Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the current COVID-19 pandemic, is one of the biggest threats to public health. However, the dynamic of SARS-CoV-2 infection remains poorly understood. Replication-competent recombinant viruses expressing reporter genes provide valuable tools to investigate viral infection. Low levels of reporter gene expressed from previous reporter-expressing recombinant (r)SARS-CoV-2 in the locus of the open reading frame (ORF)7a protein have jeopardized their use to monitor the dynamic of SARS-CoV-2 infection in vitro or in vivo. Here, we report an alternative strategy where reporter genes were placed upstream of the highly expressed viral nucleocapsid (N) gene followed by a porcine tescherovirus (PTV-1) 2A proteolytic cleavage site. The higher levels of reporter expression using this strategy resulted in efficient visualization of rSARS-CoV-2 in infected cultured cells and excised lungs or whole organism of infected K18 human angiotensin converting enzyme 2 (hACE2) transgenic mice. Importantly, real-time viral infection was readily tracked using a noninvasive in vivo imaging system and allowed us to rapidly identify antibodies which are able to neutralize SARS-CoV-2 infection in vivo. Notably, these reporter-expressing rSARS-CoV-2, in which a viral gene was not deleted, not only retained wild-type (WT) virus-like pathogenicity in vivo but also exhibited high stability in vitro and in vivo, supporting their use to investigate viral infection, dissemination, pathogenesis, and therapeutic interventions for the treatment of SARS-CoV-2 in vivo.

严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 是当前 COVID-19 大流行的病原体，是对公共卫生的最大威胁之一。然而，SARS-CoV-2 感染的动态仍然知之甚少。表达报告基因的具有复制能力的重组病毒为研究病毒感染提供了有价值的工具。以前表达报告基因的重组 (r)SARS-CoV-2 在开放阅读框 (ORF) 7a 蛋白基因座中表达的报告基因水平低，已危及它们在体外监测 SARS-CoV-2 感染动态的用途或在体内。在这里，我们报告了一种替代策略，其中将报告基因置于高表达病毒核衣壳 (N) 基因的上游，然后是猪 tescherovirus (PTV-1) 2A 蛋白水解切割位点。使用这种策略更高水平的报告基因表达导致 rSARS-CoV-2 在受感染的培养细胞和切除的肺或受感染的 K18 人血管紧张素转换酶 2 (hACE2) 转基因小鼠的整个有机体中的有效可视化。重要的是，使用无创体内成像系统可以轻松跟踪实时病毒感染，并使我们能够快速识别能够在体内中和 SARS-CoV-2 感染的抗体。值得注意的是，这些表达报告基因的 rSARS-CoV-2，其中一个病毒基因没有被删除，不仅在体内保留了野生型 (WT) 病毒样致病性，而且在体外和体内都表现出很高的稳定性，支持它们的使用研究体内 SARS-CoV-2 的病毒感染、传播、发病机制和治疗干预措施。