A versatile reverse genetics platform for SARS-CoV-2 and other positive-strand RNA viruses
Alberto A Amarilla, Julian DJ Sng, Rhys Parry, Joshua M Deerain, James R Potter, Yin Xiang Setoh, Daniel J Rawle, Thuy T Le, Naphak Modhiran, Xiaohui Wang, Nias YG Peng, Francisco J Torres, Alyssa Pyke, Jessica J Harrison, Morgan E Freney, Benjamin Liang, Christopher LD McMillan, Stacey TM Cheung, Darwin J Da Costa Guevara, Joshua M Hardy Show all
NATURE COMMUNICATIONS | NATURE RESEARCH | Published : 2021
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 comparab..View full abstract
Awarded by National Health and Medical Research Council of Australia
Awarded by Medical Research Future Fund
The authors thank Australian Infectious Diseases Research Centre for a seeding grant to A.A.K. and A.S. for establishing SARS-CoV-2 CPER and a seeding grant to R.A.H., J.H.-P., and A.S. for establishing CASV CPER. A.S. was supported by an Investigator Grant from the National Health and Medical Research Council of Australia (APP1173880). The authors acknowledge funding support from the Medical Research Future Fund (APP1202445-2020 MRFF Novel Coronavirus Vaccine Development Grant to D.W. and P.R.Y.) and Associate Prof. Keith Chappell for project support. The authors thank Clive Berghofer and Lyn Brazil (and others) for their generous philanthropic donations that funded the setup of the PC3 (BSL3) SARS-CoV-2 research facility at QIMR Berghofer MRI. The authors thank Dr I. Anraku for his assistance in managing the PC3 facility at QIMR Berghofer MRI, and Dr Viviana Lutzky for her help with proof reading. Elements of Fig. 1a were generated with BioRender.com. The authors thank Queensland Health, Brisbane, for providing the SARS-CoV-2 virus isolates.