| Assay Method Information | |
| | Assessing Antiviral Activity Against Influenza |
| Description: | To assess their antiviral activity, some compounds were tested against murine adapted human influenza (PR8) in vitro. Canine MDCK cells were grown to confluency ( 1.0×10{circumflex over ( )}4 cells/well) in 96-well plate format in Eagle's Minimal Essential Medium (EMEM) supplemented with 10% fetal bovine serum (FBS) and 100 U/ml each of penicillin and streptomycin. Wells were washed in 1×PBS and infected with an PR8 variant expressing mCherry downstream and separated by a 2A autocleavage site from the NS-1 protein at a multiplicity of 0.01 infectious unit (IU) per cell in serum free EMEM. Assays were performed in triplicate. One hour later, virus containing medium in the cells was replaced with fresh complete medium containing the indicated compounds at 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39 μM or the carrier in which the compounds are dissolved (DMSO) and supplemented with 2.5 μg/ml TPCK trypsin. Final concentration of DMSO was 0.5% in each treatment. Virus yield in the culture was determined at 3 days post infection by quantification of fluorescent (mCherry positive) cells in each well by fluorescent microscopy. Results were plotted using CDD Vault (CDD Vault was developed by Collaborative Drug Discovery, Inc., 1633 Bayshore Hwy, Suite 342, Burlingame, Calif. 94010) in order to calculate IC50s. |
| Affinity data for this assay | |
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