HBV E Antigen Research Articles

Henrylactones A–E and Anti-HBV Constituents fromIllicium henryi

Five new sesquiterpene lactones, henrylactones A-E ( 1- 5), together with ten known compounds: cycloparvifloralone ( 6), tashironin ( 7), tashironin A ( 8), neoanisatin ( 9), anisatin ( 10), anislactone B ( 11), 7- O-acetylanislactone B ( 12), merrillianolide ( 13), cyclomerrillianolide ( 14) and pseudomajucin ( 15), were isolated from the stems and roots of ILLICIUM HENRYI. Their structures were elucidated based on extensive spectroscopic data analyses. Among them, henrylactone A ( 1) is a novel sesquiterpene with a dilactone moiety and its structure was confirmed by X-ray diffraction. Sesquiterpene lactones 1- 15 were tested for their anti-hepatitis B virus (HBV) activities. The most active compound, tashironin ( 7), exhibited an IC (50) value of 0.48 mM (SI = 6.3) inhibiting on HBV surface antigen (HBsAg) secretion and an IC (50) value of 0.15 mM (SI = 20.1) inhibiting on HBV e antigen (HBeAg) secretion using HBV transfected Hep G2.2.15 cell line.

Long‐Term Benefit of Hepatitis B Vaccination among Children in Thailand with Transient Hepatitis B Virus Infection Who Were Born to Hepatitis B Surface Antigen–Positive Mothers

Transmission of hepatitis B virus (HBV) from carrier mothers to their babies appears to be one of the most important factors influencing the prevalence of chronic HBV infection in areas of high hepatitis B endemicity. Infants born to HBV surface antigen (HBsAg)-positive mothers who were or were not positive for HBV e antigen (HBeAg) or to mothers who were negative for both HBsAg and HBeAg have been followed for 17 years for serological evidence of HBV infection. These infants were divided into 2 groups on the basis of their hepatitis B vaccination protocols: group 1 received vaccine at birth and 1, 2, and 12 months later, and group 2 received vaccine at birth and 1 and 6 months later. Follow-up involved annual clinic visits, during which a blood sample was taken and analyzed for the presence of HBsAg, antibody to HBsAg, and antibody to HBV core antigen (HBcAg). Selected blood samples that tested positive for HBV markers during 2 consecutive visits separated by a long interval were further investigated by polymerase chain reaction to detect HBV DNA. Transient presence of HBsAg or transient and/or long-term presence of antibody to HBcAg suggested that this population was heavily exposed to HBV during the follow-up period. Despite these findings, no new cases of chronic HBV infection were observed. None of the subjects with transient presence of HBsAg had any clinical symptoms of liver disease. This study demonstrates the efficacy of the HBV vaccine and its ability to protect against symptomatic disease.

Anti-HBV agents. Part 2: Synthesis and in vitro anti-hepatitis B virus activities of alisol A derivatives

Chemical modifications were performed on hydroxyl groups at C-11,23,24,25 positions and C-13(17) double bond of alisol A for structure–activity relationship study. Forty-one derivatives of alisol A were synthesized and assayed for their in vitro anti-hepatitis B virus (HBV) activities and cytotoxicities. Of them, 14 compounds were active against HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) secretion in HepG 2.2.15 cells, and the most promising compound 25 exhibited high activities against secretion of HBsAg (IC 50 = 0.028 mM), HBeAg (IC 50 = 0.027 mM) and remarkable selective indices (SI HBsAg >90, SI HBeAg >93).

Virologic response to adefovir dipivoxil monotherapy is not durable in HBeAg-positive, lamivudine-resistant chronic hepatitis B patients

It has been shown that adefovir dipivoxil is an effective antiviral agent in the treatment of chronic hepatitis B (CHB), not only in wild-type hepatitis B virus (HBV) infection, but also in lamivudine-resistant (LAMV-R) cases. However, little is known about the durability of the virologic response to adefovir in LAMV-R CHB patients. Fifteen HBV e-antigen (HBeAg)-positive, LAMV-R CHB patients showed a virologic response to adefovir monotherapy. These patients received additional adefovir for at least a further 12 months. The virologic relapse rate after discontinuation of adefovir was evaluated. In addition, predictive factors associated with virologic relapse were investigated. The median level of serum HBV DNA before adefovir administration was 7,457,840 IU/mL (range 107,920-99,524,960 IU/mL). The median duration of adefovir treatment was 30 months (range 14-46 months). During a median follow-up period of 14 months after discontinuation of adefovir, the 1-, 2-, 3-, 6-, and 12-month cumulative relapse rates were 26.7%, 53.3%, 73.3%, 80%, and 80%, respectively. High pretreatment HBV DNA levels were found to be the only factor that was predictive of off-therapy relapse. Our data suggest that the adefovir-monotherapy-induced virologic response is not durable in most patients with LAMV-R HBeAg-positive CHB, especially in those with a high pretreatment HBV DNA level.

Efficacy and Safety of Adefovir Dipivoxil plus Pegylated Interferon-α2a for the Treatment of Lamivudine-Resistant Hepatitis B virus Infection in HIV-Infected Patients

Background Up to 10% of the HIV-positive population is coinfected with hepatitis B virus (HBV). Generally, combined treatment includes agents against both viruses, such as lamivudine (3TC). However, HBV resistance to 3TC is high. Adefovir dipivoxil (ADV) has shown its efficacy for treating 3TC-resistant (3TC-R) HBV in HIV-coinfected patients. ADV combined with pegylated interferon (PEG-IFN) has never been evaluated in this population. Methods HIV-HBV-coinfected patients with positive HBV e antigen (HBeAg), documented 3TC-R HBV mutation and antiretroviral treatment including 3TC were selected and received ADV (10 mg daily) and PEG-IFN-α2a (180 μg weekly) for 48 weeks. Results Of 18 eligible patients ( n=16 [89%] male, mean ±sd age 40.45 ±4.82 years), 17 were treated for 48 weeks. One stopped IFN treatment because of adverse events and continued ADV only. The median (interquartile range) HBV DNA at baseline was 8.0 (5.30–8.97) log10 copies/ml and the median (95% confidence interval [CI]) decrease after 48 and 72 weeks was 3.6 (4.9–2.4) and 1.4 (-5.0–2.2) log10 copies/ ml, respectively. None of the patients became HBeAg-negative. Median (95% CI) decrease of serum alanine aminotransferase was 27.8 (-66.2–10.5) IU/ml after 48 weeks and 93.0 (-80.0–26.1) IU/ml after 72 weeks. Conclusions ADV and PEG-IFN is safe and effective for treating 3TC-R HBV in HIV patients. However, on-treatment response was not maintained off therapy and did not lead to HBV seroconversion. The combination had no effect on HIV disease progression.

Antibody-directed complement-mediated cytotoxicity to hepatocytes from patients with chronic hepatitis B.

The susceptibility of hepatocytes from patients with chronic hepatitis B to complement-dependent cytotoxicity mediated by heterologous antibodies to hepatitis B virus core (anti-HBc) and surface (anti-HBs) antigens and to hepatic asialoglycoprotein receptor was examined using a microcytotoxicity assay. The anti-HBc-induced cytotoxicity was found to be markedly enhanced against hepatocytes isolated from patients with chronic active hepatitis (72.6 +/- 9.5% (mean +/- s.e.m.); n = 6) over that against hepatocytes from individuals with chronic persistent hepatitis or inactive liver cirrhosis (40.6 +/- 18.6%; n = 4) (P = 0.019). Overall, values of the anti-HBc-directed cytotoxicity were higher in patients positive for HBcAg in hepatocytes and seropositive for hepatitis B virus e antigen (HBeAg). Hepatocytotoxicity was also exerted by anti-HBs and anti-asialoglycoprotein receptor antibodies in the presence of complement, but it was not seemingly related to disease activity. These results indicate that hepatitis B virus core and surface antigens and asialoglycoprotein receptor at the hepatocyte surface can be recognized by antibodies, and raise the possibility that complement-dependent cytolysis may contribute to the injury of hepatitis B virus-infected hepatocytes. The data also suggest that liver cells of patients with severe chronic hepatitis might be more susceptible to anti-HBc antibody-directed complement-mediated cytotoxicity than those with inactive liver histology.

A Decline in Hepatitis B virus Surface Antigen (HBsAg) Predicts Clearance, but does not Correlate with Quantitative HBeAg or HBV DNA Levels

The elimination of hepatitis B virus surface antigen (HBsAg) is the final goal of hepatitis B treatment, but is rarely achieved. As quantitative assays for HBsAg recently became available, we have investigated whether quantitative HBsAg measurements can substitute for hepatitis B virus (HBV) DNA quantification in treatment monitoring. Within this study, 23 liver transplant patients and 18 heart transplant recipients were retrospectively analysed. Patients had been treated with famciclovir and/or lamivudine, in addition some had also received adefovir in cases of lamivudine resistance. Quantitative HBsAg and hepatitis B virus e antigen (HBeAg) levels were determined with the Architect assay. HBV DNA levels were determined with different assays available at given time points. We did not find a significant correlation between either HBsAg or HBeAg and HBV DNA levels - both in treated and untreated patients. More importantly, there was no significant concordance between an increase or decrease of HBsAg or HBeAg with HBV DNA. However, the curve and decline of quantitative HBsAg enabled prediction of eventual viral clearance. Eight patients showed a 2 log10 drop of HBsAg levels and eight patients demonstrated a reduction of HBsAg levels below 100 IU/ml; five patients fulfilled both criteria. Three of those five cleared HBsAg and became positive for antibodies against HBsAg. Quantitative HBsAg and HBeAg cannot substitute for HBV DNA quantification during the assessment of antiviral therapy; however, the decline of HBsAg does predict eventual HBsAg clearance. A 2 log10 drop to below 100 IU/ml is associated with a high likelihood of HBsAg clearance.

A valine to phenylalanine mutation in the precore region of hepatitis B virus causes intracellular retention and impaired secretion of HBe‐antigen

Hepatitis B virus (HBV) e antigen (HBeAg) is translated from precore mRNA as a precore/core protein, which is post-translationally modified to give rise to the protein that is secreted into the serum. The G1862T mutation in HBV occurs in the bulge of the encapsidation signal within the pregenomic RNA. When the precore mRNA is translated, this mutation results in a valine to phenylalanine substitution at the -3 position to the signal peptide cleavage site at the amino end of the precursor protein. The aim of this study was to determine whether this mutation could affect HBV replication and/or HBeAg expression. Following transfection of Huh 7 cells, HBV replication was followed using real time polymerase reaction (PCR) and expression of HBeAg expression was monitored using confocal microscopy. HBV replication was reduced when this mutation was introduced into genotype D but not into genotype A replication-competent constructs. Using mutant HBeAg-expressing plasmids, we demonstrated a 54% reduction in HBeAg secretion relative to the wild type. Confocal microscopy demonstrated that the mutant HBeAg accumulated in the endoplasmic reticulum, endoplasmic reticulum intermediate compartment and Golgi. These aggregates of mutant protein increased in size following treatment of the cells with a proteasome inhibitor, MG132, and had the hallmark features of aggresomes. They attracted ubiquitin, heat shock proteins and proteasomes and were isolated from the cytosol by the intermediate filaments, vimentin and cytokeratin. The formation of aggresomes, as a result of the G1862T mutation, may play a contributory role in HBV-induced liver disease.

Suboptimal Response to Adefovir Dipivoxil Therapy for Chronic Hepatitis B in Nucleoside-Naive Patients is not due to Pre-Existing Drug-Resistant Mutants

Adefovir dipivoxil (ADV) has demonstrated activity against wild-type and lamivudine-resistant hepatitis B virus (HBV). After 1 year of therapy, a median 3.5-4.0 log10 decrease in viral load is observed. Our aim was to characterize the different profiles of response to ADV in relation to the in vitro susceptibility of viral strains to ADV. In an international Phase III randomized, placebo-controlled study of ADV in patients positive for hepatitis B virus e antigen (HBeAg), different profiles of virological response to ADV 10 mg/day were identified at week 48. The top 25% patients (quartile 1, Q1) showed > 4.91 log10 reduction in serum HBV DNA at week 48, in Q2 patients demonstrated a 3.52 to 4.90 log10 reduction of viral load, whereas in Q3 a 2.22 to 3.51 log10 reduction in viral load was observed. The bottom 25% of patients (Q4) showed < 2.22 log10 reduction in HBV DNA levels. The influence of baseline characteristics and drug compliance on response was investigated. The replication capacity and drug susceptibility of HBV genomes of selected clinical isolates that were considered representative of the treatment response quartiles were analysed using a phenotypic assay. The lowest quartile of response (Q4) appears to have worse compliance. Higher alanine aminotransferase levels at baseline are associated with improved response. Phenotypic analysis of viral strains in vitro in Huh7 and HepG2 cells showed that HBV genomes remained susceptible to ADV, regardless of treatment response observed in patients. Suboptimal response to ADV might result from a host pharmacological effect or from patient compliance issues rather than from a reduced susceptibility of HBV to ADV.

Chronic HBV infection with persistently normal ALT b. not to treat

Chronic hepatitis B (CHB) is a lifelong, progressive and potentially fatal disease if not properly treated. Fortunately, successful therapy with antiviral agents resulted in sustained improvements in virologic, biochemical, histologic features of disease and clinical outcome. Although the recent development of a new and potent anti-hepatitis B virus (HBV) agents may offer many therapeutic options against HBV, there are still unsolved issues and areas for the optimal antiviral therapy. The various clinical courses and different status of the diseases, the variable response to antiviral agents, and the lack of long-term outcome of antiviral treatment contribute to the difficulties in establishment of ideal guidelines, particularly in some special patient groups [1]. It is very important to establish optimal guidelines for whom to treat and when to start, as well as how to treat in Asian patients with CHB. An individual patient’s alanine aminotransferase (ALT) level is an important factor in the decision to initiate the treatment of CHB because the elevated ALT levels indicate immune-mediated inflammation to eliminate HBV-infected hepatocytes and a higher rate of hepatitis B virus e antigen (HBeAg) seroconversion [1–3]. However, up to 40–50% of all HBeAg-positive patients may have normal ALT levels for prolonged periods [4]. The primary goal of treatment for CHB is to induce sustained suppression of HBV replication and to prevent progression of disease. APASL guidelines recommended that HBeAg-positive patients with HBV DNA levels of ≥20,000 IU/ml (≥105 copies/ml) and elevated ALT levels should be considered for treatment. However, the following issues remain unsettled: Should patients with an ALT level of less than two times upper limit of normal (ULN) be treated, and if so when and how? At that time, APASL guidelines recommended that patients with persistently normal or minimally elevated ALT should not be treated except in cirrhotic patients, but need adequate follow-up [1]. The newer therapeutic options have been shown to offer clinical benefits over lamivudine and may represent better therapeutic choices. Emerging evidence suggests that the more appropriate HBV DNA threshold for the treatment of HBeAg-negative patients is ≥2,000 IU/ml (≥104 copies/ml) [2, 3]. This review discusses the key issues and current challenges in the management of chronic HBV infection with persistently normal ALT (PNALT), which is usually defined as ALT remaining normal for at least 2 years in periodic biochemical examinations, to treat or not to treat [5].

Cerebellar abscess in a cynomolgus macaque (Macaca fascicularis)

A cynomolgus macaque (Macaca fascicularis) presented with decreased appetite, lethargy, ataxia, disorientation and visual impairment. It was used in a hepatitis B (HBV) study involving injections of HBV plasmid construct (450 microg) and aflatoxin B1 (25 microg/kg) in an effort to develop a model of liver cirrhosis and hepatocellular carcinoma. The case work-up included physical examination, including assessment of hydration, thoracic and abdominal radiographs and abdominal ultrasound. Clinical pathology included complete blood counts and tests for levels of plasma ammonia (NH(3)) and serum electrolytes, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, total bilirubin, and gamma-glutamyl transferase. Serum samples had also been serially tested for the presence of HBsAg, anti-HBc antibodies, HBV e-antigen, and HBV DNA. With a tentative diagnosis of hepatic encephalopathy, treatment with lactulose, antibiotics, and fluid therapy was initiated. Clinical pathology and diagnostic imaging performed on the animal revealed no abnormalities except for the hyperammoniemia. Absence of HBV markers in the serum indicated unsuccessful inoculation. Not fully responding to therapeutic intervention, the animal was euthanized. Necropsy revealed fibrous peritoneal adhesions and an abscess in the cerebellopontine angle. Exudate culture indicated the presence of alpha-hemolytic streptococcus, Eubacterium lentum, and Bacteroides stercoris. Brain abscesses are uncommon in non-human primates. This case of cerebellar abscess is characterized by important features similar to that found in humans, including the presenting signs and the presence of the above-cultured bacteria.

Substitution rate of the hepatitis B virus surface gene

Molecular epidemiology of hepatitis B virus (HBV) often relies on the comparison of HBV surface (S) gene sequences, although little is known about the substitution rate of the HBV S-gene. In this study, we compared HBV S-gene sequences in longitudinal sample pairs of 40 untreated, chronically HBV-infected patients, spanning 210 years of cumulative follow-up. The 40 patients included HBV e-antigen positive and negative persons; with HBV DNA levels ranging from 10(3) to 10(9) cps/mL and belonging to HBV genotypes A, B, C, D and E. In the 40 sample pairs, 70 nucleotide changes occurred in the HBV S-gene (0-8 per patient), resulting in an average substitution rate of 5.1 x 10(-4) nucleotide changes/site/year (range: 0-1.3 x 10(-2)). Surprisingly, the number of substitutions was strongly associated with the inverse level of viremia; and only weakly with the duration of follow-up: in 11 highly viremic patients (HBV DNA > or =10(8) cps/mL), only four substitutions occurred despite a cumulative observation period of 56 years (substitution rate: 1.1 x 10(-4)), while in the 10 patients with viremia below 10(4) cps/mL, 29 substitutions occurred during 30 years of follow-up (substitution rate: 14.6 x 10(-4)). We conclude that in chronic hepatitis B virus infection the rate of nucleotide substitution in the HBV S-gene is inversely related to the level of viremia and thus varies widely from person to person; hampering the phylogenetic analysis of possible chains of HBV infection.

Sequential Combination Therapy Leads to Biochemical and Histological Improvement Despite Low Ongoing Intrahepatic Hepatitis B virus Replication

We previously reported that 48 weeks of combination therapy with pegylated interferon-alpha2b (PEG-IFN-alpha2b) and adefovir dipivoxil (ADV) in patients with chronic hepatitis B led to marked decreases of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) (-2.4 log10 copies/ml). Combination therapy was followed by 96 weeks of ADV monotherapy. Here, we report on the final outcome after 144 weeks of sequential antiviral treatment. Twenty-four patients were analysed, triplet liver biopsies (taken at baseline, week 48 and week 144) were available from 16 patients. At week 144, 12/15 patients positive for hepatitis B virus e antigen (HBeAg) had lost HBeAg, alanine transaminase (ALT) levels were normal in 23 patients (96%), median serum HBV DNA had decreased by -4.9 log10 copies/ml and was undetectable (<100 copies/ml) in 11/24 individuals (46%). Median total intrahepatic HBV DNA had decreased by -2.2 log. Although no further significant cccDNA changes occurred between week 48 and week 144, two years of ADV monotherapy proved capable of controlling cccDNA levels in most patients. Analysis of intrahepatic HBV DNA species demonstrated that combination therapy with PEG-IFN-alpha2b and ADV inhibited viral productivity by 99% and subsequent ADV monotherapy by 76%, respectively. Virus suppression to undetectability within the first 12 weeks of treatment was strongly associated with long-term virological response and HBeAg and hepatitis B virus surface antigen HBsAg seroconversion. Histological improvement was determined in 11/16 patients at week 144. Two patients developed ADV resistance during the third year of treatment. Reduction of intrahepatic viral load achieved after 48 weeks of combination therapy with PEG-IFN-alpha2b and ADV was maintained in the following 96 weeks of ADV monotherapy and translated into long-term clinical benefit for most of the treated patients.

Hepatitis B Virus e Antigen Loss during Adefovir Dipivoxil Therapy Is Associated with Enhanced Virus-Specific CD4+T-Cell Reactivity

Weak T-cell reactivity to hepatitis B virus (HBV) is thought to be the dominant cause for chronic HBV infection. Treatment with adefovir dipivoxil (ADV) increases the rate of HBV e antigen (HBeAg) loss; however, the immune mechanisms associated with this treatment response are not understood. Serial analysis of HBV-specific CD4+ T-cell reactivity was performed during 48 weeks of therapy with ADV and correlated with treatment outcome for 19 HBeAg-positive patients receiving ADV (n = 13) or the placebo (n = 6). We tested T-cell reactivity to HBV at seven protocol time points by proliferation, cytokine production, and enzyme-linked immunospot assays. A panel of serum cytokines was quantitated by cytokine bead array. ADV-treated patients showed increased CD4+ T-cell responses to HBV and lower serum levels of cytokines compared to those of placebo-treated patients. Enhanced CD4+ T-cell reactivity to HBV, which peaked at treatment week 16, was confined to a subgroup of ADV-treated patients who achieved greater viral suppression (5.3 +/- 0.3 log(10) copies/ml [mean +/- standard error of the mean {SEM}] serum HBV DNA reduction from baseline) and HBeAg loss, but not to ADV-treated patients with moderate (3.4 +/- 0.2 log(10) copies/ml [mean +/- SEM]) viremia reduction who remained HBeAg positive or to patients receiving the placebo. In conclusion, T-cell reactivity to HBV increases in a proportion of ADV-treated patients and is associated with greater suppression of HBV replication and HBeAg loss.

Early upsurge in anti‐HBs titer possibly caused by the immunomodulative, not by the mutagenetic effect of interferon and ribavirin

A patient with chronic hepatitis B and C undergoing treatment with interferon and ribavirin showed an upsurge in hepatitis B virus surface antibody (anti-HBs) titer, accompanied by a decrease in hepatitis B virus surface antigen (HBsAg) during the early treatment phase. Simultaneously, elevation of alanine aminotransferase (ALT) was observed. Subsequently, the hepatitis B virus (HBV) DNA titer decreased and HBV e antigen (HBeAg) to anti-HBe seroconversion occurred. The anti-HBs titer gradually returned to the pretreatment level after cessation of ribavirin treatment and HBV-DNA became undetectable. We found no nucleotide mutations in HBV-DNA that could explain the sudden elevation in anti-HBs titer. The appearance of anti-HBs was considered to be a break in immune tolerance against some epitopes in HBsAg, possibly the r epitope, stimulated by interferon/ribavirin treatment. The immunomodulatory effect of ribavirin might have caused this unexpected early immune response to HBsAg that preceded seroconversion to anti-HBe.

RNA interference inhibits hepatitis B virus gene expression and replication in HepG2‐N10 cells

RNA interference (RNAi) refers to the phenomenon of sequence-specific degradation of homologous mRNA induced by double-stranded RNA. It has been successfully utilized to down-regulate endogenous gene expression or suppress the replication of various pathogens in mammalian cells. In this study, the effect of vector-based small interfering RNA (siRNA) promoted by pSilencer2.0-U6 inhibit hepatitis B virus (HBV) replication in cell culture was evaluated. Three fragments of short nucleic acids, respectively, targeting on S, X and C region of HBV genome were inserted into pSilencer vectors after they were annealed with their partly antisense strands. The recombination plasmids were pS, pX and pC. These expression plasmids were transfected into HepG2-N10 cells, a cell line which stably expresses hepatitis B virus surface antigen (HBsAg), hepatitis B virus e antigen (HBeAg) and adw2 subtype Dane particles. The effect of RNAi was evaluated from the changes of DNA, RNA and protein levels. Viral antigens were measured by ELISA. Viral mRNA was analyzed by RT-PCR. The covalent closed circular DNA and genome DNA of HBV secreted into the culture media were measured by quantitative real-time PCR. Analysis of variance was performed for the results. Vector-based RNA interference could potently reduce HBsAg (pS vs pN: 47%, pX vs pN: 30%, and pC vs pN: 25%, P < 0.001) and HBeAg (pX vs pN: 57% and pC vs pN: 66%, P < 0.001) expression in cell culture. Furthermore, RT-PCR analysis showed that viral mRNAs were effectively degraded, thus eliminating the messengers for protein expression as well as templates for reverse transcription (pS and pC vs pN, P < 0.001; pX vs pN, P = 0.003). Quantitative real-time PCR analysis of HBV DNA revealed that vector-based RNA interference can inhibit HBV replication efficiently (pS, pX and pC vs pN, P < 0.001). Our results indicate that RNAi can inhibit HBV gene expression and replication, and it might have the potential to revolutionize the treatment of HBV.

Nuclear HBcAg and histology activity index as independent predictors of the expression of singly spliced HBV‐RNA

Although hepatitis B virus (HBV) RNA splicing has been reported by many researchers, the clinical significance of this event remains illusive. The present study was designed to investigate the clinical roles of singly spliced HBV-RNA. Liver biopsy tissues obtained from 32 consecutive patients were subjected to reverse transcriptase-polymerase chain reaction for the detection of singly spliced and unspliced HBV-RNA. Stepwise linear regression model was used to estimate the ratio of singly spliced to unspliced (S/US) HBV-RNA in the presence of the following variables: age, gender, aspartate aminotransferase, alanine aminotransferase, total bilirubin, alpha-foetoprotein, status of HBV e antigen (HBeAg), status of antibody to HBeAg, HBV-DNA, histology activity index (HAI), fibrotic score, grade of cytoplasmic HBV core antigen (HBcAg), grade of nuclear HBcAg, genotype, status of precore-stop-mutation, basal core promoter mutation, previous lamivudine therapy and superinfection by other hepatitis viruses. The results showed that HAI (beta = -0.2616; P = 0.011) and grade of nuclear HBcAg expression (beta = 0.5599; P =0.0067) were two independent predictors for the expression of singly spliced HBV-RNA. Further categorical analysis showed that patients with HAI score <or=6 and grading of nuclear HBcAg >or=2 have significantly higher S/US ratios. In conclusion, nuclear HBcAg and HAI are two independent predictors for the expression of singly spliced HBV-RNA.

Hepatitis B virus e antigen production is dependent upon covalently closed circular (ccc) DNA in HepAD38 cell cultures and may serve as a cccDNA surrogate in antiviral screening assays

Currently available antiviral nucleoside analogs for the treatment of chronic hepatitis B virus (HBV) infections profoundly reduce virus load, but rarely cure the virus infection. This is due, at least in part, to their failure to eliminate viral covalently closed circular (ccc) DNA from the nuclei of infected hepatocytes. To screen compound libraries for antiviral drugs targeting cccDNA, we set out to develop a cell-based assay suitable for high throughput screening. Since cccDNA is time-consuming to assay, it was desirable to use a viral gene product that could serve as a reporter for intracellular cccDNA level. We predicted that the secretion of HBV e antigen (HBeAg) by HepAD38 cells, a tetracycline inducible HBV expression cell line, would be cccDNA-dependent. This is because a large portion of pre-core mRNA leader sequence in the 5′ terminus of integrated viral genome was deleted, preventing HBeAg expression from transgene, but could be restored from the 3’ terminal redundancy of pre-genomic RNA during viral DNA replication and subsequent cccDNA formation. Our experimental results showed that following induction, HepAD38 produced and accumulated cccDNA, which became detectable between 7 and 8 days. HBeAg synthesis and secretion into culture fluid were dependent upon and proportional to the level of cccDNA detected. Therefore, the secretion of HBeAg by HepAD38 cells could potentially serve as a convenient reporter for the high throughput screening of novel antiviral drugs targeting HBV cccDNA.

A New Triterpene and Anti-Hepatitis B Virus Active Compounds from Alisma orientalis

A new triterpenoid named alisol O ( 1) was isolated from the rhizomes of Alisma orientalis, together with six known compounds: alisol A 24-acetate ( 2), 25-anhydroalisol A ( 3), 13 beta,17 beta-epoxyalisol A ( 4), alisol B 23-acetate ( 5), alisol F ( 6), and alisol F 24-acetate ( 7). Based on 1D and 2D-NMR data (HMQC, HMBC, COSY, ROESY), the structure of the new compound was deduced to be 11-dehydroxy-12-dehydroalisol F-24-acetate ( 1). Compounds 2 - 7 exhibited inhibitory activity in vitro on hepatitis B virus (HBV) surface antigen (HBsAg) secretion of the Hep G2.2.15 cell line with IC (50) values of 2.3, 11.0, 15.4, 14.3, 0.6 and 7.7 microM, and on HBV e antigen (HBeAg) secretion with IC (50) values of 498.1, 17.6, 41.0, 19.9, 8.5 and 5.1 microM, respectively.

Major role of hepatitis B genotypes in liver fibrosis during coinfection with HIV

Little is know about the determinants of liver fibrosis progression and genomic variability in hepatitis B virus (HBV) in HIV/HBV-coinfected patients. A cross-sectional analysis examined common characteristics of HBV infection in an ongoing cohort study of 308 patients with both HIV-1-positive Western blot and plasma HBV surface antigen (HBsAg) seropositivity. Risk factors for liver fibrosis were studied in a subset of 104 patients for whom liver biopsy and complete HBV genomic analysis were available. Analysis was performed by exact multiple regression analysis. Mean age of the study population was 40.3 years, with a ratio male to female of 5.3 and a mean duration of HIV infection of 9.3 years. In the subset of 104 patients, plasma HBV e antigen (HBeAg) in HBV-replicative patients could not be detected in 28.4% and lamivudine-resistant mutants were detected in 67.8%. HBV genotype A was the most frequent genotype (73/104) and 25 patients were infected by the usually rare genotype G. METAVIR fibrosis score was rated F2-F4 in 70 patients. After adjustment for the most common known determinants of liver fibrosis, HBV genotype G [odds ratio (OR), 12.60; 95% confidence interval (CI), 1.72-infinite; P < 0.009], efavirenz exposure (OR, 3.55; 95% CI, 1.14-12.14; P < 0.03), and the duration of HIV infection (3.86; 95% CI, 1.27-12.64; P < 0.01) were strongly associated with the risk of grade F2-F4 fibrosis. HBV genotype G is a determinant of liver fibrosis in HIV/HBV-coinfected patients and HBV genotyping should be considered as part of the management of patients with multiple risk factors for rapid progression of liver fibrosis.