Application Of Adeno-Associated Virus In Mice Models Of Hepatitis B

Hepatitis B is an infectious disease which is caused by the hepatitis B virus (HBV), it is mainly liver inflammation and further lead to multiple organ dysfunction. HBV infection animal model provides a powerful tool for HBV research. Due to a widely range hosts, small carcinogenic, mediated exogenous genes sustained expression, adeno-associated virus (AAV) attracted widespread attention as a viral vector. In recent years, HBV persistent infection mouse model with recombinant adeno-associated virus 8 carrying 1.3 copies of HBV genome (rAAV8-1.3HBV) has been highly concerned. In this paper, rAAV8-1.3HBV as the reference, we evaluated efficacies through recombinant adeno-associated virus 1,2,5,9 carrying 1.3 copies of HBV genome(rAAVl-1.3HBV, rAAV2-1.3HBV, rAAV5-1.3HBV, rAAV9-1.3HBV) in establishing HBV cells models and mice models.The experiment mainly includes the following contents:(1) I prepared the required five serotypes of recombinant adeno-associated viruses and detected virus titer of the five viruses by real-time quantitative PCR. (2) pAAV-1.3HBV was transfected to Huh7 cells by liposome transfection method, five kinds of recombinant viruses were infected Huh7 cells, I detected expression of HBsAg and HBeAg on test cell. (3) according to the dose of 1× 1011vg (viral genome) per mouse, five kinds of recombinant viruses through intravenous injection method were injected into C57BL/6J mice and in eight weeks I detected HBV-related indicators to evaluate the mice models. I continuously detected HBeAg and HBsAg expression levels in serum; HBV DNA (serum and liver) by RT-PCR(Real-time quantitative polymerase chain reaction); HBeAg (liver) using immunohistochemistry assay and HE staining was measured in mice livers pathology.The results showed that (1) the virus titer of rAAV1-1.3HBV, rAAV2-1.3HBV, rAAV5-1.3HBV, rAAV8-1.3HBV, rAAV9-1.3HBV were 3.0 X 10",3.4X 1011,9.3X 10",7.2X 1011,4.26X 1011 vg-mL1 respectively; (2) cell supernatant of pAAV-1.3 HBV group could detect the expression of HBeAg and HBsAg, while cells supernatant of virus groups were no expression; (3) detection of HBV-related indicators showed a continuous eight weeks time, in serum,5 groups mice injected with the virus could be quantitatively detected HBeAg, HBsAg and HBV DNA; livers also could detect HBV DNA, and HBV DNA copies in 5 group livers were all significantly higher than in serum; while 8 weeks post injection, HBeAg, HBsAg, HBV DNA levels of expression in 5 groups mice were all AAV8> AAV9> AAV1> AAV5> AAV2; 5 groups mice hepatocytes could detect the expression of HBeAg high strength and a certain degree of pathological changes.Studies have shown that using AAV vectors establishing HBV models, there were differences between in vivo and in vitro. rAAV1-1.3 HBV, rAAV2-1.3 HBV, rAAV5-1.3 HBV, rAAV8-1.3 HBV, rAAV9-1.3 HBV could be set up HBV persistent infection mice models, while they could not establish HBV cells models. However mice modeling efficacies were a certain difference, due hepatotropism, AAV8 vector modeling was best, the sort of modeling efficacies were AAV8>AAV9>AAV1>AAV5>AAV2. Since modeling efficacies between AAV9 and AAV8 were similar, AAV9 as a new persistent HBV infection model could be applied to HBV-related research.