新型冠状病毒肺炎患者粪便中新型冠状病毒的分离鉴定

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（GenBank 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。

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

ObjectiveTo 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.MethodsVero 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 collectedResultsEight fecal specimens were inoculated with Vero E6 cells 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 TCID₅₀ of P1， P2 and P3 were 104.0/0.2 mL， 104.5/0.2 mL and 104.75/0.2 mL， 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（GenBank MN908947）.ConclusionThe 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.