HBeAg Secretion Research Articles

Inhibition of the Replication of Hepatitis B Virus in Vitro by Pu-erh Tea Extracts

Hepatitis B virus (HBV) is one of the most widespread viral infections in the world and poses a significant global public health problem. The implementation of effective vaccination programs has resulted in a significant decrease in the incidence of acute hepatitis B. Nevertheless, there is still a need for as many effective anti-HBV drugs as possible. In this study, the role of pu-erh tea extracts (PTE) against HBV was analyzed in vitro by using a stably HBV-transfected cell line HepG2 2.2.15. The MTT assay showed that PTE and its active components (tea polyphenols, theaflavins, and theanine) presented low cytotoxicity. ELISA analysis revealed that PTE effectively reduced the secretion of HBeAg, but any one of the active components alone showed weaker efficacy, suggesting that the anti-HBV activity of PTE might be a synergetic effect of different components. RT-PCR and luciferase assay showed that PTE suppressed HBV mRNA expression while leaving four HBV promoter transcriptional activities unchanged. Fluorescence quantitative PCR results demonstrated that PTE dramatically diminished HBV DNA produced in cell supernatants as well as encapsidated DNA in intracellular core particles. Finally, PTE significantly reduced intracellular reactive oxygen species (ROS) level. This study is the first to demonstrate that PTE possesses anti-HBV ability and could be used as a potential treatment against HBV infection with an additional merit of low cytotoxicity.

Ethanol Extract from Ampelopsis sinica Root Exerts Anti‐Hepatitis B Virus Activity via Inhibition of p53 Pathway In Vitro

Ampelopsis sinica root is widely used in Chinese folk medicine for treating liver disorders caused by the hepatitis B virus (HBV). The present study was performed in order to investigate the anti-HBV activity and mechanisms of the ethanol extract from A. sinica root (EASR) in vitro. The antiviral activity of EASR was examined by detecting the levels of HBsAg, HBeAg and extracellular HBV DNAs in stable HBV-producing human hepatoblastoma HepG2 2.2.15 cells. We found that EASR effectively suppressed the secretion of HBsAg and HBeAg from HepG2 2.2.15 cells in a dose-dependent manner, and it also suppressed the amount of extracellular HBV DNA. After EASR treatment, the percentage of apoptotic cells was found to be significantly higher than that of control by flow cytometric analysis. A luciferase reporter gene assay was used to determine the effects of EASR on the activities of HBV promoters and intracellular signaling pathways. The results showed that EASR selectively inhibited the activities of HBV promoters (Cp, S1p and Fp) and the p53 signaling pathway in HepG2 cells significantly. These data indicate that EASR exerts anti-HBV effects via inhibition of HBV promoters and the p53-associated signaling pathway, which helps to elucidate the mechanism underlying the potential therapeutic value of EASR.

Evaluation on Anti-hepatitis Viral Activity of Vitis vinifer L

Suosuo grape (Vitis vinifer L) is traditionally used as a therapeutic agent for measles and hepatitis by the ethnic Uighurs. This work aimed to investigate the anti-HBV effect of total triterpene (VTT), total flavonoids (VTF) and total polysaccharides (VTP) from Suosuo grape, and their synergistic effects were also tested. The viral antigens of cellular secretion, HBsAg and HBeAg, were determined by enzyme linked immunosorbent assay (ELISA).The quantity of HBV-DNA released in the supernatant was assayed by real-time PCR. It was found that it effectively suppressed the secretion of HBsAg and HBeAg from HepG2.2.15 cells in a dose-dependent manner, as well as the HBV DNA. The results of orthogonal design experiment showed that the combination of VTT 20 μg/mL, VTF 50 μg/mL and VTP 50 μg/mL had the best optimistic inhibitory effects on HBeAg secretion.

Effects of 1,2,4,6-tetra-O-galloyl-β-D-glucose from P. emblica on HBsAg and HBeAg secretion in HepG2.2.15 cell culture

A polyphenolic compound, 1,2,4,6-tetra-O-galloyl-β-D-glucose (1246TGG), was isolated from the traditional Chinese medicine Phyllanthus emblica L. (Euphorbiaceae) and assayed for its potential as an anti-hepatitis B virus (HBV) agent. The cytotoxicity of 1246TGG on HepG2.2.15 as well as HepG2 cells was determined by observing cytopathic effects, and the effects of 1246TGG on secretion of HBsAg and HBeAg in HepG2.2.15 cells were assayed by enzyme immunoassay. Results indicates that treatment with 1246TGG (6.25 μg/mL, 3.13 μg/mL), reduced both HBsAg and HBeAg levels in culture supernatant, yet the inhibitory effects tend to decline with the assay time. This study provides a basis for further investigation of the anti-HBV activity and possible mechanism of action of 1246TGG.

Molecular and Phylogenetic Analyses Suggest an Additional Hepatitis B Virus Genotype “I”

A novel hepatitis B virus (HBV) strain (W29) was isolated from serum samples in the northwest of China. Phylogenetic and distance analyses indicate that this strain is grouped with a series of distinct strains discovered in Vietnam and Laos that have been proposed to be a new genotype I. TreeOrderScan and GroupScan methods were used to study the intergenotype recombination of this special group. Recombination plots and tree maps of W29 and these putative genotype I strains exhibit distinct characteristics that are unexpected in typical genotype C strains of HBV. The amino acids of P gene, S gene, X gene, and C gene of all genotypes (including subtypes) were compared, and eight unique sites were found in genotype I. In vitro and in vivo experiments were also conducted to determine phenotypic characteristics between W29 and other representative strains of different genotypes obtained from China. Secretion of HBsAg in Huh7 cells is uniformly abundant among genotypes A, B, C, and I (W29), but not genotype D. HBeAg secretion is low in genotype I (W29), whose level is close to genotype A and much lower than genotypes B, C, and D. Results from the acute hydrodynamic injection mouse model also exhibit a similar pattern. From an overview of the results, the viral markers of W29 (I1) in Huh7 cells and mice had a more similar level to genotype A than genotype C, although the latter was closer to W29 in distance analysis. All evidence suggests that W29, together with other related strains found in Vietnam and Laos, should be classified into a new genotype.

In vitro anti-hepatitis B virus effect of Hypericum perforatum L.

The anti-hepatitis B virus (HBV) effects and its mechanisms of the ethanol extracts of Hypericum perforatum L. (EHP) in vitro were explored. HepG2 2.2.15 cells, a stable HBV-producing cell line, were cultured as the model system to observe the anti-HBV effect. The viral antigens of cellular secretion, HBsAg and HBeAg, were determined by enzyme linked immunosorbent assay (ELISA). The quantity of HBV-DNA released in the supernatant was assayed by real-time PCR. In order to understand the mechanisms of the suppression of HBV replication, all HBV promoters (Cp, Xp, S1p, S2p and Fp) with luciferase reporter gene were transfected into HepG2 cells respectively. Then the activities of viral promoters were examined by luciferase reporter assay. It was found EHP effectively suppressed the secretion of HBsAg and HBeAg from HepG2 2.2.15 cells in a dose-dependent manner, as well as the extracellular HBV DNA. And EHP could selectively inhibit the activity of HBV promoter Fp. Our data suggest that EHP exerts anti-HBV effects via inhibition of HBV transcription, which helps to elucidate the mechanism underlying the potential therapeutic value of EHP.

Synthesis and antiviral activity of new acrylamide derivatives containing 1,2,3-thiadiazole as inhibitors of hepatitis B virus replication

A series of new acrylamide derivatives containing 1,2,3-thiadiazole were synthesized, characterized, and evaluated for their anti-hepatitis B virus (HBV) activities in vitro. The IC 50 of compounds 9b (10.4 μg/mL), 9c (3.59 μg/mL) and 17a (9.00 μg/mL) of the inhibition on the replication of HBV DNA were higher than that of the positive control lamivudine (14.8 μg/mL). Compound 9d exhibited significant activity against secretion of HBeAg (IC 50 = 12.26 μg/mL).

Virological pattern of hepatitis B infection in an HIV-positive man with fatal fulminant hepatitis B: a case report

IntroductionThere seem to be no published data concerning the clinical impact of populations of hepatitis B virus (HBV) in the hepatic and extrahepatic compartments of HIV-infected people with severe acute hepatitis.Case presentationA 26-year-old Caucasian man presenting to our hospital with clinical symptoms suggesting acute hepatitis was found to have an acute hepatitis B profile upon admission. He developed fatal fulminant hepatitis and was found to be heavily immunocompromised due to HIV-1 infection. He had a high plasma HBV and HIV load, and analysis of the partial pre-S1/pre-S2 domain showed the presence of mixed infection with D and F genotypes. Analysis of the point mutations within this region revealed the presence of HBV strains with amino acid substitutions at the immunodominant epitopes involved in B or T cell recognition. A homogeneous population of a pre-core mutant strain harbouring the A1896G and A1899G affecting HBeAg expression was invariably found in the liver tissue, plasma and peripheral blood mononuclear cells despite active HBeAg secretion; it was the dominant strain in the liver only, and was characterised by the presence of two point mutations in the direct repeat 1 domain involved in HBV replication activity. Taken together, these mutations are indicative of a highly replicative virus capable of evading immune responses.ConclusionThis case report provides clinical evidence of a possible association between the rapid spread of highly replicative escape mutants and the development of fulminant hepatitis in a heavily immunocompromised patient. Virological surveillance of severe acute hepatitis B may be important in establishing an early treatment strategy involving antiviral drugs capable of preventing liver failure, especially in individuals for whom liver transplantation is not accepted as a standard indication.

Swerilactones A and B, Anti-HBV New Lactones from a Tradtional Chinese Herb: Swertia mileensis as a Treatment for Viral Hepatitis

Swerilactones A (1) and B (2), two novel lactones with an unprecedented 6/6/6/6/6 pentacyclic ring system, were isolated from the traditional Chinese herb of Swertia mileensis with activity against the hepatitis virus. Their structures and relative stereochemistry were elucidated based on spectroscopic methods and further confirmed by X-ray single crystal diffraction analysis. In vitro antihepatitis B virus (HBV) assay on Hep G 2.2.15 cell line showed that compound 1 inhibited HBsAg and HBeAg secretion with IC(50) values of 3.66 and 3.58 mM, respectively.

Schisanwilsonins A–G and related anti-HBV lignans from the fruits of Schisandra wilsoniana

Seven new dibenzocyclooctane lignans, schisanwilsonins A–G (1–7), were isolated from the fruits of Schisandra wilsoniana, together with five known lignans (8–12). The structures of these new compounds were elucidated by spectroscopic methods including 2D-NMR techniques. The 12 lignans were tested for anti-hepatitis B virus (HBV) activity in vitro. Schisanwilsonin D (4), schisantherin C (9), deoxyschizandrin (10) and (+)-gomisin K 3 (11) showed anti-HBV activity. 9 exhibited the most potent anti-HBV activity with potency against HBsAg and HBeAg secretion by 59.7% and 34.7%, respectively, at 50 μg/mL.

Adeno-associated virus Rep78 protein inhibits Hepatitis B virus replication through regulation of the HBV core promoter

Rep78, the rep gene product of adeno-associated virus (AAV), has been shown to inhibit the replication of several DNA viruses. This study investigated the effects of Rep78 on replication of Hepatitis B virus (HBV) and possible mechanisms of inhibition. We have shown that HBV DNA replication and secretion of HBsAg and HBeAg in HepG2 2.2.15 cells were inhibited by Rep78. We have also demonstrated, using in vitro transcription and luciferase assay, that Rep78 binds to the HBV core promoter (HBV CP) and inhibits HBV CP activity. Furthermore, after Rep78 and HBV core protein expression plasmids were co-transfected into HepG2 cells, the expression of HBV core protein was inhibited significantly. These results suggest that Rep78 can inhibit the replication of HBV, correlating strongly with suppression of HBV CP activity.

Tea polyphenols exerts anti-hepatitis B virus effects in a stably HBV-transfected cell line

In this study, the anti-HBV effects of tea polyphenols (TP) were examined. After cells were exposed to TP for 3, 6, 9 days, amounts of HBsAg, HBeAg and HBV-DNA released into the supernatant of the cultured HepG2 2.2.15 cells were detected. TP, to some extent, inhibited the secretion of HBsAg and strongly suppressed the secretion of HBeAg in a dose-dependent (P<0.01) and time-dependent manner, with 50% maximal inhibitory concentration (IC50) value being 7.34 microg/mL on the 9th day, but the time-dependence was not significant (P=0.051). Expression of HBV-DNA in the supernatant of the cell culture also was significantly decreased in a dose-dependent fashion (P<0.01). The IC50 of TP in inhibiting HBV DNA was 2.54 microg/mL. It concluded that TP possessed potential anti-HBV effects and may be used as a treatment alternative for HBV infection.

Schisanwilsonenes A−C, Anti-HBV Carotane Sesquiterpenoids from the Fruits of Schisandra wilsoniana

Three carotane-type sesquiterpenoids, schisanwilsonenes A (1), B (2), and C (3), were isolated from the fruits of Schisandra wilsoniana. Their structures and relative configurations were elucidated on the basis of spectroscopic methods including 2D-NMR techniques, and the structure of 1 was confirmed by a single-crystal X-ray diffraction experiment. Schisanwilsonene A, at 50 microg/mL, exhibited antiviral activity, inhibiting HBsAg and HBeAg secretion by 76.5% and 28.9%.

Inhibitory effect of the combination of CpG-induced cytokines with lamivudine against hepatitis B virus replication in vitro

Background Currently approved antiviral monotherapies against chronic hepatitis B fail to eradicate hepatitis B virus (HBV), to overcome the defects in HBV-specific immune responses and to prevent HBV relapse after cessation of therapy. CpG oligodesoxynucleotides (CpG ODN) are synthetic agonists of Toll-like receptor 9 and potent inducers of innate and acquired immunity. Our aim was to establish the proof of concept of the antiviral benefit of combining a nucleoside analogue with CpG-induced cytokines on HBV replication in vitro. Methods Peripheral blood mononuclear cells from HBV-negative individuals were stimulated with CpG ODN to generate CpG-induced cytokine supernatants. Proliferating HepaRG and HepG2 cells were transduced with recombinant HBV baculovirus and differentiated HepaRG cells were inoculated with HBV virions. Antiviral effects of CpG-induced cytokine with or without lamivudine were evaluated by analysing HBV DNA, HBV RNA and antigen secretion (hepatitis B surface antigen [HBsAg] and hepatitis B e antigen [HBeAg]). Results Following transduction or HBV inoculation, CpG-induced cytokines strongly inhibited HBV viral intermediates of replication, as well as HBsAg and HBeAg secretion from infected cells. Strikingly, in transduced HepaRG cells, the combination of CpG-induced cytokines with lamivudine reduced the 50% effective concentration of lamivudine by 100-fold. Importantly, the treatment of CpG-induced cytokines prior to HBV inoculation conferred a partial protection against infection to hepatocytes. Conclusions CpG-induced cytokines associated with polymerase inhibitors represent a promising combination to suppress HBV replication. Such an immunotherapeutic strategy should be evaluated in vivo to assess restoration and duration of anti-HBV-specific immune responses.

An earthworm protease cleaving serum fibronectin and decreasing HBeAg in HepG2.2.15 cells

BackgroundVirus-binding activity is one of the important functions of fibronectin (FN). It has been reported that a high concentration of FN in blood improves the transmission frequency of hepatitis viruses. Therefore, to investigate a protease that hydrolyzes FN rapidly is useful to decrease the FN concentration in blood and HBV infection. So far, however, no specific protease digesting FN in serum has been reported.MethodsWe employed a purified earthworm protease to digest serum proteins. The rapidly cleaved protein (FN) was identified by MALDI-TOF MS and western blotting. The cleavage sites were determined by N-terminus amino acid residues sequencing. The protease was orally administrated to rats to investigate whether serum FN in vivo became decreased. The serum FN was determined by western blotting and ELISA. In cytological studies, the protease was added to the medium in the culture of HepG2.2.15 cells and then HBsAg and HBeAg were determined by ELISA.ResultsThe protease purified from earthworm Eisenia fetida was found to function as a fibronectinase (FNase). The cleavage sites on FN by the FNase were at R and K, exhibiting a trypsin alkaline serine-like function. The earthworm fibronectinase (EFNase) cleaved FN at four sites, R259, R1005, K1557 and R2039, among which the digested fragments at R259, K1557 and R2039 were related to the virus-binding activity as reported. The serum FN was significantly decreased when the earthworm fibronectinase was orally administrated to rats. The ELISA results showed that the secretion of HBeAg from HepG2.2.15 cells was significantly inhibited in the presence of the FNase.ConclusionThe earthworm fibronectinase (EFNase) cleaves FN much faster than the other proteins in serum, showing a potential to inhibit HBV infection through its suppressing the level of HBeAg. This suggests that EFNase is probably used as one of the candidates for the therapeutic agents to treat hepatitis virus infection.

Synthesis and in vitro anti-hepatitis B virus activities of 4-aryl-6-chloro-quinolin-2-one and 5-aryl-7-chloro-1,4-benzodiazepine derivatives

A series of 4-aryl-6-chloro-quinolin-2-ones and 5-aryl-7-chloro-1,4-benzodiazepine were synthesized and assayed for their in vitro anti-hepatitis B virus activities and cytotoxicities for the first time. Some of the tested compounds were active against HBsAg and HBeAg secretion in Hep G2.2.15 cells. Compound 5c showed IC 50 of 0.074 and 0.449 mM on HBsAg and HBeAg secretions, respectively, which were 10 times higher than that of its analog 4c and led to better selective index (SI) values (SI = 23.2 and 3.4, respectively).

Inhibition of hepatitis B virus gene expression and replication by artificial microRNA

To investigate the inhibitory effects of hepatitis B virus (HBV) replication and expression by transfecting artificial microRNA (amiRNA) into HepG2.2.15 cells. Three amiRNA-HBV plasmids were constructed and transfected into HepG2.2.15 cells. HBV antigen secretion was detected in the cells with transient and stable transfection by time-resolved fluoroimmunoassays (TRFIA). HBV DNA replication was examined by fluorescence quantitative PCR, and the level of HBV S mRNA was measured by semi-quantitative RT-PCR. The efficiency of transient transfection of the vectors into 2.2.15 cells was 55%-60%. All the vectors had significant inhibition effects on HBsAg and HBeAg at 72 h and 96 h after transfection (P < 0.01 for all). The secretion of HBsAg and HBeAg into the supernatant was inhibited by 49.8% +/- 4.7% and 39.9% +/- 6.7%, respectively, at 72 h in amiRNA-HBV-S608 plasmid transfection group. The copy of HBV DNA within culture supernatant was also significantly decreased at 72 h and 96 h after transfection (P < 0.01 for all). In the cells with stable transfection, the secretion of HBsAg and HBeAg into the supernatant was significantly inhibited in all three transfection groups (P < 0.01 for all, vs negative control). The copies of HBV DNA were inhibited by 33.4% +/- 3.0%, 60.8% +/- 2.3% and 70.1% +/- 3.3%, respectively. In HepG2.2.15 cells, HBV replication and expression could be inhibited by artificial microRNA targeting the HBV S coding region. Vector-based artificial microRNA could be a promising therapeutic approach for chronic HBV infection.

Two New Quaternary Alkaloids and Anti-Hepatitis B Virus Active Constituents from Corydalis saxicola

Two new quaternary isoquinoline alkaloids, saxicolalines A (1) and N-methylnarceimicine (2), together with sixteen known alkaloids (3 - 18) were isolated from Corydalis saxicola Bunting. The structures of saxicolalines A (1) and N-methylnarceimicine (2) were established based on spectral methods including 1D, 2D NMR (COSY, HMQC, HMBC) and HR-ESI-MS. The anti-HBV activities of ten main alkaloids were evaluated. Among the tested compounds, dihydrochelerythrine (8) exhibited the most potent activity against HBsAg and HBeAg secretions with IC50 < 0.05 microM, SI > 3.5, respectively.

In vivo and in vitro antiviral activity of hyperoside extracted from Abelmoschus manihot (L) medik

To assess the anti-hepatitis B virus (HBV) effect of hyperoside extracted from Abelmoschus manihot (L) medik. The human hepatoma Hep G2.2.15 cell culture system and duck hepatitis B virus (DHBV) infection model were used as in vivo and in vitro models to evaluate the anti-HBV effects. In the cell model, the 50% toxic concentration of hyperoside was 0.115 g/L; the maximum nontoxic concentration was 0.05 g/L. On the maximum nontoxic concentrations, the inhibition rates of hyperoside on HBeAg and HBsAg in the 2.2.15 cells were 86.41% and 82.27% on d 8, respectively. In the DHBV infection model, the DHBV-DNA levels decreased significantly in the treatment of 0.05 g x kg(-1 ) x d(-1 ) and 0.10 g x kg(-1) x d(-1) dosage groups of hyperoside (P<0.01). The inhibition of the peak of viremia was at the maximum at the dose of 0.10 g x kg(-1 ) x d(-1) and reached 60.79% on d 10 and 69.78% on d 13, respectively. These results suggested that hyperoside is a strong inhibitor of HBsAg and HBeAg secretion in 2.2.15 cells and DHBV-DNA levels in the HBV-infected duck model.

Combination of small interfering RNA and lamivudine on inhibition of human B virus replication in HepG2.2.15 cells

To observe the inhibition of hepatitis B virus (HBV) replication and expression by combination of siRNA and lamivudine in HepG2.2.15 cells. Recombinant plasmid psil-HBV was constructed and transfected into HepG2.2.15 cells. The transfected cells were cultured in lamivudine-containing medium (0.05 micromol/L) and harvested at 48, 72 and 96 h. The concentration of HBeAg and HBsAg was determined using ELISA. HBV DNA replication was examined by real-time PCR and the level of HBV mRNA was measured by RT-PCR. In HepG2.2.15 cells treated with combination of siRNA and lamivudine, the secretion of HBeAg and HBsAg into the supernatant was found to be inhibited by 91.80% and 82.40% (2.89+/-0.48 vs 11.73+/-0.38, P<0.05; 4.59+/-0.57 vs 16.25+/-0.48, P<0.05) at 96 h, respectively; the number of HBV DNA copies within culture medium was also significantly decreased at 96 h (1.04+/-0.26 vs 8.35+/-0.33, P<0.05). Moreover, mRNA concentration in HepG2.2.15 cells treated with combination of siRNA and lamivudine was obviously lower compared to those treated either with siRNA or lamivudine (19.44+/-0.17 vs 33.27+/-0.21 or 79.9+/-0.13, P<0.05). Combination of siRNA and lamivudine is more effective in inhibiting HBV replication as compared to the single use of siRNA or lamivudine in HepG2.2.15 cells.