An Infectious cDNA Clone of SARS-CoV-2

Xuping Xie; Antonio Muruato; Kumari G Lokugamage; Krishna Narayanan; Xianwen Zhang; Jing Zou; Jianying Liu; Craig Schindewolf; Nathen E Bopp; Patricia V Aguilar; Kenneth S Plante; Scott C Weaver; Shinji Makino; James W LeDuc; Vineet D Menachery; Pei-Yong Shi

doi:10.1016/j.chom.2020.04.004

An Infectious cDNA Clone of SARS-CoV-2

Cell Host Microbe. 2020 May 13;27(5):841-848.e3. doi: 10.1016/j.chom.2020.04.004. Epub 2020 Apr 13.

Authors

Xuping Xie¹, Antonio Muruato², Kumari G Lokugamage³, Krishna Narayanan³, Xianwen Zhang⁴, Jing Zou⁴, Jianying Liu³, Craig Schindewolf³, Nathen E Bopp⁵, Patricia V Aguilar⁶, Kenneth S Plante⁷, Scott C Weaver⁸, Shinji Makino⁹, James W LeDuc¹⁰, Vineet D Menachery¹¹, Pei-Yong Shi¹²

Affiliations

¹ Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA. Electronic address: xuxie@UTMB.edu.
² Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA; Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA.
³ Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA.
⁴ Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA.
⁵ Department of Pathology, University of Texas Medical Branch, Galveston, TX, USA.
⁶ Department of Pathology, University of Texas Medical Branch, Galveston, TX, USA; World Reference Center for Emerging Viruses and Arboviruses, University of Texas Medical Branch, Galveston, TX, USA; Institute for Human Infection and Immunity, University of Texas Medical Branch, Galveston, TX, USA.
⁷ Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA; World Reference Center for Emerging Viruses and Arboviruses, University of Texas Medical Branch, Galveston, TX, USA.
⁸ Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA; World Reference Center for Emerging Viruses and Arboviruses, University of Texas Medical Branch, Galveston, TX, USA; Institute for Human Infection and Immunity, University of Texas Medical Branch, Galveston, TX, USA; Institute for Translational Sciences, University of Texas Medical Branch, Galveston, TX, USA; Department of Pathology and Center for Biodefense & Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, TX, USA; Sealy Institute for Vaccine Sciences, University of Texas Medical Branch, Galveston, TX, USA.
⁹ Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA; Institute for Human Infection and Immunity, University of Texas Medical Branch, Galveston, TX, USA; Sealy Center for Structural Biology & Molecular Biophysics, University of Texas Medical Branch, Galveston, TX, USA.
¹⁰ Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA; Galveston National Laboratory, University of Texas Medical Branch, Galveston, TX, USA.
¹¹ Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA; Institute for Human Infection and Immunity, University of Texas Medical Branch, Galveston, TX, USA; Department of Pathology and Center for Biodefense & Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, TX, USA. Electronic address: vimenach@UTMB.edu.
¹² Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA; Institute for Human Infection and Immunity, University of Texas Medical Branch, Galveston, TX, USA; Sealy Institute for Vaccine Sciences, University of Texas Medical Branch, Galveston, TX, USA; Sealy Center for Structural Biology & Molecular Biophysics, University of Texas Medical Branch, Galveston, TX, USA; Department of Pharmacology & Toxicology, University of Texas Medical Branch, Galveston, TX, USA. Electronic address: peshi@UTMB.edu.

Abstract

The ongoing pandemic of COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), underscores the urgency to develop experimental systems for studying this virus and identifying countermeasures. We report a reverse genetic system for SARS-CoV-2. Seven complimentary DNA (cDNA) fragments spanning the SARS-CoV-2 genome were assembled into a full-genome cDNA. RNA transcribed from the full-genome cDNA was highly infectious after electroporation into cells, producing 2.9 × 10⁶ plaque-forming unit (PFU)/mL of virus. Compared with a clinical isolate, the infectious-clone-derived SARS-CoV-2 (icSARS-CoV-2) exhibited similar plaque morphology, viral RNA profile, and replication kinetics. Additionally, icSARS-CoV-2 retained engineered molecular markers and did not acquire other mutations. We generated a stable mNeonGreen SARS-CoV-2 (icSARS-CoV-2-mNG) by introducing this reporter gene into ORF7 of the viral genome. icSARS-CoV-2-mNG was successfully used to evaluate the antiviral activities of interferon (IFN). Collectively, the reverse genetic system and reporter virus provide key reagents to study SARS-CoV-2 and develop countermeasures.

Keywords: COVID-19; SARS-CoV; SARS-CoV-2; antiviral; coronavirus; vaccine.

Publication types

Research Support, N.I.H., Extramural

MeSH terms

Animals
Antiviral Agents / therapeutic use
Betacoronavirus / genetics*
Betacoronavirus / pathogenicity*
COVID-19
Chlorocebus aethiops
Clone Cells
Coronavirus Infections / drug therapy
Coronavirus Infections / virology*
DNA, Complementary / genetics*
Genes, Reporter / genetics
Genome, Viral / genetics
Interferons / therapeutic use
Organisms, Genetically Modified / genetics*
Organisms, Genetically Modified / pathogenicity*
Pandemics
Pneumonia, Viral / drug therapy
Pneumonia, Viral / virology*
RNA, Viral / genetics
SARS-CoV-2
Vero Cells / virology
Virus Replication / physiology

Substances

Antiviral Agents
DNA, Complementary
RNA, Viral
Interferons

Abstract

Publication types

MeSH terms

Substances

Grants and funding