Study On The Occult HBV Infection And The Mechanisms Of Photochemical Inactivation On HBV

HBV carriers in China is7.18%, which puts forward a great challenge to the safety of blood transfusion. However, since the techniqic limitation of the enzyme immuno assays (EIA), it was used to see the detection failure because of "window period" infection, immune silent infection, and viral mutant infection.Exluding infection in window period, Occult HBV infection (OBI) presents HBsAg" but HBV DNA+in patient peripheral blood sample when testing, which was presumed asdown-regulated expression or mutation of HBsAg in infected persons for some reasons. Since it would be failed to bedetected by HBsAg EIA, nucleic acid testing (NAT) is widely used in blood screening for one of the sake of OBI. Nevertheless, recent studies showed that there still was a certain risk of failure to detect OBI with NAT. In order to further ensure the blood safety, methylene blue photochemical (MB-P) method has been used for the viral inactivation in plasma, the mechanisms of which are that under the action of light, methylene blue molecules are excited and can inactivate pathogens in the blood by destroying the structures of them. It is known that the method targets viral nucleic acids and envelope as its inactivation points, which can inactivate both enveloped and non-enveloped viruses, however the inactivation mechanisms, especially the effect on the structures of the viruses remains to be elucidated. In this paper, some preliminary studies were carried out on the mechanisms of occult HBV and MB-P viral inactivation treatment on HBV.In the first part, in order to investigate the prevalent rate of OBI in non-remunerative volunteer blood donors in Shanghai, and to analyze the relationships between the markers of HBV in serum, the amounts of HBV DNA, the genotypes and amino-acid mutants in S region with OBI,250,360primarily screened non-remunerate volunteer blood donation samples were collected from October2011to July2012. All the samples were tested by NAT either qualitatively and quantitatively, and the samples of which were HBsAg"DNA+were further tested for the five serological markers and analyzed the genotypes and mutations in amino-acid sequences in S region by DNA extraction, Nest PCR, cloning and sequencing. The results showed that there were197/250,360donations with HBsAg" and DNA+. The HBV DNA load in these samples were between0and7.11×103IU/ml by quantitative method, and48.78%samples were with HBV DNA load lower than100IU/ml.The197samples were tested for five serological markers of HBV, of which25samples (25/197,12.7%) were without any markers, suspecting as infection in window period or primary OBI, it would not be determined until further investigation for a follow up; of which172samples (172/197,87.3%) showed HBsAb+or HBcAb+, which were considered as OBI, so it was concluded that the prevalent rate of OBI in Shanghai was0.069%(172/250,360), which was lower than other regions in China.47samples in172were selected for analysis of mutations in S region of HBV genome, However, only17samples (17/47,36.17%) were amplified for S region by Nest PCR, of which14were C genotype,3were B genotype. The results showed that the C genotype had a higher proportion than B in OBI. The mutation rate for C and B genotype were35.71%(5/14) and33.33%(1/3) respectively, and the high mutation rate in126aa and130aa were found, which was correlated with other literatures. The lower HBV DNA load of OBI samples requests the higher sensitivity of the NAT. It suggested that even if the NAT was carried out, it would still miss some OBI samples.In the second part, in order to explore the mechanisms of methylene blue photochemical inactivation treatment (MB-P) and to provide a theoretical basis for the use of the method in blood safety for reducing the infectious risks of blood transfusion, the effects of the immunization of inactivated HBV on mice were studied. In view of the MB-P mechanism on the viral nucleic acid has been elucidated, and MB-P inactivation method has been validated by laboratory, animal models and clinical trials, however there is less reports on the effects of MB-P on the antigenicity of the HBV envelope, which will be studied preliminarily here. HBV from supernatant of HepG2.2.15(experimental group) and HepG2(control group) were purified and concentrated by sucrose gradient centrifugation and ultrafiltration centrifugation method. Purified HBV were inactivated by methylene blue photochemical inactivation treatment. The antigenicity of inactivated HBV were tested by ELISA, which showed that the inactivated HBV still retained its antigencity. PBMC which were isolated from blood were incubated with inactivated HBV, IFN-γ secretion which was tested by ELISA after3days incubation was increasing. Balb/c mice were immunized with inactivated HBV every two weeks for4times by intraperitoneal injection or in different points of thigh groin. After the initial immunization, blood was collected for4times in orbital venous plexus of the mice before each immunization and in20days after the last immunization, and serum were separated from blood. HBsAb induced by inactivated HBV which were tested by ELISA were increasing over time, and the amounts of HBsAb were more than10mIU/ml in the fourth week generally, which was concluded that the inactivated HBV could induce strong humoral immunization. Later, to explore the Thl/Th2balance, specific T cells proliferation and cytotoxicity, blood was collected when the eyes of mice were removed in20days after the last immunization, serum was separated too. The serum was used for the analyisis of cytokines of Thl/Th2(IL-2,1L-4,1L-5, IFN-γ,TNF) by flow cytometry method, and the induced cytokines were increased significantly. Mice were dissected for the spleens, which were prepared for suspension in single cells. After that the cells were used for HBsAg specific cellular proliferation and cytotoxicity experiments. It was concluded that inactivated HBV could elicit a stronger HBsAg specific cytotoxic activity, however there was not a significant difference of the proliferation in two groups. Although the MB-P could destroy the HBV nucleic acid, in which way the HBV infection activity was lost, The experimental results showed that MB-P inactivitation method had no effect on the antigenicity of HBV, on the other hand, inactivated HBV could improve the humoral and cellular immunnization in mice, which would help us for further understanding of the inactivation mechanisms of MB-P.