Abstract
We established a haemadsorption-based colorimetric assay system, which may be used to screen a large number of anti-influenza compounds. Madin Darby canine kidney (MDCK) cells infected with influenza virus were cultured in the presence of test compounds and after 3 days of infection guinea pig erythrocytes (GPE) were added to infected MDCK cells. After 4 washings of the cells the adsorbed GPE were lysed with distilled water, and peroxidase activities contained in GPE were measured using o-phenylendia-mine and H2O2 as substrates. The peroxidase activity that was read as optical density of oxidized o-phenylendiamine (quinoid form) correlated well with the dose of virus and day of infection in MDCK cells. Fifty per cent inhibitory concentration (IC50) of 1-(β-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide (ribavirin) and adamantanamine hydrochloride (amantadine) against influenza virus A/H3N2/lshikawa and influenza B/Singapore, the IC50s of ribavirin to influenza A and B were 0.5 μg ml−1 to 1.0 μg ml−1 respectively. On the other hand, the IC50 of amantadine for influenza A was 1 μg ml−1 but was more than 100 μg ml−1 for influenza B virus. By this method, ribavirin, 5-ethynyl-1-β-D-ribofuranosylimidazole-4-carboxamide (EICAR), 3-(β-D-ribofuranosyl)-4-hydroxypyrazole-5-carboxamide (pyrazofurin) have shown inhibitory effects for virus replication at lower concentrations than their cytotoxic doses. On the other hand carbocyclic citidine (carbodine) was cytotoxic at the concentration of virus inhibition. Ribavirin and pyrazofurin showed one tenth or less EC50 for influenza A and B viruses by the haemadsorption-based colorimetric assay compared with the ECso by TCID50 method. It is possible to estimate the inhibitory effect of antiviral compounds against influenza viruses rapidly and quantitatively by this colorimetric assay system.