Overcoming Culture Restriction for SARS-CoV-2 in Human Cells Facilitates the Screening of Compounds Inhibiting Viral Replication

Ramírez, Santseharay; Fernandez-Antunez, Carlota; Galli, Andrea; Underwood, Alexander; Pham, Long V.; Ryberg, Line Abildgaard; Feng, Shan; Pedersen, Martin Schou; Mikkelsen, Lotte S.; Belouzard, Sandrine; Dubuisson, Jean; Sølund, Christina; Weis, Nina; Gottwein, Judith M.; Fahnøe, Ulrik; Bukh, Jens

doi:10.1128/aac.00097-21

Cited by 73 publications

(84 citation statements)

References 87 publications

(131 reference statements)

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“…The virus seed stock used in this study was a passage 4 virus stock derived from the virus isolate SARS-CoV-2/human/Denmark/DK-AHH1/2020 [ 15 ]. Passage 1 and 2 were carried out in Vero E6 cells maintained in DMEM supplemented with 10% fetal bovine serum (Sigma), 100 U/mL penicillin and 100 µg/mL streptomycin (Sigma) [ 15 ]. Passage 3 and 4 were carried out in Vero (WHO) cells maintained in serum-free medium as described above.…”

Section: Methodsmentioning

confidence: 99%

“…SARS-CoV-2 infectivity titers were determined in a 96-well-based TCID 50 assay evaluated by immunostaining of SARS-CoV-2 Spike protein subunit 1 (S1) [ 15 , 17 , 18 ]. Briefly, a 10-fold virus dilution series was prepared in DMEM and 100 µL per well were added in four replicates to Vero E6 cells seeded the previous day in a flat-bottom 96-well plate (Nunc) at 1 × 10 4 cells/well.…”

Section: Methodsmentioning

confidence: 99%

“…Viral RNA was extracted as described above for RNA titration. Reverse transcriptase polymerase chain reaction (RT-PCR) and PCR conditions and primers are described elsewhere [ 15 ]. Amplicons were purified with the DNA clean and concentrator-25 kit (Zymo Research) according to the manufacturer’s instructions.…”

Section: Methodsmentioning

confidence: 99%

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SARS-CoV-2 Production in a Scalable High Cell Density Bioreactor

et al. 2021

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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has demonstrated the value of pursuing different vaccine strategies. Vaccines based on whole viruses, a widely used vaccine technology, depend on efficient virus production. This study aimed to establish SARS-CoV-2 production in the scalable packed-bed CelCradleTM 500-AP bioreactor. CelCradleTM 500-AP bottles with 0.5 L working volume and 5.5 g BioNOC™ II carriers were seeded with 1.5 × 108 Vero (WHO) cells, approved for vaccine production, in animal component-free medium and infected at a multiplicity of infection of 0.006 at a total cell number of 2.2–2.5 × 109 cells/bottle seven days post cell seeding. Among several tested conditions, two harvests per day and a virus production temperature of 33 °C resulted in the highest virus yield with a peak SARS-CoV-2 infectivity titer of 7.3 log10 50% tissue culture infectious dose (TCID50)/mL at 72 h post-infection. Six harvests had titers of ≥6.5 log10 TCID50/mL, and a total of 10.5 log10 TCID50 were produced in ~5 L. While trypsin was reported to enhance virus spread in cell culture, addition of 0.5% recombinant trypsin after infection did not improve virus yields. Overall, we demonstrated successful animal component-free production of SARS-CoV-2 in well-characterized Vero (WHO) cells in a scalable packed-bed bioreactor.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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SARS-CoV-2 Production in a Scalable High Cell Density Bioreactor

et al. 2021

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“…In these experiments the Danish SARS-CoV-2 isolate SARS-CoV-2/human/Denmark/DK-AHH1/2020 was used employing a 96-well plate based concentration-response antiviral treatment assay, allowing for multiple replicates per concentration, as described in "Materials and methods" and Supplementary Information (Figures S3 and S4) 20,21 . Seven replicates were measured at each concentration and a range of concentrations was evaluated to increase data accuracy when compared to the plaque-reduction assay, which was carried out in duplicates.…”

Section: Efficacy Of a Annua Extracts In Concentration-response Antiviral Treatment Assays In Veroe6 Cellsmentioning

confidence: 99%

“…The SARS-CoV-2/human/Denmark/DK-AHH1/2020 virus for cell culture studies was obtained following inoculation of VeroE6 cells with patient swab sample, virus propagation in VeroE6 cells and generation of a sequence confirmed 2nd viral passage stock with an infectivity titer of 5.5 log TCID50/mL as described in Ramirez et al 20 .…”

Section: Compoundsmentioning

confidence: 99%

In vitro efficacy of artemisinin-based treatments against SARS-CoV-2

Zhou

Gilmore

Ramírez

et al. 2021

Sci Rep

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Effective and affordable treatments for patients suffering from coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are needed. We report in vitro efficacy of Artemisia annua extracts as well as artemisinin, artesunate, and artemether against SARS-CoV-2. The latter two are approved active pharmaceutical ingredients of anti-malarial drugs. Concentration–response antiviral treatment assays, based on immunostaining of SARS-CoV-2 spike glycoprotein, revealed that treatment with all studied extracts and compounds inhibited SARS-CoV-2 infection of VeroE6 cells, human hepatoma Huh7.5 cells and human lung cancer A549-hACE2 cells, without obvious influence of the cell type on antiviral efficacy. In treatment assays, artesunate proved most potent (range of 50% effective concentrations (EC50) in different cell types: 7–12 µg/mL), followed by artemether (53–98 µg/mL), A. annua extracts (83–260 µg/mL) and artemisinin (151 to at least 208 µg/mL). The selectivity indices (SI), calculated based on treatment and cell viability assays, were mostly below 10 (range 2 to 54), suggesting a small therapeutic window. Time-of-addition experiments in A549-hACE2 cells revealed that artesunate targeted SARS-CoV-2 at the post-entry level. Peak plasma concentrations of artesunate exceeding EC50 values can be achieved. Clinical studies are required to further evaluate the utility of these compounds as COVID-19 treatment.

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Jumping from Fragment to Drug via Smart Scaffolds

Farahani¹,

França²,

Alapour³

et al. 2022

ChemMedChem

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A focused drug repurposing approach is described where an FDA‐approved drug is rationally selected for biological testing based on structural similarities to a fragment compound found to bind a target protein by an NMR screen. The approach is demonstrated by first screening a curated fragment library using 19F NMR to discover a quality binder to ACE2, the human receptor required for entry and infection by the SARS‐CoV‐2 virus. Based on this binder, a highly related scaffold was derived and used as a “smart scaffold” or template in a computer‐aided finger‐print search of a library of FDA‐approved or marketed drugs. The most interesting structural match involved the drug vortioxetine which was then experimentally shown by NMR spectroscopy to bind directly to human ACE2. Also, an ELISA assay showed that the drug inhibits the interaction of human ACE2 to the SARS‐CoV‐2 receptor‐binding‐domain (RBD). Moreover, our cell‐culture infectivity assay confirmed that vortioxetine is active against SARS‐CoV‐2 and inhibits viral replication. Thus, the use of “smart scaffolds” based on binders from fragment screens may have general utility for identifying candidates of FDA‐approved or marketed drugs as a rapid repurposing strategy. Similar approaches can be envisioned for other fields involving small‐molecule chemical applications.

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Overcoming Culture Restriction for SARS-CoV-2 in Human Cells Facilitates the Screening of Compounds Inhibiting Viral Replication

Cited by 73 publications

References 87 publications

SARS-CoV-2 Production in a Scalable High Cell Density Bioreactor

SARS-CoV-2 Production in a Scalable High Cell Density Bioreactor

In vitro efficacy of artemisinin-based treatments against SARS-CoV-2

Jumping from Fragment to Drug via Smart Scaffolds

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