A versatile reverse genetics platform for SARS-CoV-2 and other positive-strand RNA viruses
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Published:2021-06-08
Issue:1
Volume:12
Page:
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ISSN:2041-1723
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Container-title:Nature Communications
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language:en
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Short-container-title:Nat Commun
Author:
Amarilla Alberto A., Sng Julian D. J., Parry RhysORCID, Deerain Joshua M., Potter James R., Setoh Yin XiangORCID, Rawle Daniel J.ORCID, Le Thuy T., Modhiran NaphakORCID, Wang Xiaohui, Peng Nias Y. G.ORCID, Torres Francisco J.ORCID, Pyke AlyssaORCID, Harrison Jessica J., Freney Morgan E., Liang BenjaminORCID, McMillan Christopher L. D.ORCID, Cheung Stacey T. M., Guevara Darwin J. Da Costa, Hardy Joshua M.ORCID, Bettington Mark, Muller David A., Coulibaly FasséliORCID, Moore Frederick, Hall Roy A., Young Paul R., Mackenzie Jason M.ORCID, Hobson-Peters JodyORCID, Suhrbier AndreasORCID, Watterson DanielORCID, Khromykh Alexander A.ORCID
Abstract
AbstractThe 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.
Funder
Australian Infectious Diseases Research Centre, COVID-19 seed grant.
Publisher
Springer Science and Business Media LLC
Subject
General Physics and Astronomy,General Biochemistry, Genetics and Molecular Biology,General Chemistry
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