tetano
Editor, Senior Moderator
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[SUP] #[/SUP][SUP] 1 [/SUP], Julian D J Sng[SUP] #[/SUP][SUP] 1 [/SUP], Rhys Parry[SUP] #[/SUP][SUP] 1 [/SUP], Joshua M Deerain[SUP] #[/SUP][SUP] 2 [/SUP], James R Potter[SUP] #[/SUP][SUP] 1 [/SUP], Yin Xiang Setoh[SUP] #[/SUP][SUP] 1 3 [/SUP], Daniel J Rawle[SUP] 4 [/SUP], Thuy T Le[SUP] 4 [/SUP], Naphak Modhiran[SUP] 1 [/SUP], Xiaohui Wang[SUP] 1 [/SUP], Nias Y G Peng[SUP] 1 [/SUP], Francisco J Torres[SUP] 1 [/SUP], Alyssa Pyke[SUP] 5 [/SUP], Jessica J Harrison[SUP] 1 [/SUP], Morgan E Freney[SUP] 1 [/SUP], Benjamin Liang[SUP] 1 [/SUP], Christopher L D McMillan[SUP] 1 [/SUP], Stacey T M Cheung[SUP] 1 [/SUP], Darwin J Da Costa Guevara[SUP] 1 [/SUP], Joshua M Hardy[SUP] 6 [/SUP], Mark Bettington[SUP] 7 [/SUP], David A Muller[SUP] 1 [/SUP], Fasséli Coulibaly[SUP] 6 [/SUP], Frederick Moore[SUP] 5 [/SUP], Roy A Hall[SUP] 1 8 [/SUP], Paul R Young[SUP] 1 8 [/SUP], Jason M Mackenzie[SUP] 9 [/SUP], Jody Hobson-Peters[SUP] 10 11 [/SUP], Andreas Suhrbier[SUP] 12 13 [/SUP], Daniel Watterson[SUP] 14 15 [/SUP], Alexander A Khromykh[SUP] 16 17 [/SUP]
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.
. 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[SUP] #[/SUP][SUP] 1 [/SUP], Julian D J Sng[SUP] #[/SUP][SUP] 1 [/SUP], Rhys Parry[SUP] #[/SUP][SUP] 1 [/SUP], Joshua M Deerain[SUP] #[/SUP][SUP] 2 [/SUP], James R Potter[SUP] #[/SUP][SUP] 1 [/SUP], Yin Xiang Setoh[SUP] #[/SUP][SUP] 1 3 [/SUP], Daniel J Rawle[SUP] 4 [/SUP], Thuy T Le[SUP] 4 [/SUP], Naphak Modhiran[SUP] 1 [/SUP], Xiaohui Wang[SUP] 1 [/SUP], Nias Y G Peng[SUP] 1 [/SUP], Francisco J Torres[SUP] 1 [/SUP], Alyssa Pyke[SUP] 5 [/SUP], Jessica J Harrison[SUP] 1 [/SUP], Morgan E Freney[SUP] 1 [/SUP], Benjamin Liang[SUP] 1 [/SUP], Christopher L D McMillan[SUP] 1 [/SUP], Stacey T M Cheung[SUP] 1 [/SUP], Darwin J Da Costa Guevara[SUP] 1 [/SUP], Joshua M Hardy[SUP] 6 [/SUP], Mark Bettington[SUP] 7 [/SUP], David A Muller[SUP] 1 [/SUP], Fasséli Coulibaly[SUP] 6 [/SUP], Frederick Moore[SUP] 5 [/SUP], Roy A Hall[SUP] 1 8 [/SUP], Paul R Young[SUP] 1 8 [/SUP], Jason M Mackenzie[SUP] 9 [/SUP], Jody Hobson-Peters[SUP] 10 11 [/SUP], Andreas Suhrbier[SUP] 12 13 [/SUP], Daniel Watterson[SUP] 14 15 [/SUP], Alexander A Khromykh[SUP] 16 17 [/SUP]
Affiliations
- PMID: 34103499
- DOI: 10.1038/s41467-021-23779-5
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.