Journal: Current Research in Virological Science

Article Title: Identification of broad anti-coronavirus chemical agents for repurposing against SARS-CoV-2 and variants of concern

doi: 10.1016/j.crviro.2022.100019

Figure Lengend Snippet: Workflow of the screening procedure Initially, we assessed a library of 5440 compounds for efficacy against hCoV-229E-GFP. This yielded 53 hits, which were tested for efficacy against hCoV-OC43 and SARS-CoV-2 in different cell lines, including Vero-E6, Huh7-ACE2, A549-ACE2 and HeLa-ACE2. In parallel, we determined the EC 50 , TC 50 and the ratio TC 50 /EC 50 , i.e., the quasi therapeutic index (TI). A selection of hits in advanced clinical state (approved and systemically administrable) was tested for SARS-CoV-2 inhibition in differentiated nasal and bronchial airway epithelia grown at air-liquid interface. Methylene blue (MB) and mycophenolic acid (MPA) and posaconazole (POS) were the most potent inhibitors of SARS-CoV-2 infectious particle formation. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The SARS-qRNAs were in vitro transcribed from a synthetic SARS-CoV-2 M-gene DNA template (Microsynth AG, Balgach Switzerland) under control of a T7 promoter by using the HiScribe™ T7 High Yield RNA Synthesis Kit (NEB, E2040).

Techniques: Selection, Inhibition

Journal: Current Research in Virological Science

Article Title: Identification of broad anti-coronavirus chemical agents for repurposing against SARS-CoV-2 and variants of concern

doi: 10.1016/j.crviro.2022.100019

Figure Lengend Snippet: MB and MPA inhibit hCoV-229E-GFP, OC43 and SARS-CoV-2 infection A) Dose-response curves of MB, MPA and POS treated Huh7 cells infected with hCoV-229E-GFP at 50 FFU. After 48h of infection, cells were fixed and imaged. Curves were fitted through the data points using a 3-parameter log-logistic function with a lower limit of 0. Raw data from single wells are shown. Three technical replicates are shown. Note the concentration-dependent inhibition of viral plaque formation by MB, MPA and POS. B) Broad hCoV inhibition profiles of MB, MPA and POS at 1.67 ​μM. Indicated cell lines were infected with either hCoV-OC43 (68h) or SARS-CoV-2 (24 or 48h) in presence of MB, MPA or POS, and stained for newly synthesized viral nucleoprotein. The infection index was between 10 and 30%, except for Vero E6, where it was around 5%. Data points representing infected cell counts are from raw data of individual wells. Bar heights represent the mean of four technical replicates and whiskers represent the standard deviation. Blue data points representing cell counts show means ​± ​standard deviation from four technical replicates. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The SARS-qRNAs were in vitro transcribed from a synthetic SARS-CoV-2 M-gene DNA template (Microsynth AG, Balgach Switzerland) under control of a T7 promoter by using the HiScribe™ T7 High Yield RNA Synthesis Kit (NEB, E2040).

Techniques: Infection, Concentration Assay, Inhibition, Staining, Synthesized, Standard Deviation

Journal: Current Research in Virological Science

Article Title: Identification of broad anti-coronavirus chemical agents for repurposing against SARS-CoV-2 and variants of concern

doi: 10.1016/j.crviro.2022.100019

Figure Lengend Snippet: MB and remdesivir synergize against hCoV-229E infection, and independently reduce the intracellular load of replicated OC43 and SARS-CoV-2 plus strand RNA, alongside with POS and MPA A) Overview of a 96-well plate infected with hCoV-229E-GFP and treated with combinations of remdesivir (1 ​nM–250 ​nM) and MB (100 ​nM–1 ​μM) in 2-, 4-, 5-, 10-fold serial dilutions. B) Quantification of plaque counts in presence of the indicated concentrations of MB and remdesivir in Huh7 (left panel) and H1299 (right panel). C) Huh7 cells were infected with OC43 for 2 or 24h in presence of remdesivir (1 ​μM), POS (10 ​μM), MB (2 ​μM) or MPA (2 ​μM). Cells were fixed and stained against OC43 ORF1a (+)RNA using RNA FISH with branched DNA signal amplification. Nuclei were stained with DAPI. OC43 (+)RNA dots were segmented and quantified per cell. Infected cells contained at least one viral RNA per cell; replicating cells were defined as containing more than seventy-five viral RNAs per cell, i.e., the maximal detection limit for counting individual mRNA puncta in infected Huh7 cells (see similar numbers for A549 ​cells in, Suomalainen et al., 2020 ). Images are maximum projections. Scale bar, 10 ​μm. Bar graphs in panels C) and D) denote not infected cells (grey bars), infected cells containing individual (+)RNA puncta (light green) and cells containing replicated viral RNA (dark green). D) Huh7-ACE2 cells were infected with SARS-CoV-2 for 2 or 8h in presence of remdesivir (1 ​μM), POS (10 ​μM), MB (2 ​μM) or MPA (2 ​μM). Cells were fixed, stained against SARS-CoV-2 ORF1a (+)RNA and processed as described in (C). Images are maximum projections. Scale bar, 10 ​μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The SARS-qRNAs were in vitro transcribed from a synthetic SARS-CoV-2 M-gene DNA template (Microsynth AG, Balgach Switzerland) under control of a T7 promoter by using the HiScribe™ T7 High Yield RNA Synthesis Kit (NEB, E2040).

Techniques: Infection, Staining, Amplification

Journal: Current Research in Virological Science

Article Title: Identification of broad anti-coronavirus chemical agents for repurposing against SARS-CoV-2 and variants of concern

doi: 10.1016/j.crviro.2022.100019

Figure Lengend Snippet: MB, MPA and POS inhibit SARS-CoV-2 infection of nasal HAEEC. Antiviral effects of drug treatments represented as means ​± ​SEM of two independent replicates. Baseline levels represent the apical means ​± ​SEM of virus titer from the DMSO control sample at 1d pi. The same DMSO control was used for the pre- and post-exposure treatments. A) Nasal HAEEC grown at ALI were inoculated apically with 1000 FFU of SARS-CoV2 Wuhan (day 0), and treated with drugs in the basolateral medium in a pre-infection regimen, starting at 2h prior to virus inoculation (left) or in a post-infection regimen starting at 1 ​d pi (right). MB and MPA were administrated daily until day 3 ​at 10 ​μM or until day 6 ​at 1 ​μM. B ) POS (5 and 20 ​μM) was administrated daily until day 8. Remdesivir (10 ​μM) and DMSO served respectively as positive and negative drug treatment control and were administrated daily until day 8. SARS-CoV-2 released to the apical side was collected daily by apical washing and quantified by virus TCID 50 titration.

Article Snippet: The SARS-qRNAs were in vitro transcribed from a synthetic SARS-CoV-2 M-gene DNA template (Microsynth AG, Balgach Switzerland) under control of a T7 promoter by using the HiScribe™ T7 High Yield RNA Synthesis Kit (NEB, E2040).

Techniques: Infection, Titration

Journal: Current Research in Virological Science

Article Title: Identification of broad anti-coronavirus chemical agents for repurposing against SARS-CoV-2 and variants of concern

doi: 10.1016/j.crviro.2022.100019

Figure Lengend Snippet: MPA, MB, and POS inhibit SARS-CoV-2 alpha, beta, gamma and delta variant infections of nasal HAEEC Antiviral effects of drug treatment represented as means ​± ​SEM of two independent replicates. Baseline levels represent the apical means ​± ​SEM of virus titer from the DMSO control sample at 1d pi. Note that the official WHO nomenclature of the SARS-CoV2 variants (alpha, beta, gamma, delta) refer to the commonly used PANGO lineages (B.1.1.7, B.1.351, P.1, and B.1.617.2). Nasal HAEEC grown at ALI were inoculated apically with 1000 FFU of SARS-CoV2 alpha, beta, gamma or delta variants (day 0) and subjected to drug treatment in the basolateral medium, in a post-infection regimen starting at 1 ​d pi. MB (10 ​μM), MPA (10 ​μM), and POS (20 ​μM) were administered daily until day 8. Remdesivir (10 ​μM) and DMSO served as drug treatment controls. SARS-CoV-2 released to the apical side was collected daily by apical washing and quantified by virus TCID 50 titration.

Article Snippet: The SARS-qRNAs were in vitro transcribed from a synthetic SARS-CoV-2 M-gene DNA template (Microsynth AG, Balgach Switzerland) under control of a T7 promoter by using the HiScribe™ T7 High Yield RNA Synthesis Kit (NEB, E2040).

Techniques: Variant Assay, Infection, Titration