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Duck hepatitis B virus inactivation and 8-methoxypsoralen photoadduct formation in human platelet concentrates
1Department of Laboratory Medicine, University of California, San Francisco 94143-0100, USA.
Abstract:
Photochemical inactivation (PCI) of virus and bacteria in platelet concentrates (PC) has been demonstrated using 8-methoxypsoralen (8-MOP) and long-wavelength UV light (UVA). To study inactivation of blood-borne virus, we have employed duck hepatitis B virus (DHBV), a model for human hepatitis B virus. A specific hepatocyte culture infectivity assay, with PCR detection, could measure 5-6 log10 virus kill. The DHBV inactivation in PC was dependent on UVA dose, was enhanced when plasma was reduced from 100% to 20% and was limited by 8-MOP solubility in the reduced-plasma medium. Optimum conditions for PCI were 100 micrograms/mL 8-MOP in 20% plasma and 80% synthetic platelet storage medium. A radiolabeling assay for 8-MOP photoadducts in hepatocytes seeded into PC confirmed that DHBV inactivation reflected DNA modification and indicated that adduct formation was insensitive to minor variations in conditions. Kinetic modeling indicated that optimum adduct formation was a compromise between 8-MOP dark binding and optical transmittance and that plasma proteins competed for 8-MOP binding. The PCI results in various media correlated with corresponding DNA modification densities and were compared to statistical models incorporating DHBV characteristics and predictions of 8-MOP crosslink formation between DNA strands. Behavior was consistent with one or a small number of lethal modifications per DNA strand, including monoadducts, but probably not crosslinks alone. A minor subpopulation of DHBV was found to be somewhat more difficult to inactivate, consistent with three-fold lower modification, due possibly to single-stranded DNA character or host repair of photoadducts.
Insights
Photochemical inactivation (PCI) effectively reduces duck hepatitis B virus (DHBV) in platelet concentrates using 8-methoxypsoralen (8-MOP) and UV light. Optimal conditions involve reduced plasma, enhancing viral DNA modification for safe blood products.
Area of Science:
- Biotechnology
- Virology
- Blood Product Safety
Background:
- Photochemical inactivation (PCI) using 8-methoxypsoralen (8-MOP) and UVA light is established for virus and bacteria in platelet concentrates (PC).
- Duck hepatitis B virus (DHBV) serves as a model for human hepatitis B virus, enabling study of blood-borne virus inactivation.
Purpose of the Study:
- To investigate the inactivation kinetics of DHBV in platelet concentrates using PCI.
- To determine optimal conditions for DHBV inactivation and understand the underlying mechanisms of viral DNA modification.
Main Methods:
- Utilized a hepatocyte culture infectivity assay with PCR detection to quantify DHBV kill.
- Assessed DHBV inactivation in PC under varying UVA doses, plasma concentrations, and 8-MOP concentrations.
- Employed radiolabeling assays to detect 8-MOP photoadducts and kinetic modeling to analyze modification processes.
Main Results:
- DHBV inactivation was dose-dependent on UVA and enhanced by reducing plasma from 100% to 20%.
- Optimal PCI conditions were identified as 100 µg/mL 8-MOP in 20% plasma and 80% synthetic platelet storage medium.
- Viral inactivation correlated with DNA modification, with results consistent with lethal monoadducts rather than crosslinks, though a resistant subpopulation was noted.
Conclusions:
- PCI with 8-MOP and UVA is effective for DHBV inactivation in PC, with optimized conditions improving efficiency.
- DNA modification is the primary mechanism of inactivation, influenced by plasma protein competition and medium composition.
- Further research may be needed to address the minor resistant DHBV subpopulation.