Journal: Nature Communications

Article Title: STAT2 signaling restricts viral dissemination but drives severe pneumonia in SARS-CoV-2 infected hamsters

doi: 10.1038/s41467-020-19684-y

Figure Lengend Snippet: a Schematic representation of SARS-CoV-2 inoculation schedule. WT (blue), STAT2 −/− (red), and IL28R-a −/− (purple) hamster strains were inoculated intranasally with 2 × 10 5 TCID 50 of passage 4 or 2 × 10 6 of passage 6 SARS-CoV-2. Outcomes derived from inoculation with passage 4 or passage 6 SARS-CoV-2 is designated by circles (P4, n = 3) or squares (P6, n = 4). On the indicated days post inoculation (p.i.), organs and blood were collected to determine viral RNA levels and infectious viral load. Viral loads in the indicated organs were quantified by RT-qPCR ( b , d – f ) or virus titration ( c ). b , e Viral RNA levels in the lungs (day 2 and day 3 p.i. of each genotype ( n = 3); day 4 p.i. of each genotype ( n = 7)) ( b ) or the indicated organs on day 4 p.i. ( n = 4 for each genotype) ( e ) were normalized against β-actin mRNA levels and transformed to estimate viral genome equivalents (vge) content per weight of the tissue (Fig. ). c Infectious viral loads in the lung on day 4 p.i. are expressed as the number of infectious virus particles per 100 mg of lung tissue ( n = 7 for each genotype). d Viral RNA levels in the blood (day 2 and day 3 p.i. of each genotype ( n = 3); day 4 p.i. of each genotype ( n = 7)) were calculated from a standard of infectious virus and expressed as TCID 50 equivalents per ml blood. Dotted lines indicate lower limit of quantification (LLOQ) or lower limit of detection (LLOD). f Viral RNA levels in hamsters after treatment with a previously described single-domain antibody. Hamsters were either left untreated (blue, n = 5) or treated with VHH-72-Fc (green, n = 4) and sacrificed on day 4 p.i. Viral RNA levels were determined in the lungs, normalized against β-actin, and fold changes were calculated using the 2 (−ΔΔCq) method compared to the mean of untreated control. g Inhibition of JAK/STAT signaling by Ruxolitinib can rescue SARS-CoV-2 virus replication in human airway epithelial cells from the antiviral effect of type I IFN. Calu-3 (human airway epithelial) cells were left untreated or treated with Ruxolitinib (4 µM), IFN-α (10 IU/ml), or a combination of both ( n = 8 for each condition). Treatment was initiated 4 h before infection and was continued through the whole experiment. Cultures were infected with P6 SARS-CoV-2 (MOI of 0.1), and 48 h p.i., cell culture supernatant was collected, RNA was extracted, and the amount of vRNA was quantified using RT-qPCR. A serial dilution of the same virus stock was used to generate a standard curve for absolute quantification. The data shown are mean ± SEM. Statistical significance between groups was calculated by Kruskal–Wallis with two-sided Dunn’s post hoc test ( b – d , g ) or by an unpaired two-sided t test ( f ). P values: * P = 0.010 ( c ), *** P = 0.0009 and * P = 0.02 ( d ), **** P < 0.0001 ( f ), * P = 0.022 and * P = 0.013 (left to right in g ); ns not significant.

Article Snippet: Calu-3 (human airway epithelial) cells were plated at 5 × 10 4 cells/well in a 96-well plate and incubated overnight with 4 µM of the JAK1/2 inhibitor Ruxolitinib (Toronto Research Chemicals, ON, Canada).

Techniques: Derivative Assay, Quantitative RT-PCR, Titration, Transformation Assay, Inhibition, Infection, Cell Culture, Serial Dilution

Journal: Scientific Reports

Article Title: Transcriptomics-based drug repositioning pipeline identifies therapeutic candidates for COVID-19

doi: 10.1038/s41598-021-91625-1

Figure Lengend Snippet: Haloperidol inhibits viral replication of SARS-CoV-2 in the Calu-3 lung cell line. ( A ) Calu-3 cells were infected with SARS-CoV-2 at an MOI of 0.05 for 72 h. Viral replication levels were determined by RT-qPCR from supernatant RNA using specific primers for the E gene. Viral RNA levels relative to DMSO are graphed. Error bars represent 3 or 4 independent experiments. One-way ANOVA analysis was used to determine significance. ( B ) Microscopy: Calu-3 cells were infected with SARS-CoV-2 at an MOI of 0.05 for 72 h. Cells were fixed with paraformaldehyde and used for immunofluorescence analysis with dsRNA antibody (SCICONS) and DAPI stain. Images were acquired and analyzed using ImageXpress Micro Confocal High-Content Imaging System.

Article Snippet: The inhibitory effects of haloperidol, clofazimine, valproic acid, and fluticasone were evaluated in SARS-CoV-2 infected Calu-3 cells (human lung epithelial cell line), with remdesivir also tested as a positive control.

Techniques: Infection, Quantitative RT-PCR, Microscopy, Immunofluorescence, Staining, Imaging

Journal: Emerging Microbes & Infections

Article Title: HA N193D substitution in the HPAI H5N1 virus alters receptor binding affinity and enhances virulence in mammalian hosts

doi: 10.1080/22221751.2024.2302854

Figure Lengend Snippet: Growth curves of the rCT/W811-HA 193N and rCT/W811-HA 193D viruses in avian- and mammalian-origin cells. Cell monolayers of DF-1 (A), CEF (B), MDCK (C), A549 (D), Calu-3 (E), and NHBE (F) were infected with the rCT/W811-HA 193N and rCT/W811-HA 193D viruses at an MOI of 0.01 for 72 h. TCID 50 virus titres were measured in the supernatants at the indicated time points. The data are presented as the mean values of three inoculated wells ± the SEM for each time point and are representative of three independent experiments. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001.

Article Snippet: Human lung epithelial Calu-3 and A549 cells were purchased from Korean Cell Line Bank (KCLB) (Seoul, Korea, no. 30055 and 10185), grown and maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS, 100 IU/mL of penicillin, and 100 μg/mL of streptomycin.

Techniques: Infection, Virus