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A versatile reverse genetics platform for SARS-CoV-2 and other positive-strand RNA viruses.
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.
  • Amarilla AA; School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD, Australia.
  • Sng JDJ; School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD, Australia.
  • Parry R; School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD, Australia.
  • Deerain JM; Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC, Australia.
  • Potter JR; School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD, Australia.
  • Setoh YX; School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD, Australia.
  • Rawle DJ; Microbiology and Molecular Epidemiology Division, Environmental Health Institute, National Environmental Agency, Singapore, Singapore.
  • Le TT; QIMR Berghofer Medical Research Institute, Herston, QLD, Australia.
  • Modhiran N; QIMR Berghofer Medical Research Institute, Herston, QLD, Australia.
  • Wang X; School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD, Australia.
  • Peng NYG; School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD, Australia.
  • Torres FJ; School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD, Australia.
  • Pyke A; School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD, Australia.
  • Harrison JJ; Queensland Health Forensic & Scientific Services, Queensland Department of Health, Coopers Plains, QLD, Australia.
  • Freney ME; School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD, Australia.
  • Liang B; School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD, Australia.
  • McMillan CLD; School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD, Australia.
  • Cheung STM; School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD, Australia.
  • Guevara DJDC; School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD, Australia.
  • Hardy JM; School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD, Australia.
  • Bettington M; Infection & Immunity Program, Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, Australia.
  • Muller DA; School of Medicine, University of Queensland, Kelvin Grove, QLD, Australia.
  • Coulibaly F; School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD, Australia.
  • Moore F; Infection & Immunity Program, Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, Australia.
  • Hall RA; Queensland Health Forensic & Scientific Services, Queensland Department of Health, Coopers Plains, QLD, Australia.
  • Young PR; School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD, Australia.
  • Mackenzie JM; Australian Infectious Diseases Research Centre, Global Virus Network Centre of Excellence, Brisbane, QLD, Australia.
  • Hobson-Peters J; School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD, Australia.
  • Suhrbier A; Australian Infectious Diseases Research Centre, Global Virus Network Centre of Excellence, Brisbane, QLD, Australia.
  • Watterson D; Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC, Australia. jason.mackenzie@unimelb.edu.au.
  • Khromykh AA; School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD, Australia. j.peters2@uq.edu.au.
Nat Commun ; 12(1): 3431, 2021 06 08.
Article in English | MEDLINE | ID: covidwho-1262001
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.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: Reverse Genetics / SARS-CoV-2 Type of study: Prognostic study Limits: Animals / Humans Language: English Journal: Nat Commun Journal subject: Biology / Science Year: 2021 Document Type: Article Affiliation country: S41467-021-23779-5

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Full text: Available Collection: International databases Database: MEDLINE Main subject: Reverse Genetics / SARS-CoV-2 Type of study: Prognostic study Limits: Animals / Humans Language: English Journal: Nat Commun Journal subject: Biology / Science Year: 2021 Document Type: Article Affiliation country: S41467-021-23779-5