ABSTRACT
Objective To characterize the profile and clinical significance of hepatitis B virus (HBV) quasispecies in patients infected with hepatitis B virus based on the sequence of reverse transcriptase (RT) region.Methods Fifty HBV infected treatment-naive patients were enrolled and divided into three groups,asymptomatic carriers (ASC) group (10 cases),chronic hepatitis B (CHB) group (30 cases) and liver cirrhosis (LC) group (10 cases).HBV genomes were extracted from serum samples.The sequence of RT region was amplified by polymerase chain reaction (PCR) and cloned into vectors.Fifteen to thirty clones per sample were selected,sequenced and analyzed by bioinformatics software.The mean values among groups were compared by analysis of variance.The median values among groups were compared by nonparametric statistics.The enumeration data were analyzed by x2 test.Results Totally 1221 HBV RT region nucleotide sequences were obtained (152from ASC patients,780 from CHB patients and 289 from LC patients).Genotype distribution showed no difference among three groups.However,the quasispecies complexity showed significant differences among the three groups,LC group ＞CHB group＞ ASC group (F=33.400,P＜0.05).The quasispecies diversity was LC group ＞CHB group＞ ASC group,and that of LC group was significantly different from the other two groups (F=18.070,P＜0.05),while there was no significant difference between CHB and ASC patients.Conclusions The HBV isolated from patients in immune clearance phase have higher variability than those isolated from patients in immune tolerance phase.The longer the infection persists and the more severe the disease is,the more variable HBV quasispecies are.
ABSTRACT
Objective To characterize serum hepatitis B virus(HBV)full-length genome quasispecies and to investigate its ralationship with severe exacerbation of chronic hepatitis B(CHB).Methods HBV full-length genome was amplified and cloned from four treatment naive CHB patients and four treatment naive CSHB patients.Fourteen to sixteen clones per sample were selected,sequenced and analyzed by bioinformatics software.The measurement data was compared by independent-samples t test and count data was analyzed by x2 test. Results Totally 120 HBV fulllength genome sequences were obtained.All the patients had either genotype B or C virus monoinfection.One hundred percent clones(60/60)from CSHB patients showed mutations including G1896A,A1762T/G1764A(one patient even carried A1762T/G1764A/C1766T mutations),T1753C/G and start codon mutations in preS2,preS1,which were more common than those from CHB patients(46/60,76.7%;x2=15.85,P<0.01).The quasispecies complexity and diversity were higher in CSHB patients than CHB patients within full-length genome,S,X,P genes and reverse transcriptase region,but lower within C gene at both nucleotide and amino acid levels.But the difference were not statistically significant in all regions.Conclusion The mutation frequency and quasispeeies heterogeneity in HBV genome are higher in CSHB patients than in CHB patients,which may play a role in the severe exacerbation of CHB and needs further investigation in large scale studies.
ABSTRACT
Objective To investigate the association of hepatitis B virus(HBV) S gene quasispecies with the outcome of HBV infection.Methods Serum samples were collected from three chronic HBV carriers, three chronic hepatitis B and three chronic severe hepatitis B patients.All subjects were male and with HBV genotype C.HBV S gene was amplified, and 20 clones of HBV fragment were randomly selected and sequenced from each sample.SPSS 15.0 software was adopted for analysis.Results Quasispecies complexity of HBV S gene in chronic HBV carriers and chronic hepatitis B tended lower than that of the severe chronic hepatitis B, but the difference was not of statistical significance (P＞0.05).In T cell epitope 45, 47, 85 amino acid sites of the HBV S gene, the constitution of quasispecies in the chronic hepatitis B was more complex than that of the HBV carriers (P=0.01), but compared with the severe chronic hepatitis, the difference was not significant (P=0.06).The computer model showed that both the dominant clones and the non dominant clones could effectively bind to the receptors of cytotoxic T lymphocytes.Conclusion Quasispecies in some T cell epitopes of HBV S gene may be related with the clinical outcome of hepatitis B.