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 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 () and Alexander A. Khromykh ()
Additional contact information
Alberto A. Amarilla: University of Queensland
Julian D. J. Sng: University of Queensland
Rhys Parry: University of Queensland
Joshua M. Deerain: University of Melbourne
James R. Potter: University of Queensland
Yin Xiang Setoh: University of Queensland
Daniel J. Rawle: QIMR Berghofer Medical Research Institute
Thuy T. Le: QIMR Berghofer Medical Research Institute
Naphak Modhiran: University of Queensland
Xiaohui Wang: University of Queensland
Nias Y. G. Peng: University of Queensland
Francisco J. Torres: University of Queensland
Alyssa Pyke: Queensland Department of Health
Jessica J. Harrison: University of Queensland
Morgan E. Freney: University of Queensland
Benjamin Liang: University of Queensland
Christopher L. D. McMillan: University of Queensland
Stacey T. M. Cheung: University of Queensland
Darwin J. Da Costa Guevara: University of Queensland
Joshua M. Hardy: Monash University
Mark Bettington: University of Queensland
David A. Muller: University of Queensland
Fasséli Coulibaly: Monash University
Frederick Moore: Queensland Department of Health
Roy A. Hall: University of Queensland
Paul R. Young: University of Queensland
Jason M. Mackenzie: University of Melbourne
Jody Hobson-Peters: University of Queensland
Andreas Suhrbier: QIMR Berghofer Medical Research Institute
Daniel Watterson: University of Queensland
Alexander A. Khromykh: University of Queensland

Nature Communications, 2021, vol. 12, issue 1, 1-15

Abstract: 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.

Date: 2021
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DOI: 10.1038/s41467-021-23779-5

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