Establishment Of Chronically Infected-drug Assessment Mouse Model Mediated By RAAV8-1.3HBV

ObjectiveHepatitis B virus (HBV) is one of the major pathogens of acute and chronichepatitis of human beings. About one-third of people around the world have beeninfected with HBV. China, belonging to the high endemic area of HBV infection, hashepatitis B surface antigen (HBsAg) positive rate close to10%. At present, HBVrelated animal models are the main application to the research for HBV, especiallyreferring widely used mice. The frequently used mice models for HBV study include:HBV transgenic mouse model and hydrodynamic injection of HBV mouse model.However, because of species differences of HBV, all of the mice models inferredabove show their flaws and limits of application.Our research group was successfully perpared recombinant adeno-associated virus8carrying1.3copies of HBV genome (rAAV8-1.3HBV, ayw type) in2010. Weinjected C57BL/6mice by tail vein and detected persistent expression of HBV in thismodel intially. For further evaluating the rate of success of this model and exploringwhether the model can be used as new anti-HBV drug screening model, we designand conduct this experiment.Methods1. The method of building and evaluating mouse model Selecting30male C57BL/6mice between4-6weeks, we injected rAAV8-1.3HBVby tail vein to these mice according to the dose of1x1011vg. The expressions ofHBeAg and HBsAg in serum were qualitative detected after giving virus for2weeksand4weeks. The expressions of HBeAg, HBsAg and HBV DNA in serum werequantitative detected after giving virus for6weeks. Combining quantitative,qualitative detection of serological results, the successful mice models in30wereselected for further used in follow-up experiments.2. Plans for drug treated grouping and administrating drugsThe selected chronically infected mice models mediated by rAAV8-1.3HBV wereset as the high and low dose of ETV, the high and low dose of LAM, normal salinetreated and untreated control, respectively. The drug treated groups wereadministrated by oral for10days and withdrawal for15days.3. The effective indications for drug evaluationWe qualitative detected the expressions of HBeAg, HBsAg and HBV DNA inserum at the points of drug administration for5days,10days and withdrawl for15days. The conclusion was drawed though contracting the situations before and afterdrug administration, as well as before and after drug withdrawal using statisticalanalysis.4. The detecting methodsThe expression of HBV DNA in serum was detected by quantitative real time PCR.The expressions of HBsAg and HBeAg in serum were detected by qualitative andquantitative ELISA.Results1. Evaluation of the successful rate of mouse model1.1Qualitative ELISA28in30of models were positive of HBeAg expression on14thday after virus injection, while all of the30mice were positive of HBsAg.29in30of models were positive of HBeAg expression on28thday after virus injection,while all mice models were positive of HBsAg.1.2Fluorescence quantitative PCR for HBV DNA There were27in30micemodels above5.0in the expression level of HBV DNA (lg (IU/mL)) in serum on the42ndday. 1.3Quantitative ELISA There were3out of30mice with relatively lowexpression level of HBeAg and HBsAg in serum on the42ndday after virus injection.Correspondingly, the remaining27mice have the average level of13.22±1.78S/COreferring HBeAg, while the average level of93.24±1.78S/CO referring HBsAg.Combination of HBV DNA expression and both the qualitative and quantitativeELISA results, eventually we selected27in30mice models using in the further testwith the successful rate of90%.2. The effects of the expression level of HBV DNA in serum with drugsAfter processing with drugs in mice models, the level of HBV DNA were decreasedobviously (P＜0.05) in low dose ETV treated group compared with NS group on the10thday with drugs. While significantly increased (P＜0.05) were shown after15days drug withdrawal compared with the10thday with drug in low ETV-treatedgroup. There were apparently decreased (P＜0.01) in high dose of ETV treatedgroup since the5thday with drugs compared with NS group, as well as the10thdaywith drugs.The level of HBV DNA were decreased obviously (P＜0.05) in low dose LAMtreated group compared with NS group on the10thday with drugs. Whilesignificantly increased (P＜0.05) were observed after15days drug withdrawalcompared with the10thday with drug in low LAM-treated group. Additionally, therewere apparently decreased (P＜0.05) in high dose of LAM treated group since the10thday with drugs compared with NS group.3. The effects of the expression level of HBeAg and HBsAg in serum with drugsThere was no apparent change in the level of HBeAg and HBsAg in serum in thewhole process, including the day before giving drugs, the10thday with drugs and the15thwithout drugs, among all of the groups.ConclusionsAfter offering Entecavir and Lamivudine to the mouse model of HBV persistentinfection mediated by rAAV8-1.3HBV, there was apparently decreased on the level ofHBV DNA, while no change was shown in the expression of HBeAg and HBsAg in serum. As a result, this mouse model can effectively reflect the anti-HBV effects ofnucleotide analogues and has the features of simple preparation and high successfulrate, which implies that this kind of mouse model should be extended to further use asanti-HBV drug screening model for new drugs of nucleotide analogues.