A New Model Mimicking Persistent HBV E Antigen-negative Infection Using Covalently Closed Circ?lar DNA In Immunocompetent Mice

Background and ObjectiveDespite the availability of an effective vaccine,hepatitis B virus(HBV)infection remains a major health problem.There are approximately 248 million individuals infected chronically with HBV worldwide.Hepatitis B virus infection has two common phenotypes,thirty years ago,the wild-type HBe Ag-positive virus was the primary epidemic strain;in recent years,the prevalence of HBe Ag-negative strains is increasing globally.Among the two types of CHB,there are major differences in terms of epidemiology,pathogenesis,natural clinical course,prognosis,and treatment.Compared to HBe Ag-positive CHB patients,HBe Ag-negative patients exhibit low sustained response rates to antiviral therapy,resulting in increased treatment times.Moreover,HBe Ag-negative CHB has a greater chance of developing into HCC.Therefore,the development of HBe Ag-negative HBV pathogenesis and its treatment of drugs is significant.Experimental animal model plays an important role in disease pathogenesis and drug development.However,research on HBV infections,treatment of CHB,and antiviral drug screening still rely on an HBe Ag-positive animal model.Previously,no animal model mimicked the clinic course of HBe Ag-negative HBV infection.Therefore,the aim of this study is to establish a mouse model which can simulating HBe Ag-negative HBV infection.Methods1.53 cases of HBV patients HBs Ag and HBe Ag carrying status were analysed.The HBV genotype was analyzed by SNa Pshot.2.The 3.2-kb full-length genome of the HBe Ag-negative virus was amplified from a clinical sample by PCR.Subsequently,full-length DNA from the HBe Ag-negative virus was cloned into the pEASY-Blunt Simple Cloning vector through molecular biology methods.The results of cloning were verified by sequencing.The sequencing result was subjected to a BLAST search at GenBank.3.The plasmid was digested with the BspQI restriction enzyme then purification.The linear target fragment was retrieved after agarose gel electrophoresis.Linear HBV was circularized by T4 DNA ligase,covalently closed circular(cccDNA)can be obtained in vitro.The results of circularized were verified by restriction enzyme digestion and sequencing.4.C57BL/6J male mice was injected with circularized HBe Ag-negative HBV DNA and p AAV-HBV 1.2,respectively,by the hydrodynamic method.Serum samples were collected via the tail vein at 1 and 3 days,and at 1,2,3,4,5,6,7,8,9and 10 weeks after injection.After 3 and 10 weeks,three mice from each group were sacrificed by cervical dislocation,serum and liver tissue were collected.5.The levels of HBs Ag and HBe Ag in serum were detected using a radioimmunoassay diagnostic kit.Serum levels of ALT were detected by a mouse ELISA kit.The location and expression of HBs Ag and HBc Ag in liver tissue were detected by IHC staining.Histopathological changes in mouse hepatic tissue assessed by hematoxylin eosin(HE)staining.HBV DNA was detected in serum by real-time fluorescent quantitative PCR(qPCR).cccDNA in liver was detected by rolling circle amplification(RCA)-PCR.Results1.Among the 53 patients,twenty-seven(50.9%)of the HBV-infected patients were HBe Ag-negative.One patient presented the A genotype,whereas 38 were infected with hepatitis B,and 14 had the C type.2.The clone sequence was 98% identical to GenBank sequence JX661478.1 which is the HBV sequence.The four overlapping open reading frames of gene were complete.And the mutation rate was insignificant except for G1896 A,G1899A,A1762 T,and T1764 G point mutations in the C region;The presence of double mutations in the A1762 T and T1764 G in Basal core promoter region(BCP)reduced HBe Ag expression point mutations;The presence of the G1896 A mutation in pre C region prematurely terminated HBe Ag expression.3.The result of sequencing and digestion confirmations showed that linear HBV was circularized to cccDNA.The cyclized product has no base deletion and insertion of exogenous genes.4.In mice injected with HBe Ag-negative cccDNA,the HBV infection rate was 100% within 5 weeks after injection.HBs Ag increased up to 1 week,at which point levels peaked and dropped quickly thereafter.In 60% of injected mice,HBs Ag and HBc Ag persisted for more than 10 weeks.All mice,HBe Ag negative;107 copies / ml and 108 copies / ml HBV DNA were detected in the serum and liver.Moreover,cccDNA persisted in the liver tissue of HBe Ag-negative mice.In p AAV-HBV 1.2 injected mice,HBsAg,HBe Ag or HBs Ag,HBc Ag can be detected in serum and liver.ConclusionA model of HBe Ag-negative persistent infection was successfully established in immunocompetent mice for the first time.Compared to pAAV-HBV1.2-injected mice,the infection persistence and levels of serum virological(No containing HBe Ag)and biochemical markers were no significant difference.This model will be usefu for mechanistic studies on HBe Ag-negative infection and will facilitate the evaluation of new antiviral drugs.