Antiviral Assay for Rabies Virus

In a previously developed antiviral assay [25 (link)], the fluorescent viral signal was quantified with a standard plate reader in 96-well plates. Similarly, in this study, hit compounds were serially diluted 2-fold, starting from 50 µM to yield 8 concentrations. A total of 15,000 BHK-21 cells were incubated with compounds overnight at 37 °C and 5% CO₂ in a 150 µL assay medium. After the incubation, mCherry-SAD-B19 or CVS-11 strains were added at an MOI of 0.01 TCID₅₀/cell in a volume of 50 µL and further incubated at 37 °C for 5 days. For the CVS-11 strain infection, the cells were fixed with 80% cold acetone for 30 min at 5 dpi. Following a 3-time PBS washing, RABV N protein was stained using a 1:100 diluted FITC Anti-Rabies Monoclonal Globulin (Fujirebio, Gent, Belgium). The fluorescence intensity from antibody staining (CVS-11) or mCherry expression by the virus (mCherry-SAD-B19) was quantified using a microplate reader (SPARK, Tecan, Mechelen, Belgium). Cell viability was determined by an MTS assay as described previously [26 (link)]. The program GraphPad Prism 9.0 was used to calculate the half maximal effective concentration (EC₅₀), the 50% cytotoxic concentration (CC₅₀), and the selectivity index (SI = CC₅₀/EC₅₀).