MTT colorimetric assay system for the screening of anti-orthomyxo- and anti-paramyxoviral agents

Abstract

A rapid and sensitive method was developed for screening potential antiviral agents against orthomyxo- and paramyxoviruses, using the MTT method 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 the measles virus (MSV). Test compounds were diluted and plated in 96-well 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 MTT assay was carried out after the degeneration of virus-infected cells became evident. The optical density (OD) of formazan was determined using a computer-controlled microplate reader. With this assay system, the EC50 values of Ribavirin (used as the reference compound) were 3.7 micrograms/ml for Fluv. A, 4.5 micrograms/ml for RSV, and 12.3 micrograms/ml for MSV, respectively. These EC50 values were equivalent to those obtained using the plaque reduction assay. The confluent cell culture system was inadequate for antiviral assays against RSV and MSV when the MTT method was used, because the inhibition of formazan formation was not observed in viral-infected cells. Moreover, the suspension method is more sensitive to the cytotoxicity of antiviral agents than the confluent cell culture system.