由于获得疫苗的机会有限,COVID-19大流行继续威胁着全球的医疗保健系统,次优治疗方案,以及新的和更具传染性的SARS-CoV-2变种的不断涌现。病毒基因和突变的反向遗传学研究已被证明对推进基础病毒研究非常有价值,导致治疗学的发展。通过将COVID-19相关病毒分离株(DK-AHH1)的序列克隆到细菌人工染色体(BAC)中,我们开发了一种功能性且用途广泛的全长SARS-CoV-2感染系统。将体外转录本RNA转染到VeroE6细胞中后回收的病毒显示出与DK-AHH1病毒分离株相似的生长动力学和remdesivir易感性。报告基因的插入,绿色荧光蛋白,和纳米荧光素酶进入ORF7基因组区域导致高水平的报告活性,这促进了高通量处理实验。我们发现假定的冠状病毒remdesivir抗性相关取代F480L和V570L-和自然发现的多态性A97V,P323L,和N491S,所有nsp12患者均未降低SARS-CoV-2对瑞德西韦的易感性.缺失穗(S)的纳米荧光素酶报告基因克隆,信封(E),膜(M)蛋白表现出高水平的瞬时复制,被Remdesivir抑制了,因此可以作为一种有效的非感染性亚基因组复制子系统。开发的SARS-CoV-2反向遗传学系统,包括重组体,以修饰具有自主基因组RNA复制的感染性病毒和非感染性亚基因组复制子,将允许高通量细胞培养研究-提供对这种冠状病毒的基本生物学的基本理解。我们已经证明了该系统在快速引入nsp12突变并研究其对remdesivir疗效的影响方面的实用性,在世界范围内用于治疗COVID-19。我们的系统提供了一个平台,可以有效地测试药物的抗病毒活性和SARS-CoV-2突变体的表型。
The COVID-19 pandemic continues to threaten healthcare systems worldwide due to the limited access to vaccines, suboptimal treatment options, and the continuous emergence of new and more transmissible SARS-CoV-2 variants. Reverse-genetics studies of viral genes and mutations have proven highly valuable in advancing basic virus research, leading to the development of therapeutics. We developed a functional and highly versatile full-length SARS-CoV-2 infectious system by cloning the sequence of a COVID-19 associated virus isolate (DK-AHH1) into a bacterial artificial chromosome (BAC). Viruses recovered after RNA-transfection of in vitro transcripts into Vero E6 cells showed growth kinetics and remdesivir susceptibility similar to the DK-AHH1 virus isolate. Insertion of reporter genes, green fluorescent protein, and
nanoluciferase into the ORF7 genomic region led to high levels of reporter activity, which facilitated high throughput treatment experiments. We found that putative coronavirus remdesivir resistance-associated substitutions F480L and V570L-and naturally found polymorphisms A97V, P323L, and N491S, all in nsp12-did not decrease SARS-CoV-2 susceptibility to remdesivir. A
nanoluciferase reporter clone with deletion of spike (S), envelope (E), and membrane (M) proteins exhibited high levels of transient replication, was inhibited by remdesivir, and therefore could function as an efficient non-infectious subgenomic replicon system. The developed SARS-CoV-2 reverse-genetics systems, including recombinants to modify infectious viruses and non-infectious subgenomic replicons with autonomous genomic RNA replication, will permit high-throughput cell culture studies-providing fundamental understanding of basic biology of this coronavirus. We have proven the utility of the systems in rapidly introducing mutations in nsp12 and studying their effect on the efficacy of remdesivir, which is used worldwide for the treatment of COVID-19. Our system provides a platform to effectively test the antiviral activity of drugs and the phenotype of SARS-CoV-2 mutants.