Inhibition of various steps in the replication cycle of vesicular stomatitis virus contributes to its photoinactivation by AlPcS4 or Pc4 and red light.

Vesicular stomatitis virus (VSV) was used as a model virus to study the processes involved in photoinactivation by aluminum phthalocyanine tetrasulfonate (AlPcS4) or silicon phthalocyanine HOSiPcOSi(CH3)2(CH2)3N(CH3)2 (Pc4) and red light. Previously a very rapid decrease in the intracellular viral RNA synthesis after photodynamic treatment was observed. This decrease was correlated to different steps in the replication cycle. Binding of VSV to host cells and internalization were only slightly impaired and could be visualized by electron microscopy. The capability of the virus to fuse with membranes in an acidic endosomal environment was studied using both pyrene-labeled liposomes and a hemolysis assay as a model. These tests indicate a rapid decrease of fusion capacity after AlPcS4 treatment, which correlated with the decrease in RNA synthesis. For Pc4 treatment no such correlation was found. The fusion process is the first step in the replication cycle, affected by AlPcS4 treatment, but also in vitro RNA polymerase activity was previously shown to be inhibited. Inactivation of VSV by Pc4 treatment is apparently caused by damage to a variety of viral components. Photodynamic treatment of virus suspensions with both sensitizers causes formation of 8-oxo-7,8-dihydroguanosine in viral RNA as measured by HPLC with electrochemical detection. This damage might be partly responsible for inhibition of the in vitro viral RNA polymerase activity by photodynamic treatment.

Shelf-life of photodynamically sterilized red cell concentrates with various numbers of white cells.

BACKGROUND: Phthalocyanines are useful sensitizers for the photodynamic sterilization of red cell concentrates. The use of the phthalocyanine Pc4 (HOSiPcOSi(CH3)2(CH2)3N(CH3)2) and red light is very efficient in killing various viruses. The addition of scavengers of Type I photodynamic reactions and the use of cremophor to deliver Pc4 give protection to the red cells. STUDY DESIGN AND METHODS: Various red cell components, either white cell-enriched, buffy coat-removed, or white cell-reduced, have been used to study the effect of photodynamic treatment with Pc4 on hemoglobin and potassium leakage and on ATP and glucose levels after prolonged storage. RESULTS: After treatment, storage interval-dependent damage to the red cells could be observed. In components with 26 x 10(9) white cells per L, virus inactivation was less efficient than that in components with no or 2 x 10(9) white cells per L. Similarly, red cells were less affected by the treatment in components with a large number of white cells. Pretreatment storage and use within 1 week after photodynamic treatment induce less damage to the red cells at the moment of transfusion. CONCLUSION: Various improvements in the treatment protocol may ultimately lead to the implementation of photodynamic treatment in transfusion practice. In this respect, the white cell content of the red cell concentrates should be taken into account.

Primary targets for photoinactivation of vesicular stomatitis virus by AIPcS4 or Pc4 and red light.

Phthalocyanines are useful sensitizers for the photodynamic sterilization of red blood cell concentrates. The mechanism of photoinactivation of lipid-enveloped viruses is not completely understood. Vesicular stomatitis virus (VSV) was used as a model virus to study the primary targets of photoinactivation by aluminum phthalocyanine tetrasulfonate (AIPcS4) or silicon phthalocyanine HOSiPcOSi(CH3)2(CH2)3N(CH3)2 (Pc4) and red light. Inactivation conditions for VSV in buffer were determined using an end point dilution assay, and viral RNA synthesis in host cells was measured to determine the loss of infectivity in a direct way. The very rapid decrease in the viral RNA synthesis after photodynamic treatment was correlated with respect to different potential primary targets that are involved in different steps of the viral replication cycle. Damage to the viral proteins, induced by treatment with AIPcS4 or Pc4 and analyzed by gel electrophoresis, could not account for the observed loss of infectivity. Binding of VSV to host cells was only slightly impaired after photodynamic treatment with both sensitizers and could therefore not be responsible for the rapid decrease in viral RNA synthesis in cells. A very strong inhibition of viral RNA polymerase activity after treatment with AIPcS4 and red light was detectable using an in vitro assay. This decrease correlated well with the loss of infectivity, indicating that either the RNA or the viral RNA polymerase is the primary target for photoinactivation of VSV with AIPcS4. Treatment with Pc4 did not cause inhibition of viral RNA polymerase activity to an extent that could account for the observed very rapid loss of infectivity. It was therefore concluded that neither the viral proteins nor the binding to the host cells nor the RNA or RNA polymerase are the primary targets for photoinactivation of VSV by Pc4.