Antisense oligonucleotide therapy of hepadnavirus infection.
Explore the source record for details and available documents.
Animals↗
SEARCH · Search PubMed
Results for “Hepadnaviridae”
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Development of viral disinfectant assays for duck hepatitis B virus using cell culture/PCR.
Human hepatitis B virus (HBV) is a worldwide public health problem with chronic carriers at risk for developing cirrhosis and hepatocellular carcinoma. Accidental nosocomial infections from inadequately disinfected equipment or exposure to blood and body fluids from patients are major routes. To solve such problems, disinfectants to inactivate HBV must be validated. Duck hepatitis B virus (DHBV) is accepted as a surrogate for HBV, due to their similar sensitivities to disinfectants and its safety. Ducklings are used for disinfectant efficacy assays; however, the same virus titer is obtained using duck embryonic hepatocytes. Viral titration in disinfectant efficacy assay is conducted using Southern hybridization of infected duck serum. However, this test requires radioisotopes. Therefore, disinfectant assessment protocols were developed using duck embryonic hepatocytes with polymerase chain reaction (PCR) or nested PCR. The ease of handling, lowered cost and enhanced sensitivity make PCR desirable. Chicken embryonic hepatocytes were applied to DHBV disinfectant efficacy assay. Results were consistent and could be used under certain conditions. The virucidal activities of two quaternary ammonium chloride disinfectants, n-alkyl dimethyl benzyl ammonium chloride and alkyl dimethyl benzyl ammonium chloride (10C-12C) were compared and effective concentrations were 1200 and 1800 ppm, respectively. Efficacies of these disinfectants were validated using real-time quantitative PCR. Results confirmed that the efficacy of n-alkyl dimethyl benzyl ammonium chloride was higher than alkyl dimethyl benzyl ammonium chloride (10C-12C). This assay was useful for rapid discrimination of killing potentials of disinfectants. In conclusion, these assays can be applied to other viruses that are unable to cause CPE in cell cultures and broadened the utility of DHBV as animal model for HBV.
Animals↗
A quantitative competitive PCR assay for the covalently closed circular form of the duck hepatitis B virus.
A crucial step in the establishment and maintenance of a hepadnavirus infection is the formation of a pool of covalently closed circular viral genomes in the nucleus. Changes in the size of this pool occur when an infection is established, when acute infections are resolved, and when chronic infections are treated with antiviral drugs. However, the lack of a quantitative assay for the cccDNA form of the virus has hampered study of the biology of this replication intermediate. In response to this need we have devised a sensitive and accurate competitive PCR assay that is highly selective for the cccDNA form of the duck hepatitis B virus. Since only small amounts of DNA are needed for the assay, cccDNA pool sizes can be monitored in live animals using DNA derived from needle biopsies of infected liver.
Animals↗
The SATE pronucleotide approach applied to acyclovir: part II. Effects of bis(SATE)phosphotriester derivatives of acyclovir on duck hepatitis B virus replication in vitro and in vivo.
The in vitro and in vivo antiviral activities of two mononucleoside phosphotriester derivatives of acyclovir (ACV) incorporating S-acyl-2-thioethyl (SATE) groups are reported using the duck model of hepatitis B (DHBV). In primary duck hepatocyte cultures, the described phosphotriesters significantly inhibited the replication of DHBV at submicromolar concentrations. They were found to be more potent than the parent nucleoside. This result was in agreement with our data concerning the anti-HBV activity of these pronucleotides in HepG2.2.15 cells (previous paper). In vivo, the studied SATE pronucleotide was also found to be more efficient than ACV in infected ducklings upon short-term oral therapy, while intraperitoneal treatment showed high anti-DHBV activity with both ACV and its SATE pronucleotide in this animal model. These findings demonstrate the potential of SATE pronucleotides of ACV as anti-HBV agents.
Acyclovir↗
Decrease of wild-type and precore mutant duck hepatitis B virus replication during lamivudine treatment in white Pekin ducks infected with the viruses.
BACKGROUND/AIMS: Lamivudine, an antiviral agent, has been used in the treatment of chronic hepatitis B, but little is known about its effect on intrahepatic replication of hepatitis B virus. We investigated the effect of lamivudine on the replication of wild-type and precore mutant duck hepatitis B virus (DHBV) in the liver and serum of DHBV carrier ducks. METHODS: Chronic carrier ducks with either wild-type or precore mutant DHBV were treated for 2 weeks with either low-dose (20 mg/kg, p.o., b.i.d.) or high-dose lamivudine (100 mg/kg, p.o., b.i.d.) or were untreated. Serum levels of DHBV DNA were examined serially by slot-blot hybridization. A second group of chronic carrier ducks was treated for 12 weeks with lamivudine (100 mg/kg, p.o., b.i.d.) or was untreated. The amount of DHBV DNA in serum and its various replicative intermediates in the liver were serially examined by slot-, Southern, and Northern blot methods. RESULTS: In the 2-week treatment study, concentration of DHBV DNA in serum treated with low- and high-dose lamivudine was reduced to 10.8% and 1.1% of the control level in wild-type DHBV carriers, and to 2.3% and 0.48% in precore mutant DHBV carriers, respectively. In the 12-week treatment study, concentration of DHBV DNA in serum at the end of treatment was reduced to <0.65% and <5.36% in wild-type and precore mutant DHBV carriers, respectively. Southern and Northern blot analyses revealed that the various replicative forms of DHBV DNA in the liver were decreased in all treated ducks, but, covalently closed circular DNA and RNA intermediates tended to remain unchanged. CONCLUSIONS: Our results showed that lamivudine could reduce both wild-type and precore mutant DHBV levels in the liver through inhibition of the reverse transcription step, but complete elimination of the viruses from liver is difficult even by relatively long-term lamivudine monotherapy, suggesting a need for some additional therapy to obtain complete clearance.
Animals↗
Antiviral effect of adefovir in combination with a DNA vaccine in the duck hepatitis B virus infection model.
BACKGROUND/AIMS: Combination of antiviral drugs with immunotherapeutic approaches may be a promising approach for the treatment of chronic hepatitis B. We used the duck HBV (DHBV) infection model to evaluate the efficacy of the combination of adefovir with DNA-immunization by comparison with the respective monotherapies. METHODS: Pekin ducks chronically infected with DHBV received adefovir treatment alone or in association with intramuscular immunization with a plasmid (pCI-preS/S) expressing the DHBV large envelope protein. Ducks immunized with pCI-preS/S plasmid alone and two control groups receiving empty plasmid injections or no treatment were followed in parallel. RESULTS: All animals treated with adefovir showed a marked drop in viremia titers during drug administration, followed by a rebound of viral replication after drug withdrawal. Eight weeks after the third DNA boost, the median of viremia within the duck group receiving the combination therapy tended to be lower compared to that of the other groups. In addition, our results suggest a trend to an additive effect of adefovir and DNA vaccine since a 51% decrease in DHBV DNA was observed in autopsy liver samples from combination therapy group, whereas pCI-preS/S or adefovir monotherapies decreased intrahepatic viral DNA by 38 and 14%, respectively. This effect was sustained since it was observed 12 weeks after the end of therapy. CONCLUSIONS: Our results suggest that combination of adefovir with DNA-vaccine may be able to induce a sustained antiviral effect in vivo.
Adenine↗
Mechanisms governing hepadnaviral nucleocapsid assembly.
Reverse transcription of an RNA pregenome is the central step in the replication cycle of the hepatitis B viruses. This reaction takes place within the viral nucleocapsid composed of the core protein, product(s) of the P (pol) gene and the RNA pregenome. As the enzymatic activities required reside in the P-protein it plays a major role in the hepadnaviral life cycle. This article summarizes recent data on structure and function of the hepadnaviral P-protein and discusses its important role in the early steps of nucleocapsid assembly.
Capsid↗
The problem of antiviral therapy for chronic hepadnavirus infections.
Attempts at antiviral therapy of patients with active liver disease as a consequence of chronic hepatitis B virus infection have been moderately successful. The molecular and cellular basis for a successful outcome in these patients is not understood and the same therapies do not appear to benefit carriers that still have fairly normal livers and only a moderate hepatitis as a result of the immune response to the infection. Most carriers fall into this latter classification, at least during the early years of infection, and a therapy that could be successfully applied before extensive liver damage had occurred would presumably reduce the risk of subsequent liver damage and the progression to primary hepatocellular carcinoma. Traditionally, it has been assumed that the primary reason that individuals become chronically infected is that the cytotoxic T-cell response and/or antibody-dependent killing of infected hepatocytes is insufficient to clear the infection. Less attention has been focused on the role of the antibody response in the generation of virus-neutralizing antibodies as the possible major deficiency predisposing some individuals to become carriers. However, carriers normally are antigenemic for HBsAg and virus, and carriers with only antibodies to these structures in their circulation are virtually unknown. In addition, it is usually assumed that the hepatocyte, the major target of infection, does not spontaneously turn over and that, in the absence of an immune response to the infected cell, hepatocellular viability is unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Pathobiology of hepatitis delta virus.
Early observations in humans and infectivity studies in the chimpanzee suggested a direct cytotoxic effect as the major pathogenetic factor in delta hepatitis. Data acquired in liver transplant patients, as well as additional experimental and clinical evidence, have modified this view and indicate instead that liver damage depends on several factors. To explain the array of different clinical presentations, the replication strategy of hepatitis delta virus, the helper function and genetic heterogeneity of the co-infecting Hepadnavirus, and the response of the host immune system should all be taken into consideration.
Animals↗
Gene expression and active virus replication in the liver after injection of duck hepatitis B virus DNA into the peripheral vein of ducklings.
BACKGROUND/AIMS: Duck hepatitis B virus is a member of the hepadnavirus family, which possesses strong hepatotropism. Duck hepatitis B virus DNA serves as a replicative template for producing biologically active virus particles after transfection into cell lines established from human hepatocellular carcinoma or into duck liver by direct injection of calcium phosphate-precipitated DNA. Our aim was to develop a new method of liver-specific gene expression after intravenous DNA delivery. METHODS/RESULTS: We inoculated duck hepatitis B virus DNA with and without cationic liposomes, Lipofectin or LipofectAMINE, as DNA carries. Two weeks after a single intravenous injection of 10 or 50 micrograms of plasmid DNA containing a head-to-tail dimer of duck hepatitis B virus DNA into 25 one-day old ducklings, duck hepatitis B virus RNA transcripts including the pregenome replicative intermediate were detected by Northern blot in the liver of eight ducks (100%) of the Lipofectin group, five ducks (63%) of the LipofectAMINE group, and three ducks (50%) of the group which received DNA without carrier. Duck hepatitis B virus RNA transcription was almost exclusively liver specific, even though the liposomes had no tissue specificity. Replicative forms of duck hepatitis B virus DNA were detected in the liver and DHBsAg was observed in the cytoplasm of the hepatocytes by immunostaining. The serum of transfected ducklings contained virus particles which were infectious in other ducklings. CONCLUSION: The efficient and liver-specific expression of inoculated DNA was due to the amplification of nucleic acids by active virus replication process under the control of hepatocyte specific regulation.
Animals↗
DHBV manipulation and prediction of the outcome of infection.
BACKGROUND/AIMS: The immunological response of ducks to acute infection with duck hepatitis B virus (DHBV) has not been fully characterised. In this study the relationship between viral dose and the outcome of infection in immune competent 26-day-old ducks was examined. METHODS: Indirect ELISA assays were developed to detect the presence of antibody to DHB surface antigen and DHB core antigen. A DHBV serum pool was titrated in 1-day-old and 26-day-old ducklings. RESULTS: The ID50 dose of the ducks injected at 26 days of age was found to be 1000 times that of the ducks injected on day of hatch. The antibody responses and serum DHBV DNA were followed in eight ducks inoculated with DHBV positive serum when 26 days of gene and in three ducks infected with DHBV on day of hatch. The three ducks infected on day of hatch were viraemic by day 7 and remained highly viraemic throughout the experimental period. In the older ducks, inoculation with 1000ID50 resulted in the development of chronic carriage, while inoculation with either 100 or 10ID50 doses resulted in acute infection with or without viraemia. These ducks were able to clear the infection from their circulation, but only 50% cleared DHBV from the liver within the experimental period. All infected ducks developed anti-core activity. Only non-viraemic ducks developed anti-surface activity. CONCLUSION: DHBV infection can be established in immune competent adolescent ducks, with variable disease outcomes comparable to HBV infection in humans.
Acute Disease↗
Progress in DNA vaccine for prophylaxis and therapy of hepatitis B.
Increasing lines of evidence suggest that DNA vaccine is of interest to fight chronic hepatitis B virus (HBV) infection. We used the Pekin duck infected by duck HBV (DHBV), closely related to the human virus, which is an attractive model allowing study of protective and therapeutic effectiveness of DNA vaccines against hepatitis B. Immunisation with a plasmid encoding the DHBV large (L) envelope protein induced a strong, specific, highly neutralising and long-lasting anti-preS humoral response in uninfected ducks. Importantly, maternal antibodies elicited by such DNA immunisation were vertically transmitted and protected progeny against viral challenge. Therapeutic immunisation of chronic DHBV-carrier ducks with this plasmid DNA led to the dramatic and sustained decrease in viral replication and even to clearance of intrahepatic viral covalently close circular DNA (cccDNA) pool in some animals. Our recent combination therapy data showed even a more pronounced antiviral effect of DNA vaccine to DHBV envelope protein when associated with antiviral drug (lamivudine) treatment. Therefore, DNA-based vaccine appears as a promising new approach for prophylaxis and therapy of hepatitis B.
Animals↗
Comparison of the kinetics of the specific cellular immune response to duck hepatitis B virus in infected and immune ducks.
The kinetics of the cell mediated immune response by ducks acutely and chronically infected with, or immune to infection by duck hepatitis B virus (DHBV) was determined. This was measured by an antigen specific blastogenesis assay to duck hepatitis B surface antigen (DHBsAg) and duck hepatitis B core antigen (DHBcAg) using peripheral blood mononuclear cells (PBMC). The three outcomes of acute infection by DHBV were either clearance from both serum and liver, clearance from serum but not liver, or the development of persistent viraemia. Acutely infected ducks that failed to clear the infection also failed to develop a significant cellular immune response to both antigens. Ducks with chronic infection acquired as neonates or as the result of the failure to clear acute infection had an increasing cellular immune response over time. Two groups of immune ducks were examined. These were either ducks that had become immune following infection or that had been vaccinated. Both groups of ducks demonstrated significant cellular responses following challenge with DHBV irrespective of the level of their responses before challenge. However, there was a reduction in the response of their PBMC over a 4-week-period postchallenge. The range of cellular immune responses to DHBV antigens observed in this study has a number of counterparts in hepatitis B infection of humans. Coupled with the defined clinical outcomes that can be established in the duck/DHBV model, further study of the cellular immune response to DHBV is warranted.
Animals↗
Multiple rearrangements and activated expression of c-myc induced by woodchuck hepatitis virus integration in a primary liver tumour.
Woodchuck hepatitis virus (WHV) is a small, partially double-stranded DNA virus. Like the related human hepatitis B virus (HBV), WHV induces acute and chronic hepatitis and hepatocellular carcinoma (HCC) in its natural host. WHV DNA integration into c-myc and N-myc, resulting in deregulated expression of these genes, has been described previously in woodchuck HCC. We have analysed a woodchuck liver tumour in which WHV DNA was integrated in the c-myc gene. The virus insertion provoked multiple alterations in one c-myc allele, probably involving secondary deletions and mutations. Integrated viral DNA, including promotor and enhancer sequences, acted as an insertional mutagen, leading to enhanced expression of heterogenous c-myc transcripts ranging from 7.2 to 14 kb in size, strikingly longer than normal 2.3-kb c-myc RNA. These results provide an additional example in which the oncogenic activation of a myc gene by cis-acting effect of WHV insertion may play a critical role in virus-induced woodchuck HCC.
Animals↗
Perspectives for the treatment of hepatitis B virus infections.
Primarily resulting as a spin-off of the search for effective anti-HSV or anti-HIV agents, several compounds have been identified as effective and promising candidate anti-HBV drugs, i.e. famciclovir (penciclovir), BMS-200475, lamivudine (3TC), (-)FTC, L(-)Fd4C, L-FMAU, DAPD (DXG), bis(POM)-PMEA and bis(POC)-PMPA. They all inhibit HBV replication in Hep G2 2.2.15 at concentrations that are well below the cytotoxicity threshold. All these nucleoside analogues require three phosphorylation steps to be active, in their triphosphate form, as inhibitors of the HBV DNA polymerase, except for PMEA (adefovir) and PMPA (tenofovir), which need only two phosphorylation steps, to PMEApp and PMPApp, respectively, to interact as chain terminators with the HBV DNA polymerase reaction. Several of these compounds (for example, famciclovir, lamivudine and adefovir) have proven to be efficacious in the duck and/or woodchuck hepatitis models, and, accordingly, famciclovir, lamivudine and adefovir have also proven to be effective (i.e. in reducing HBV DNA levels) in patients with chronic HBV infection. Yet, famciclovir and lamivudine may lead to the emergence of resistance mutations (i.e. L528M and M552V/I) in the HBV DNA polymerase upon long-term treatment. These penciclovir- and lamivudine-resistant HBV mutants still retain susceptibility to adefovir, which, in turn, has so far not been found to engender resistance mutations in HBV. As has become obvious from the experience with the treatment of HIV infections, future HBV chemotherapy may reside in combination drug therapy so as to achieve the highest possible virus reduction, thereby minimizing the likelihood of drug resistance development.
Acute Disease↗
Why are hepadnaviruses DNA and not RNA viruses?
Virion assembly in hepadnaviruses is a two-step process leading to (1) the packaging of viral pregenomic RNA and reverse transcriptase into nucleocapsids and (2) the assembly of nucleocapsids with envelope components, which results in the formation of mature virus particles. Characteristically, both steps are intimately coupled to viral DNA synthesis. While assembly of nucleocapsids is coupled to the protein priming of reverse transcription, virion formation is linked to genome maturation.
DNA, Viral↗
DNA-designed avian IgY antibodies: novel tools for research, diagnostics and therapy.
We have recently demonstrated, using the duck Hepatitis B virus (DHBV) model, closely related to human HBV, that following DNA immunization of breeding ducks with a plasmid encoding the targeted protein, specific and biologically active IgY (egg yolk immunoglobulines) are vertically transmitted from their serum into the egg yolk from which they can be extracted and purified. Thus an egg can be considered as a small "factory" for antibody production, since about 60-100 mg of purified IgY can be obtained from each egg yolk of a DNA-immunized duck. One of the major advantages of this new method of "DNA-designed" IgY antibodies is their production via immunization with a gene vector that expresses a corresponding antibody in situ in the cells of an avian host. Therefore this approach allows direct generation of antibodies from plasmid DNA and avoids the costly and tedious preparation of purified antigens required for conventional antibody production. In addition, duck IgY are of remarkable high affinity, avidity and are highly neutralizing. Moreover, the epitope pattern of IgY generated by DNA immunization of ducks is closely related to that observed in viral infection. Such duck IgY are also of particular value as immunodiagnostic tools, since they do not cross-react serologically with mammalian immunoglobulins and complement. Because IgY are resistant to the gastric barrier, the recently described DNA-designed IgY specific to H. pylori Urease B can be of particular interest for passive immunotherapy of gastrointestinal tract infections. Another interesting application is the recent generation in our laboratory of DNA-designed IgY antibodies specific to HBsAg mutants. These antibodies are currently being used to design new diagnostic assay for detection of HBV mutants that are undetectable by actual tests. Moreover, this approach allowing a quick and inexpensive production of a new generation of antibodies will provide pertinent tools to link the fields of genomics and protcomics.
Animals↗
Identification of hepatitis B virus genome in faecal sample from wild living chimpanzee (Pan troglodytes troglodytes) in Gabon.
Non-invasive faecal sampling in the equatorial forest in Gabon allowed the first identification of the hepatitis B virus (HBV-Ch(RC170)) genome in samples collected from wild chimpanzees (Pan troglodytes troglodytes). The HBV-Ch(RCl70)sequence clustered with 100% bootstrap support with previous viral sequences obtained from Pan troglodytes subspecies. This is the first evidence of HBV infection in wild apes and confirms that the HBV-like strains thus far characterized in captive apes are directly related to those circulating in the wild.
Animals↗