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.
| Authors | Amarilla, Alberto A; Sng, Julian D J; Parry, Rhys; Deerain, Joshua M; Potter, James R; Setoh, Yin Xiang; Rawle, Daniel J; Le, Thuy T; Modhiran, Naphak; Wang, Xiaohui; Peng, Nias Y G; Torres, Francisco J; Pyke, Alyssa; Harrison, Jessica J; Freney, Morgan E; Liang, Benjamin; McMillan, Christopher L D; Cheung, Stacey T M; Guevara, Darwin J Da Costa; Hardy, Joshua M; Bettington, Mark; Muller, David A; Coulibaly, Fasséli; Moore, Frederick; Hall, Roy A; Young, Paul R; Mackenzie, Jason M; Hobson-Peters, Jody; Suhrbier, Andreas; Watterson, Daniel; Khromykh, Alexander A |
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| Journal | Nature Communications |
| Pages | 3431 |
| Volume | 12 |
| Date | 1/01/2021 |
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| URL | http://www.ncbi.nlm.nih.gov/pubmed/?term=10.1038/s41467-021-23779-5 |