Inhibition of human and woodchuck hepatitis virus DNA polymerase by the triphosphates of acyclovir, 1-(2′-deoxy-2′-fluoro-β-d-arabinofuranosyl)-5-iodocytosine and E-5-(2-bromovinyl)-2′-deoxyuridine

The triphosphates of acyclovir (ACV), 1-(2′-deoxy-2′-fluoro-β-d-arabinofuranosyl)-5-iodocytosine (FIAC) and E-5-(2-bromovinyl)-2′-deoxyuridine (BVdU) have been examined for their inhibitory effects on the endogenous DNA polymerase reactions of human hepatitis B virus (HBV) and woodchuck hepatitis virus (WHV). All three triphosphates (ACVTP, FIACTP and BVdUTP) inhibited the HBV and WHV DNA polymerases by competing with the corresponding natural substrates. FIACTP was the most potent inhibitor of HBV and WHV DNA polymerase while ACVTP was the least effective inhibitor. The inhibitory properties of these compounds were compared with those of the 5′-triphosphates of 1-β-arabinofuranosylcytosine (ara-CTP) and 1-β-arabinofuranosylthymine (ara-TTP). The 50% inhibitory doses for HBV and WHV DNA polymerases were in the following order: FIACTP < BVdUTP < ara-TTP < ACVTP < ara-CTP. BVdUTP appeared to be an efficient alternate substrate to dTTP for HBV DNA polymerase while FIACTP was much less efficient when substituted for dCTP. ACVTP did not act as an alternate substrate to dGTP and appeared to prevent DNA chain elongation.     

Protocatechuic aldehyde inhibits hepatitis B virus replication both in vitro and in vivo

Natural compounds provide a large reservoir of potentially active anti-hepatitis B virus (HBV) agents. We examined the direct effects of protocatechuic aldehyde (PA; derived from the Chinese herb, Salvia miltiorrhiza) on HBV replication in HepG2 2.2.15 cell line and duck hepatitis B virus (DHBV) replication in ducklings in vivo. The extracellular HBV DNA, hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) concentrations in cell culture medium were determined by quantitative real-time PCR and ELISA, respectively. DHBV in duck serum was analyzed by dot blot. PA appeared to downregulate the secretion of HBsAg and HBeAg as well as the release of HBV DNA from HepG2 2.2.15 in a dose- and time-dependent manner at concentrations between 24 and 48 microg/mL. PA (25, 50, or 100 mg/kg, intraperitoneally, twice daily) also reduced viremia in DHBV-infected ducks. We provide the first evidence that PA, a novel anti-HBV substance derived from traditional Chinese herb S. miltiorrhiza, can efficiently inhibits HBV replication in HepG2 2.2.15 cell line in vitro and inhibit DHBV replication in ducks in vivo. PA therefore warrants further investigation as a potential therapeutic agent for HBV infections.     

依肝达对鸭乙型肝炎病毒DNA的抑制作用

目的研究壮药依肝达体内抗鸭乙型肝炎病毒(DHBV)的作用。方法 1日龄北京雏鸭24只,接种鸭DHBV 7 d后,随机分为依肝达高、低剂量组(相当于10、5.0 g·kg-1生药量)、DHBV组和拉米夫定阳性对照组。各组均ig给药,bid,共10 d。采用斑点杂交法观察用药前与用药后第0、5、10日及停药后3 d血清中DHBV-DNA的表达。结果高剂量依肝达于给药后第5、10日,低剂量依肝达于给药后第10日,显著抑制感染鸭血清DHBV-DNA的复制,停药3 d后未见明显反跳。结论依肝达可抑制鸭体内DHBV的复制。     

Effects of Phyllanthus plant extracts on duck hepatitis B virus in vitro and in vivo.

The effects of extracts of five Australian Phyllanthus species (P. hirtellus, P. gunnii, P. gasstroemii, P. similis and P. tenellus), other plant extracts and the antiviral drug foscarnet on duck hepatitis B virus (DHBV) endogenous DNA polymerase (DNAp) activity were compared. All 5 Phyllanthus species caused 50% inhibition at concentrations of dry weight between 350-800 micrograms/ml, which is comparable with the effect described for P. amarus on the DNAp of human and woodchuck hepatitis B viruses. Incubation of P. hirtellus with 100 ID50 DHBV neutralized infection. However, neither P. gasstroemi extract, given by intraperitoneal injection (i.p.) at a dose of 20 mg/kg 3 times per week to ducklings early in the incubation period, or P. hirtellus extract, given to established DHBV carrier ducklings, prevented or eliminated infection.     

Expression of the active human and duck hepatitis B virus polymerases in heterologous system of Pichia methanolica

We expressed the Hepatitis B virus polymerase (HBV P protein) using a recently introduced yeast system, Pichia methanolica. HBV (1-680 amino acids) and Duck Hepatitis B virus (DHBV, 1-780 amino acids) polymerase were expressed and showed DNA dependent DNA polymerase (DDDP). The DHBV polymerase had RNA dependent DNA polymerase (RDDP) and RNase H activities. We present a new simplified way of obtaining active viral P protein using the yeast expression system. The viral P proteins proved to be stable and were not aggregated in the yeast system.     

Mutations in the conserved woodchuck hepatitis virus polymerase FLLA and YMDD regions conferring resistance to lamivudine

During more than 104 weeks of treatment with lamivudine (3TC) in chronic woodchuck hepatitis virus (WHV) carrier woodchucks, viral recrudescence occurred. Analysis of WHV DNA polymerase from woodchuck serum samples by PCR followed by DNA sequencing demonstrated that all samples were wild type at the conserved YMDD motif in domain C. Four of the six 3TC-treated woodchucks showed a mixture of the wild-type Ala (GCT) and the mutant Thr (ACT) at the conserved amino acid residue 566 (FLLA) in domain B of the WHV polymerase region. The appearance of the A566T mutation was temporally associated with viral recrudescence. This change is analogous with the amino acid 181 (FLLA) in HBV where 3TC selects for a change from Ala to Thr in humans. In the woodchuck, the Ala to Thr change in the polymerase gene results in a mutation of the WHV surface protein (amino acid 377) from Trp (TGG) to an opal codon (TGA), which may prematurely terminates the polypeptide. Three WHV molecular infectious clones were constructed to study this mutation in greater detail in vitro: A566T, analogous to A181T in HBV; M589V, analogous to the M204V in HBV; and the double mutant A566T/M589V, analogous to A181T/M204V in HBV. These mutants exhibited drug-sensitivity and replication profiles that paralleled those reported for analogous HBV variants. In transfected Huh7 cells, WHV containing the M589V mutation conferred at least 100-fold increased resistance to 3TC, but replicated approximately 5-fold less efficiently than wild-type virus as judged by both extracellular virus production and intracellular DNA replicative forms. In contrast, A566T mutant was approximately 10-fold more resistant to 3TC, replicated intracellularly as well as wild type, but produced 10-fold lower levels of virions than wild type. These findings are consistent with the observation that the A566T mutation alters the overlapping WHV surface antigen reading frame. WHV carrying mutations in the conserved YMDD motif, while not directly selected during lamivudine therapy in WHV carrier woodchucks, are replication competent in cell culture indicating the potential for their emergence in treated animals. These results further illustrate the utility of the WHV/woodchuck model to studies of HBV-drug resistance.     

In vitro and in vivo anti-hepatitis B virus activities of a plant extract from Geranium carolinianum L

Natural products provide a large reservoir of potentially active agents with anti-hepatitis B virus (HBV) activity. We examined the effect of the polyphenolic extract from Geranium carolinianum L. (PPGC) on HBV replication both in vitro and in vivo. In the human HBV-transfected liver cell line HepG(2) 2.2.15, PPGC effectively suppressed the secretion of the HBV antigens in a dose-dependent manner with IC(50) values of 46.85 microg/ml for HBsAg and 65.60 microg/ml for HBeAg at day 9. Consistent with the HBV antigen reduction, PPGC (100 microg/ml) also reduced HBV DNA level by 35.9%. In the duck hepatitis B virus (DHBV) infected ducks, after PPGC was dosed intragastricly (i.g.) once a day for 10 days, the plasma DHBV DNA level was reduced, with an ED(50) value of 47.54 mg/kg. In addition, Southern blot analysis confirmed the in vivo anti-HBV effect of PPGC in ducks and PPGC also reduced the plasma and the liver DHBV DNA level in a dose-dependent manner. Furthermore, significant improvement of the liver was observed after PPGC treatment, as evaluated by the histopathological analysis.     

Antiviral effects of PNA in duck hepatitis B virus infection model

AIM: To study the efficacy of antiviral treatment with PNA for the duck model of HBV (DHBV)-infected ducks. PNA is a 2-amine-9-(2,3-dideoxy-2,3-dihydro-beta-D-arabinofuranosyl)-6-methoxy-9H-purine. METHODS: The Sichuan Mallard ducklings in the hepatitis B virus model were treated with PNA, a new antiviral agent. DHBV DNA from the blood serum and liver tissues were measured at 0, 5, and 10 d during the treatment and at 3 d withdrawal by real-time PCR. The duck hepatitis B surface antigen (DHBsAg) in the liver cells was observed by Immunohistochemistry (IHC). Pathological changes in the liver tissues were also observed. Control group I was administered with distilled water and control group II was administered with 3-thiacytidine. Treatment group I was administered with PNA at a dose of 40 mg/kg and treatment group II was administered perorally (po) with PNA at a dose of 80 mg/kg. Treatment group III was administered with PNA at a dose of 20 mg/kg and treatment group IV was intravenously administered with PNA at a dose of 40 mg/kg. Each group contained 15 ducklings. RESULTS: PNA can significantly lower the DHBV replication levels in serum and liver. Compared with control group II, there were no significant differences in inhibiting efficacy in treatment groups I and III (P>0.05) and there were significant differences in inhibiting efficacy in treatment groups II and IV (P<0.05). Interestingly, significant differences were observed at 3 d withdrawal. The DHBV replication levels in each group slightly increased at 3 d withdrawal, but rebounded slightly in the PNA treatment groups than in control group II (P<0.05). The DHBV replication levels in the treatment groups were lower than in control group I. The DHBV replication levels in sera had a positive relationship with that in the liver, but the DHBV replication levels in the liver was lower than that in sera. Pathological changes in the treatment groups were obviously improved and the changes were associated with liver viral DNA levels. CONCLUSION: The results demonstrate that PNA is a strong inhibitor of DHBV replication in the DHBV-infected duck model.     

Clevudine for the treatment of chronic hepatitis B virus infection

Chronic hepatitis B virus (HBV) infection is a major health problem that is responsible for < or = 1 million deaths and 500,000 cases of hepatocellular carcinoma worldwide each year. Drugs that are currently approved by the FDA for the treatment of chronic HBV consist of two groups: the immunomodulators, such as conventional IFN-alpha and pegylated IFN-alpha2a; and nucleoside/nucleotide analogues, such as lamivudine, adefovir dipivoxil and entecavir. However, due to the limitations of these agents, newer agents with improved efficacy are currently being developed. One nucleoside/nucleotide analogue that is drawing a wide range of interest is clevudine, which is an analogue of the unnatural beta-L configuration. In the woodchuck hepatitis virus (WHV), clevudine 10 mg/kg has proven to be effective in suppressing viral replication with < or = 9 log10 decreases in WHV. At this dose, a significant reduction of intrahepatic WHV RNA and covalently closed circular WHV DNA levels can also be observed. Treatment with clevudine 10 mg/kg can confer additional antiviral benefit in the form of a more sustained reduction in WHV replication, serum woodchuck hepatitis surface antigen and intrahepatic woodchuck hepatitis core antigen expression following the withdrawal of clevudine. In humans, clevudine 10, 50, 100 or 200 mg/day for 28 days can reduce the median HBV DNA by -2.5, -2.7, -3 and -2.6 log10, respectively. More importantly, this suppression of antiviral activity is maintained at 12 and 24 weeks post treatment. Based on the early results of clevudine, more large-scale human studies with clevudine monotherapy or combination therapy is eagerly awaited.     

Clevudine for the treatment of chronic hepatitis B virus infection

Chronic hepatitis B virus (HBV) infection is a major health problem that is responsible for ≤ 1 million deaths and 500,000 cases of hepatocellular carcinoma worldwide each year. Drugs that are currently approved by the FDA for the treatment of chronic HBV consist of two groups: the immunomodulators, such as conventional IFN-α and pegylated IFN-α2a; and nucleoside/nucleotide analogues, such as lamivudine, adefovir dipivoxil and entecavir. However, due to the limitations of these agents, newer agents with improved efficacy are currently being developed. One nucleoside/nucleotide analogue that is drawing a wide range of interest is clevudine, which is an analogue of the unnatural β-L configuration. In the woodchuck hepatitis virus (WHV), clevudine 10 mg/kg has proven to be effective in suppressing viral replication with ≤ 9 log₁₀ decreases in WHV. At this dose, a significant reduction of intrahepatic WHV RNA and covalently closed circular WHV DNA levels can also be observed. Treatment with clevudine 10 mg/kg can confer additional antiviral benefit in the form of a more sustained reduction in WHV replication, serum woodchuck hepatitis surface antigen and intrahepatic woodchuck hepatitis core antigen expression following the withdrawal of clevudine. In humans, clevudine 10, 50, 100 or 200 mg/day for 28 days can reduce the median HBV DNA by -2.5, -2.7, -3 and -2.6 log₁₀, respectively. More importantly, this suppression of antiviral activity is maintained at 12 and 24 weeks post treatment. Based on the early results of clevudine, more large-scale human studies with clevudine monotherapy or combination therapy is eagerly awaited.     

Anti-hepatitis B virus activity of wogonin in vitro and in vivo

The traditional Chinese medicine Scutellaria radix has been used for thousands of years, mainly for the treatment of inflammatory conditions including hepatitis. The major active constituent, wogonin (WG), isolated from S. radix has attracted increasing scientific attention in recent years due to its potent biological activities. However, pharmacologic studies have primarily been focused on wogonin's anti-inflammatory and anti-cancer activities. In this study, we have investigated wogonin's anti-hepatitis B virus (HBV) activity both in vitro and in vivo. In the human HBV-transfected liver cell line HepG2.2.15, wogonin effectively suppressed the secretion of the HBV antigens with an IC(50) of 4 microg/ml at day 9 for both HBsAg and HBeAg. Consistent with the HBV antigen reduction, wogonin also reduced HBV DNA level in a dose-dependent manner. Duck hepatitis B virus (DHBV) DNA polymerase was dramatically inhibited by wogonin with an IC(50) of 0.57 microg/ml. In DHBV-infected ducks wogonin dosed i.v. once a day for 10 days reduced plasma DHBV DNA level with an ED(50) of 5mg/kg. The in vivo anti-HBV effect of wogonin in ducks was confirmed by Southern blotting of DHBV DNA in the liver. Histopathological evaluation of the liver revealed significant improvement by wogonin. In addition, in human HBV-transgenic mice, wogonin dosed i.v. once a day for 10 days significantly reduced plasma HBsAg level. Immunohistological staining of the liver confirmed the HBsAg reduction by wogonin. In conclusion, our results demonstrate that wogonin possesses potent anti-HBV activity both in vitro and in vivo. Currently, wogonin is under early development as an anti-HBV drug candidate.     

Antiviral therapeutic efficacy of foscarnet in hepatitis B virus infection

Foscarnet (PFA), a viral DNA polymerase inhibitor, is a clinical agent for herpes viruses. The goal of the study was to evaluate the therapeutic efficacy of PFA in hepatitis B virus (HBV) infection. Intravenous infusion of PFA (1 g/day) for 4 weeks significantly reduced serum HBeAg (p<0.01) and HBV DNA copies (p<0.05) in 31 patients who were diagnosed with active chronic HBV infection (CHB) and had not received antiviral treatment previously. Alanine aminotransaminase (ALT), aspartate aminotransaminase (AST) and gamma glutamyl transpeptidase (gamma-GT) of the patients declined (p<0.001, 0.001 and 0.01, respectively). Kidney function (blood creatinine and urea nitrogen) remained unchanged. Another 21 lamivudine-resistant CHB patients with mutations at the tyrosine-methionine-aspartate-aspartate motif (YMDD) displayed a response to PFA similar to that mentioned above, with reductions in HBeAg (p<0.05), HBV DNA (p<0.01) and liver enzymes (ALT and AST, p<0.001; gamma-GT, p<0.05). Moreover, PFA reduced serum HBeAg (p<0.01), HBV DNA (P<0.05), AST (p<0.05) and ALT (p<0.02) in a cohort of 13 severe CHB patients with advanced liver damage. PFA was also evaluated in vitro and in vivo. PFA inhibited HBV DNA replication in HBV-transfected human HepG2 cells (2.2.15 cells) with reduced amount of HBV RC-DNA and DS-DNA. In the duck HBV-infected ducklings, PFA reduced viral DNA and duck HBsAg in the serum (p<0.01 for both).     

乙肝病毒携带产妇乳汁乙肝病毒标志物检测及分析

目的 通过检测乙肝血清学指标阳性的产妇乳汁中乙肝两对半及外周血HBV-DNA定量,以探讨两者之间的关系及其对于母乳喂养的影响.方法 对42例血清乙肝指标阳性的产妇,应用酶联免疫吸附法和荧光定量法检测乳汁中乙肝两对半及血HBV-DNA,42例血清乙肝指标阴性产妇作为对照组.结果 42例HBV携带孕妇产后乳汁乙肝两对半HbsAg检出率为33.3%.42例HBV携带孕妇HBV-DNA阳性率为28.57%.对照组乳汁HbsAg检出率为0,HBV-DNA阳性率为0.结论 乙肝小三阳或仅HbsAg阳性的孕妇乳汁乙肝病毒检出率低,母乳喂养相对安全,并且其乳汁的传染性明显低于乙肝大三阳孕妇.乳汁乙肝两对半全阴且血清HBV-DNA处于正常范围的产妇提倡母乳喂养.     

蛞蝓多糖抗鸭乙型肝炎病毒作用

目的研究蛞蝓多糖抗鸭乙型肝炎病毒（DHBV）的作用。方法采用先天感染DHBV的北京雏鸭为模型,随机分设0．9％氯化钠注射液模型对照组、拉米夫定组、蛞蝓多糖低、中、高3个剂量组,每组8只雏鸭均以20mg·kg^-1·d^-1灌胃给药10d。斑点杂交法观察用药前与用药后5d、10d及停药后5d血清中DHBV-DNA表达。结果蛞蝓多糖不同剂量组对DHBV均有一定抑制作用。其中蛞蝓多糖的中、高剂量组于用药后第5天血清DHBV．DNA的吸光度A值为（0.66±0．14）、（0．73±0．26）,第10天为（0．47±0．18）、（0．54±0．10）,较0．9％氯化钠注射液对照组（1．61±0．54）、（1．20±0．51）明显降低。蛞蝓多糖的中、高剂量组对DHBV．DNA的抑制率于用药后第5天为24．14％、23．96％,第10天为45．98％、43．75％,与0．9％氯化钠注射液对照组相比明显提高。结论蛞蝓多糖具有降低血DHBV—DNA含量,抑制乙型肝炎病毒作用。     

山西省乙型肝炎病毒基因型分布及临床意义研究

目的了解山西省乙型肝炎病毒(HBV)基因型流行病学分布特征,并分析不同基因型与HBV感染谱的相关性。方法用S基因巢式聚合酶链反应-限制片段长度多态性(PCR-RFLP)的基因型分型方法,对44例携带者、70例慢性乙型肝炎、34例慢性重症乙型肝炎、29例肝硬化患者感染的HBV进行分型,并分析了不同感染谱中HBV基因型的分布。结果①177例感染者HBV基因型C型130例(73.5%)和B型43例(24.3%),偶见D型1例(0.6%),未定型3例(1.7%)。②各种乙型肝炎患者的HBV基因型以C型为主,HBV携带者的基因型主要为B基因型。结论山西省乙型肝炎病毒感染者以C型和B型为优势基因型,与我国北方地区HBV基因型分布相似,但亦有少数其他基因型存在。HBV基因型C可能是乙型肝炎病情进展的相关因素。     

乙型肝炎病毒基因型分布及临床意义

目的 了解珠海地区乙型肝炎病毒(HBV)基因型流行病学分布特征,并分析不同基因型与HBV感染谱的相关性.方法 采用聚合酶链反应-限制片段长度多态性(PCR-RFLP)的基因型分型方法对62例HBV携带者及63例慢性乙型肝炎、7例慢性重症乙型肝炎、5例肝硬化患者感染的HBV进行分型,并分析不间感染谱中HBV基因型的分布.结果 137例感染者HBV基因型B型72例(52.6%),C型65例(47.4%),慢性肝炎患者、重症肝炎、肝硬化患者的C基因型比例[45例(69.2%)、5例(7.7%)、4例(6.2%)]明显高于B基因型[18例(25.0%)、2例(2.8%)、1例(1.4%)],差异均有统计学意义(均P＜0.05);HBV携带者B基因型比例[51例(70.8%)]明显高于C基因型[11例(16.9%)],差异有统计学意义(P＜0.05).C基因型慢性肝炎患者的HBV-DNA、平均ALT水平高于B基因型慢性肝炎患者,但差异均无统计学意义(P＞0.05).结论 珠海地区HBV感染者以B型和C型为优势基因型,C基因型可能是乙型肝炎病情进展的相关因素.

Abstract：

Objective To understand the distribution of hepatitis B virus (HBV) genotypes in Zhuhai area and to analyze the relationship between HBV genotypes and patients with hepatitis B. Methods A polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis assay amplifying S-segment sequences of HBV-DNA were used for analyzing the distribution of HBV genotypes among 62 HBV carriers (ASC group),63 patients with chronic hepatitis B (CHB group),7 patients with severe chronic hepatitis B (CSH group)and 5 cases liver cirrhosis (LC group). Results The proportion of HBV genotyjpes B and C was 52.6% and 47.4%. There were no significant differences of indexes related to liver function damage (ALT) or serum HBV DNA levels between subtype B and subtype C infected patients. Conclusion The genotypes B and C are two predominant genotypes in Zhuhai area;genotype C may be associated with the development of hepatitis.     

慢性乙型肝炎血清HBVDNA基因含量与临床的相关性研究

目的探讨慢性乙型肝炎（慢乙肝）患者病毒复制水平与HBV标志物（HBV—M）模式及肝损害程度的关系。方法360例慢性乙型肝炎（轻度160例、中度140例、重度60例）患者血清HBVDNA含量采用荧光标记（AmpliSensor）定量PCR方法检测,HBV—M检测采用ELISA法。结果血清HBVDNA含量与HBV—M模式有关,血清HBeAg阳性组HBVDNA含量（106．35±1．84）显著高于HBeAg阴性组（104．73±1．88）（P〈0．01）。慢乙肝轻度、中度患者HBVDNA含量[（105．58±1．92）,（106．27±2．05）]与慢乙肝重度患者HBVDNA含量（105．73±1．90）相比较无显著性差异（P〉0．05）,且不同HBVDNA水平患者的TBil、AI』、AST差别无显著性（P〉0．05）。结论血清HBeAg的存在影响HBVDNA的水平变化,随着肝损害程度的加重,慢性乙型肝炎轻度、中度、重度患者血清HBVDNA基因含量未发生显著变化,同时血清HBVDNA含量与TBil、ALT、AST水平无明显关系。     

慢性HBV感染患者HBV DNA前C区变异的临床意义

目的:探讨慢性HBV感染患者HBVDNA前C区变异的临床意义。方法:采用ELISA测定99例HBV感染患者血清肝炎病毒标志物,并采用RT-PCR检测HBVDNA前C区变异。结果:99例HBV感染患者中,乙型肝炎病毒携带26例,慢性乙型病毒性肝炎61例,慢性重型病毒性肝炎(乙型)11例和急性乙型病毒性肝炎1例。乙型肝炎病毒携带,慢性乙型病毒性肝炎和慢性重型病毒性肝炎中,变异株组HBVDNA前C区变异发生率与混合株组、野生株组相比,差异均无显著性(P>0.05)。与混合株和野生株两组相比,HBVDNA变异株组肝功能变化差异均无显著性(P>0.05)。结论:HBVDNA前C区变异可发生于HBV感染的不同临床状态。HBVDNA前C区变异可能与肝炎程度和病情进展无明显关系。