Optimization of an infectious subgenomic amplicons reverse genetics protocol for the rescue of synthetic coronaviruses

Reverse genetics (rg) systems are indispensable tools for investigating the pathogenesis of RNA viruses, facilitating vaccine design, and advancing antiviral therapeutic strategies. In this study, we optimized the Infectious Subgenomic Amplicons (ISA) method for generating synthetic r-wt SARS-CoV-2 Wuhan-Hu-1. This system was validated by demonstrating the successful rescue of infectious viral particles from overlapping DNA fragments and their propagation in vitro. Sequencing confirmed 100 % identity of the recovered virus with the Wuhan-Hu-1 reference genome. Importantly, in vivo experiments using K18-hACE2 mice revealed that the r-wt SARS-CoV-2 Wuhan-Hu-1 strain caused clinical symptoms, weight loss, and mortality comparable to those induced by a virulent SARS-CoV-2 field variant. This ISA rg method offers a rapid and reproducible approach to generating synthetic coronaviruses, with potential applications in pathogenesis studies, antiviral testing, and vaccine development.