Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA.
Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA; Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA.
Cell Host Microbe. 2020 May 13;27(5):841-848.e3. doi: 10.1016/j.chom.2020.04.004. Epub 2020 Apr 13.
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 × 10 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.
持续的 COVID-19 大流行是由严重急性呼吸系统综合症冠状病毒 2（SARS-CoV-2）引起的，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。我们报告了一种 SARS-CoV-2 的反向遗传系统。跨越 SARS-CoV-2 基因组的七个互补 DNA（cDNA）片段被组装成全长 cDNA。全长 cDNA 转录的 RNA 在电穿孔进入细胞后具有高度感染性，产生 2.9×10 个噬菌斑形成单位（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）表现出相似的蚀斑形态、病毒 RNA 谱和复制动力学。此外，icSARS-CoV-2 保留了工程化的分子标记，并且没有获得其他突变。我们通过将该报告基因引入病毒基因组的 ORF7 中，生成了稳定的 mNeonGreen SARS-CoV-2（icSARS-CoV-2-mNG）。icSARS-CoV-2-mNG 成功用于评估干扰素（IFN）的抗病毒活性。总之，该反向遗传系统和报告病毒提供了研究 SARS-CoV-2 和开发对策的关键试剂。
