A versatile reverse genetics platform for SARS-CoV-2 and other positive-strand RNA viruses
Alberto A. Amarilla, Julian D. J. Sng, Rhys Parry, Joshua M. Deerain, James R. Potter, Yin Xiang Setoh, Daniel J. Rawle, Thuy T. Le, Naphak Modhiran, Xiaohui Wang, Nias Y. G. Peng, Francisco J. Torres, Alyssa T. Pyke, Jessica J. Harrison, Morgan E. Freney, Benjamin Liang, Christopher L. D. McMillan, Stacey T. M. Cheung, Darwin J. Da Costa Guevara, Joshua M. Hardy, Mark Bettington, David A. Muller, Fasséli Coulibaly, Frederick Moore, Roy A. Hall, Paul R. Young, Jason M. Mackenzie, Jody Hobson‐Peters, Andreas Suhrbier, Daniel Watterson, Alexander A. Khromykh
Abstract
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.