| Objective: Chronic hepatitis B virus infection is a worldwide public health threat that can lead to liver cirrhosis,liver cancer.HBV is divided into ten different genotypes denoted A-J;genotype C is the main epidemic genotype in northern China.Currently the main treatment strategies for HBV infection are interferon therapy and nucleos(t)ide analogs(NAs)antiviral treatment.In recent years,multidrug-resistant(MDR)HBV infection is increasing year by year,which bring greater challenge to clinical treatment.HBV stable replication mouse model is an important tool for studying the mechanism of t HBV infection and evaluating the effectiveness of anti-HBV drugs.The aim of this study is to establish the wild-type and MDR HBV genotype C stable replication mouse models and to evaluate NAs effects using these models.Methods:(1)The recombinant adeno-associated virus(r AAV)plasmids which containing 1.3 copies of HBV genome(serotype adr,WT and MDR)were constructed as p AAV2neo-1.3HBV-C-WT and p AAV2neo-1.3HBV-CMDR.Then the plasmids were transfected into human hepatocellular carcinoma cell lines Huh7 in vitro,the HBV DNA replication level and HBV antigen(HBs Ag,HBe Ag)secretion were assayed to confirm the plasmid constructions.After that,the recombinant viruses r AAV8-1.3HBV-C-WT and r AAV8-1.3HBV-C-MDR were packaged and purified.(2)High titers of the recombinant virus r AAV8-1.3HBV-C-WT were injected into the 6-8 weeks old C57BL/6 mice,and the HBV-C-WT replicating mouse models(n=26)were established.After 5 weeks of stable replication of HBV in mouse models,two of them were randomly selected for liver tissue HE staining and immunohistochemical analysis.The remaining mouse models were randomly divided into four treatment groups(n=6): ETV group(0.1 mg/kg),TDF group(310 mg/kg),ETV(0.1 mg/kg)+ TDF(310 mg/kg)group,and nomal saline(NS)control group.After 2 weeks of continuous administration,the drug was stopped for 5 weeks.The HBV DNA load and HBs Ag,HBe Ag secretion in serum(retinal vein collection)were detected at different time points.(3)The HBV-C-MDR stable replication mouse models(n=30,experimental group)were established using the above-described methods,and the HBV-C-WT stable replication mouse models(n=30,control group)were established as control group.After 6 weeks of stable replication of HBV in mouse models,both groups of mouse models in experimental group and control group were randomly divided into 5 treatment groups(n=6): ETV group(0.1 mg/kg),TDF group(310 mg/kg),ETV(0.1 mg/kg)+ADV(2.1 mg/kg),ETV(0.1 mg/kg)+ TDF(310 mg/kg)group,and NS control group.After 2 weeks of continuous administration,the drug was stopped for 8 weeks.The HBV DNA load and HBs Ag,HBe Ag secretion in serum(retinal vein collection)were detected at different time points.NAs were administered to evaluate the established mouse model.Results:(1)The mean levels of HBV DNA in the supernatant of Huh7 cells 72 hours post-tranfection with p AAV2neo-1.3HBV-C-WT and p AAV2neo-1.3HBV-C-MDR were 8.71 lg IU/ml and 8.74 lg IU/ml respectively;the mean OD(A450nm)levels of HBs Ag were 3.50 and 3.38,respectively;and the mean OD(A450nm)levels of HBe Ag were 3.41 and 3.33,respectively.These results indicated the successful construction of the recombinant plasmids and they could replicate in Huh7 cells with high replication/expression.(2)After the injection of r AAV8-1.3HBV-C-WT,the mouse models were observed for 5 weeks,and the HBV DNA load,HBs Ag level and HBe Ag level in serum were detected.The results were as follow: the HBV DNA was 3.90-4.54 lg IU/ml,HBs Ag and HBe Ag OD(A450nm)maintained at more than 2,which indicated that the mouse models were successfully established.One week after drug administration,HBV DNA load,HBs Ag level,and HBe Ag level were measured.The results showed that HBV DNA decreased by 1.84,2.23 and 2.08 lg IU/ml respectively in the administration groups with statistically significance(P <0.05).There was no significant difference in the NS group after and before drug administration.The HBV DNA load relaped(>1.0,lg IU/ml)after 1-2 week of drug withdrawal.The HBs Ag and HBe Ag level were highly expressed [OD(A450nm)>2] throughout the experiment.(3)The model showed stable replication for 6 weeks of observation window,with HBV DNA level of 3.14-5.49 lg IU/ml,HBs Ag OD(A450nm)level of 1.21-4.00,and HBe Ag level of 1.67-4.00.The HBV DNA replication was supressed by NAs adminstration,with decrease of 0.91,0.90,0.88,0.93 lg IU/ml for ETV,TDF,ETV+ADV,ETV+TDF respectively(P <0.05).However,the decrease in HBV DNA level was more significant in the NS group(2.00 lg IU/ml,P <0.05).The viral replication rebounded after the suspension of NAs in the following 8 weeks of observation window.The serum HBV DNA of HBV-C-WT replicative mouse model(positive control group)was stable in the range of 3.65-6.05 lg IU/ml;the replication was supressed by 2 weeks of NAs adminstration,with decrease of 3.66,3.48,3.18,2.89 lg IU/ml for ETV,TDF,ETV+ADV,ETV+TDF respectively(P <0.05);while no significant decrease was observed in NS group(P >0.05).The HBV DNA load relaped(>2.0,lg IU/ml)after 1-2 weeks of drug withdrawal.The HBs Ag and HBe Ag level were highly expressed [OD(A450nm)>2] throughout the experiment.Conclusion: In this study,recombinant plasmids p AAV2neo-1.3HBV-C-WT and p AAV2neo-1.3HBV-C-MDR were successfully constructed.By in vivo transduction with the recombinant virus r AAV8-1.3HBV-C-WT,a mouse model that stably expressed and replicated HBV-C-WT has been successfully established.And it was preliminarily proved that NAs could effectively inhibit the replication of HBV-C-WT in the mouse model.We also tried establishment of the HBV-C-MDR replication mouse model,but the results were not fully consistent with expectation and further study is needed to improve the project.Taken together,the establishment of the HBV-C replication mouse model provides a research tool for screening new and effective anti-Chinese epidemic HBV-C drugs and treatment methods. |
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