A versatile reverse genetics platform for SARS-CoV-2 and other positive-strand RNA viruses
| Autor: | Daniel J. Rawle, Jessica J. Harrison, Nias Y. G. Peng, Julian D. J. Sng, Darwin J. Da Costa Guevara, Naphak Modhiran, Thuy T. Le, Benjamin Liang, David A. Muller, Fasséli Coulibaly, Joshua M. Deerain, Morgan E. Freney, Alberto A. Amarilla, Rhys Parry, Xiaohui Wang, Stacey T. M. Cheung, Daniel Watterson, Christopher L. D. McMillan, Jody Hobson-Peters, Yin Xiang Setoh, Jason M. Mackenzie, James R. Potter, Francisco J. Torres, Mark Bettington, Joshua M. Hardy, Alyssa T. Pyke, Roy A. Hall, Paul R. Young, Alexander A. Khromykh, Andreas Suhrbier, Frederick Moore |
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| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
Science viruses 030106 microbiology General Physics and Astronomy Alphavirus Genome Viral Virus-host interactions Virus Replication Polymerase Chain Reaction General Biochemistry Genetics and Molecular Biology Virus Article 03 medical and health sciences chemistry.chemical_compound Mice Viral Proteins Complementary DNA Chlorocebus aethiops Animals Humans Amino Acid Sequence Vero Cells Polymerase Furin Multidisciplinary biology Base Sequence SARS-CoV-2 RNA virus diseases General Chemistry biology.organism_classification Virology Reverse genetics Reverse Genetics 030104 developmental biology Culicidae HEK293 Cells RAW 264.7 Cells chemistry Viral replication Mutation biology.protein NIH 3T3 Cells Receptors Virus DNA |
| Zdroj: | Nature Communications Nature Communications, Vol 12, Iss 1, Pp 1-15 (2021) |
| ISSN: | 2041-1723 |
| Popis: | 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. Here the authors describe a simple reverse genetics method that relies on overlapping cDNA fragments for generation of positive-strand viruses including SARS-CoV-2 and characterize them in vitro and in vivo. |
| Databáze: | OpenAIRE |
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