An Infectious cDNA Clone of SARS-CoV-2
An Infectious cDNA Clone of SARS-CoV-2
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DOI：
10.1016/j.chom.2020.04.004
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发表时间：
2020-05-13
影响因子：
30.3
通讯作者：
Shi, Pei-Yong
中科院分区：
文献类型：
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作者：
Xie, Xuping;Muruato, Antonio;Shi, Pei-Yong
由严重急性呼吸综合征冠状病毒2 （SARS-CoV-2）引起的2019冠状病毒病（COVID-19）大流行正在持续，这突显了开发研究该病毒和确定对策的实验系统的紧迫性。我们报告了SARS-CoV-2的反向遗传系统。跨越SARS-CoV-2基因组的7个互补DNA （cDNA）片段被组装成一个全基因组cDNA。从全基因组cDNA转录的RNA电穿孔进入细胞后具有高度传染性，产生2.9 x 10(6) PFU /mL的病毒。与临床分离株相比，传染性克隆衍生的SARS-CoV-2 （icSARS-CoV-2）表现出相似的斑块形态、病毒RNA谱和复制动力学。此外，icSARS-CoV-2保留了工程分子标记，没有获得其他突变。我们通过将该报告基因导入病毒基因组的ORF7，生成了稳定的mNeonGreen SARS-CoV-2 （icSARS-CoV-2-mNG）。icSARS-CoV-2-mNG成功用于评价干扰素（IFN）的抗病毒活性。反向遗传系统和报告病毒共同为研究SARS-CoV-2和制定对策提供了关键试剂。
The ongoing pandemic of COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), underscores the urgency to develop experimental systems for studying this virus and identifying countermeasures. We report a reverse genetic system for SARS-CoV-2. Seven complimentary DNA (cDNA) fragments spanning the SARS-CoV-2 genome were assembled into a full-genome cDNA. RNA transcribed from the full-genome cDNA was highly infectious after electroporation into cells, producing 2.9 x 10(6) plaque-forming unit (PFU)/mL of virus. Compared with a clinical isolate, the infectious-clone-derived SARS-CoV-2 (icSARS-CoV-2) exhibited similar plaque morphology, viral RNA profile, and replication kinetics. Additionally, icSARS-CoV-2 retained engineered molecular markers and did not acquire other mutations. We generated a stable mNeonGreen SARS-CoV-2 (icSARS-CoV-2-mNG) by introducing this reporter gene into ORF7 of the viral genome. icSARS-CoV-2-mNG was successfully used to evaluate the antiviral activities of interferon (IFN). Collectively, the reverse genetic system and reporter virus provide key reagents to study SARS-CoV-2 and develop countermeasures.