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

Author

Listed:
  • Alberto A. Amarilla

    (University of Queensland)

  • Julian D. J. Sng

    (University of Queensland)

  • Rhys Parry

    (University of Queensland)

  • Joshua M. Deerain

    (University of Melbourne)

  • James R. Potter

    (University of Queensland)

  • Yin Xiang Setoh

    (University of Queensland
    National Environmental Agency)

  • Daniel J. Rawle

    (QIMR Berghofer Medical Research Institute)

  • Thuy T. Le

    (QIMR Berghofer Medical Research Institute)

  • Naphak Modhiran

    (University of Queensland)

  • Xiaohui Wang

    (University of Queensland)

  • Nias Y. G. Peng

    (University of Queensland)

  • Francisco J. Torres

    (University of Queensland)

  • Alyssa Pyke

    (Queensland Department of Health)

  • Jessica J. Harrison

    (University of Queensland)

  • Morgan E. Freney

    (University of Queensland)

  • Benjamin Liang

    (University of Queensland)

  • Christopher L. D. McMillan

    (University of Queensland)

  • Stacey T. M. Cheung

    (University of Queensland)

  • Darwin J. Da Costa Guevara

    (University of Queensland)

  • Joshua M. Hardy

    (Monash University)

  • Mark Bettington

    (University of Queensland)

  • David A. Muller

    (University of Queensland)

  • Fasséli Coulibaly

    (Monash University)

  • Frederick Moore

    (Queensland Department of Health)

  • Roy A. Hall

    (University of Queensland
    Global Virus Network Centre of Excellence)

  • Paul R. Young

    (University of Queensland
    Global Virus Network Centre of Excellence)

  • Jason M. Mackenzie

    (University of Melbourne)

  • Jody Hobson-Peters

    (University of Queensland
    Global Virus Network Centre of Excellence)

  • Andreas Suhrbier

    (QIMR Berghofer Medical Research Institute
    Global Virus Network Centre of Excellence)

  • Daniel Watterson

    (University of Queensland
    Global Virus Network Centre of Excellence)

  • Alexander A. Khromykh

    (University of Queensland
    Global Virus Network Centre of Excellence)

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.

Suggested Citation

  • 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 & Francisc, 2021. "A versatile reverse genetics platform for SARS-CoV-2 and other positive-strand RNA viruses," Nature Communications, Nature, vol. 12(1), pages 1-15, December.
    • Handle: RePEc:nat:natcom:v:12:y:2021:i:1:d:10.1038_s41467-021-23779-5
    DOI: 10.1038/s41467-021-23779-5
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    Cited by:

    1. Taha Y. Taha & Irene P. Chen & Jennifer M. Hayashi & Takako Tabata & Keith Walcott & Gabriella R. Kimmerly & Abdullah M. Syed & Alison Ciling & Rahul K. Suryawanshi & Hannah S. Martin & Bryan H. Bach , 2023. "Rapid assembly of SARS-CoV-2 genomes reveals attenuation of the Omicron BA.1 variant through NSP6," Nature Communications, Nature, vol. 14(1), pages 1-13, December.
    2. Wen Juan Tu & Michelle Melino & Jenny Dunn & Robert D. McCuaig & Helle Bielefeldt-Ohmann & Sofiya Tsimbalyuk & Jade K. Forwood & Taniya Ahuja & John Vandermeide & Xiao Tan & Minh Tran & Quan Nguyen & , 2023. "In vivo inhibition of nuclear ACE2 translocation protects against SARS-CoV-2 replication and lung damage through epigenetic imprinting," Nature Communications, Nature, vol. 14(1), pages 1-21, December.

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