A simple reverse genetics method to generate recombinant coronaviruses

Engineering recombinant viruses is a pre-eminent tool for deciphering the biology of emerging viral pathogens such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the large size of coronavirus genomes renders the current reverse genetics methods challenging. Here, we describe a simple method based on infectious subgenomic amplicons (ISA) technology to generate recombinant infectious coronaviruses with no need for reconstruction of the complete genomic cDNA and apply this method to SARS-CoV-2 and also to the feline enteric coronavirus. In both cases we rescue wild-type viruses with biological characteristics similar to original strains. Specific mutations and fluorescent red reporter genes can be readily incorporated into the SARS-CoV-2 genome enabling the generation of a genomic variants and fluorescent reporter strains for in vivo experiments, serological diagnosis, and antiviral assays. The swiftness and simplicity of the ISA method has the potential to facilitate the advance of coronavirus reverse genetics studies, to explore the molecular biological properties of the SARS-CoV-2 variants, and to accelerate the development of effective therapeutic reagents.

Automated summary

This study describes a simple method using infectious subgenomic amplicons (ISA) technology to generate recombinant infectious coronaviruses without the need for reconstruction of the complete genomic cDNA. This method has been successfully applied to SARS-CoV-2 and feline enteric coronavirus, enabling research on coronavirus reverse genetics, exploration of the molecular biological properties of SARS-CoV-2 variants, and acceleration of the development of effective therapeutic reagents.