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  • A versatile reverse genetics platform for SARS-CoV-2 and other positive-strand RNA viruses.

A versatile reverse genetics platform for SARS-CoV-2 and other positive-strand RNA viruses.

Nature communications (2021-06-10)
Alberto A Amarilla, Julian D J Sng, Rhys Parry, Joshua M Deerain, James R Potter, Yin Xiang Setoh, Daniel J Rawle, Thuy T Le, Naphak Modhiran, Xiaohui Wang, Nias Y G Peng, Francisco J Torres, Alyssa Pyke, Jessica J Harrison, Morgan E Freney, Benjamin Liang, Christopher L D McMillan, Stacey T M Cheung, Darwin J Da Costa Guevara, Joshua M Hardy, Mark Bettington, David A Muller, Fasséli Coulibaly, Frederick Moore, Roy A Hall, Paul R Young, Jason M Mackenzie, Jody Hobson-Peters, Andreas Suhrbier, Daniel Watterson, Alexander A Khromykh
摘要

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.

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Sigma-Aldrich
抗肌动蛋白抗体 兔抗, affinity isolated antibody, buffered aqueous solution
Sigma-Aldrich
Anti-Norovirus (MNV-1) Antibody, clone 5C4.10, clone 5C4.10, from mouse