The adsorption of cationic organic dyes (methylene blue, thionine, and thiopyronine) on Qf3 bacteriophage was studied by UV-visible and fluorescence spectroscopy. The dyes have shown a strong affinity to the virus and some have been used as sensitizers for photo-induced inactivation of virus. In the methylene blue concentration range of 0.1-5 ,uM and at high ratios of dye to virus (greater than 1000 dye molecules per virion), the dyes bind as aggregates on the virus. Aggregation lowers the efficiency of photoinactivation because of self-quenching of the dye. At lower ratios of dye to virus (lower than 500 dye molecules per virion), the dye binds to the virus as a monomer. Fluorescence polarization and time-resolved studies of the fluorescence support the conclusions based on fluorescence quenching. Increasing the ionic strength (adding NaCl) dissociates bound dye aggregates on the virus and releases monomeric dye into the bulk solution.Two broad techniques have been used for eliminating viral contamination in blood products: removal and inactivation. The former has been partially achieved by washing, filtration, or adsorption (1). The latter has utilized heat, intrinsically active chemical agents, photo-and y-irradiation without chemical addition, and photo-irradiation in the presence of sensitizing agents that are usually inactive until irradiated (2-6). For inactivation to preserve sensitive bystander molecules or cells, a very high degree of specificity for the virus must be obtained. In general, photo-irradiation in the presence of sensitizing agents is the only method that has yet approached the requisite specificity, in part because the sensitizer can be concentrated on the virus before it is activated. The sensitizing agent must have a high binding constant and bind specifically to a viral component: its lipid envelope (when present), its protein coat, or its genome. It must also have a path by which it can reach this component, as well as time to do so. Any coreactant (e.g., oxygen) must also have a path by which it can reach the reaction site. Inactivation may occur either because photoinduced damage renders the virus unable to enter a prospective host cell or because the virus is unable to replicate after entry because its genome has been rendered defective.We have selected methylene blue (MB) and its derivatives thionine (Th) and thiopyronine (TP) as prototype sensitizing agents ( Fig. 1) for virus inactivation. These dyes have high in vitro binding affinity to nucleic acids, a feature that appears to confer their microbiocidal and virucidal activity (7,8). MB is known as an antivirucidal compound, and it is at least competitive with other agents in minimizing undesirable effects. Its chemical and biological properties have been thoroughly studied (7), the optical absorption is optimum for the blood banking application, and it is currently being used clinically for photoinactivation of fresh frozen plasma in Europe (9). Inferring from the high binding affinity of MB, Th, and TP to nucleic acid...
Concentration effects of methylene blue (MB) and oxygen on the photoinactivation rate of Q beta bacteriophage were examined. The effect of initial virus concentration was verified on the similar f2 phage. The inactivation rate, kappa, is an increasing function of MB and O2 concentration and shows saturation with respect to MB concentration. Thus the results suggest that MB must adsorb to Q beta sites and oxygen must be present for photoinactivation to occur. The inactivation rate is independent of the initial number of phage particles present before inactivation, indicating that inactivation does not depend upon interaction among viral particles or on surface effects. The results indicate that at least two different viral phenotypes exist within the wild-type Q beta and f2 populations: one susceptible and the other resistant.
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