Establishment of the Haemadsorption-Based Colorimetric Assay for the Screening of Anti-Influenza Compounds

Abstract

SummaryWe 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 presenct"~ftestcompounds and after 3 days of infection gUin'e~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-phenylendiamine and H 2 0 2 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 (ICso) of 1-(f3-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide (ribavirin) and adamantanamine hydrochloride (amantadine) against influenza virus A1H3N2Ilshikawa and influenza B/Singapore, the ICsosof ribavirin to influenza A and B were O.5J.Lg mr " to 1.0J.Lg mr ' respectively. On the other hand, the IC so of amantadine for influenza A was 1 J.Lg ml" but was more than 100 J.Lg mr" for influenza B virus. By this method, ribavirin, 5-ethynyl-1-f3-Dribofuranosylimidazole-4-carboxamide (EICAR), 3-(f3-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 EC so for influenza A and B viruses by the haemadsorption-based colorimetric assay compared with the EC so by TCID so method. It is possible to estimate the inhibitory effect of antiviral compounds

Approaches to Antiviral Chemotherapy for Acute Respiratory Infections

“…One of these is based on the haemadsorption of influenza virus, devised by Takeuchi et al (1993). One of these is based on the haemadsorption of influenza virus, devised by Takeuchi et al (1993).…”