@article{fac4656af1be4118a421938b041577e7,
title = "A versatile reverse genetics platform for SARS-CoV-2 and other positive-strand RNA viruses",
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.",
keywords = "RNA viruses, Viral vector, covid-19, SARS coronavirus, CoV-2, Reverse genetics",
author = "Amarilla, {Alberto A.} and Sng, {Julian D.J.} and Rhys Parry and Deerain, {Joshua M.} and Potter, {James R.} and Setoh, {Yin Xiang} and Rawle, {Daniel J.} and Le, {Thuy T.} and Naphak Modhiran and Xiaohui Wang and Peng, {Nias Y.G.} and Torres, {Francisco J.} and Alyssa Pyke and Harrison, {Jessica J.} and Freney, {Morgan E.} and Benjamin Liang and McMillan, {Christopher L.D.} and Cheung, {Stacey T.M.} and Guevara, {Darwin J.Da Costa} and Hardy, {Joshua M.} and Mark Bettington and Muller, {David A.} and Fass{\'e}li Coulibaly and Frederick Moore and Hall, {Roy A.} and Young, {Paul R.} and Mackenzie, {Jason M.} and Jody Hobson-Peters and Andreas Suhrbier and Daniel Watterson and Khromykh, {Alexander A.}",
note = "Funding Information: 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. Publisher Copyright: {\textcopyright} 2021, The Author(s). Copyright: Copyright 2021 Elsevier B.V., All rights reserved.",
year = "2021",
month = dec,
doi = "10.1038/s41467-021-23779-5",
language = "English",
volume = "12",
journal = "Nature Communications",
issn = "2041-1723",
publisher = "Nature Publishing Group",
number = "1",
}