A colorimetric LDH assay for the titration of infectivity and the evaluation of anti-viral activity against ortho- and paramyxoviruses

A rapid and precise screening assay was developed for in vitro evaluation of anti-orthomyxo- and anti-paramyxovirus agents. The procedure is spectrophotometrical assessment for viability of cells via extracellular leakage of lactic dehydrogenase (LDH). HMV-II cells, a human melanoma cell line was found to be suitable for the titration of virus infectivity and screening of anti-viral agents for orthomyxo- and paramyxoviruses. Comparative titration of infectivity of stock viruses by the LDH and the MTT in site reduction of 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) methods with HMV-II cells as well as plaque titration with MDCK, Vero and HeLa cells was carried out. The LDH method was comparable or more sensitive for influenza viruses (FLUV)-A,B,C, parainfluenza viruses (PFLUV)-1, 2 and less sensitive for PFLUV-3, mumps virus (MPSV), measles viruses (MLSV) and respiratory syncytial virus (RSV) than the plaque titration. The 50% effective concentration (EC(50)) of 1-beta-D-ribofuranosyl-1,2,4-triazol-3-carboxamide (ribavirin) and 5-ethynyl-1-beta-D-ribofuranosyl-imidazole-4-carboxamide (EICAR) against orthomyxo- and paramyxoviruses were examined comparatively by the LDH, MTT and plaque reduction (PR) methods. The EC(50) values of FLUV-C and PFLUV-1 mere able to be evaluated only by the LDH but not by the MTT and PR methods. The LDH method with HMV-II cells simplifies the assay procedure and permits the evaluation of a large number of compounds for anti-orthomyxo- and anti-paramyxoviruses activity in vitro.