| Objective:To investigate the quasispecies distribution of hepatitis B virus in HBV genome in patients with coexistence of HBsAg and HBsAb and to learn the pathogenesis of coexistence of HBsAg and HBsAb from the perspective of HBV quasispecies heterogeneity.Methods:Three patients with coexistence of HBsAg and HBsAb were studied and two patients with HBsAg positive were enrolled as control.HBV-DNA was extracted from serum sample and the HBV-DNA genome of hepatitis B virus was amplified by PCR. The PCR products were purified and then cloned and sequenced. The differences of quasispecies characteristics between the experimental group and the control group, which including HBV phylogenetic tree,quasispecies complexity and diversity and the change in varied degrees, were analyzed using bibinformatics methods.The variation of single amino acid in the pre-S1, pre-S2 and S proteins of the two groups HBV quasispecies was compared. Compared two groups of HBcAg, LHBsAg and HBxAg amino acid mutation index and single locus entropy change.And to gather statistics on the insertion and deletion of all clones.Results:1.50 strains of cloning were gotten,each patient were 8 to 13 clone strains.In the experimental group,two patients were C genetype and one patient was B genetype. In the control group, B genetype and C genetype each with one patient respectively.The quasispecies complexity of HBV genome of experimental group is more complicated than control group. Experimental group patients quasispecies complexity of function section of LHBsAg (amino acid level) and MHBsAg (nucleotides level) is more complicated than control group, the difference was statistically significant (P<0.05).2.Phylogenetic tree analysis revealed that the different types of clones were clustered together, and all the clones were clustered together according to genetype. The genotype of C quasispecies, two patients from coexistence of HBsAg and HBsAb were clustered together, and isolated from HBsAg positive patients.3.Coexistence of HBsAg and HBsAb HBV quasispecies versus to HBsAg positive HBV quasispecies, the diversity of maximum net change of value appears in the HBx gene (d (aa) and dN) and CP/C gene (d (nt) and dS).4.For the HBsAg "hot spots" aal26, only patients N23 of C genotype with 4 clones had the mutation. Patients N24 of C genotype with 3 clones had the mutation in the pre-S2 aa33 site,and all clones of genotype B patients in the experimental group occurred mutation in the site, all clones of patients in the control group were did not occur mutation in the site.5.In LHBsAg, entropy of genotype B and C genotype patients in the experimental group was significantly higher than that of control group (nonparametric test, P< 0.05). For genotype C patients in the experimental group, entropy of pre-C/C protein was significantly lower than that in the control group (nonparametric test, P< 0.05), the entropy of HBx protein was significantly higher than that of control group (nonparametric test, P< 0.05).Conclusios:1.HBV exists in infected patients as a form of quasispecies,the quasispecies constitution of HBV in the coexistence HBsAg and HBsAb patients is more complex than the HBsAg positive patients.2.Different types of clonal strains have independent origins. In patients of coexistence of HBsAg and HBsAb with C genotype of HBV quasispecies,may have similar evolutionary pattern.3. Aa33 on Pre-S2 protein is an important B lymphocyte epitopes, which mutation may be an important factor causing the coexistence of HBsAg and HBsAb.4.The coexistence of HBsAg and HBsAb patients of HBV may improve the frequency of amino acid variation in the LHBsAg epitope, in order to change the LHBsAg antigenicity and the binding force with the antibody, which leads to the emergence of the coexistence of HBsAg and HBsAb. |
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