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 (EC50) 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 EC50 values of FLUV-C and PFLUV-1 were 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.
Mori
S
S
Department of Microbiology, Fukushima Medical College, Fukushima.
Watanabe
W
W
Shigeta
S
S
D000998:Antiviral Agents / Q000494:pharmacology*; D003124:Colorimetry; D003588:Cytopathogenic Effect, Viral / Q000187:drug effects; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000032:analysis*; D008546:Melanoma, Experimental / Q000201:enzymology; D009975:Orthomyxoviridae / Q000187:drug effects* / Q000472:pathogenicity*; D010252:Paramyxoviridae / Q000187:drug effects* / Q000472:pathogenicity*; D012254:Ribavirin / Q000494:pharmacology; D012263:Ribonucleosides / Q000494:pharmacology; D013778:Tetrazolium Salts; D013844:Thiazoles; D014407:Tumor Cells, Cultured; D010948:Viral Plaque Assay
D000998:Antiviral Agents; D012263:Ribonucleosides; D013778:Tetrazolium Salts; D013844:Thiazoles; C059077:5-ethynyl-1-ribofuranosylimidazole-4-carboxamide; D012254:Ribavirin; D007770:L-Lactate Dehydrogenase; C022616:thiazolyl blue