Generation of a SARS-CoV-2 Reverse Genetics System and Novel Human Lung Cell Lines That Exhibit High Virus-Induced Cytopathology
The global COVID-19 pandemic continues with continued cases worldwide and the emergence of new SARS-CoV-2 variants. In our study, we have developed novel tools with applications for screening antivirals, identifying virus–host dependencies, and characterizing viral variants. Using reverse genetics,...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-05-01
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| Series: | Viruses |
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| Online Access: | https://www.mdpi.com/1999-4915/15/6/1281 |
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| author | Juveriya Qamar Khan Megha Rohamare Karthic Rajamanickam Kalpana K. Bhanumathy Jocelyne Lew Anil Kumar Darryl Falzarano Franco J. Vizeacoumar Joyce A. Wilson |
| author_facet | Juveriya Qamar Khan Megha Rohamare Karthic Rajamanickam Kalpana K. Bhanumathy Jocelyne Lew Anil Kumar Darryl Falzarano Franco J. Vizeacoumar Joyce A. Wilson |
| author_sort | Juveriya Qamar Khan |
| collection | DOAJ |
| description | The global COVID-19 pandemic continues with continued cases worldwide and the emergence of new SARS-CoV-2 variants. In our study, we have developed novel tools with applications for screening antivirals, identifying virus–host dependencies, and characterizing viral variants. Using reverse genetics, we rescued SARS-CoV-2 Wuhan1 (D614G variant) wild type (WTFL) and reporter virus (NLucFL) using molecular BAC clones. The replication kinetics, plaque morphology, and titers were comparable between viruses rescued from molecular clones and a clinical isolate (VIDO-01 strain). Furthermore, the reporter SARS-CoV-2 NLucFL virus exhibited robust luciferase values over the time course of infection and was used to develop a rapid antiviral assay using remdesivir as proof-of-principle. In addition, as a tool to study lung-relevant virus–host interactions, we established novel human lung cell lines that support SARS-CoV-2 infection with high virus-induced cytopathology. Six lung cell lines (NCI-H23, A549, NCI-H1703, NCI-H520, NCI-H226, and HCC827) and HEK293T cells were transduced to stably express ACE2 and tested for their ability to support virus infection. A549ACE2 B1 and HEK293TACE2 A2 cell lines exhibited more than 70% virus-induced cell death, and a novel lung cell line, NCI-H23ACE2 A3, showed about ~99% cell death post-infection. These cell lines are ideal for assays relying on live–dead selection, such as CRISPR knockout and activation screens. |
| first_indexed | 2024-03-11T01:49:59Z |
| format | Article |
| id | doaj.art-559ad5e3ebf943dcb8f26ba43a055a2e |
| institution | Directory Open Access Journal |
| issn | 1999-4915 |
| language | English |
| last_indexed | 2024-03-11T01:49:59Z |
| publishDate | 2023-05-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Viruses |
| spelling | doaj.art-559ad5e3ebf943dcb8f26ba43a055a2e2023-11-18T13:01:34ZengMDPI AGViruses1999-49152023-05-01156128110.3390/v15061281Generation of a SARS-CoV-2 Reverse Genetics System and Novel Human Lung Cell Lines That Exhibit High Virus-Induced CytopathologyJuveriya Qamar Khan0Megha Rohamare1Karthic Rajamanickam2Kalpana K. Bhanumathy3Jocelyne Lew4Anil Kumar5Darryl Falzarano6Franco J. Vizeacoumar7Joyce A. Wilson8Department of Biochemistry, Microbiology, and Immunology, University of Saskatchewan, Saskatoon, SK S7N 5E5, CanadaDepartment of Biochemistry, Microbiology, and Immunology, University of Saskatchewan, Saskatoon, SK S7N 5E5, CanadaDivision of Oncology, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5E5, CanadaDivision of Oncology, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5E5, CanadaVaccine and Infectious Disease Organization (VIDO), University of Saskatchewan, Saskatoon, SK S7N 5E3, CanadaDepartment of Biochemistry, Microbiology, and Immunology, University of Saskatchewan, Saskatoon, SK S7N 5E5, CanadaVaccine and Infectious Disease Organization (VIDO), University of Saskatchewan, Saskatoon, SK S7N 5E3, CanadaDivision of Oncology, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5E5, CanadaDepartment of Biochemistry, Microbiology, and Immunology, University of Saskatchewan, Saskatoon, SK S7N 5E5, CanadaThe global COVID-19 pandemic continues with continued cases worldwide and the emergence of new SARS-CoV-2 variants. In our study, we have developed novel tools with applications for screening antivirals, identifying virus–host dependencies, and characterizing viral variants. Using reverse genetics, we rescued SARS-CoV-2 Wuhan1 (D614G variant) wild type (WTFL) and reporter virus (NLucFL) using molecular BAC clones. The replication kinetics, plaque morphology, and titers were comparable between viruses rescued from molecular clones and a clinical isolate (VIDO-01 strain). Furthermore, the reporter SARS-CoV-2 NLucFL virus exhibited robust luciferase values over the time course of infection and was used to develop a rapid antiviral assay using remdesivir as proof-of-principle. In addition, as a tool to study lung-relevant virus–host interactions, we established novel human lung cell lines that support SARS-CoV-2 infection with high virus-induced cytopathology. Six lung cell lines (NCI-H23, A549, NCI-H1703, NCI-H520, NCI-H226, and HCC827) and HEK293T cells were transduced to stably express ACE2 and tested for their ability to support virus infection. A549ACE2 B1 and HEK293TACE2 A2 cell lines exhibited more than 70% virus-induced cell death, and a novel lung cell line, NCI-H23ACE2 A3, showed about ~99% cell death post-infection. These cell lines are ideal for assays relying on live–dead selection, such as CRISPR knockout and activation screens.https://www.mdpi.com/1999-4915/15/6/1281SARS-CoV-2human lung cell linesCOVID-19reverse geneticsreporter viruses |
| spellingShingle | Juveriya Qamar Khan Megha Rohamare Karthic Rajamanickam Kalpana K. Bhanumathy Jocelyne Lew Anil Kumar Darryl Falzarano Franco J. Vizeacoumar Joyce A. Wilson Generation of a SARS-CoV-2 Reverse Genetics System and Novel Human Lung Cell Lines That Exhibit High Virus-Induced Cytopathology Viruses SARS-CoV-2 human lung cell lines COVID-19 reverse genetics reporter viruses |
| title | Generation of a SARS-CoV-2 Reverse Genetics System and Novel Human Lung Cell Lines That Exhibit High Virus-Induced Cytopathology |
| title_full | Generation of a SARS-CoV-2 Reverse Genetics System and Novel Human Lung Cell Lines That Exhibit High Virus-Induced Cytopathology |
| title_fullStr | Generation of a SARS-CoV-2 Reverse Genetics System and Novel Human Lung Cell Lines That Exhibit High Virus-Induced Cytopathology |
| title_full_unstemmed | Generation of a SARS-CoV-2 Reverse Genetics System and Novel Human Lung Cell Lines That Exhibit High Virus-Induced Cytopathology |
| title_short | Generation of a SARS-CoV-2 Reverse Genetics System and Novel Human Lung Cell Lines That Exhibit High Virus-Induced Cytopathology |
| title_sort | generation of a sars cov 2 reverse genetics system and novel human lung cell lines that exhibit high virus induced cytopathology |
| topic | SARS-CoV-2 human lung cell lines COVID-19 reverse genetics reporter viruses |
| url | https://www.mdpi.com/1999-4915/15/6/1281 |
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