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Objective To investigate the effects of adefovir resistance related mutations in hepatitis B virus (HBV) reverse transcription (RT) region on the viral replication and hepatitis B surface antigen (HBsAg) secretion. Methods Twelve adefovir treated chronic hepatitis B (CHB) patients who experienced a viral breakthrough were enrolled in this study. The RT region was amplified by polymerase chain reaction (PCR) using HBV DNA extracted from sera as the template. PCR products were then sequenced and analyzed to find out mutation patterns. Full-length HBV genome was amplified from 4 representative serum samples followed by direct sequencing. The dominant strain was cloned into vector PHY106 to construct a recombinant plasmid containing the 1.1 unit of HBV genome Which was transfected into Huh7 cells. HBsAg and hepatitis B e antigen (HBeAg) expression were determined by enzyme-linked immunosorbent assay (ELISA), meanwhile intracellular HBV DNA level was determined by quantitative real-time PCR. Furthermore strain harboring rtA181T/sW172 · mutation and strain without rtA181 mutation were cotransfected into Huh7 cells. HBsAg and intracellular HBV DNA were also determined after transfection. Results Ten out the 12 patients enrolled in this study exhibited mutations conferring resistance to adefovir. The rtA181T mutation was detected in 5 cases, and the rtA181T/S+rtN236T mutation was observed in 4 cases. Different mutants showed variable HBsAg secretion competency in vitro. Despite the defect of HBsAg secretion of the rtA181T/sW172 · mutant, the replication efficiency was almost the same in different mutants. When the strains with and without rtA181 mutation were cotranfected into cells, the HBsAg level increased in accordance with the amount of stains without rtA181 mutation. However, the intracellular HBV DNA level was not changed significantly. Conclusions The rtA181 mutation is common in patients with adefovir resistance, of which the rtA181T mutation is the major pattern. In vitro analysis reveals that the rtA181T/sW172 · mutant is defective in HBsAg secretion which could be rescued by coexistence of wild-type strains. The replication efficiency in various mutants shows no obvious differences.
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OBJECTIVE: To study the efficacy and safety of Fuzheng Huayu Capsule (FZHY Capsule) against liver fibrosis with chronic hepatitis B. METHODS: Multicentric, randomized, double blinded and paralleled control led trial was conducted on patients (aged between 18 and 65) with liver fibrosis in chronic hepatitis B Indexes observed: (1) hepatic histological changes and HBV markers were observed at 0 and 24th week during the treatment; serological indexes (HA, LN, P-III-P, IV-C) were determined and B ultrasound examination of spleen and liver was taken at 0, 12th, 24th week; liver function (during the period of follow-up, liver function and serological indexes for liver fibrosis were evaluated) were observed at 0, 6th, 12th, 18th, 24th week; (2) indexes for safety: blood and urine routine tests, renal function and ECG were examined. RESULTS: (1) Enrollment and demographic data: There was no significant difference between the trial (110 cases) and control group (106 cases) in demographic feature, vital signs, course of illness, history for drug anaphylaxis, history of previous therapy, liver function, serological indexes for liver fibrosis, liver histological examination (99 cases for test group, 96 cases for control group), HBV markers, and renal function, etc. (2) Histological pathological examination: 93 cases of liver histological examination were taken, of these 50 cases for the trial group and 43 cases for control group which turned out to be at S mean value of 2.33 and 2.11 respectively pretreatment according to criteria for liver fibrosis staging. Post-treatment, the trial showed a significant decrease with S value of 1.80 compared to that of pretreatment; however, there was no significant improvement in control group before and after the treatment with S mean value of 2.14. There was significant difference in reversing rate (decrease at least 1 stage according to criteria for liver fibrosis staging) between the trial (52%) and control (23.3%) after liver biopsy. The trial had a rather good effect on improving inflammatory activity and was superior to control group with a marked decrease of mean value of inflammatory activity and score of inflammation (P<0.05). (3)Serological indexes for liver fibrosis: There was a significant decrease in HA, LN, P-III-P, IV-C content in test group after 12 and 24 weeks' treatment compared to that of pretreatment; the differences of HA, LN, P-III-P, IV-C between 12, 24 weeks' treatment and pretreatment were significantly greater than control group (P<0.01 or 0.05); the effectual was defined as 2 of 4 indexes lowered more than 30% of the baseline, according to this criteria, the trial was 72.7%, while control group 27.4% (P<0.01). (4)Liver function: Obvious improvement of serum Alb, ALT, AST, GGT was seen in 2 groups; compared with control group, marked improvement of GGT and Alb in the trial (P<0.05); the effective rate of serum ALT in the trial group was 72.7%, while control 59.4%. (5)No changes of significant difference between pre- and post-treatment in routine tests for blood and urine, renal function and ECG, etc. There was also no difference in the stable rate of ALT and serological indexes for liver fibrosis between the trial and control group 12 weeks after withdrawal (P<0.05). CONCLUSION: Fuzheng Huayu Capsule has good effect on alleviating liver fibrosis in chronic hepatitis B without any adverse effect and is superior to Heluo Shugan Capsule. Fuzheng Huayu Capsule is a safe and effective medicine for the treatment of liver fibrosis in chronic hepatitis B.
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Objective To compare the structural genome variations of lamivudine resistant HBV strain from a patient with acute exacerbation after lamivudine therapy with pretretment HBV strain. Methods Full length genomes of two HBV strains before lamivudine treatment and after occurring severe acute exacerbation during lamivudine treatment were cloned and sequenced in a patient with chronic hepatitis B. Nucleotide sequence and predicted amino acid sequences of 4 open reading frames in two HBV strains were analyzed. Results Compared with pretreatment full length HBV genome, there were 30 amino acid substitutions in four ORFs of lamivudine resistant HBV strain, including substitution of valine for methionine at residue 550(M550V)accompanied by substitution of methionine for leucine at residue 526(L526M). The numbers of amino acid substitutions in ORF P, X, S, Precore/Core were 10(1.19%), 4(2.60%), 8( 2.00%) and 8 (3.77%) respectively. The total variation rate along the whole HBV genome was 1.86%. A G→A change at nucleotide position 1898 resulted in a precore stop codon and led to lack of HBeAg expression; At the preS2 initiation codon ATG was changed to ATA which caused the loss of preS2 protein expression. Some amino acid changes occurred within the known CTL, B or T cells epitopes.Conclusions In a patient with severe acute exacerbation during lamivudine therapy, together with 29 additional amino acid substitutions were detected outside the YMDD motif in the lamivudine resistant HBV strain.
