Serum HBV RNA Level Reflects the Potency of Nucleos(t)ide Analogue Y.W. Huang 1,2 , K. Chayama 3,4 , M. Tsuge 3,4 , S. Takahashi 3,4 , T. Hatakeyama 3,4 , M.Y. Lai 2,5 , H.L. You 1 , J.T. Hu 1 , C.J. Liu 2,5 , P.J. Chen 2,5 , D.S. Chen 2,5 , S.S. Yang 1 , J.H. Kao 5, 6 1 Liver Unit, Cathay General Hospital Medical Center, 2 Background and aims: Serum HBV RNA is detectable in patients treated with lamivudine (LMV) or entecavir (ETV) (Hatakeyama, 2007 and Huang, 2008) . The aim of this study was to determine the clinical significance of serum HBV RNA levels in patients treated with nucleos(t)ide analogues of different potency. Methods: Serum HBV RNA was serially determined in 20 patients treated with nucleos(t)ide analogues for 48 to 52 weeks (9 with adefovir (ADV), 5 with LMV, and 6 with ETV). Serum HBV RNA was quantified by reverse transcription of HBV nucleic acid extract with subsequent real-time PCR. Results: HBV RNA was detectable in 11 patients as follows: 2 of 9 in ADV (22%), 3 of 5 in LMV (60%), and 6 of 6 in ETV (100%) (p = 0.002). Mean log serum HBVDNA levels at baseline were 10.1 ± 0.6 for ADV, 6.6 ± 2.0 for LMV, and 9.5 ± 0.9 for ETV, which were comparable between less potent ADV and most potent ETV (p = 0.14). During antiviral therapy, peak log HBV RNA level of patients with ETV was significantly higher than that of those with ADV or LMV (3.2 ± 0.9 vs. Background: In the phase III clinical trials, clevudine 30mg for 6 months showed potent antiviral activity along with a marked post-treatment antiviral effect. The objective of this study is to compare the anti-HBV activity of combination of clevudine and vaccine over clevudine alone in chronic hepatitis B (CHB) patients in a randomised way. Methods: The patients are received clevudine for 24 weeks and then combination of clevudine and vaccine for another 24 weeks or clevudine alone for 48 weeks. Eligible patients were treatment-naïve HBeAg(+) CHB patients with HBV DNA levels 500,000 copies/mL. The primary endpoint is the proportion of patients with HBeAg loss. Preliminary results are presented here. Results: Thirty-one patients have completed week 24 visits and from them, 15 patients (11 in clevudine alone and 4 in combination group) have completed week 36 visits. At week 24, 26% of patients had HBeAg loss. At week 36, 46% in clevudine alone and 25% in combination group (3 months on combination after clevudine monotherapy) had HBeAg loss. At week 24, 63% of patients had negative HBV DNA by Amplicor PCR (<300 copies/mL). At week 36, all of patients in both groups had negative HBV DNA by PCR and 73% in clevudine alone and 80% in combination group had normal ALT. Conclusion: Clevudine demonstrated good serologic response as well as significant viral suppression and ALT normalization. With this data, we conclude that combination therapy of clevudine and vaccine for short period does not show the superiority over clevudine alone. Background/Aims: To determine the reasonable number of clones for HBV quasispecies analysis. Methods: 16 chronic hepatitis B patients were enrolled with HBVDNA levels range from 4~10 log copies/ml. HBVDNA was extracted. HBV reverse transcriptase (RT) gene encompassing the overlapping surface S gene was amplified by polymerase chain reaction, then cloned and sequenced. Ten positive clones for each sample were sequenced in the first group, and then additional ten positive clones were sequenced in other groups until up to thirty clones. The characteristics of HBV quasispecies including Shannon entropy and genetic distance were calculated. Results: The Shannon entropy and genetic distance of 10 clones group was higher than those of 20 and 30 clones group, either in RT gene or in S gene (p<0.01). While the Shannon entropy and genetic distance of 20 clones group showed on difference with those of 30 clones group, neither in RT gene nor in S gene (p>0.05). The number of different quasispecies detected in 30 clones group was higher than that of 20 and 10 clones group (p<0.01). The Shannon entropy and genetic distance in three different clones group had no correlation with HBV DNA levels (p>0.05). Conclusion: Although the number of different quasispecies detected was increased with the augmentation of clone number, the quasispecies characteristic didn't changed significantly when the clone number more than 20. The information contained in 20 clones per sample could well represent the quasispecies characteristics. The clone number was not necessary modulated according to different HBV DNA levels. Background: Recent studies reported that basal core promoter mutation (A1762T and G1764A) was associated with more aggressive progression of liver disease from inactive carrier to active hepatitis, and eventually to liver cirrhosis and HCC. But the effect of the double mutations on the activity of enhancer II/basal core promoter is still uncertain. Objectives: To evaluate the influence of nt1762 A/T and nt1764 G/A mutations on HBV enhancer II/basal core promoter activity. Methods: The PCR fragments of HBV enhancer II/basal core promoter (nt1601 to nt1815) from the serum-derived genotype B HBV DNAs of one HBV carrier aged 66 and one HBV related hepatocellular carcinoma patient aged 26 were introduced into the pGL3-Basic-Vector from Promega via restriction sites of Xho I and Hind III. The nt1762 A to T and T to A, the nt1764 G to A and A to G mutations were carried out by GeneTailor Site-Directed Mutagenesis System from Invitrogen. The promoter activity was evaluated by comparing firefly luciferase measurement with Renilla luciferase as the internal control using the Dual-Luciferase Reporter Assay System from Promega. Results: The luciferase reporter assay results indicated that the 1762 T to A combined with 1764 A to G mutations increase (P<0.001) while the 1762 A to T combined with 1764 G to A mutations decrease (P<0.05) the HBV enhancer II/basal core promoter activity significantly. Conclusions: Associated with increased risk of hepatocellular carcinoma, A1762T and G1764A double mutations of hepatitis B virus reduce the enhancer II/basal core promoter activity. Background/Aims: A substantial proportion of chronic hepatitis B (CHB) patients with mildly elevated alanine aminotransferase (ALT) have significant fibrosis. We evaluated the factors associated with significant fibrosis and clinical outcomes in these patients. Methods: One hundred five CHB patients with ALT less than two times the upper limit of normal underwent liver biopsy. Multiple clinical, biochemical and virologic variables were evaluated to determine the predictors of significant fibrosis and progressive liver disease. Results: There were 27 patients in the low normal ALT group, 21 in the high normal ALT group, 37 in the low elevated ALT group, and 20 in the high elevated ALT group. Fifty eight patients (55.2%) had significant fibrosis ( stage 3) and 35 (21.0%) had significant inflammation ( grade 3). The age, platelet count and grade of inflammation were factors associated with significant fibrosis. Progressive liver disease was observed in 23 (27.4%) of the 84 followed-up patients. The stage of fibrosis, ALT group and antiviral therapy were significant predictive factors for progressive liver disease. Conclusion: Liver biopsies should be recommended in patients over 35 years with mildly elevated ALT levels, and antiviral therapy should be considered in patients with significant fibrosis to prevent progressive liver disease. Background: Four nucleos(t)ide analogues (NAs) are currently approved for the treatment of HBV infection in China. However, long-term benefits are limited by the emergence of drug-resistant viruses. Methods: Patients accepted the examination based on physician's instruction. HBV reverse transcriptase gene was amplified from serum via nested PCR and sequenced directly. Results: Well-recognized drug-resistant mutations were detected in 567 of 2,000 patients. In patients receiving NA monotherapy, corresponding drug-resistant mutations were detected in 214/381 for lamivudine (LAM), 35/333 receiving adefovir (ADV), 0/57 for entecavir (ETV), and 9/17 for telbivudine (L-dT). The mutations were detected in 272/615 patients receiving 31 kinds of sequential/combined usages of the 4 NAs. M204I (32%), M204V+L180M V173L (32%), and M204I+L180M (21%) were identified as major mutant patterns of LAM monotherapy. N236T A181 substitution was the dominant ADV-resistant mutation. T184 substitution was the dominant ETV-resistant mutation always accompanied with LAM-resistant mutation. L-dT-resistant mutation was M204I L180M exclusively. ADV-resistant mutation was frequently seen in LAM-resistant patients receiving ADV sequential therapy rather than those receiving ADV add-on therapy. Controversial LAM/ADV-resistant mutations including A181T, V214A, Q215S and I233V were detected in some patients singly or with the well-recognized drug-resistant mutations. Interestingly, the drug-resistant mutations were also observed in a few of patients naïve to NAs. Conclusions: The exploration of HBV drug-resistant mutation profile in large clinical samples furthers our understanding of HBV drug-resistant status in China with implications for administrating anti-HBV therapy more reasonably.