MTT COLORIMETRIC ASSAY SYSTEM FOR THE SCREENING OF ANTIORTHOMYXOVIRALAND ANTI-PARAMYXOVIRAL AGENTS

A rapid and sensitive method was developed for screening potential ant iviral agents against orthomyxo- and paramyxoviruses, using the MTT me thod with cell culture suspensions. The cell lines used for the assay were as follows: MDCK cells for the influenza A virus (Fluv. A), HeLa cells for the respiratory syncytial virus (RSV), and Vero cells for th e measles virus (MSV). Test compounds were diluted and plated in 96-we ll round-bottomed microtiter plates. Trypsinized cell suspensions and viruses were added to each well, the plates were then centrifuged (700 x g, 5 min, room temperature), and incubated for several days. The MT T assay was carried out after the degeneration of virus-infected cells became evident. The optical density (OD) of formazan was determined u sing a computer-controlled microplate reader. With this assay system, the EC(50) values of Ribavirin (used as the reference compound) were 3 .7 mu g/ml for Fluv. A, 4.5 mu g/ml for RSV, and 12.3 mu g/ml for MSV, respectively. These EC(50) values were equivalent to those obtained u sing the plaque reduction assay. The confluent cell culture system was inadequate for antiviral assays against RSV and MSV when the MTT meth od was used, because the inhibition of formazan formation was not obse rved in viral-infected cells. Moreover, the suspension method is more sensitive to the cytotoxicity of antiviral agents than the confluent c ell culture system.