An in vivo duck hepatitis B virus model recapitulates key aspects of nucleic acid polymer treatment outcomes in chronic hepatitis B patients.

Nucleic acid polymers (NAPs) are an attractive treatment modality for chronic hepatitis B (CHB), with REP2139 and REP2165 having shown efficacy in CHB patients. A subset of patients achieve functional cure, whereas the others exhibit a moderate response or are non-responders. NAP efficacy has been difficult to recapitulate in animal models, with the duck hepatitis B virus (DHBV) model showing some promise but remaining underexplored for NAP efficacy testing. Here we report on an optimized in vivo DHBV duck model and explore several characteristics of NAP treatment. REP2139 was efficacious in reducing DHBV DNA and DHBsAg levels in approximately half of the treated ducks, whether administered intraperitoneally or subcutaneously. Intrahepatic or serum NAP concentrations did not correlate with efficacy, nor did the appearance of anti-DHBsAg antibodies. Furthermore, NAP efficacy was only observed in experimentally infected ducks, not in endogenously infected ducks (vertical transmission). REP2139 add-on to entecavir treatment induced a deeper and more sustained virological response compared to entecavir monotherapy. Destabilized REP2165 showed a different activity profile with a more homogenous antiviral response followed by a faster rebound. In conclusion, subcutaneous administration of NAPs in the DHBV duck model provides a useful tool for in vivo evaluation of NAPs. It recapitulates many aspects of this class of compound's efficacy in CHB patients, most notably the clear division between responders and non-responders.

Nucleic acid polymer REP 2139 and nucleos(T)ide analogues act synergistically against chronic hepadnaviral infection in vivo in Pekin ducks.

Nucleic acid polymer (NAP) REP 2139 treatment was shown to block the release of viral surface antigen in duck HBV (DHBV)-infected ducks and in patients with chronic HBV or HBV/hepatitis D virus infection. In this preclinical study, a combination therapy consisting of REP 2139 with tenofovir disoproxil fumarate (TDF) and entecavir (ETV) was evaluated in vivo in the chronic DHBV infection model. DHBV-infected duck groups were treated as follows: normal saline (control); REP 2139 TDF; REP 2139 + TDF; and REP 2139 + TDF + ETV. After 4 weeks of treatment, all animals were followed for 8 weeks. Serum DHBsAg and anti-DHBsAg antibodies were monitored by enzyme-linked immunosorbent assay and viremia by qPCR. Total viral DNA and covalently closed circular DNA (cccDNA) were quantified in autopsy liver samples by qPCR. Intrahepatic DHBsAg was assessed at the end of follow-up by immunohistochemistry. On-treatment reduction of serum DHBsAg and viremia was more rapid when REP 2139 was combined with TDF or TDF and ETV, and, in contrast to TDF monotherapy, no viral rebound was observed after treatment cessation. Importantly, combination therapy resulted in a significant decrease in intrahepatic viral DNA (>3 log) and cccDNA (>2 log), which were tightly correlated with the clearance of DHBsAg in the liver. CONCLUSION: Synergistic antiviral effects were observed when REP 2139 was combined with TDF or TDF + ETV leading to control of infection in blood and liver, associated with intrahepatic viral surface antigen elimination that persisted after treatment withdrawal. Our findings suggest the potential of developing such combination therapy for treatment of chronically infected patients in the absence of pegylated interferon. (Hepatology 2018;67:2127-2140).

Effect of alcohol extract of Acanthus ilicifolius L. on anti-duck hepatitis B virus and protection of liver.

ETHNOPHARMACOLOGICAL RELEVANCE: Acanthus ilicifolius L. is an important medicinal mangrove plant. It is popularly used for its anti-inflammatory, antioxidant activity and hepatoprotective effects. The present study was conducted to evaluate the effect of treatment with alcohol extract of Acanthus ilicifolius L. on duck hepatitis B. MATERIALS AND METHODS: One-day-old Guangxi shelducks injected intraperitoneally with strong positive duck hepatitis B virus (DHBV) serum were used to establish a duck hepatitis B animal model in the study. The ducks were respectively administered in different groups with low-, middle- and high-dose alcohol extracts of Acanthus ilicifolius L., the positive control drug acyclovir (ACV) and double-distilled water. The levels of serum DHBV DNA were detected by fluorescence quantitative PCR (FQ-PCR). Duck hepatitis B surface antigen (DHBsAg) and duck hepatitis B e antigen (DHBeAg) OD values in the serum were measured by ELISA. The activity of Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST) in the serum was measured, and the livers were taken for histopathological examination. RESULTS: The levels of serum DHBV DNA and the values of DHBsAg and DHBeAg OD were not significant in any of the dose extract groups. However, the ALT activity was obviously lower in the middle- and high-dose extract groups. It was also found that a high dose of alcohol extract could reduce the activity of AST significantly and significantly improve hepatic pathological effects. CONCLUSIONS: High-dose alcohol extract of Acanthus ilicifolius L. has an obvious protective effect on the liver function and liver tissue. However, the present study finds that Acanthus ilicifolius L. cannot inhibit the replication of duck hepatitis B virus.

Anti-hepatitis B virus activity of α-DDB-FNCG, a novel nucleoside-biphenyldicarboxylate compound in vitro and in vivo.

A novel codrug, α-DDB-FNCG, was synthesized through coupling of α-biphenyl dimethyl dicarboxylate (α-DDB) and the nucleoside analogue FNCG, via an ester bond. The anti-HBV activity and hepatoprotective effects of this compound were investigated both in vitro and in vivo. In HBV-transfected HepG2.2.15 cell line, the secretion of HBsAg and HBeAg as well as the levels of extracellular and intracellular viral DNA were determined by ELISA and real-time fluorescent quantitative Polymerase Chain Reaction (FQ-PCR), respectively. In DHBV-infected ducks, the viral DNA levels in serum and liver were determined by FQ-PCR. In addition, the levels of alanine transaminase (ALT) and aspartate aminotransferase (AST) in both serum and liver were also examined. The improvement of ducks' livers was evaluated by histopathological analysis. It has been demonstrated that α-DDB-FNCG could suppress the levels of HBV antigens and viral DNA in a time- and dose-dependent manner in the HepG2.2.15 cell line. Furthermore, this codrug could also significantly inhibit the viral DNA replication and reduce the ALT and AST levels in both serum and liver of DHBV-infected ducks, with improved hepatocellular architecture in drug-treated ducks. In short, these results suggest that α-DDB-FNCG could be a promising candidate for further development of new anti-HBV agents with hepatoprotective effects.

In vitro and in vivo anti-hepatitis B virus activities of the lignan niranthin isolated from Phyllanthus niruri L.

ETHNOPHARMACOLOGICAL RELEVANCE: Niranthin is a lignan isolated from Phyllanthus niruri L. This plant has long been used in folk medicine for liver protection and antihepatitis B in many Asian countries. This study was designed to evaluate the anti-hepatitis B virus activity of niranthin using HepG2.2.15 cells and duck hepatitis B virus (DHBV) infected ducks as in vitro and in vivo models. MATERIALS AND METHODS: Niranthin was isolated from Phyllanthus niruri L. (Euphorbiaceae) by extraction and chromatographic procedures and the anti-hepatitis B virus activity was evaluated both in vitro and in vivo. The human HBV-transfected liver cell line HepG2.2.15 was used in vitro assay. And the in vivo anti-hepatitis B virus activity was evaluated on the expression of HBV replication, HBsAg, HBeAg, ALT and AST on day 0, 7, 14, 17 after niranthin was dosed intragastricly (i.g.) once a day for 14 days at the dosages of 25, 50 and 100 mg/kg/day in the duck hepatitis B virus (DHBV) infected ducks. RESULTS: In the human HBV-transfected liver cell line HepG2.2.15, the secretion of HBsAg and HBeAg were significantly decreased after treatment with niranthin for 144 h, with IC50 values for HBsAg of 15.6 µM, IC50 values for HBeAg of 25.1 µM. In DHBV-infected ducklings, niranthin significantly reduced the serum DHBV DNA, HBsAg, HBeAg, ALT and AST. Furthermore, analysis of the liver pathological changes confirmed the hepatoprotective effect of niranthin. CONCLUSION: The experimental data demonstrated that niranthin exhibits anti-hepatitis B virus activity both in vitro and in vivo.

Isothiafludine, a novel non-nucleoside compound, inhibits hepatitis B virus replication through blocking pregenomic RNA encapsidation.

AIM: To investigate the action of isothiafludine (NZ-4), a derivative of bis-heterocycle tandem pairs from the natural product leucamide A, on the replication cycle of hepatitis B virus (HBV) in vitro and in vivo. METHODS: HBV replication cycle was monitored in HepG2.2.15 cells using qPCR, qRT-PCR, and Southern and Northern blotting. HBV protein expression and capsid assembly were detected using Western blotting and native agarose gel electrophoresis analysis. The interaction of pregenomic RNA (pgRNA) and the core protein was investigated by RNA immunoprecipitation. To evaluate the anti-HBV effect of NZ-4 in vivo, DHBV-infected ducks were orally administered NZ-4 (25, 50 or 100 mg·kg⁻¹·d⁻¹) for 15 d. RESULTS: NZ-4 suppressed intracellular HBV replication in HepG2.2.15 cells with an IC50 value of 1.33 µmol/L, whereas the compound inhibited the cell viability with an IC50 value of 50.4 µmol/L. Furthermore, NZ-4 was active against the replication of various drug-resistant HBV mutants, including 3TC/ETV-dual-resistant and ADV-resistant HBV mutants. NZ-4 (5, 10, 20 µmol/L) concentration-dependently reduced the encapsidated HBV pgRNA, resulting in the assembly of replication-deficient capsids in HepG2.2.15 cells. Oral administration of NZ-4 dose-dependently inhibited DHBV DNA replication in the DHBV-infected ducks. CONCLUSION: NZ-4 inhibits HBV replication by interfering with the interaction between pgRNA and HBcAg in the capsid assembly process, thus increasing the replication-deficient HBV capsids. Such mechanism of action might provide a new therapeutic strategy to combat HBV infection.

Isolation and identification of an anti-hepatitis B virus compound from Hydrocotyle sibthorpioides Lam.

ETHNOPHARMACOLOGICAL RELEVANCE: Hydrocotyle sibthorpioides (Apiaceae) have been used as a folk remedy for the treatment of fever, edema, detoxication, throat pain, psoriasis and hepatitis B virus infections in China. The aim of this study is to isolate and identify an anti-HBV compound from this herb. MATERIALS AND METHODS: A compound (saponin) was isolated from the active ethanol extract using bioassay-guided screening. The structure of the saponin was elucidated by spectroscopic methods and compared with published data. The anti-HBV activity of the saponin was evaluated by detecting the levels of HBV antigens, extracellular HBV DNA, nuclear covalent closed circular DNA (cccDNA) and five HBV promoters in HepG2.2.15 cells. In addition, the levels of serum HBsAg/HBeAg, DHBV DNA, ALT/AST and hepatic pathological changes were analyzed in DHBV-infected ducks. RESULTS: The chemical analysis indicated that the saponin isolated from Hydrocotyle sibthorpioides is asiaticoside. The pharmacodynamics experimental studies showed that asiaticoside effectively suppressed the levels of HBsAg/HBeAg, extracellular HBV DNA and intracellular cccDNA in a dose-dependent manner. Furthermore, experiments demonstrated that asiaticoside markedly reduced viral DNA transcription and replication by inhibiting the activities of core, s1, s2, and X gene promoters. In addition, asiaticoside markedly reduced DHBV replication without any obvious signs of toxicity. The levels of serum DHBV DNA, HBsAg/HBeAg were increased 3 days after drug withdrawal, but the levels rebounded slightly in the asiaticoside treatment groups compared with the 3TC treatment group. Moreover, analysis of the serum ALT/AST levels and the liver pathological changes indicated that asiaticoside could alleviate liver damage. CONCLUSIONS: Our results show that asiaticoside could efficiently inhibit HBV replication both in vitro and in vivo, and asiaticoside may be a major bioactive ingredient in Hydrocotyle sibthorpioides.

Antiviral activity of methyl helicterate isolated from Helicteres angustifolia (Sterculiaceae) against hepatitis B virus.

The anti-HBV effect of methyl helicterate (MH), a triterpenoid isolated from the Chinese herb Helicteres angustifolia, was explored both in vitro and in vivo. In the HBV-transfected cell line HepG2.2.15, the secretion of HBsAg/HBeAg, the levels of HBV DNA and cccDNA, and the amount of viral RNA were significantly decreased after treatment with MH for 144h. In addition, MH had no inhibitory effect on the mitochondrial DNA content. In DHBV-infected ducklings, MH significantly reduced the serum DHBV DNA, liver total viral DNA, and cccDNA levels. Furthermore, analysis of the liver pathological changes confirmed the hepatoprotective effect of MH. These results indicate that MH efficiently inhibits HBV replication both in vitro and in vivo and that MH may be a major bioactive ingredient in H. angustifolia.

Nucleic acid polymers prevent the establishment of duck hepatitis B virus infection in vivo.

Nucleic acid polymers (NAPs) are novel, broad-spectrum antiviral compounds that use the sequence-independent properties of phosphorothioate oligonucleotides (PS-ONs) as amphipathic polymers to block amphipathic interactions involved in viral entry. Using the duck hepatitis B virus (DHBV) model of human hepatitis B virus infection, NAPs have been shown to have both entry and postentry antiviral activity against DHBV infection in vitro in primary duck hepatocytes (PDH). In the current study, various NAPs were assessed for their prophylactic activity in vivo against DHBV infection in ducks. The degenerate NAP REP 2006 prevented the development of widespread and persistent DHBV infection in 14-day-old ducks, while the acidic-pH-sensitive NAP REP 2031 had little or no prophylactic effect. REP 2006 displayed significant toxicity in ducks, which was attributed to CpG-mediated proinflammation, while REP 2031 (which has no CpG motifs) displayed no toxicity. A third NAP, REP 2055, which was designed to retain amphipathic activity at acidic pH and contained no CpG motifs, was well tolerated and displayed prophylactic activity against DHBV infection at doses as low as 1 mg/kg of body weight/day. These studies suggest that NAPs can be easily and predictably tailored to retain anti-DHBV activity and to have minimal toxic effects in vivo. Future studies are planned to establish the therapeutic efficacy of NAPs against persistent DHBV infection.

Anti-HBV effect of individual traditional Chinese herbal medicine in vitro and in vivo: an analytic review.

Traditional Chinese herbal medicine (TCHM) has been widely used in the treatment of chronic hepatitis B (CHB) in China. The systematic analysis of clinical research of TCHM against CHB revealed its potential but not confirmed its therapeutic effect. To understand the detailed antiviral effect of TCHM against HBV infection, we systematically analysed the anti-HBV effect of individual Chinese herbs on the basis of the research on individual TCHM in vitro and in vivo, which were published from 1995 to 2012. Among 171 herbal components isolated from 76 Chinese herbs, we found 13 compounds and 9 extracts isolated from 18 Chinese herbs showing strong inhibitory effect on HBV DNA, HBeAg or HBsAg release with low cytotoxicity in HepG2.2.15 cells, and agents from 12 Chinese herbs showing the highest inhibition rates of plasma DHBV DNA of more than 50% in DHBV-infected ducks. In addition, the two compounds chrysophanol 8-O-beta-D-glucoside isolated from Rheum palmatum and wogonin isolated from Scutellaria baicalensis were found to display strong anti-HBV activity. Interestingly, compounds isolated from 5 of these effective anti-HBV Chinese herbs were found to show strong antibacterial or antifungal activity also. This review summarizes and analyses the studies on the anti-HBV effect of individual TCHM in cell and animal models, providing potential perspective in the understanding of TCHM in the treatment of hepatitis B and the development of new anti-HBV drugs from TCHM.

Inhibitory effect of total saponins isolated from Taraphochlamys affinis on duck hepatitis B virus replication.

It has been previously shown that Taraphochlamys affinis possessed anti-hepatitis B virus (HBV) activities. To identify the active ingredients, the total saponins (TSTA) were isolated from T. affinis and the inhibitory effect of TSTA on HBV in the duck HBV model was examined. The results showed that serum levels of DHBV-DNA decreased in all ducks treated with TSTA (1.0 and 2.0 g x kg(-1) x d(-1)) and lamivudine (3TC) (50 mg x kg(-1) x d(-1)) during treatment, but 7 days after the cessation of treatment (p7) with 3TC, the viral replication level returned to the pretreatment baseline. Contrariwise in ducks treated with TSTA, the effect of DHBV DNA inhibition lasted. Compared with model control group,the alanine aminotransferase (ALT), aspartate aminotransferase (AST) and duck hepatitis B surface antigen (DHBsAg) values of 1.0 and 2.0 g x kg(-1) x d(-1)-dose TSTA groups were significantly lower on 7, 14 days after the treatment (d7, d14) and p7, and at p7, the ALT and DHBsAg levels of 2.0 g x kg(-1) x d(-1)-dose TSTA group was significantly lower than that of 3TC group. Furthermore, significant histological improvement was noted in ducklings of TSTA treatment group 7 days after the withdrawal. The study results demonstrate that TSTA possesses potent anti-HBV activity.

Synthesis, stability and pharmacological evaluation of a novel codrug consisting of lamivudine and ursolic acid.

A novel codrug (LMX) was obtained from lamivudine (LMV) and ursolic acid (UA) coupled with ethyl chloroacetate through an amide and ester linkage. The structure of LMX was confirmed by ¹H NMR, ¹³C NMR, IR and HRMS. Herein, the in vitro non-enzymatic and enzymatic hydrolysis and in vivo pharmacological activities of LMX were studied. The kinetics of hydrolysis of LMX was studied in aqueous solution of pH 1-10, 80% buffered human plasma and in the presence of lipase from Porcine pancreas (EC 3.1.1.3) at 37°C. It is found that LMX hydrolysis rate was significantly faster in lipase with half-life of 1.4h compared to pH 7.4 phosphate buffer (t(1/2) 11.2h) and buffered human plasma (t(1/2) 5.4h). The decomposition rates in aqueous solution (pH 1-10) showed a U-shaped curve. LMX was comparatively stable between pH 3 and 6 (half-life >40 h). Pharmacological studies indicated that LMX had the dual action of anti-hepatitis B virus activity and hepatoprotective effects against acute liver injury. These findings suggest that LMX could be a promising candidate agent for the treatment of hepatitis.

Helioxanthin analogue 8-1 inhibits duck hepatitis B virus replication in cell culture.

BACKGROUND: Current approved anti-HBV treatment cannot completely eliminate HBV infection, and emergence of resistant virus is an important treatment issue. Effective anti-HBV agents with different mechanisms of action on novel target sites are needed for the treatment of HBV infection and for combating the resistant virus, alone or in combination with current anti-HBV strategies. Helioxanthin analogue 8-1 displayed potent anti-HBV activity in human HBV in vitro and in animal models, with a unique antiviral mechanism. Its antiviral activity in other HBV system needs further study. METHODS: The anti-duck hepatitis B virus (DHBV) activity of 8-1, an analogue of a natural product, helioxanthin, was studied in the DHBV inducible cell line, dstet5, in comparison to and in combination with the nucleoside analogue, lamivudine (3TC). RESULTS: Helioxanthin analogue 8-1 exhibited anti-DHBV activity as demonstrated by quantification of viral DNA, RNA, covalently closed circular DNA and protein synthesis. Analogue 8-1 did not affect the stability of cellular macromolecules and did not have a sustained antiviral effect after drug removal. When DHBV replication was induced, virus-harbouring cells were more susceptible to the cytotoxicity of 8-1 than non-induced cells. CONCLUSIONS: 8-1 exhibited effective inhibition on DHBV replication. The combination of 8-1 with 3TC resulted in additional anti-DHBV activity. Viral induced cells displayed higher susceptibility to 8-1 treatment than non-induced cells. HBV X protein might not be an essential factor in the initiation of the biological activity of 8-1, as demonstrated by its absence in DHBV. These findings warrant further development of 8-1 for the treatment of chronic hepatitis B and its associated diseases.

Synthesis and biological evaluation of heat-shock protein 90 inhibitors: geldanamycin derivatives with broad antiviral activities.

BACKGROUND: Previous studies have suggested that geldanamycin (GA) inhibits the replication of several viruses in vitro. Here, we aimed to synthesize and evaluate the antiviral activity of 17-amino-17-demethoxygeldanamycin derivatives. METHODS: A series of 17-substituted and 17-,19-disubstituted GA derivatives were screened for antiviral activities against eight different viral strains, including herpesvirus, hepatitis virus, retrovirus and picornavirus. RESULTS: Most of the tested compounds showed inhibitory activity against the viruses and showed reduced cytotoxicity in vitro as compared with the parent compound GA. In vivo efficacy evaluation results showed that compound 6 noticeably inhibited duckling hepatitis B virus DNA replication in duckling serum after oral administration. Viral rebound did not occur after termination of the treatment. The modified GA derivatives also showed median lethal dose values that were higher than that of the parent GA in mice intraperitoneally treated with the study compounds. CONCLUSIONS: Targeting heat-shock protein 90 could be a new antiviral approach that is not prone to the development of drug resistance. The 17-amino-17-demethoxygeldanamycin derivatives could be novel agents with potential antiviral activity.

The persistence in the liver of residual duck hepatitis B virus covalently closed circular DNA is not dependent upon new viral DNA synthesis.

Residual hepatitis B virus (HBV) DNA can be detected following the resolution of acute HBV infection. Our previous work using duck hepatitis B virus (DHBV) infected ducks, indicated that ~80% of residual DHBV DNA in the liver is in the covalently closed circular DNA (cccDNA) form, suggesting that viral DNA synthesis is suppressed. The current study asked more directly if maintenance of residual DHBV cccDNA is dependent upon ongoing viral DNA synthesis. Ducks that recovered from acute DHBV infection were divided into 2 groups and treated with the antiviral drug, Entecavir (ETV), or placebo. No major differences in the stability of cccDNA or levels of residual cccDNA were observed in liver biopsy tissues taken 95 days apart from ETV treated and placebo control ducks. The data suggest that residual DHBV cccDNA is highly stable and present in a cell population with a rate of turnover similar to normal, uninfected hepatocytes.

The woodchuck: a model for therapeutic vaccination against hepadnaviral infection.

Interferon-alpha and nucleoside analogues are available for the treatment of chronic hepatitis B virus (HBV) infection but do not lead to a satisfactory result. New findings about the immunological control of HBV during acute infection suggest the pivotal role of T-cell mediated immune responses. Several preclinical and clinical trials were undertaken to explore the possibility of stimulating specific immune responses in chronically infected animals and patients by vaccination. However, vaccination with commercially available HBV vaccines in patients and immunization in woodchucks with core or surface proteins of woodchuck hepatitis virus (WHV) did not result in effective control of HBV and WHV infection, suggesting that new formulations of therapeutic vaccines are needed. Some new approaches combining antiviral treatments with nucleoside analogues, DNA vaccines and protein vaccines were tested in the woodchuck model. It could be shown that therapeutic vaccinations are able to stimulate specific B- and T-cell responses and to achieve transient suppression of viral replication. These results suggest the great potential of therapeutic vaccination in combination with antivirals to reach an effective and sustained control of HBV infection.

Anti-hepatitis B virus activity of chlorogenic acid, quinic acid and caffeic acid in vivo and in vitro.

Chlorogenic acid and its related compounds are abundant plant polyphenols that have a diverse antiviral activity. In this study, HepG2.2.15 cells and duck hepatitis B virus infection model were used as in vitro and in vivo models to evaluate their anti-HBV activity. In the cell model, all the three compounds inhibited HBV-DNA replication as well as HBsAg production. Chlorogenic acid and caffeic acid also reduced serum DHBV level in DHBV-infected duckling model. Moreover, the anti-HBV activity of crude extracts of coffee beans, which have a high content of chlorogenic acid, was studied. Both the extracts of regular coffee and that of decaffeinated coffee showed inhibitory effect on HBV replication.

The effect of Gankang Suppository on duck hepatitis B virus, serum biochemistry and liver histology in ducklings.

To examine the effect of Gankang Suppository on duck hepatitis B virus (DHBV), the serum biochemistry and hepatic histology in an animal model of DHBV infection, a model of DHBV infection was established by infecting 1-day-old Yingtaogu ducklings with DHBV-positive serum. The successful model was confirmed by PCR assay and 48 ducklings infected with DHBV were randomly divided into 3 groups: a Gankang Suppository treatment group, an acyclovir (ACV) group and a DHBV model group (control), with each group having 16 animals. All the animals were given the medicines for 4 weeks in a row. The serum of the animals was taken 14 and 28 days after the medication and 7 days after drug discontinuation. Real-time PCR was performed to detect the copy numbers of DHBV DNA in the serum. ALT and AST were dynamically monitored. The ducklings were sacrificed on the 7th day after the discontinuation of the treatment and livers were harvested and examined for inflammation and degeneration of liver cells by using HE staining. The results showed that on day 14, 28 after the treatment and day 7 after the withdrawal, the logarithmic values (log) of DHBV DNA copy numbers in ducklings of Gankang Suppository treatment group were significantly lower than that before the treatment (P=0.0092, P=0.0070, P=0.0080, respectively). Compared with DHBV model control group, the ALT level was significantly decreased (P=0.0020, P=0.0019, respectively) on day 28 after the treatment and on day 7 after the withdrawal. The AST level was also reduced on day 14 after the treatment (P=0.0298). Compared with the ACV control group, the level of ALT was lower on day 7 after the withdrawal (P=0.0016). Histologically, the hepatocyte swelling, vacuolous degeneration and acidophilic degeneration in Gankang Suppository treatment group were alleviated 7 days after the withdrawal as compared with model control group (P=0.0282, P=0.0084, P=0.0195, respectively). It is concluded that Gankang Suppository can effectively suppress DHBV replication, reduce the levels of serum ALT and AST and improve hepatic histology.

[Establishment of an in vivo model for duck hepatitis B virus infection using Hubei duckling].

OBJECTIVE: To develop a standard duck hepatitis B virus (DHBV) animal model using a local Hubei species of duck, Ma Ya, and use it as an in vivo experimental system to study antiviral strategies against hepatitis B. METHODS: Two-day-old Ma Ya ducklings were experimentally infected via intraperitoneal injection with the DHBV inocula which was collected from the transfected culture supernatant of 1.5-fold-overlength genome recombinant plasmid. Blood samples were taken twice or thrice a week during post-inoculation for 50 days. Viremia was quantified by serum real-time PCR to show the peak. Antiviral treatment of the DHBV-infected ducklings was started 3 d post-inoculation. The animals received oral administration of lamivudine (3TC) at a dose of 25 mg/kg/d for 5 d, followed by a maintenance therapy thrice weekly for 3 more weeks. Serum was quantified to show the viremia peak and liver biopsy specimens were analysed by Southern blotting and in-situ hybridization at the end of antiviral drug treatment. RESULTS: The experimental infection rate of 2-day-old ducklings was 87.5%. Viremia started to be detectable on day 7 and reached a peak on day 11 post-inoculation, followed by a decrease and fluctuations. Four weeks of oral administration of 3TC led to a significant decrease in viremia peak during. This effect was not sustained, as a rebound in viremia was observed after drug withdrawal. Similarly, the analysis of liver biopsies at the end of 3TC treatment showed a marked decrease in DHBV DNA. However, after drug withdrawal a rebound of intrahepatic DHBV DNA was observed in duck livers. CONCLUSION: The Hubei duck model with experimental DHBV infection of transfected supernatant is more suitable for the hepadnavirus biologic research due to its stability and practicability.