Comparison of Methylene Blue and Methylene Violet for Photoinactivation of Intracellular and Extracellular Virus in Red Cell Suspensions

Andrey Skripchenko, Daniel Robinette and Stephen J. Wagner*

*To whom correspondence should be addressed at: Jerome H. Holland Laboratory, American Red Cross Biomedical Services, 15601 Crabbs Branch Way, Rockville, MD 20855, USA. Fax: 301-738-0704.

ABSTRACT

Previous studies with methylene blue (MB) in red cell suspensions have demonstrated that extracellular, but not intracellular, virus can be readily photoinactivated. To test if the resistance of intracellular virus to inactivation is related to the permanent positive charge of the phenothiazine, a series of uncharged phenothiazine dyes, methylene violet (MV), monodemethylated MV and didemethylated MV, were studied. Values of the sensitivity of intracellular relative to extracellular vesicular stomatitis virus (VSV) inactivation for the three dyes (D_{10 extracellular}/D_{10 intracellular}) in buffer were 1.0, 0.60 and 0.33, respectively. In contrast, intracellular virus was resistant to inactivation with MB, with a D_{10 extracellular}/D_{10 intracellular} of 0.05 in buffer. Because virucidal activity of MV was inhibited by the presence of plasma, the red cells (30% hematocrit) were repeatedly washed prior to photoinactivation and storage. Under conditions where MB and MV inactivated approximately 5 log₁₀ of extracellular VSV, intracellular VSV was inactivated by more than 4 log₁₀ with MV compared to 0.88 log₁₀ with MB. These phototreatment conditions did not significantly affect red cell morphology, extracellular pH, ATP or 2,3-diphosphoglycerol levels during 42 days of 1-6°C storage. There was enhanced potassium efflux and hemolysis over values obtained from untreated controls; the extent of change from controls was comparable for each phototreatment. These results indicate that the uncharged phenothiazine dye, MV, can inactivate both intracellular and extracellular virus yet exhibit similar in vitro red cell storage properties as MB phototreatment.