In vivo inhibition of duck hepatitis B virus replication and gene expression by phosphorothioate modified antisense oligodeoxynucleotides.

Antisense oligodeoxynucleotide strategies have been employed in a variety of eukaryotic systems both to understand normal gene function and to block gene expression. Pharmacologically, 'code blockers' are ideal agents for antitumour and antimicrobial treatments because of their specific mode of action. Here we report the inhibition of duck hepatitis B virus (DHBV) by antisense oligodeoxynucleotides in primary duck hepatocyte cultures in vitro as well as in DHBV-infected Pekin ducks in vivo. The most effective antisense oligodeoxynucleotide was directed against the 5' region of the pre-S gene and resulted in a complete inhibition of viral replication and gene expression in vitro and in vivo. These results demonstrate the application of antisense oligodeoxynucleotides in vivo and exemplify their potential as human antiviral therapeutics.     

A technique for liver biopsy in Pekin ducks

The duck hepatitis B virus (DHBV), a member of the hepadna-virus group, has become a useful animal model for exploring important aspects in this family of viruses such as viral replication, course of infection, and the response to antiviral therapy. In chronically DHBV infected ducks, repeated analyses of liver tissue are important in defining the degree of viral replication and liver injury. We describe a technique for repeated liver biopsy using a Keyes skin punch biopsy. This technique provided sufficient quantities of liver tissue for serial analyses with minimal hemorrhage in 18 Pekin ducks. This procedure offers a safe and reliable method of obtaining serial liver biopsies.     

Production of infectious duck hepatitis B virus in a human hepatoma cell line.

The differentiated human hepatoma cell line Hep-G2 was transfected with cloned duck hepatitis B virus (DHBV) DNA. Introduction of closed circular DNA into the human liver cells resulted in the production of viral proteins: core antigen was detected in the cytoplasm, and e antigen, a related product, was secreted into the medium. Moreover, viral particles were released into the tissue culture medium which were indistinguishable from authentic DHBV by density, antigenicity, DNA polymerase activity, and morphology. Intravenous injection of tissue culture-derived DHBV particles into Pekin ducks established DHBV infection. In conclusion, transfection of human hepatoma cells with cloned DHBV DNA results in the production of infectious virus, as occurs with cloned human hepatitis B virus DNA. Human liver cells are therefore competent to support production of the avian and mammalian hepadnaviruses, indicating that liver-specific viral gene expression is controlled by evolutionarily conserved mechanisms. This new DHBV transfection system offers the opportunity to rapidly produce mutated DHBV which then can be further investigated in Pekin ducks.     

Covalently closed circular DNA is the predominant form of duck hepatitis B virus DNA that persists following transient infection

Residual hepatitis B virus (HBV) DNA can be detected in serum and liver after apparent recovery from transient infection. However, it is not known if this residual HBV DNA represents ongoing viral replication and antigen expression. In the current study, ducks inoculated with duck hepatitis B virus (DHBV) were monitored for residual DHBV DNA following recovery from transient infection until 9 months postinoculation (p.i.). Resolution of DHBV infection occurred in 13 out of 15 ducks by 1-month p.i., defined as clearance of DHBV surface antigen-positive hepatocytes from the liver and development of anti-DHBV surface antibodies. At 9 months p.i., residual DHBV DNA was detected using nested PCR in 10/11 liver, 7/11 spleen, 2/11 kidney, 1/11 heart, and 1/11 adrenal samples. Residual DHBV DNA was not detected in serum or peripheral blood mononuclear cells. Within the liver, levels of residual DHBV DNA were 0.0024 to 0.016 copies per cell, 40 to 80% of which were identified as covalently closed circular viral DNA by quantitative PCR assay. This result, which was confirmed by Southern blot hybridization, is consistent with suppressed viral replication or inactive infection. Samples of liver and spleen cells from recovered animals did not transmit DHBV infection when inoculated into 1- to 2-day-old ducklings, and immunosuppressive treatment of ducks with cyclosporine and dexamethasone for 4 weeks did not alter levels of residual DHBV DNA in the liver. These findings further characterize a second form of hepadnavirus persistence in a suppressed or inactive state, quite distinct from the classical chronic carrier state.     

Phosphonylmethoxyalkyl derivatives of purine as inhibitors of human hepatitis B virus DNA synthesis

A selected number of antiviral compounds which have been previously shown to inhibit the replication of DNA viruses or retroviruses were examined for their inhibitory effects on human hepatitis B virus (HBV) DNA synthesis. The assay system was based on the use of a human hepatoblastoma cell line (HB611) that continuously synthesizes HBV DNA. The following phosphonylmethoxyalkyl-purine derivatives were found to inhibit HBV DNA synthesis: 9-(2-phosphonyl-methoxyethyl)-2',6'-diaminopurine (PMEDAP), (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA) and 9-(2-phosphonylmethoxyethyl)adenine (PMEA). PMEDAP, HPMPA and PMEA not only inhibit HBV DNA synthesis in HB611 cells but also duck hepatitis B virus (DHBV) DNA and core antigen synthesis in primary duck hepatocytes.     

The duck hepatitis B virus pre-C region encodes a signal sequence which is essential for synthesis and secretion of processed core proteins but not for virus formation.

Analysis of the serum of duck hepatitis B virus (DHBV)-infected ducks has revealed the presence of C-terminally truncated viral core proteins (e antigens). These proteins are glycosylated and therefore were not released from infected cells by lysis but rather by active secretion, indicating that the DHBV core protein can be synthesized alternatively as a cytoplasmic or a secretory protein. Transient expression of cloned wild-type DHBV DNA and of a specifically designed viral mutant in a human hepatoma cell line (Hep-G2) showed that the DHBV core gene promoter is active in differentiated human liver cells and that synthesis and secretion of the processed core proteins are dependent on the expression of the pre-C region, a small open reading frame which precedes the core gene. In addition, these experiments showed that the mechanism of core protein processing and secretion is conserved between DHBV and the human hepatitis B virus and therefore might be important for the hepatitis B virus life cycle in general. In spite of this, intrahepatic injection of the pre-C mutant into uninfected ducks resulted in viremia without concomitant e-antigen synthesis, indicating that virus formation is independent of pre-C expression.     

Protective Efficacy of DNA Vaccines against Duck Hepatitis B Virus Infection

The efficacy of DNA vaccines encoding the duck hepatitis B virus (DHBV) pre-S/S and S proteins were tested in Pekin ducks. Plasmid pcDNA I/Amp DNA containing the DHBV pre-S/S or S genes was injected intramuscularly three times, at 3-week intervals. All pre-S/S and S-vaccinated ducks developed total anti-DHBs and specific anti-S antibodies with similar titers reaching 1/10,000 to 1/50,000 and 1/2,500 to 1/4,000, respectively, after the third vaccination. However, following virus challenge, significant differences in the rate of virus removal from the bloodstream and the presence of virus replication in the liver were found between the groups. In three of four S-vaccinated ducks, 90% of the inoculum was removed between <5 and 15 min postchallenge (p.c.) and no virus replication was detected in the liver at 4 days p.c. In contrast, in all four pre-S/S-vaccinated ducks, 90% of the inoculum was removed between 60 and 90 min p.c. and DHBsAg was detected in 10 to 40% of hepatocytes. Anti-S serum abolished virus infectivity when preincubated with DHBV before inoculation into 1-day-old ducklings and primary duck hepatocyte cultures, while anti-pre-S/S serum showed very limited capacity to neutralize virus infectivity in these two systems. Thus, although both DNA vaccines induced high titers of anti-DHBs antibodies, anti-S antibodies induced by the S-DNA construct were highly effective in neutralizing virus infectivity while similar levels of anti-S induced by the pre-S/S-DNA construct conferred only very limited protection. This phenomenon requires further clarification, particularly in light of the development of newer HBV vaccines containing pre-S proteins and a possible discrepancy between anti-HBs titers and protective efficacy.     

In Vitro Anti-hepatitis B Virus Activity of 2',3'-Dideoxyguanosine

2',3'-dideoxyguanosine(DoG) has been demonstrated to inhibit duck hepatitis B virus(DHBV) replication in vivo in a duck model of HBV infection. In the current study, the in vitro antiviral effects of DoG on human and animal hepadnaviruses were investigated. Our results showed that DoG effectively inhibited HBV, DHBV, and woodchuck hepatitis virus(WHV)replication in hepatocyte-derived cells in a dose-dependent manner, with 50% effective concentrations(EC50) of 0.3 ± 0.05, 6.82 ± 0.25, and 23.0 ± 1.5 lmol/L, respectively. Similar to other hepadnaviral DNA polymerase inhibitors,DoG did not alter the levels of intracellular viral RNA but induced the accumulation of a less-than-full-length viral RNA species, which was recently demonstrated to be generated by RNase H cleavage of pgRNA. Furthermore, using a transient transfection assay, DoG showed similar antiviral activity against HBV wild-type, 3TC-resistant rtA181 V, and adefovirresistant rtN236T mutants. Our results suggest that DoG has potential as a nucleoside analogue drug with anti-HBV activity.     

Sulfamoylbenzamide Derivatives Inhibit the Assembly of Hepatitis B Virus Nucleocapsids

Chronic hepatitis B virus (HBV) infection, a serious public health problem leading to cirrhosis and hepatocellular carcinoma, is currently treated with either pegylated alpha interferon (pegIFN-α) or one of the five nucleos(t)ide analogue viral DNA polymerase inhibitors. However, neither pegIFN-α nor nucleos(t)ide analogues are capable of reliably curing the viral infection. In order to develop novel antiviral drugs against HBV, we established a cell-based screening assay by using an immortalized mouse hepatocyte-derived stable cell line supporting a high level of HBV replication in a tetracycline-inducible manner. Screening of a library consisting of 26,900 small molecules led to the discovery of a series of sulfamoylbenzamide (SBA) derivatives that significantly reduced the amount of cytoplasmic HBV DNA. Structure-activity relationship studies have thus far identified a group of fluorine-substituted SBAs with submicromolar antiviral activity against HBV in human hepatoma cells. Mechanistic analyses reveal that the compounds dose dependently inhibit the formation of pregenomic RNA (pgRNA)-containing nucleocapsids of HBV but not other animal hepadnaviruses, such as woodchuck hepatitis virus (WHV) and duck hepatitis B virus (DHBV). Moreover, heterologous genetic complementation studies of capsid protein, DNA polymerase, and pgRNA between HBV and WHV suggest that HBV capsid protein confers sensitivity to the SBAs. In summary, SBAs represent a novel chemical entity with superior activity and a unique antiviral mechanism and are thus warranted for further development as novel antiviral therapeutics for the treatment of chronic hepatitis B.     

Glycine decarboxylase mediates a postbinding step in duck hepatitis B virus infection

Envelope protein precursors of many viruses are processed by a basic endopeptidase to generate two molecules, one for receptor binding and the other for membrane fusion. Such a cleavage event has not been demonstrated for the hepatitis B virus family. Two binding partners for duck hepatitis B virus (DHBV) pre-S envelope protein have been identified. Duck carboxypeptidase D (DCPD) interacts with the full-length pre-S protein and is the DHBV docking receptor, while duck glycine decarboxylase (DGD) has the potential to bind several deletion constructs of the pre-S protein in vitro. Interestingly, DGD but not DCPD expression was diminished following prolonged culture of primary duck hepatocytes (PDH), which impaired productive DHBV infection. Introduction of exogenous DGD promoted formation of protein-free viral genome, suggesting restoration of several early events in viral life cycle. Conversely, blocking DGD expression in fresh PDH by antisense RNA abolished DHBV infection. Moreover, addition of DGD antibodies soon after virus binding reduced endogenous DGD protein levels and impaired production of covalently closed circular DNA, the template for DHBV gene expression and genome replication. Our findings implicate this second pre-S binding protein as a critical cellular factor for productive DHBV infection. We hypothesize that DCPD, a molecule cycling between the cell surface and the trans-Golgi network, targets DHBV particles to the secretary pathway for proteolytic cleavage of viral envelope protein. DGD represents the functional equivalent of other virus receptors in its interaction with processed viral particles.     

不同种雏鸭建立鸭乙肝病毒感染模型及抗病毒效果的实验

目的探讨不同种雏鸭建立鸭乙肝病毒感染模型的影响因素,观察应用该模型抗病毒的效果。方法采集鸭血清,应用PCR方法定性检测鸭血清中病毒DNA;定量PCR方法检测鸭血清中病毒DNA载量变化;用抗病毒药物处理,观察其在鸭DHBV感染模型中的抗病毒效果。结果不同种鸭DHBV自然感染率不同,樱桃谷鸭为8.75%,湖北麻鸭两个批次分别为17.80%和10.68%;静脉注射和腹腔注射两途径均能致雏鸭感染DHBV,静脉注射感染率80%,腹腔注射感染率65%;鸭感染DHBV后,体内病毒载量维持在106～108copies/mL,可持续20 d以上;抗病毒药物处理后,在不同DHBV模型中其抗病毒效果变化趋势一致。结论鸭的种类和人工感染途径可影响DHBV感染率;雏鸭感染DHBV后其体内有持续性的病毒血症;DHBV感染模型是药物抗病毒研究较好模型。     

In vitro experimental infection of primary duck hepatocyte cultures with duck hepatitis B virus.

Duck hepatitis B virus (DHBV) obtained from the serum of congenitally infected ducks was used to infect primary duck hepatocyte cultures 1 to 4 days after plating. Virus replication was demonstrated by the appearance, beginning at 2 days after infection, of intracellular covalently closed-circular and single-stranded DHBV DNA replicative intermediates which were not present in the inoculating virus preparation. With increasing time after infection there was further amplification of intracellular relaxed circular, covalently closed-circular, and single-stranded DHBV DNA. Cultures of primary duck hepatocytes are competent for infection with DHBV only during the first 4 days of culture. Synthesis of DHBV core antigen and DHBV surface antigen was detected by immunofluorescence in 10% of the hepatocytes in culture. De novo synthesis and release of infectious virus was also demonstrated. Therefore, all stages of viral replication were carried out by these experimentally infected primary hepatocyte cultures. This system makes it possible to study DHBV replication in vitro.     

利用鸭乙型肝炎病毒感染模型评价血液成分中病毒的灭活效果

目的 利用鸭乙型肝炎病毒(DHBV)感染动物模型,评价亚甲蓝光化学病毒灭活方法对血液成分中DNA病毒的灭活效果。方法 将超离纯化的DHBV分别加入人血浆或人红细胞,经亚甲蓝光化学灭活病毒,将含不同基因组拷贝数DHBV的血浆成分经静脉感染1 d龄雏鸭。采用放射性核素核酸杂交法对血清中DHBV DNA进行检测,计算病毒灭活处理前、后人血浆及人红细胞中DHBV的半数感染计量(ID50)。结果 结果显示加入DHBV的血浆在未经灭活处理前对1 d龄雏鸭的ID50值为103.33,而经病毒灭活处理后ID50值为1010拷贝,灭活处理可使病毒感染性滴度下降达6个Log;加入DHBV的红细胞灭活前ID50值为103.35,经灭活处理后ID50值为108.35拷贝,灭活处理使病毒感染性滴度下降5个Log。结论 利用DHBV感染动物模型,可以检测到少量病毒在自然感染宿主体内的感染性,可用于评判血液成分中病毒灭活方法的效果,亚甲蓝光化学处理对血浆中DNA病毒的灭活效果较好于对红细胞中DNA病毒的灭活作用。     

Antiviral effect of Phyllanthus nanus ethanolic extract against hepatitis B virus (HBV) by expression microarray analysis

Ethanolic extract of Phyllanthus nanus (P. nanus) treatment exhibited potent antiviral activity against Hepatitis B virus (HBV). The effects of these extracts on HBV in the HBV genome integrated cell lines--Alexander cells and HepG2 2.2.15 cells were examined. Experimental results showed that the ethanolic extract of P. nanus produced suppressive effect on HBsAg secretion and HBsAg mRNA expression. The extract also inhibited HBV replication as measured by HBV DNA level in vitro. In addition, using a duck HBV (DHBV) primary culture model, the P. nanus ethanolic extract suppressed viral replication of DHBV in DHBV infected primary duck hepatocytes. The gene expression pattern in Alexander cells that had been treated with the ethanolic extract of P. nanus was also revealed by microarray techniques. The microarray results indicated that there was up-regulation of expression of several genes, including annexin A7 (Axn7). The subcellular localization of Axn7 and anti-HBV effect of Axn7 over-expression in Alexander cells were also investigated. Results showed that expression of Axn7-GFP fusion protein are localized around the secretory vesicles and could cause a decrease in HBsAg secretion in Alexander cells. Axn7 protein might play an important role in the medicinal effect of the active principle(s) of P. nanus.