Nat Commun


. 2021 Jun 8;12(1):3431.
doi: 10.1038/s41467-021-23779-5.
A versatile reverse genetics platform for SARS-CoV-2 and other positive-strand RNA viruses


Alberto A Amarilla # 1 , Julian D J Sng # 1 , Rhys Parry # 1 , Joshua M Deerain # 2 , James R Potter # 1 , Yin Xiang Setoh # 1 3 , Daniel J Rawle 4 , Thuy T Le 4 , Naphak Modhiran 1 , Xiaohui Wang 1 , Nias Y G Peng 1 , Francisco J Torres 1 , Alyssa Pyke 5 , Jessica J Harrison 1 , Morgan E Freney 1 , Benjamin Liang 1 , Christopher L D McMillan 1 , Stacey T M Cheung 1 , Darwin J Da Costa Guevara 1 , Joshua M Hardy 6 , Mark Bettington 7 , David A Muller 1 , Fasséli Coulibaly 6 , Frederick Moore 5 , Roy A Hall 1 8 , Paul R Young 1 8 , Jason M Mackenzie 9 , Jody Hobson-Peters 10 11 , Andreas Suhrbier 12 13 , Daniel Watterson 14 15 , Alexander A Khromykh 16 17



Affiliations

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.