期刊
VIRUSES-BASEL
卷 15, 期 6, 页码 -出版社
MDPI
DOI: 10.3390/v15061281
关键词
SARS-CoV-2; human lung cell lines; COVID-19; reverse genetics; reporter viruses
类别
The study developed novel tools for screening antivirals, identifying virus-host dependencies, and characterizing viral variants. The researchers used reverse genetics to rescue SARS-CoV-2 wild type and reporter virus, and found comparable replication kinetics and characteristics between these viruses and a clinical isolate. They also established human lung cell lines that support SARS-CoV-2 infection and have high virus-induced cytopathology, making them ideal for live-dead selection assays.
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 similar to 99% cell death post-infection. These cell lines are ideal for assays relying on live-dead selection, such as CRISPR knockout and activation screens.
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