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Generation of a SARS-CoV-2 reverse genetics system and novel human lung cell lines that exhibit high virus-induced cytopathology (preprint)
biorxiv; 2023.
Preprint
in English
| bioRxiv | ID: ppzbmed-10.1101.2023.03.08.531833
ABSTRACT
The global COVID-19 pandemic continues with an increasing number of 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 rescued molecular clones and a clinical isolate (VIDO strain), thus providing confidence that the rescued viruses can be used as effective replication tools. 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 and are currently being used in CRISPR knockout and activation screens in our lab.
Full text:
Available
Collection:
Preprints
Database:
bioRxiv
Main subject:
Tumor Virus Infections
/
Death
/
COVID-19
/
Graft vs Host Disease
Language:
English
Year:
2023
Document Type:
Preprint
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