Sequence Analysis Of A HBV Wild Strain And Its Preliminary Application

ObjectiveHepatitis B virus?HBV?is a hepatotropic DNA virus with strong tissue and species specificity.Therefore,the establishment of an ideal animal model of HBV becomes a difficult problem in the world.Traditional animal models,such as chimpanzees,groundhog,tree shrew and duck,play an important role in the study of HBV infection,but there are limits.In order to establish a better animal model of HBV,HBV genome is also an significant factor in addition to host animal participation.In the reported animal model,no detailed analysis of relevant genetic background was found,which limited the better utilization of the model.Therefore,in the premise of the quality assurance of the animal,the basic information of HBV sequence is analyzed and it lays a foundation for better modeling and utilization of the model.The purpose of this study was to characterize the natural genetic variation of a natural HBV strain and to study its function in vivo.It provides a methodology for the individualization of HBV model,and lays a foundation for individualized treatment and accurate treatment of HBV.Methods1.Elect the special patient serum DNA from 53 patients.Full-length HBV gene was amplified by PCR.pEASY-HBV HBeAg negative plasmid was built by molecular biology method and determined by sanger sequencing.2.The genic sequence was compared to related sequences using alignment and phylogenetic analysis,and the location and variation sites of the sequence were obtained.3.Eight mice were selected randomly for virus quality detection,mainly including detection of M-Ect,MHV,MNV and MSV by ELISA.4.CBA/CaJ male and female mice at six and twelve weeks of age were selected,and then the body and main organ weights,blood physiological and biochemical parameters and proportion of B cells,T cells,NK cells and granulocyte ratio were observed respectively.5.HBV full-length gene was amplificated through PCR.The product was digested by BspQI enzyme and then purified recycle product.Next,use T₄DNA ligase in 16?tocircumscrib it into covalently closed circular DNA?cccDNA?,then cyclization product purification recycling.The product was identified by Eco RI,PSAD enzyme and sanger sequencing.6.HBV cccDNA,pAAV/HBV1.2 plasmid and normal saline were injected into the CBA/CaJ mice respectively through hydrodynamic method.Serum samples were collected via the tail vein at 0.5,1,2,3,4,5,6,8,9,14,18,22,24,and 26 weeks after injection.7.The expression of serum specific markers HBsAg and HBeAg were detected by radioimmunoassay.Results1.A representative sample from a man?B type and HBe Ag negative?was selected from 53 patients,and the plasmids of pEASY-HBV HBeAg negative were constructed and sequenced successfully.2.The sequence had a full genome length of 3215 nucleotides and the four open reading frames?ORF?are complete.The phylogenetic tree showed that the genotype was B,sub-genotype was B2.In the genome,there were two main mutation sites in the basal core promoter region?BCP?:ntA1762T,ntG1764A.In the PC region,there are two main mutation sites:ntG1896A and ntG1899A.ntG1896A mutation causes the amino acid to become premature stop codon.The amino acid sites of 122,125,127 and 160 in S gene were identified as Lys,Thr,Pro and Lys.These sites indicated the serotype was adw2 and there was no reported immune escape sites in S gene.The P gene showed no reported resistance mutation sites.3.The results of M-Ect,MHV,MNV and MSV were all negative.4.Compared with the same age of male and female mice,body weight,heart,liver,kidney weight of males were significantly higher than females?P<0.01?.Mice with the same genders but different weeks,the body weight of male mice at 12 weeks was significantly higher than 6 weeks?P=0.000?.There were no significant differences in female mice.Compared the male and female mice at six weeks,MCV,RDW,PLT,P-LCR,BUN and CHOL of male were significantly higher than females?P<0.01?,while MCHC of male was significantly lower than female?P=0.000?.At twelve weeks,ALKP of male mice was lower than female?P=0.001?,while CHOL was higher than female?P=0.000?,there was no significant differences for other indices.Mice with the same genders and different weeks,MCV,RDW,MCH and ALKP of twelve weeks mice were significantly lower than six weeks?P<0.01?,while BUN was higher than six weeks mice?P<0.01?.All mice had a certain proportion of immune cells.5.Enzyme digestion showed that linear DNA has been circumscribed into cccDNA successfully;The sequencing results showed that the cccDNA did not have the deletion,alteration or insertion of exogenous genes.6.CBA/Ca J mice injected with cccDNA,HBsAg persisted for 26 weeks and HBeAg was still negative.CBA/CaJ mice injected with PAAV-HBV 1.2plasmid,HBsAg and HBeAg were detected in serum for 26 weeks.Conclusions1.ntA1762T,ntG1764A decreased the expression of HBeAg and ntG1896A stop the expression of HBeAg.The obtained HBV is a representative HBeAg negative,B genotype,and adw serum type strain.It is an important reference sequence for HBV HBeAg negative strains in China.2.CBA/CaJ mice are qualified.3.The basic biological data of CBA/CaJ mice were established.The results suggested that the cellular immune system for the CBA/CaJ mice is intact,and these indicators were affected by age and gender.4.The HBV HBeAg negative infection model was successfully established in the CBA/CaJ mice with immunological activity.