Characterization of the antiviral effects of methylene blue (MB). (A and B) Hepatitis C virus (HCV) infection in the presence of MB at 4°C (A) and 37°C (B). Luc-Jc1 virus was added to complete Dulbecco’s modified Eagle’s medium (DMEM) supplemented with MB to achieve the indicated concentrations. The medium was exchanged after 4 hours. Huh7-5 cells were lysed, and luciferase activity was determined after 48 hours at 37°C. In addition, cytotoxicity of Epigallocatechin gallate and MB was determined using the Rotitest Vital assay (A, right). Mean values ± standard deviation (SD) of 3 independent experiments performed in duplicate are depicted. (C) Hepatitis C virus infection in the presence of MB and histidine-tryptophan-ketoglutarate (HTK). Luc-Jc1 supplemented with MB was then mixed with HTK (1:1). After 10, 30, and 60 minutes, the infection mixture was added to Huh-7.5 cells at an initial dilution of 1:5 and was then serially diluted (1:3). The medium was exchanged for complete DMEM after 4 hours of incubation at 37°C. After 72 hours, Huh-7.5 cells were fixed with methanol and stained with a NS5A primary antibody. Infected wells were counted, and the median tissue culture infective dose (TCID₅₀/mL) was calculated. Mean values ± SD of 1 representative experiment performed in 6 technical replicates are depicted. Experiment was performed 3 times with 6 replicates each. (D) Infectivity of different HCV genotypes in the presence of 1 µM MB and histidine-tryptophan-ketoglutarate (HTK). Huh7-5 cells were used. Data were normalized to the respective HTK control. Mean percentages ± SD are depicted. Data represent 3 independent experiments. * p < 0.05, **p < 0.01, ***p < 0.005. Abbreviations: ns, not significant; RLU, relative luciferase units.