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  • 彭浩然,李成忠,唐海琳,薛建亚,陈志辉,梁雪松,朱咏梅,夏炳辉,朱勇喆,吴俊杰,赵兰娟,任浩,江亮亮,何燕华,戚中田,赵平.上海2株严重急性呼吸综合征冠状病毒2的分离与鉴定[J].第二军医大学学报,2020,41(4):365-370    [点击复制]
  • PENG Hao-ran,LI Cheng-zhong,TANG Hai-lin,XUE Jian-ya,CHEN Zhi-hui,LIANG Xue-song,ZHU Yong-mei,XIA Bing-hui,ZHU Yong-zhe,WU Jun-jie,ZHAO Lan-juan,REN Hao,JIANG Liang-liang,HE Yan-hua,QI Zhong-tian,ZHAO Ping.Isolation and identification of two strains of severe acute respiratory syndrome coronavirus 2 from coronavirus disease 2019 patients in Shanghai[J].Acad J Sec Mil Med Univ,2020,41(4):365-370   [点击复制]
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上海2株严重急性呼吸综合征冠状病毒2的分离与鉴定
彭浩然,李成忠,唐海琳,薛建亚,陈志辉,梁雪松,朱咏梅,夏炳辉,朱勇喆,吴俊杰,赵兰娟,任浩,江亮亮,何燕华,戚中田,赵平
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(1. 海军军医大学(第二军医大学)海军医学系生物医学防护教研室, 上海 200433;
2. 海军军医大学(第二军医大学)长海医院感染科, 上海 200433;
3. 海军军医大学(第二军医大学)长海医院呼吸与危重症医学科, 上海 200433
*通信作者)
摘要:
目的 从新型冠状病毒肺炎(COVID-19)患者鼻/咽拭子样本中分离、培养严重急性呼吸综合征冠状病毒2(SARS-CoV-2)。方法 将来自上海市COVID-19患者的3份鼻/咽拭子样本以TPCK胰酶处理,然后接种Vero E6细胞;待大部分细胞出现明显病变时,取细胞培养上清用qRT-PCR法检测病毒核酸,并用反转录PCR扩增病毒受体结合区(RBD)基因片段;将病毒扩增培养后感染接种于96孔板中的Vero E6细胞,观察细胞病变效应,并用免疫荧光法检测病毒蛋白。结果 2份COVID-19患者鼻/咽拭子样本接种的Vero E6细胞出现明显细胞病变效应,细胞培养上清中检测出新复制产生的SARS-CoV-2核酸,扩增出的RBD序列与早期分离出的SARS-CoV-2相应序列完全一致;病毒感染的Vero E6细胞病变迅速,并能与SARS-CoV-2核衣壳蛋白(N蛋白)单克隆抗体、刺突蛋白(S蛋白)单克隆抗体及COVID-19患者恢复期血清发生反应。结论 从2份COVID-19患者鼻/咽拭子样本中成功分离出SARS-CoV-2,为后续开展SARS-CoV-2感染与致病机制研究、防治药物与疫苗的研发奠定了基础。
关键词:  严重急性呼吸综合征冠状病毒2  新型冠状病毒肺炎  鼻拭子  咽拭子  病毒分离
DOI:10.16781/j.0258-879x.2020.04.0365
投稿时间:2020-03-28修订日期:2020-04-08
基金项目:国家重点研发计划(2016YFC1200401),国家科技重大专项(2017ZX10304403-003).
Isolation and identification of two strains of severe acute respiratory syndrome coronavirus 2 from coronavirus disease 2019 patients in Shanghai
PENG Hao-ran,LI Cheng-zhong,TANG Hai-lin,XUE Jian-ya,CHEN Zhi-hui,LIANG Xue-song,ZHU Yong-mei,XIA Bing-hui,ZHU Yong-zhe,WU Jun-jie,ZHAO Lan-juan,REN Hao,JIANG Liang-liang,HE Yan-hua,QI Zhong-tian,ZHAO Ping
(1. Department of Biomedical Defense, Faculty of Naval Medicine, Naval Medical University (Second Military Medical University), Shanghai 200433, China;
2. Department of Infectious Diseases, Changhai Hospital, Naval Medical University (Second Military Medical University), Shanghai 200433, China;
3. Department of Respiratory and Critical Care Medicine, Changhai Hospital, Naval Medical University (Second Military Medical University), Shanghai 200433, China
*Corresponding authors)
Abstract:
Objective To isolate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from nasal/throat swabs of coronavirus disease 19 (COVID-19) patients. Methods Three nasal/throat swab samples from COVID-19 patients in Shanghai were treated with TPCK trypsin and were used to treat Vero E6 cells inoculated in 96-well plates. When most of the cells showed obvious cytopathy, the cell culture supernatants were collected. We then detected the viral nucleic acid by fluorescent quantitative real-time polymerase chain reaction, and amplified the gene fragment of the virus receptor binding domain (RBD) by reverse transcription polymerase chain reaction. After amplification and culture, the virus was used to infect the Vero E6 cells inoculated in 96-well plates. The cytopathy was observed and the virus protein was detected by immunofluorescence. Results The Vero E6 cells that cultured with two of three nasal/pharyngeal swab samples showed obvious cytopathic effect and newly synthesized viral nucleic acid was detected in the supernatants of the cell culture. The amplified RBD sequence was completely consistent with the corresponding fragment of SARS-CoV-2 isolated earlier. Virusinfected Vero E6 cells showed cytopathies rapidly and could react with the monoclonal antibody against nucleocapsid protein (N protein) and spike protein (S protein) of SARS-CoV-2, and convalescence sera of COVID-19 patients. Conclusion Two SARS-CoV-2 strains were successfully isolated from two nasal/throat swab samples of COVID-19 patients in Shanghai, which provides evidence for the mechanism research on the infection and pathogenesis of SARS-CoV-2 as well as the development of drugs and vaccines against SARS-CoV-2.
Key words:  severe acute respiratory syndrome coronavirus 2  coronavirus disease 2019  nasal swab sample  throat swab sample  virus isolation