The production and application of SARS-CoV-2 reverse genetic systems to facilitate vaccine development and biosafe drug discovery platforms.

针对SARS-CoV-2的疫苗和抗病毒药物的开发受到高度遏制的要求的阻碍，这种要求仅限于全球少数实验室。我们最近建立了一种快速程序来生产SARS-CoV-2 RNA基因组的DNA副本，可通过称为“反向遗传学”的过程用于基因工程新病毒。我们现在建议设计SARS-CoV-2基因组，以产生1)表达绿色荧光蛋白（turboGFP）的SARS-CoV-2病毒和2)表达turboGFP而不是结构上重要的刺突蛋白的失活生物安全病毒（复制子）。当将复制子引入合适的细胞时，复制子会持续繁殖，但不会产生感染性病毒颗粒。然后，这两种病毒的工程形式可用于开发基于高通量荧光的检测方法，以监测病毒进入细胞及其复制。第一种方法可用于识别由候选疫苗诱导的抗体，这些抗体可中和病毒的传染性，也可用于识别允许逃避抗体反应的病毒突变。复制子可以在较低的控制水平上用于快速筛选抗病毒化合物，这将使更多的实验室能够参与药物发现工作。

The development of vaccines and antiviral drugs against SARS-CoV-2 is hampered by the requirement to grow the virus under high-containment, which is limited to few laboratories worldwide. We have recently established a rapid procedure to produce a DNA copy of the SARS-CoV-2 RNA genome, that can be used to genetically engineer new viruses via a process termed "reverse genetics". We now propose to engineer the SARS-CoV-2 genome to produce 1) a SARS-CoV-2 virus that expresses a green fluorescent protein (turboGFP) and 2) a disabled biosafe form of the virus (replicon) that expresses turboGFP instead of the structurally important spike protein. When introduced into suitable cells, the replicon reproduces continously but does not produce infectious virus particles. The two engineered forms of the virus can then be used to develop high-throughput fluorescence based assays to monitor the entry of the virus into cells and its replication. The first can be used to identify antibodies induced by vaccine candidates that neutralise virus infectivity, and also viral mutations that allow escape from the antibody response. The replicon can be used for rapid screening of antiviral compounds, at a lower containment level, which would allow many more laboratories to participate in drug discovery efforts.