R17 bacteriophage in buffer was inactivated using 1,9-dimethylmethylene blue (DMMB) and 655 nm of irradiation. The photoinactivation by DMMB was concentration-dependent. The participation of reactive oxygen species (ROS) in this process was investigated by using a quencher or enhancer. The photoinactivation of R17 bacteriophage by DMMB was suppressed by sodium azide (singlet oxygen quencher) and promoted by the substitution of H₂O for deuterium oxide (D₂O), which is known to prolong the lifespan of singlet oxygen. These results suggest that virus inactivation by DMMB and 655 nm of irradiation proceeds via a singlet oxygen-mediated pathway.

Photoinactivation of vesicular stomatitis virus (VSV) in stroma-free hemoglobin (SFH) was carried out with DMMB: 6.43 log₁₀ of VSV was inactivated by lμM DMMB and 1.71 J/cm² irradiation at 655 nm. VSV was more sensitive to inactivation by 655 nm light with lμM DMMB than the same concentration of methylene blue. Under conditions which inactivated 6 log₁₀ of VSV, the methemoglobin content (Met-Hb [%]) and P₅₀ (50% saturated oxygen pressure) of hemoglobin (Hb) were not changed by lμM DMMB phototreatment. The migration of Hb during electrophoresis was not changed by the DMMB phototreatment. These results indicate that the virucidal phototreatment with DMMB does not damage Hb.

In contrast to the results obtained with DMMB at 655 nm, irradiation of SFH with DMMB at 580 nm resulted in a significant increase of Met-Hb (%) under conditions which only inactivated 1.19 log₁₀ of VSV. Irradiation at 580 nm primarily activates the dimer and higher-order aggregates of the dyes, while irradiation at 655 nm primarily activates the monomer. These results indicate that, compared with the dimer, the monomer of DMMB can effectively inactivate viruses without damaging Hb.

The effects of the virucidal phototreatment with the monomer DMMB on superoxide dismutase (SOD) and catalase (CAT), which are important antioxidants in red cells, in SFH were studied. Under conditions that inactivated 6 log₁₀ of VSV, there was no significant decline in SOD or CAT activity. These results suggest that the antioxidant systems that protect against superoxide and hydrogen peroxide in SFH were preserved after virucidal phototreatment with DMMB.

DMMB phototreatment is a promising method for the sterilization of SFH, since it maintains not only the oxidative state of Hb but also antioxidant systems under virucidal conditions.