pyrazofurin and thiazolyl-blue

The detection of lactate dehydrogenase (LDH) can be used to evaluate efficiently anti-influenza A virus agents. LDH levels in the virus-infected Madin-Darby canine kidney cell cultures were significantly higher than in controls, were in proportion to the degree of virus infection, and corresponded to a decrease in mitochondrial dehydrogenase activity as assayed using a tetrazolium colorimetric assay (MTT method). The EC50 value and cytotoxicity of ribavirin, 3-deazaguanine, pyrazofurin, and carbodine against influenza A virus as measured by the LDH detection method was equivalent to that derived by the MTT method.

Topics: Amides; Animals; Antiviral Agents; Cell Line; Colorimetry; Coloring Agents; Cytidine; Dogs; Guanine; Influenza A virus; L-Lactate Dehydrogenase; Pyrazoles; Ribavirin; Ribonucleosides; Ribose; Tetrazolium Salts; Thiazoles

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.

Topics: Amides; Animals; Antiviral Agents; Cell Line; Chlorocebus aethiops; Colorimetry; Coloring Agents; Cytidine; Dogs; HeLa Cells; Humans; Influenza A virus; Measles virus; Microbial Sensitivity Tests; Orthomyxoviridae; Pyrazoles; Respiratory Syncytial Viruses; Respirovirus; Ribavirin; Ribonucleosides; Ribose; Sensitivity and Specificity; Tetrazolium Salts; Thiazoles; Vero Cells; Virus Replication