Vector nova red kit

Overnight, Huh-7.5 cells (1.5 × 10⁴ cells/well) seeded in a 96-well plate were inoculated for 3 h with HCVcc containing a supernatant at a MOI of 0.1. Next, the virus was removed, and the cells were overlaid with fresh medium with different concentrations of compounds or sofosbuvir. Three days post infection, the cells were washed with PBS, fixed with methanol for 30 min, and permeabilized in 0.5% Triton X100 in PBS for 5 min followed by another wash with PBS, and immunostaining (IPMA) to detect pseudo-plaques was performed. An anti-core antibody (Hep C cAg (C7-50); Santa Cruz Biotechnology, Dallas, TX, USA; 1:300 dilution) was used as the primary antibody, and anti-mouse HRP labeled antibody (1:1000 dilution) was used as the secondary antibody. HCV-positive pseudo-plaques were detected using the Vector Nova Red kit (Vector Laboratories Ltd., Peterborough, UK), and IC₅₀ was calculated as the concentration at which the number of pseudo-plaques (foci) was reduced by 50% compared to infected control cells using the GraphPad Prism software.

Confluent monolayers of MDCK cells in 12-well plates were inoculated with influenza A virus for 1 h at 37 °C. After removal of the virus, the cells were washed with serum-free medium and overlaid with fresh serum-free medium supplemented with 1.2% Avicel (FMC BioPolymer, Philadelphia, PA, USA), 2 µg/mL TPCK-trypsin, and inhibitors at different concentrations. Three days post-infection, cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, and virus plaques were detected by immunostaining with anti-M1 antibody (diluted 1:1000 in PBS, 1% Tween 20, 5% FBS) followed by anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (diluted 1:1000 in PBS containing 1% Tween 20 and 5% FBS). Plaques were visualized using the Vector Nova-Red kit (Vector Laboratories Ltd., Peterborough, UK) and counted. The 50% inhibitory concentration (IC₅₀) was determined as the concentration at which the plaques area is reduced by 50% compared to untreated infected control. This value was calculated in both virus-induced CPE by MTS assay and immunohistochemical method in plaque reduction assay.

Monolayers of A549 cells seeded in 12-well plates were inoculated with TBEV at MOI of 0.001. Virus inoculum was removed after 2 h at 37 °C, the cells were washed and overlaid with 1% carboxymethylcellulose in Modified Eagle’s Medium (MEM) medium with DMSO or tested compounds. After three days, cells were washed with phosphate-buffered saline (PBS) and fixed with methanol. The infected virus foci were visualized by immunostaining with the monoclonal anti-Flavivirus group antigen antibody (4G2) (Absolute Antibody, Oxford, UK; diluted 1:1500 in PBS, 0,05% Tween 80, 5% FBS) as a primary antibody and anti-mouse secondary antibody conjugated with horseradish peroxidase (HRP) (diluted 1:2000) in PBS containing 0,05% Tween 80 and 5% FBS. Plaques were detected using a Vector Nova Red kit (Vector Laboratories Ltd., Peterborough, UK) according to the manufacturer’s protocol and counted. If necessary, one-way ANOVA with Dunnett’s post-test was performed in some experiments for statistical analysis using the GraphPad Prism software (version 5.01, GraphPad Software, San Diego, CA, USA).