[Effect of extracts from Radix Trichosanthis on the expression of HBsAg and HBeAg in HepG2.2.15 cells].

OBJECTIVE: To investigate the effect of extracts with water and alcohol from Radix Trichosanthis on the cell survival and the expression of hepatitis B surface antigen (HBsAg) and hepatitis B e-antigen (HBeAg) in HepG2.2.15 cell supernatant. METHODS: The cell survival rate of HepG2.2.15 cells was detected by MTT assay. The HBsAg and HBeAg in HepG 2.2.15 cell supernatant were evaluated by enzyme linked immunosorbent assay. RESULTS: The water and alcohol soluble extracts from Radix Trichosanthis significantly inhibited the levels of HBsAg and HBeAg in HepG2.2.15 cells in a time-and-concentration-dependent manner. However, the therapeutic index of extracts with water from Radix Trichosanthis was better than that in the alcohol group. CONCLUSION: The activity of water-soluble extract from Radix Trichosanthis is stronger on anti-hepatitis B virus than that of the alcohol-soluble extract.

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

AIM: To assess the anti-hepatitis B virus (HBV) effect of hyperoside extracted from Abelmoschus manihot (L) medik. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

In vitro antiviral activity of 1,2,3,4,6-penta-O-galloyl-beta-D-glucose against hepatitis B virus.

This study examined the antiviral activity of the root of Paeonia lactiflora PALL. Among the solvent fractions of the crude drug, the ethyl acetate fraction showed anti-hepatitis B virus (HBV) activity (IC50, 8.1 microg/ml) in an HBV-producing HepG2.2.15 cell culture system. The active anti-HBV principle was isolated and identified as 1,2,3,4,6-penta-O-galloyl-beta-D-glucose (PGG) from the crude drug by activity-guided fractionation. PGG isolated from P. lactiflora was examined for the inhibition of HBV multiplication by measurement of HBV DNA and hepatitis B surface antigen (HBsAg) levels in the extracellular medium of HepG2.2.15 cells after 8-d treatment. PGG decreased the level of extracellular HBV (IC50, 1.0 microg/ml) in a dose-dependent manner. PGG also reduced the HBsAg level by 25% at a concentration of 4 microg/ml. The gallate structure of PGG may play a critical role in the inhibition of anti-HBV activity. These results suggest that PGG could be a candidate for developing an anti-HBV agent.

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.

Effect of Oenanthe javanica flavone on human and duck hepatitis B virus infection.

AIM: To study the antiviral effect of Oenanthe javanica flavones (OjF) on human hepatoma HepG2.2.15 culture system and duck hepatitis B virus (DHBV) infection. METHODS: (1) After incubation for 24 h, the 2.2.15 cells were treated with different concentrations of OjF for 12 d. The cell alteration was observed by microscope. The presence of HBsAg and HBeAg were measured using the enzyme immunoassay kit after 2.2.15 cells were treated with OjF for 9 d. (2) Ducklings infected with DHBV intravenously were divided into 5 groups and treated with OjF, acyclovir (ACV), and normal saline respectively for 10 d. All the ducklings were bled before, during, and after treatments at different times, and serum levels of DHBV-DNA were detected by a dot-blot hybridization assay. RESULTS: (1) The 50% toxic concentration (TC50) of OjF was 2.28 g/L. The maximum nontoxic concentration (TC0) was 1.00 g/L. In nontoxic concentrations, OjF significantly inhibited HBsAg and HBeAg in 2.2.15 cells after 9 d of treatment (P<0.05, P<0.01). (2) The DHBV-DNA levels decreased significantly after the treatment with 0.50 and 1.00 g/kg of OjF (P<0.01). The inhibition of the peak of viremia was maximum at a dose of 1.00 g/kg and reached 54.3% on d 5 and 64.5% on d 10, respectively. CONCLUSION: The results demonstrate that OjF is a strong inhibitor of HBsAg and HBeAg secretion in 2.2.15 cells and DHBV-DNA levels in the HBV-infected duck model.

[The inhibitive effects of the ethanol extract from Radix et Rhizoma Rhei on the secretion of HBsAg and HBeAg].

OBJECTIVE: To study the anti-HBV effects of the ethanol extract from Radix et Rhizoma Rhei. METHODS: The influence of the ethanol extract from Radix et Rhizoma Rhei on the secretion of HBeAg and HBsAg was observed through the culture of the 2.2.15 cell with the ethanol extract. RESULTS: 11 days after the ethanol extract's action on the 2.2.15 cell, its 50% concentration dose (CD50) is 39.69 g/L; inhibiting dose (ID50) to HBsAg and HBeAg are 3.29 g/L and 2.34 g/L respectively, and TI 12.06 and 16.96 respectively. CONCLUSION: The ethanol extract from Radix et Rhizoma Rhei can markedly inhibit the secretion of HBsAg and HBeAg in 2.2.25 cell lines.

Screening of 25 compounds isolated from Phyllanthus species for anti-human hepatitis B virus in vitro.

Using an HBV-producing cell line and inhibition of the expression of the HBsAg and HBeAg as antiviral indicators, a study was conducted on 25 compounds isolated from four Phyllanthus (Euphorbiaceae) plants, including P. amarus Schum. & Thonn., P. multi florus Willd., P. tenellus Roxb. and P. virgatus Forst. f. It was found that niranthin (1), nirtetralin (3), hinokinin (5) and geraniin (13) at the non-cytotoxic concentration of 50 micro m, suppressed effectively both HBsAg and HBeAg expression, with the highest inhibition at 74.3%, 45.3%; 69.6%, 33.9%; 68.1%, 52.3%; 32.1%, 46.6%, respectively. Of these, niranthin (1) showed the best anti-HBsAg activity, while the most potent anti-HBeAg activity was observed with hinokinin (5).

Priming Th1 immunity to viral core particles is facilitated by trace amounts of RNA bound to its arginine-rich domain.

Particulate hepatitis B core Ag (C protein) (HBcAg) and soluble hepatitis B precore Ag (E protein) (HBeAg) of the hepatitis B virus share >70% of their amino acid sequence and most T and B cell-defined epitopes. When injected at low doses into mice, HBcAg particles prime Th1 immunity while HBeAg protein primes Th2 immunity. HBcAg contains 5-20 ng RNA/microg protein while nucleotide binding to HBeAg is not detectable. Deletion of the C-terminal arginine-rich domain of HBcAg generates HBcAg-144 or HBcAg-149 particles (in which >98% of RNA binding is lost) that prime Th2-biased immunity. HBcAg particles, but not truncated HBcAg-144 or -149 particles stimulate IL-12 p70 release by dendritic cells and IFN-gamma release by nonimmune spleen cells. The injection of HBeAg protein or HBcAg-149 particles into mice primes Th1 immunity only when high doses of RNA (i.e., 20-100 microg/mouse) are codelivered with the Ag. Particle-incorporated RNA has thus a 1000-fold higher potency as a Th1-inducing adjuvant than free RNA mixed to a protein Ag. Disrupting the particulate structure of HBcAg releases RNA and abolishes its Th1 immunity inducing potency. Using DNA vaccines delivered intradermally with the gene gun, inoculation of 1 microg HBcAg-encoding pCI/C plasmid DNA primes Th1 immunity while inoculation of 1 microg HBeAg-encoding pCI/E plasmid DNA or HBcAg-149-encoding pCI/C-149 plasmid DNA primes Th2 immunity. Expression data show eukaryotic RNA associated with HBcAg, but not HBeAg, expressed by the DNA vaccine. Hence, codelivery of an efficient, intrinsic adjuvant (i.e., nanogram amounts of prokaryotic or eukaryotic RNA bound to arginine-rich sequences) by HBcAg nucleocapsids facilitates priming of anti-viral Th1 immunity.