[Dynamic detection of duck hepatitis B virus cccDNA in serum of ducks with liver injury].

OBJECTIVE: To confirm whether DHBV cccDNA could be detected in serum of DHBV-infected ducks after liver injury. METHODS: Twenty four ducks with persistent DHBV infection were divided into 4 groups with the following treatments: A, D-galactosamine (D-GalN, 2.2 g/kg) and lipopolysaccharide (LPS, 100 µg/kg); B, 10 mg/kg of carbon tetrachloride (CCl4) every 12 h twice following D-GalN and LPS; C, 15 mg/kg of CCl4 every 12 h twice following D-GalN and LPS; D, normal saline as normal control (NC). At 0 h, 24 h, 36 h and 48 h post-treatment, sera were collected from each duck for determination of serum DHBV load, DHBV cccDNA and alanine aminotransferase (ALT). And ducks were eventually sacrificed to obtain liver specimens for pathological assessment of liver lesions and determination of intrahepatic total DHBV DNA and DHBV cccDNA. RESULTS: (1) No obvious pathological change was observed in the liver of ducks from NC group while the indices of liver injury were significantly different between Groups A, B and C; (2) DHBV cccDNA was undetectable in the sera of ducks from NC and A group at all time points. In contrast, DHBV cccDNA, varying from 3.17 × 10(3) copies/ml to 1.72 × 10(4) copies/ml, was detected in the sera of 2 ducks from Group B and 3 ducks from Group C at 36 h post-treatment. The occurrence of DHBV cccDNA in serum was significantly correlated with the degree of liver injury while no significant association with serum ALT level and DHBV load as well as with the level of intrahepatic total DHBV DNA and DHBV cccDNA was observed. CONCLUSION: DHBV cccDNA may be detected in the serum when the liver of duck is seriously damaged. The incidence of DHBV cccDNA occurrence in the serum is significantly associated with the severity of liver injury.

Effect of oxymatrine on the replication cycle of hepatitis B virus in vitro.

AIM: To determine the antiviral mechanism or target of oxymatrine against hepatitis B virus (HBV). METHODS: HepG2.2.15 cells were incubated with culture medium containing 500 microg/mL of oxymatrine for 2 and 5 d. The surface antigen of HBV (HBsAg) and e antigen of HBV (HBeAg) in supernatant were determined by ELISA. HBV DNA in supernatant, and intracellular covalently closed circular DNA (cccDNA), relaxed circular DNA (rcDNA) and pregenomic RNA (pgRNA) were quantified by specific real-time polymerase chain reaction (PCR) or reverse transcription (RT)-PCR. RESULTS: Treatment with oxymatrine for 2 d and 5 d reduced the production of HBV by the cell line, as indicated by the decline of HBsAg (22.67%, t = 5.439, P = 0.0322 and 22.39%, t = 5.376, P = 0.0329, respectively), HBeAg (55.34%, t = 9.859, P = 0.0101 and 43.97%, t = 14.080, P = 0.0050) and HBV DNA (40.75%, t = 4.570, P = 0.0447 and 75.32%, t = 14.460, P = 0.0047) in the supernatant. Intracellular cccDNA was also markedly reduced by 63.98% (t = 6.152, P = 0.0254) and 80.83% (t = 10.270, P = 0.0093), and intracellular rcDNA by 34.35% (t = 4.776, P = 0.0413) and 39.24% (t = 10.050, P = 0.0097). In contrast, intracellular pgRNA increased by 6.90-fold (t = 8.941, P = 0.0123) and 3.18-fold (t = 7.432, P = 0.0176) after 500 microg/mL of oxymatrine treatment for 2 d and 5 d, respectively. CONCLUSION: Oxymatrine may inhibit the replication of HBV by interfering with the process of packaging pgRNA into the nucleocapsid, or inhibiting the activity of the viral DNA polymerase.

[The spontaneous YMDD mutation rate in chronic hepatitis B patients].

OBJECTIVE: To investigate the spontaneous YMDD mutation rate. METHODS: Serum samples collected from 196 untreated chronic HBV patients were detected by primer-specific real-time PCR. RESULTS: Among 196 patients, spontaneous YMDD variants were detected in 21 subjects (20 YVDD mutants and 1 YIDD mutant). YMDD variants account for more than 50%, 25% to 50%, 9% to 25% of total virus load in 1, 5 and 15 patients, respectively. Gender, age, HBeAg status, serum viral load, the state of disease and duration of infection were not associated with spontaneous YMDD mutation. Genotype B had higher spontaneous YMDD rate than genotype C (20.00% vs 7.38%, x(2) = 6.28, P < 0.05). CONCLUSION: Spontaneous YMDD variants exist in chronic hepatitis B patients, Genotype B is associated with higher spontaneous YMDD rate.

[Comparative study on clearance dynamics of hepatitis B virion and cccDNA in blood].

OBJECTIVE: To confirm whether it is feasible to detect HBV cccDNA based on an analysis of its stability and clearance dynamics in the blood of ducks. METHODS: Twelve 1-week-old ducklings were randomized into group A and group B, and each duckling was intravenously injected with 1 milliliter of serum containing 1.2 x 10(9) copies of HBV virion (group A) or plasmid pBS HBV 3.6 II (group B). At 0.25, 2, 4, 6, 8, 12, 24 and 32 h post-injection, serum HBV DNA level in each duckling was quantified by Real-time fluorescent PCR. RESULTS: Both of HBV virion and plasmid pBS HBV 3.6 II could be detected from 0.25 h to 24 h after intravenous administration, and declined approximately by 1 log(10) every 2 hours within 8 h and was more slowly cleared at 8 h, and became undetectable at 32 h. Both of them are characteristic of one-phase exponential decay, as indicated by the relative index of the one-phase exponential curve ranging from 0.9593 to 0.9976. The mean half-life of HBV virion and plasmid (cccDNA) was 4.536 +/- 0.769 h (range: 3.5 - 5.4 h) and 4.5 +/- 0.8 h (range: 3.9 - 6.1 h) respectively. No significant difference was observed in the half-life between HBV virion and plasmid/cccDNA (t = 0.0523, P = 0.9593). CONCLUSIONS: HBV cccDNA is similar to HBV virion in its characteristics of clearance dynamics in blood. Thus it is feasible to detect serum HBV cccDNA. The clearance half-life of Hepatitis B virus in serum is not as long as previously believed at 1.0 - 1.2 d.