Molecular Mechanisms Of Methylene Blue Photochemical Viral Inactivation Treatment On Hepatitis C Virus In Plasma

Transfusion is one of the most essential parts of modern medicine. But in blood transfusion, there are certain risks of viral infections, which could cause serious diseases, for example, transfusion transmitted HIV, HBV, and HCV. Avoiding transfusion transmitted diseases and ensuring the safety of blood transfusion have become the focus not only of the medical field but also of our whole society.Nowadays, it's not able to distinguish all the virus contaminated bloods or blood components products, due to the shortcomings of detection methods of virus in blood products and the window period of virus infection. So it's impossible to eliminate all the transfusion transmitted diseases just by blood screening measures (e.g.ELISA). Virus inactivation technology of blood components has been considered widely as one of the most important guarantees for the safety of transfusion. Hence the research on the methods of virus inactivation and its molecular mechanisms are widely concerned.One of the proven effective virus inactivation methods in latest years is Methylene blue photochemical treatment (MB-P), which can inactivate lipid-coated viruses and some nonlipid-coated viruses effectively.MB-P has been used successfully in the inactivation of viruses contaminating clinical single bag of plasma .Its efficacy and safety has been widely proven. The molecular mechanism of MB-P, however, is not yet clear. Clarifying the molecular mechanism of virus inactivation by MB-P can improve its methodology, provide theoretical references for other inactivation methods as well.HCV (hepatitis C virus, HCV) is chosen to study the molecular mechanism of MB-P, mainly because HCV is one of the most important pathogens causing serious transfusion transmitted diseases in China. And there are no effective cell culture systems for HCV and no other animal model except chimpanzee model. Meanwhile HCV, as a lipid-coated virus, can clarify more appropriately the inactivation mechanism of lipid-coated viruses.In our study, PCR and fluorescence quantitative PCR technology were employed to demonstrate the degradation of HCV RNA, as well as the relationship of HCV RNA degradation and its structural changes.Published results of our laboratory show that MB-P treatment causes the degradation of HCV NS5 gene. According to HCV genome, the more conservative genes of HCV genome, HCV 5'NCR (5'Non-coding region) and neighboring Core gene (844bp) were chosen as PCR templates, as well as HCV gene NS₂ (973bp). HCV positive plasma samples were reserved at different time points before and after MB-P treatment. Then PCR and fluorescence quantitative PCR were employed to detect the quantity of HCV RNA by amplifying 5'NCR + Core gene and NS2 gene. The results show: in earlier phase of MB-P treatment, the quantity of HCV RNA decreases rapidly, it drops from 5.5×10⁵ copies/ml to 8.2×10³ copies/ml in 20 minutes; and the speed of degradation slows down in later phase, the quantity of HCV RNA reduces to 2.8×10³ copies/ml after 60minutes of MB-P treatment . During the whole process, HCV RNA reduces in a logarithmic pattern. This result shows that MB-P treatment aims at HCV RNA.To demonstrate the role of the other viral structures and viral proteins during inactivation, in vitro transcripted 5'NCR and core gene RNA is inactivated, which is transcripted in vitro from a reconstructed and identified pBluescript II SK(+) inserted with HCV5'NCR and core gene. And so did the in vitro transcripted RNA of NS2 gene. After diluted with HES 20(20-hydroxyethyl starch and sodium chloride injection) or PBS buffer to the same concentration of HCV in plasma (≥10⁵copies) , in vitro transcripted HCV RNA is treated , in the absence of other viral structures and viral proteins, by MB-P treatment same as the foregoing .The result shows that after MB-P treatments, the quantity of in vitro transcripted HCV RNA also decreases. It implies that in vitro transcripted HCV RNA also degrades by MB-P treatment, its quantity drops from 3.2×10⁵copies/ml to 6.2×10⁴ copies/ml in 20minutes, and to 2.0×10⁴ copies/ml in 60 minutes. It implies that MB-P is able to affect directly nucleic acid strand, and inactivate eventually viruses. However, the inactivation rate of virus in plasma is faster than that of in vitro transcripted HCV RNA dissolved in HES or PBS. This result implies that the other viral structures and viral proteins maybe affect the inactivation efficiency of MB-P on HCV RNA.Our research proves for the first time that MB-P can degrade efficiently HCV RNA both of 5'-NCR+ C and NS2 gene. The conclusion is (1) when exposed in light, Methylene blue can act on viral nucleic acid strands, and inactivate viruses finally;(2) treated by MB-P, HCV RNA decreased in a logarithmic pattern;(3)the inactivation efficiency of in vitro transcripted HCV RNA is lower than that of HCV RNA in plasma.