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In a photodynamic virus inactivation procedure for human fresh frozen plasma the plasma is exposed to visible light in the presence of 1 microM methylene blue. This procedure is known to inactivate HIV-1 by at least 10(6.32) TCID50/ml within 10 minutes. To elucidate the mechanism of photodynamic inactivation of HIV-1 by methylene blue/light treatment, reverse transcriptase (RT), the HIV-1 associated protein p24, and viral RNA were examined. In the dark, methylene blue up to 10 microM has no inhibitory effect on recombinant RT. In the presence of light, recombinant RT inactivation was dependent on illumination time and the concentration of methylene blue. After photoinactivation of the whole virus by methylene blue/light treatment, RT activity was also almost completely inhibited. Simultaneously, it was found by Western blotting that HIV-1 p24 and gp120 are altered in size, possibly due to protein cross-linking. In addition, it was shown by polymerase chain reaction (PCR) inhibition assay that HIV-1 inactivation leads to destruction of its RNA. In summary, methylene blue/light treatment acts on HIV-1 at different target sites: the envelope and core proteins, and the inner core structures RNA and RT.

Methylene blue (MB) and its derivatives azure A, B, C and thionine are photoactive and, in principle, are suitable for photodynamic virus inactivation of blood and blood products, such as therapeutic plasma. Methylene blue was selected for plasma decontamination because it is being clinically used and because of its known toxicological and other properties. The standard procedure for photodynamic treatment of single units of fresh plasma involves illumination with visible light at an MB concentration of 1 microM. Polymerase chain reaction analysis revealed that, in addition to model viruses, the bloodborne viruses hepatitis B virus, hepatitis C virus, human immune deficiency virus-1 and probably also the nonenveloped parvovirus B19 are sensitive to MB/light treatment. The procedure is further improved when the fluorescent tubes routinely used for illumination are replaced by more intense light sources, e.g. light-emitting diodes or low-pressure sodium lamps. Surprisingly, the improved virus kill is accompanied by reduced damage to plasma proteins.