Texas Biomedical Research Institute, San Antonio, Texas, USA.
Department of Veterinary Microbiology, University of Ilorin, Ilorin, Nigeria.
mBio. 2020 Sep 25;11(5):e02168-20. doi: 10.1128/mBio.02168-20.
Infectious coronavirus (CoV) disease 2019 (COVID-19) emerged in the city of Wuhan (China) in December 2019, causing a pandemic that has dramatically impacted public health and socioeconomic activities worldwide. A previously unknown coronavirus, severe acute respiratory syndrome CoV-2 (SARS-CoV-2), has been identified as the causative agent of COVID-19. To date, there are no U.S. Food and Drug Administration (FDA)-approved vaccines or therapeutics available for the prevention or treatment of SARS-CoV-2 infection and/or associated COVID-19 disease, which has triggered a large influx of scientific efforts to develop countermeasures to control SARS-CoV-2 spread. To contribute to these efforts, we have developed an infectious cDNA clone of the SARS-CoV-2 USA-WA1/2020 strain based on the use of a bacterial artificial chromosome (BAC). Recombinant SARS-CoV-2 (rSARS-CoV-2) was readily rescued by transfection of the BAC into Vero E6 cells. Importantly, BAC-derived rSARS-CoV-2 exhibited growth properties and plaque sizes in cultured cells comparable to those of the natural SARS-CoV-2 isolate. Likewise, rSARS-CoV-2 showed levels of replication similar to those of the natural isolate in nasal turbinates and lungs of infected golden Syrian hamsters. This is, to our knowledge, the first BAC-based reverse genetics system for the generation of infectious rSARS-CoV-2 that displays features similar to those of a natural viral isolate. This SARS-CoV-2 BAC-based reverse genetics will facilitate studies addressing several important questions in the biology of SARS-CoV-2, as well as the identification of antivirals and development of vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19 disease. The pandemic coronavirus (CoV) disease 2019 (COVID-19) caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is a major threat to global human health. To date, there are no approved prophylactics or therapeutics available for COVID-19. Reverse genetics is a powerful approach to understand factors involved in viral pathogenesis, antiviral screening, and vaccine development. In this study, we describe the feasibility of generating recombinant SARS-CoV-2 (rSARS-CoV-2) by transfection of a single bacterial artificial chromosome (BAC). Importantly, rSARS-CoV-2 possesses the same phenotype as the natural isolate and This is the first description of a BAC-based reverse genetics system for SARS-CoV-2 and the first time that an rSARS-CoV-2 isolate has been shown to be phenotypically identical to a natural isolate in a validated animal model of SARS-CoV-2 infection. The BAC-based reverse genetics approach will facilitate the study of SARS-CoV-2 and the development of prophylactics and therapeutics for the treatment of COVID-19.
2019 年 12 月，一种新型冠状病毒（CoV）疾病（COVID-19）在中国武汉市爆发，导致了一场全球性的大流行，对全球公共卫生和社会经济活动产生了巨大影响。一种以前未知的冠状病毒，严重急性呼吸综合征冠状病毒 2（SARS-CoV-2），已被确定为 COVID-19 的致病因子。迄今为止，尚无美国食品和药物管理局（FDA）批准的疫苗或疗法可用于预防或治疗 SARS-CoV-2 感染和/或相关的 COVID-19 疾病，这引发了大量科学努力来开发控制 SARS-CoV-2 传播的对策。为了为这些努力做出贡献，我们基于细菌人工染色体（BAC）开发了一种新型冠状病毒（SARS-CoV-2）美国-WA1/2020 株的传染性 cDNA 克隆。通过将 BAC 转染到 Vero E6 细胞中，很容易挽救出重组 SARS-CoV-2（rSARS-CoV-2）。重要的是，BAC 衍生的 rSARS-CoV-2 在培养细胞中的生长特性和蚀斑大小与天然 SARS-CoV-2 分离株相当。同样，rSARS-CoV-2 在感染的金黄地鼠的鼻甲骨和肺部中的复制水平与天然分离株相似。据我们所知，这是第一个基于 BAC 的用于生成具有与天然病毒分离株相似特征的传染性 rSARS-CoV-2 的反向遗传学系统。这种 SARS-CoV-2 BAC 反向遗传学将有助于研究 SARS-CoV-2 生物学中的几个重要问题，以及鉴定用于治疗 SARS-CoV-2 感染和相关 COVID-19 疾病的抗病毒药物和疫苗。由严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）引起的大流行冠状病毒（CoV）疾病 2019（COVID-19）对全球人类健康构成重大威胁。迄今为止，尚无针对 COVID-19 的批准预防剂或疗法。反向遗传学是一种了解病毒发病机制、抗病毒筛选和疫苗开发相关因素的有力方法。在这项研究中，我们描述了通过转染单个细菌人工染色体（BAC）生成重组 SARS-CoV-2（rSARS-CoV-2）的可行性。重要的是，rSARS-CoV-2 具有与天然分离株相同的表型，这是第一个基于 BAC 的 SARS-CoV-2 反向遗传学系统的描述，也是第一个证明 rSARS-CoV-2 分离株在 SARS-CoV-2 感染的验证动物模型中与天然分离株表型相同的分离株。基于 BAC 的反向遗传学方法将有助于 SARS-CoV-2 的研究和 COVID-19 治疗的预防剂和疗法的开发。
