4.6 Article

Generation and Characterization of Recombinant SARS-CoV-2 Expressing Reporter Genes

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JOURNAL OF VIROLOGY
卷 95, 期 7, 页码 -

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AMER SOC MICROBIOLOGY
DOI: 10.1128/JVI.02209-20

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SARS-CoV-2; coronavirus; fluorescent; luciferase; reporter virus; reverse genetics

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The emergence of SARS-CoV-2 has had a devastating impact on global public health and economies, with limited therapeutic options available. Researchers have developed reporter-expressing rSARS-CoV-2 constructs to track viral infections, showing promise in identifying neutralizing antibodies or antiviral drugs.
The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen responsible for coronavirus disease 2019 (COVID-19), has devastated public health services and economies worldwide. Despite global efforts to contain the COVID-19 pandemic, SARS-CoV-2 is now found in over 200 countries and has caused a death toll of over 1 million human lives as of November 2020. To date, only one Food and Drug Administration (FDA)-approved therapeutic drug (remdesivir) and a monoclonal antibody (MAb), bamlanivimab, are available for the treatment of SARS-CoV-2. As with other viruses, studying SARS-CoV-2 requires the use of secondary approaches to detect the presence of the virus in infected cells. To overcome this limitation, we have generated replication-competent recombinant SARS-CoV-2 (rSARS-CoV-2) constructs expressing fluorescent (Venus or mCherry) or bioluminescent (Nluc) reporter genes. Vero E6 cells infected with reporter-expressing rSARS-CoV-2 can be easily detected via fluorescence or luciferase expression and display a good correlation between reporter gene expression and viral replication. Moreover, rSARS-CoV-2 expressing reporter genes has plaque sizes and growth kinetics comparable to those of wild-type virus, rSARS-CoV-2/WT. We used these reporter-expressing rSARS-CoV-2 constructs to demonstrate their feasibility to identify neutralizing antibodies (NAbs) or antiviral drugs. Our results demonstrate that reporter-expressing rSARS-CoV-2 represents an excellent option to identify therapeutics for the treatment of SARS-CoV-2, where reporter gene expression can be used as a valid surrogate to track viral infection. Moreover, the ability to manipulate the viral genome opens the feasibility of generating viruses expressing foreign genes for their use as vaccines for the treatment of SARS-CoV-2 infection. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen that causes coronavirus disease 2019 (COVID-19), has significantly impacted the human health and economic status worldwide. There is an urgent need to identify effective prophylactics and therapeutics for the treatment of SARS-CoV-2 infection and associated COVID-19. The use of fluorescent -protein-or luciferase-expressing reporter viruses has significantly advanced viral research. Here, we generated recombinant SARSCoV-2 (rSARS-CoV-2) constructs expressing fluorescent (Venus and mCherry) or luciferase (Nluc) reporter genes and demonstrated that these viruses represent an excellent option to track viral infections in vitro. Importantly, reporter-expressing rSARS-CoV-2 constructs display growth kinetics and plaque phenotypes similar to those of their wild-type counterpart (rSARS-CoV-2/WT), demonstrating their usefulness for identifying drugs and/or neutralizing antibodies (NAbs) for the therapeutic treatment of SARS-CoV-2. Henceforth, these reporter-expressing rSARS-CoV-2 constructs can be used to interrogate large libraries of compounds and/or monoclonal antibodies (MAb), in high-throughput screening settings, to identify those with therapeutic potential against SARS-CoV-2.

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