Adolescent ; Adult ; Aged ; DNA Mutational Analysis ; DNA, Viral ; genetics ; Drug Resistance, Viral ; drug effects ; genetics ; Female ; Hepatitis B virus ; drug effects ; genetics ; isolation & purification ; Hepatitis B, Chronic ; genetics ; virology ; Humans ; Male ; Middle Aged ; Young Adult

Fifteen known compounds were isolated from Swertia delavayi by silica gel, Sephadex LH-20 and Rp-18 column chromatographies. Based on extensive spectroscopic analysis (MS, 1H, 13C-NMR), their structures were identified aserythrocentaurin (1), erythrocentaurindimethylacetal (2), sweroside (3), swertiamarin (4), gentiopicroside (5), swertiakoside A (6), 2'-O-acetylswertiamarin (7), 4'-O-[(Z) -coumaroyl] swertiamarin (8), 1,5,8-trihydroxy-3-methoxyxanthone (9), 8-O-β-D-glucopyranosyl-1-hydroxy-2,3, 5-trimethoxyxanthone (10), 8-O-[β-D-xyl- opyranosyl-(1 --> 6)-β-D-glucopyranosyl]-7,8-dihydroxy-3-methoxyxanthone (11), isovitexin (12), β-sitosterol (13), daucosterol (14), and oleanolic acid (15). Among them, ten ones (14, 7-11, 13) were obtained from S. delavayi for the first time. The isolates were evaluated for their anti-HBV activities in HepG 2. 2. 15 cell line in vitro. The results showed that compound 1, 2, 6, 7, 9 and 12 exhibited significant inhibitory activity on HBV DNA replication with IC50 values from 0.05 to 1.46 mmol x L(-1).
Antiviral Agents ; chemistry ; isolation & purification ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Hepatitis B virus ; drug effects ; genetics ; Magnetic Resonance Imaging ; Molecular Structure ; Spectrometry, Mass, Electrospray Ionization ; Swertia ; chemistry

<b>OBJECTIVEb>To establish a new method for rapidly selecting anti-hepatitis B virus drugs in clinical therapy.

<b>METHODSb>The full-length hepatitis B virus (HBV) genomes from 8 patients with chronic hepatitis B (CHB) were generated by polymerase chain reaction (PCR). All patients were resistant to lamivudine therapy. Their HBV DNA fragments were inserted into Sap I site of pHY106 eukaryotic expression vector separately. The recombinant plasmids containing 1.1 copies of HBV genome were transfected into Huh7 cell line; the levels of HBsAg, HBeAg and HBV DNA in supernatants of Huh7 cells were measured by ELISA and real-time quantitative PCR, and intracellular HBV replicative intermediates were detected by Southern blot. Antiviral effects of lamivudine and adefovir were evaluated in this vitro system.

<b>RESULTSb>The 8 recombinant plasmids containing a full-length genome of clinical HBV isolates could replicate and be expressed in Huh 7 cells. There were 6 isolates with polymerase YVDD mutations and 2 isolates with polymerase YIDD mutations. Adefovir, but not lamivudine, inhibited the HBV replication and gene expression in vitro. Furthermore, adefovir inhibited HBV replication in these CHB patients.

<b>CONCLUSIONb>The method described here enables a rapid selection of anti-HBV drugs in clinical therapy and is very useful in antiviral therapy for CHB patients.

Adult ; Antiviral Agents ; pharmacology ; Drug Evaluation, Preclinical ; Drug Resistance, Viral ; Female ; Hepatitis B ; virology ; Hepatitis B virus ; drug effects ; genetics ; isolation & purification ; Humans ; Male ; Middle Aged ; Virosomes ; Young Adult

<b>OBJECTIVEb>To evaluate clinical applicability of a novel technique that can quantify the lamivudine-resistant mutants of hepatitis B virus (HBV) in the serum of patients utilizing gene microarray technology.

<b>METHODSb>The oligonucleotide microarray was designed to detect 3 important mutational positions. Fifty-one patients who were receiving lamivudine therapy were selected as subjects. The oligonucleotide microarray and traditional sequencing were applied to detect the lamivudine resistant mutation, the monitoring lasted for 24 months. Then the clinical result was analyzed and the obtained data were compared between the two methods.

<b>RESULTSb>Lamivudine resistant mutation was detected in 39 percent of the patients during the 2 years period. The results of the oligonucleotide microarray technique was consistent to the results of traditional sequencing in accuracy and the miroarray was more sensitive in detection of the mixed infection.

<b>CONCLUSIONb>Application of the oligonucleotide microarray for quantitative detection of lamivudine-resistant mutation of HBV is feasible.

Antiviral Agents ; therapeutic use ; Drug Resistance, Viral ; Female ; Hepatitis B ; drug therapy ; virology ; Hepatitis B virus ; drug effects ; genetics ; isolation & purification ; Humans ; Lamivudine ; therapeutic use ; Male ; Mutation ; Oligonucleotide Array Sequence Analysis ; methods

<b>OBJECTIVEb>To analyze antiviral effects of entecavir in patients with hepatitis B virus-related cirrhosis.

<b>METHODSb>104 patients of hepatitis B virus-related cirrhosis with no previous history of antiviral therapy were treated with entecavir 0.5 mg once daily. 37 patients were taken hepatic histologic examination before and after the treatment.

<b>RESULTSb>Mean reductions of serum HBV DNA was 5.1 log10 96 weeks after the treatment, HBV DNA became undetectable in 98.1% patients, and ALT became normal in 80.7% patients; HBeAg seroconversion occurred in 13.9% of the 72 HBeAg positive patients; 61.5% of these patients were infected with genotype C HBV, and 26.9% were infected with genotype B HBV. The genotype of HBV was not associated with the therapeutical effect. Child-pugh score was associated with the progression of the disease: the proportion of patients with disease progression was highest in Child-Pugh C grade patients and lowest in Child-Pugh A grade patients. The level of the HBV DNA load was positively correlated with Knodell HAI score at the baseline and 96 weeks after the treatment.

<b>CONCLUSIONb>Entecavir treatment results in suppression of HBV replication and delayed progression of fibrosis in patients with hepatitis B virus-related cirrhosis.

Adult ; Alanine Transaminase ; blood ; Antiviral Agents ; therapeutic use ; DNA, Viral ; blood ; Female ; Genotype ; Guanine ; analogs & derivatives ; therapeutic use ; Hepatitis B e Antigens ; blood ; Hepatitis B virus ; drug effects ; genetics ; isolation & purification ; Hepatitis B, Chronic ; complications ; drug therapy ; virology ; Humans ; Liver Cirrhosis ; drug therapy ; etiology ; virology ; Male ; Middle Aged ; Time Factors ; Treatment Outcome ; Virus Replication ; drug effects

BACKGROUND AND AIMS: Telbivudine is an L-nucleoside analogue with potent antiviral activity against hepatitis B virus (HBV). Clinical trials have shown that telbivudine is more potent than lamivudine for suppressing virus. METHODS: A total 101 Korean patients among 1,367 patients who participated in the phase III GLOBE trial were randomized in this study. All 101 HBeAg positive or HBeAg negative patients were assigned to treatment with 600 mg of telbivudine or 100 mg of lamivudine once daily. The primary efficacy endpoint (the "therapeutic response") was defined as suppression of the serum HBV DNA to less than 5 log10 copies/mL coupled with either normalization of the serum alanine aminotransferase level or loss of HBeAg. The secondary endpoints included the histologic response, serum HBV DNA reduction, serum alanine aminotransferase normalization and HBeAg loss for the HBeAg positive patients. This analysis includes the data collected at 52 weeks of treatment. RESULTS: Fifty four of 101 patients were assigned to telbivudine treatment and 47 patients were assigned to lamivudine treatment. At week 52, significantly more patients who were treated with telbivudine than those treated with lamivudine had a therapeutic response (83% vs 62%, respectively, P=0.017), their mean serum HBV DNA levels were more reduced (6.6 vs 5.6 log10 copies/mL, respectively, P=0.027), and they more often achieved PCR-undetectable levels of serum HBV DNA (74% vs 34%, P<0.0001). No virologic resistance to telbivudine was detected (0% vs 18%, respectively, P=0.001). Telbivudine was well tolerated and it had a safety profile comparable to lamivudine. CONCLUSIONS: Patients treated with telbivudine achieved earlier and more profound viral suppression than those treated with lamivudine.
Adolescent ; Adult ; Alanine Transaminase/analysis ; Antiviral Agents/administration & dosage/adverse effects/*therapeutic use ; Drug Resistance, Viral ; Female ; Hepatitis B e Antigens/analysis ; Hepatitis B virus/drug effects/genetics/isolation & purification ; Hepatitis B, Chronic/*drug therapy/virology ; Humans ; Korea ; Lamivudine/administration & dosage/adverse effects/therapeutic use ; Male ; Middle Aged ; Nucleosides/administration & dosage/adverse effects/*therapeutic use ; Pyrimidinones/administration & dosage/adverse effects/*therapeutic use ; Treatment Outcome

<b>OBJECTIVEb>To evaluate the sensitivity and specificity of nested PCR combined with pyrosequencing in the detection of HBV drug-resistance gene.

<b>METHODSb>RtM204I (ATT) mutant and rtM204 (ATG) nonmutant plasmids mixed at different ratios were detected for mutations using nested-PCR combined with pyrosequencing, and the results were compared with those by conventional PCR pyrosequencing to analyze the linearity and consistency of the two methods. Clinical specimens with different viral loads were examined for drug-resistant mutations using nested PCR pyrosequencing and nested PCR combined with dideoxy sequencing (Sanger) for comparison of the detection sensitivity and specificity.

<b>RESULTSb>The fitting curves demonstrated good linearity of both conventional PCR pyrosequencing and nested PCR pyrosequencing (R(2)>0.99, P<0.05). Nested PCR showed a better consistency with the predicted value than conventional PCR, and was superior to conventional PCR for detection of samples containing 90% mutant plasmid. In the detection of clinical specimens, Sanger sequencing had a significantly lower sensitivity than nested PCR pyrosequencing (92% vs 100%, P<0.01). The detection sensitivity of Sanger sequencing varied with the viral loads, especially in samples with low viral copies (HBV DNA ≤3log10 copies/ml), where the sensitivity was 78%, significantly lower than that of pyrosequencing (100%, P<0.01). Neither of the two methods yielded positive results for the negative control samples, suggesting their good specificity.

<b>CONCLUSIONb>Compared with nested PCR and Sanger sequencing method, nested PCR pyrosequencing has a higher sensitivity especially in clinical specimens with low viral copies, which can be important for early detection of HBV mutant strains and hence more effective clinical management.

Adolescent ; Adult ; Aged ; DNA, Viral ; genetics ; Drug Resistance, Viral ; genetics ; Female ; Hepatitis B virus ; drug effects ; genetics ; isolation & purification ; Humans ; Male ; Middle Aged ; Phosphoric Acids ; Plasmids ; Polymerase Chain Reaction ; Reagent Kits, Diagnostic ; Sensitivity and Specificity ; Young Adult

Adult ; Antiviral Agents ; therapeutic use ; DNA, Viral ; blood ; Female ; Follow-Up Studies ; Hepatitis B Surface Antigens ; blood ; Hepatitis B e Antigens ; blood ; Hepatitis B virus ; drug effects ; genetics ; isolation & purification ; Hepatitis B, Chronic ; blood ; drug therapy ; virology ; Humans ; Interferon-alpha ; therapeutic use ; Liver Function Tests ; Male ; Polymerase Chain Reaction ; methods ; Recombinant Proteins ; Time Factors ; Treatment Outcome

<b>OBJECTIVEb>To study the relationships between intrahepatic HBV DNA level and serum HBV DNA level, between intrahepatic HBV DNA level and hepatitis B e antigen (HBeAg) level in patients with chronic hepatitis B (CHB), and assess the valuation of pretreatment liver HBV DNA level in antivirus therapy.

<b>METHODSb>Liver specimens taken from 41 HBeAg-positive CHB patients before antivirus treatment were divided into two parts, one for histological examination, and the other for intrahepatic HBV DNA quantified detection by PCR-fluorescence. At the same time, serum levels of HBV DNA and HBeAg were detected. The patients were classified into two groups according to the pretreatment intrahepatic HBV DNA level (< or = 10(4)fg/cm(3) in group A, >10(4)fg/cm(3) in group B) and accepted interferon alpha-1b (3MU every day for 26 weeks) in combination with lamivudine (100mg per day for 52 weeks). During the treatment, the serum levels of alanine aminotransferase (ALT), HBV DNA and HBeAg seroconversion rate were monitored.

<b>RESULTSb>(1) The level of liver HBV DNA was much higher than that of serum HBV DNA (4.081 +/-1.127 vs 3.163 +/-1.010, t = 2.218, P < 0.05). Liver HBV DNA level had positive correlation to serum HBV DNA level (r = 0.840, t = 4.322, P < 0.001) and serum HBeAg level (r = 0.459, t = 3.056, P < 0.005). (2) Intrahepatic HBV DNA level was negative correlation to the severity of liver damage (chi(2) = 3.874, P < 0.05). (3) Serum HBV DNA level in all the patients reduced remarkedly after therapy, especially in group A. At the end of 52 weeks, the rates of HBeAg and anti-HBe seroconversion in group A were higher than those in group B (68.4% vs 36.4%, chi(2) = 4.194, P < 0.05; 73.7% vs 40.9%, chi(2) = 4.447, P<0.05).

<b>CONCLUSIONSb>Intrahepatic HBV DNA is a more valuable marker than serum HBV DNA or HBeAg to assess HBV replication, and can reflect the status of body immunity indirectly. It may be a useful indicator for the efficacy of antivirus treatment.

Adolescent ; Adult ; Antiviral Agents ; therapeutic use ; DNA, Viral ; analysis ; blood ; Female ; Hepatitis B e Antigens ; blood ; Hepatitis B virus ; genetics ; isolation & purification ; Hepatitis B, Chronic ; drug therapy ; virology ; Humans ; Interferon-alpha ; therapeutic use ; Lamivudine ; therapeutic use ; Liver ; virology ; Male ; Middle Aged ; Treatment Outcome ; Viral Load ; Virus Replication ; drug effects

<b>OBJECTIVEb>The aim was to build a PCR-RFLP method for detecting rtN236T mutants and to observe their kinetics in chronic hepatitis B (CHB) patients.

<b>METHODSb>Seven CHB patients who had suboptimal viral response or viral breakthrough under adefovir mono-therapy were studied. Part of the HBV reverse transcriptional gene from serial sera samples was sequenced with PCR products or cloned HBV DNA; mutations at rt236 were simultaneously analyzed by a PCR-RFLP assay. Genetic diversity of HBV was observed by calculating Hamming distance within domains B, C and D of RT.

<b>RESULTSb>Three patients had viral breakthrough and one with suboptimal viral response had adefovir-resistance mutants, one had rtA181V mutation and three had rtN236T mutation. A novel PCR-RFLP assay based on restriction enzyme HpaI or DraI for on the detection of rtN236T mutant was established, which detected 10% minor strains with 100% specificity. Mutants (rtA181V or rtN236T) appeared 0-8 months earlier than the viral breakthrough, then afterwards became the dominant ones. In one patient after stopping the adefovir therapy, 3 months later a wild type virus re-took again the mutant one (rtN236T); in one patient who developed a rt236T mutant after 132 weeks of adefovir treatment, a novel mutant (rtN236V) appeared and then became the dominant one while adefovir treatment continued.

<b>CONCLUSIONSb>A rapid and easy method was established to detect rtN236T mutants. Mutants for adefovir-resistance accumulated rapidly then became dominant, but they could be taken over again by a wild type or novel mutant HBV.

Adenine ; analogs & derivatives ; pharmacology ; Adult ; Antiviral Agents ; pharmacology ; DNA, Viral ; genetics ; Drug Resistance, Viral ; drug effects ; genetics ; Hepatitis B ; virology ; Hepatitis B virus ; drug effects ; genetics ; isolation & purification ; Humans ; Male ; Middle Aged ; Mutation ; Organophosphonates ; pharmacology ; Polymerase Chain Reaction ; methods ; Polymorphism, Restriction Fragment Length