A versatile reverse genetics platform for SARS-CoV-2 and other positive-strand RNA viruses

Amarilla, Alberto A.; Sng, Julian D. J.; Parry, Rhys; Deerain, Joshua M.; Potter, James R.; Setoh, Yin Xiang; Rawle, Daniel J.; Le, Thuy T.; Modhiran, Naphak; Wang, Xiaohui; Peng, Nias Y. G.; Torres, Francisco J.; Pyke, Alyssa; Harrison, Jessica J.; Freney, Morgan E.; Liang, Benjamin; McMillan, Christopher L. D.; Cheung, Stacey T. M.; Guevara, Darwin J. Da Costa; Hardy, Joshua M.; Bettington, Mark; Muller, David A.; Coulibaly, Fasséli; Moore, Frederick; Hall, Roy A.; Young, Paul R.; Mackenzie, Jason M.; Hobson-Peters, Jody; Suhrbier, Andreas; Watterson, Daniel; Khromykh, Alexander A.

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We demonstrate that despite the large size of the viral RNA genome (~30 kb), infectious full-length cDNA is readily assembled in vitro by a circular polymerase extension reaction (CPER) methodology without the need for technically demanding intermediate steps. Overlapping cDNA fragments are generated from viral RNA and assembled together with a linker fragment containing CMV promoter into a circular full-length viral cDNA in a single reaction. Transfection of the circular cDNA into mammalian cells results in the recovery of infectious SARS-CoV-2 virus that exhibits properties comparable to the parental virus in vitro and in vivo. CPER is also used to generate insect-specific Casuarina virus with ~20 kb genome and the human pathogens Ross River virus (Alphavirus) and Norovirus (Calicivirus), with the latter from a clinical sample. Additionally, reporter and mutant viruses are generated and employed to study virus replication and virus-receptor interactions.