence test,virulence test and whole genome sequencing.50%tissue culture infective dose(TCID50)was calculated after the cell cultures of each generation were collected[Results]Eight fecal specimens were inoculated with Vero E6cells after treatment and cultured for 48 h.One specimen showed obvious cytopathic effect on Vero E6 cells.One SARS-CoV-2 out of 8 fecal samples from COVID-19 patients were isolated,and separation rate was 12.5%.The TCID50of P1,P2 and P3 were 104.0/0.2 m L,104.5/0.2 m L and 104.75/0.2 m L,respectively.Only one of the 8 stool samples had SARS-CoV-2 virus replication and amplification,and the Ct value of the nucleic acid detection was about 10.The sequence of the isolation was more than 99.99%homologous with that of Wuhan-Hu-1(Gen Bank MN908947).[Conclusion]The SARS-CoV-2 strain is isolated from the fecal samples of COVID-19 cases and is confirmed by genomic sequencing and immunofluorescence test,which indicates the presence of live virus in feces of COVID-19 cases.

关 键 词：新型冠状病毒 粪便 Vero E6细胞 聚合酶链反应 全基因组测序 

分 类 号：R185[医药卫生—流行病学]