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1.
Chinese Journal of Virology ; (6): 382-386, 2014.
Article in Chinese | WPRIM | ID: wpr-280356

ABSTRACT

Rolling circle amplification (RCA) is a newly developed experimental technique that can specific ally amplify circular DNA. Since 2008, RCA has been extensively used in hepatitis B virus (HBV) research, such as the amplification of the full-length sequence of the HBV genome, and the analysis of the drug-resistant mutations of HBV covalently closed circular DNA (cccDNA), amongst others. To create an easy assay for the analysis of duck hepatitis B virus (DHBV) cccDNA, this study established an RCA-based method. DHBV cccDNA was amplified from the DHBV DNA samples of duck liver with four pairs of sulfur-modified primers, which were designed according to the highly conserved sequence of DHBV using sera DHBV DNA as the negative control. DHBV cccDNA was detected in the obtained RCA products by the sequencing of RCA amplicons that were amplified with primer pairs on both sides of the gap of DH BV relaxed circular DNA, rather than by digesting RCA products with a restriction enzyme. The liver and sera DHBV DNA samples of 39 ducks infected with DHBV were examined with the RCA-based DHBV cccDNA detection method, and the results showed that while DHBV cccDNA was detected from all 39 liver DHBV DNA samples, no DHBV cccDNA was found in any of the sera DHBV DNA samples. These results suggest that the method established in the study is highly specific and sensitive for the detection of DHBV cccDNA. The establishment of this RCA-based DHBV method for cccDNA detection lays the groundwork for using a DHBV model to study the role of cccDNA in the pathogenesis of hepatitis B and to evaluate the effect of anti-virus therapies.


Subject(s)
Animals , DNA Primers , Genetics , DNA, Circular , Genetics , DNA, Viral , Genetics , Ducks , Hepadnaviridae Infections , Virology , Hepatitis B Virus, Duck , Genetics , Liver , Virology , Polymerase Chain Reaction , Methods , Poultry Diseases , Virology
2.
Chinese Journal of Virology ; (6): 180-184, 2013.
Article in Chinese | WPRIM | ID: wpr-339955

ABSTRACT

Brown ducks carrying DHBV were widely used as hepatitis B animal model in the research of the activity and toxicity of anti-HBV dugs. Studies showed that the ratio of DHBV carriers in the brown ducks in Guilin region was relatively high. Nevertheless, the characters of the DHBV genome of Guilin brown duck remain unknown. Here we report the cloning of the genome of Guilin brown duck DHBV and the sequence analysis of the genome. The full length of the DHBV genome of Guilin brown duck was 3 027bp. Analysis using ORF finder found that there was an ORF for an unknown peptide other than S-ORF, PORF and C-ORF in the genome of the DHBV. Vector NTI 8. 0 analysis revealed that the unknown peptide contained a motif which binded to HLA * 0201. Aligning with the DHBV sequences from different countries and regions indicated that there were no obvious differences of regional distribution among the sequences. A fluorescence quantitative PCR for detecting DHBV was establishment based on the recombinant plasmid pGEM-DHBV-S constructed. This study laid the groundwork for using Guilin brown duck as a hepatitis B animal model.


Subject(s)
Animals , Base Sequence , China , Epidemiology , Cloning, Molecular , Ducks , Genome, Viral , Hepadnaviridae Infections , Diagnosis , Virology , Hepatitis B Virus, Duck , Classification , Genetics , Molecular Sequence Data , Phylogeny , Polymerase Chain Reaction , Methods , Poultry Diseases , Diagnosis , Virology
3.
Chinese Journal of Virology ; (6): 681-688, 2012.
Article in Chinese | WPRIM | ID: wpr-339985

ABSTRACT

Duck hepatitis B virus (DHBV) belongs to the Avihepadnavirus genus of the Hepadnaviridae, and it not only has the same replication pattern, but also has the similar genomic and antigenic structures to Hepatitis B virus (HBV). The genome of DHBV is a partially double-stranded closed circular DNA. The genome consists of three distinct open reading frames (ORFs): ORF-PreS/S, ORF-PreC/C and ORF-P, which all locate on the negative DNA strand and encode four separate proteins. The ORF-PreS/S encodes envelope proteins L and S, and the ORF-PreC/C and ORF-P encode capsid proteins C and polymerase proteins P, respectively. The characteristics of genome structure,viral proteins features and functions were described in this review in order to provide useful information for the further study of DHBV and the duck model infected by DHBV.


Subject(s)
Animals , Ducks , Hepadnaviridae Infections , Virology , Hepatitis B Virus, Duck , Chemistry , Genetics , Hepatitis, Viral, Animal , Virology , Open Reading Frames , Protein Structure, Tertiary , Viral Proteins , Chemistry , Genetics
4.
Article in English | WPRIM | ID: wpr-274337

ABSTRACT

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.


Subject(s)
Animals , Antigens, Surface , Blood , Antiviral Agents , Pharmacology , DNA, Viral , Blood , Drugs, Chinese Herbal , Pharmacology , Hepadnaviridae Infections , Drug Therapy , Virology , Hepatitis B Virus, Duck , Allergy and Immunology , Hepatitis, Viral, Animal , Drug Therapy , Virology , Liver , Metabolism , Pathology , Liver Function Tests , Saponins , Pharmacology , Virus Replication
5.
Article in Chinese | WPRIM | ID: wpr-332586

ABSTRACT

<p><b>OBJECTIVE</b>To construct a lamivudine-resistant plasmid containing 1.2 unit genome of duck hepatitis B virus and identify its replication and drug-resistance in avian LMH hepatica cells.</p><p><b>METHODS</b>The recombinant plasmid PBS-DHBV1.2 was constructed using the 1.2-genome length DHBV DNA sequence from a dimer DHBV genome with pcDNA3.1 as the template. With site-directed mutagenesis, we obtained PBS-DHBV1.2-M512V plasmids with polymerase gene mutation from PBS-DHBV1.2. Two constructed plasmids were transiently transfected into LMH cells using FuGENETM6 transfection reagent and cultured in the medium containing different concentrations of lamivudine. Southern blot hybridization was performed to detect DHBV replication intermediates.</p><p><b>RESULTS</b>PCR amplification, restriction digestion and plasmid sequencing all confirmed successful construction of PBS-DHBV1.2-M512V recombinant plasmid. Southern blot analysis identified the presence of all the expected DHBV replication intermediates in LMH cells. The replication capacity of the mutant plasmid was decreased by 2.7 times compared with that of the wild plasmid. The IC(50) of lamivudine was 37.12∓8.81 ng/ml for the mutant, greater than that of the wild plasmid (10.90∓4.80 ng/ml).</p><p><b>CONCLUSION</b>Compared with the wild plasmid, the mutant plasmid has a lower replication capacity and sensitivity to lamivudine in vitro.</p>


Subject(s)
Antiviral Agents , Pharmacology , Drug Resistance, Viral , Genetics , Hepatitis B Virus, Duck , Genetics , Lamivudine , Pharmacology , Mutagenesis, Site-Directed , Plasmids
6.
Acta Pharmaceutica Sinica ; (12): 1440-1444, 2009.
Article in Chinese | WPRIM | ID: wpr-344057

ABSTRACT

The aim of this study is to investigate the effect of hyperin on the cccDNA of duck hepatitis B virus and its immunological regulation. Duck hepatitis B virus (DHBV) infection model and normal mouse spleen lymphocyte were used to evaluate the anti-HBV and immunoregulation effects. The DHBV-DNA of serum was detected at different time points by using serum DOT-BLOT hybridization. Polymerase chain reaction (PCR) was used for the determination of nuclear covalent closed circular DNA (cccDNA). Cytokine secretion was determined by ELISA method. DHBV-DNA were inhibited by hyperin (25 or 50 mg x kg(-1)), while cccDNA of liver could be eliminated efficiently by hyperin (25 or 50 mg x kg(-1), P < 0.05, P < 0.01). The T helper 1 effector cytokine was markedly enhanced by hyperin (25 or 50 microg x mL(-1), P < 0.01). In conclusion, hyperin has anti-HBV activity via multiple targets and pathways, and cccDNA may be one of the important targets.


Subject(s)
Animals , Antiviral Agents , Pharmacology , DNA, Circular , Metabolism , DNA, Viral , Metabolism , Hepadnaviridae Infections , Virology , Hepatitis B Virus, Duck , Genetics , Hepatitis, Viral, Animal , Virology , Interferon-gamma , Bodily Secretions , Interleukin-12 , Bodily Secretions , Liver , Virology , Lymphocytes , Bodily Secretions , Mice , Quercetin , Pharmacology , Spleen , Pathology , Virology
7.
Article in Chinese | WPRIM | ID: wpr-265340

ABSTRACT

<p><b>OBJECTIVE</b>We have evaluated the direct effect of ampelopsis (APS) on duck hepatitis B virus (DHBV) replication in ducklings in vivo.</p><p><b>METHOD</b>One-day-old ducklings were infected with DHBV. After infection for 7 days, the animals were treated with APS at dosages of 70, 150, 300 mg x kg(-1) of body weight via the oral route. The drug was given twice per day for 10 days continuously, and normal saline was used as control. The blood was drawn from the posterior tibial vein of all ducks before treatment (T0), after the medication for 5 (T5), 10 (T10) days and withdrawal of the drug for 3 days (P3). DHBV DNA in duck serum was detected by dot blot.</p><p><b>RESULT</b>The duck serum DHBV-DNA levels were reduced in the group of APS (150, 300 mg x kg(-1)) after treated for 5 and 10 days and the levels of DHBV-DNA did not markedly relapse in both groups of APS after withdrawal of the drug for 3 days. We provide the first evidence that APS can efficiently inhibits DHBV replication in ducks in vivo.</p><p><b>CONCLUSION</b>APS therefore warrants further investigation as a potential therapeutic agent for HBV infections.</p>


Subject(s)
Ampelopsis , Chemistry , Animals , Antiviral Agents , Pharmacology , DNA, Viral , Metabolism , Drugs, Chinese Herbal , Pharmacology , Ducks , Blood , Virology , Hepatitis B Virus, Duck , Metabolism , Physiology , Virus Replication
8.
Chinese Journal of Virology ; (6): 296-302, 2009.
Article in Chinese | WPRIM | ID: wpr-297959

ABSTRACT

Previously, we have established an epsilon library and selected out a series of RNA aptamers with higher affinity to P protein based on the in vitro Systematic Evolution of Ligands by Exponential Enrichment (SELEX) in duck hepatitis B virus (DHBV) system. In order to study the structural elements within the epsilon that is essential for initiating priming of HBV reverse transcriptase (P protein), all selected aptamers were subjected to in vitro priming assay and RNA secondary structure probing. We found that all those aptamers supporting priming had an undamaged bulge, while those lacking of the bulge no more support priming. Our results suggest an undamaged bulge within Depsilon is indispensable for initiating priming of P protein.


Subject(s)
Base Sequence , Hepatitis B Virus, Duck , Chemistry , Genetics , Molecular Sequence Data , Nucleic Acid Conformation , RNA, Viral , Chemistry , Genetics , RNA-Directed DNA Polymerase , Genetics , Metabolism , Reverse Transcription , Sequence Alignment , Viral Proteins , Genetics , Metabolism
9.
Article in Chinese | WPRIM | ID: wpr-254127

ABSTRACT

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>


Subject(s)
Animals , Animals, Newborn , DNA, Viral , Genetics , Metabolism , Disease Models, Animal , Ducks , Hepadnaviridae Infections , Blood , Drug Therapy , Virology , Hepatitis B Virus, Duck , Genetics , Hepatitis, Viral, Animal , Blood , Drug Therapy , Virology , Lamivudine , Pharmacology , Liver , Pathology , Virology , Reverse Transcriptase Inhibitors , Pharmacology , Viremia , Blood
10.
Afro-Arab Liver Journal. 2007; 6 (1-2): 49-57
in English | IMEMR | ID: emr-81611

ABSTRACT

Entry and intracellular transport of hepatitis B viruses have several unusual, largely unknown aspects. In this study, we explored the mode of virus entry using the duck hepatitis B virus [DHBV] and the primary hepatocyte infection model. Upon internalization, viral particles were enriched in an endosomal compartment, as revealed by biochemical and ultrastructural analysis. Virus-containing vesicles harbored early endosome markers. Kinetic analysis revealed time-dependent partial translocation of viral DNA from endosomes into the cytosol. This was strongly reduced by inhibition of vacuolar ATPase; [vATPase] activity with bafilomycin A1 and resulted in abortive infection and prevention of cccDNA formation. Inactivation of vATPase induced accumulation and stabilization of incoming viral particles in endosomes, presumably by blocking endosomal carrier vesicle-mediated cargo transport and sorting. Although neutralization of the endomembrane organelles alone led to stabilization of incoming viral particles, it did not inhibit virus infection. In line with this, a pH-dependent ectopic virus fusion at the plasma membrane could not be artificially induced. This provided further evidence for a pH-neutral translocation mechanism. Endosomal membrane potential was required for viral infection because cotreatment of cells with monensin partially overcame the inhibitory effect of bafilomycin A1. In conclusion, hepatitis B viral infection is mediated by a novel cellular entry mechanism with features different from that of all other known viruses


Subject(s)
Hepatocytes , Hepatitis B Virus, Duck , Endosomes , Cytosol , Hydrogen-Ion Concentration , Hepadnaviridae Infections , Membrane Proteins , Hepadnaviridae
12.
Article in Chinese | WPRIM | ID: wpr-269090

ABSTRACT

<p><b>OBJECTIVE</b>To study the viral inhibitory effect of Shenling Yigan Granule (SYG) on duck hepatitis B virus (DHBV) in vivo.</p><p><b>METHODS</b>Chongqing ducks infected with DHBV were used. They were randomly divided into five groups, the small-, medium- and high-dose (1.6 g/kg, 3.2 g/kg, 6.4 g g/kg) SYG groups, the lamivudine positive control group, and the model group. The changes of serum DHBV-DNA, DHB-sAg contents and hepatic pathology were observed.</p><p><b>RESULTS</b>The serum content of DHBV-DNA in the three SYG groups and the positive control group was significantly decreased (P < 0.05), while it was rebounded in the latter at day 7 after stopped lamivudine administration. The change of DHBsAg level was insignificantly in all groups. And the hepatic pathological change in the SYG groups and positive control group was slighter than that in the model control group, but showed insignificant difference in comparison between the SYG groups and the model group (P > 0.05).</p><p><b>CONCLUSION</b>SYG has certain in vivo inhibitory effects on DHBV-DNA.</p>


Subject(s)
Animals , Antiviral Agents , Pharmacology , Therapeutic Uses , Drugs, Chinese Herbal , Pharmacology , Therapeutic Uses , Ducks , Hepadnaviridae Infections , Drug Therapy , Hepatitis B Virus, Duck , Hepatitis, Viral, Animal , Drug Therapy , Phytotherapy , Random Allocation
13.
Chinese Journal of Hepatology ; (12): 738-741, 2006.
Article in Chinese | WPRIM | ID: wpr-260611

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effect of apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G) mediated antiviral activity against hepatitis B virus (HBV) and duck hepatitis B virus (DHBV).</p><p><b>METHODS</b>Total RNA was extracted from peripheral blood mononuclear cells (PBMCs), RT-PCR product was cloned into the EcoR I/Hind III restriction sites of the CMV-driven expression vector fused with a hemagglutinin fusion epitope tag at its carboxyl terminal. Replication competent 1.3 fold over-length HBV was constructed with full-length HBV of ayw subtype. The mammalian hepatoma cell HepG2 was cotransfected with the replication competent 1.3 fold over-length HBV and various amounts of CMV-driven expression vector encoding APOBEC3G-HA. Levels of HBsAg and HBeAg in the media of the transfected cells were determined by ELISA, HBV DNA. RNA from intracellular core particles was examined using Northern and Southern blot analyses. Chicken hepatoma cell LMH was cotransfected with head-to-tail dimer of an EcoR I monomer of DHBV and various amounts of CMV-driven expression vector encoding APOBEC3G-HA. DHBV DNA from intracellular core particles was examined using Southern blot analysis.</p><p><b>RESULTS</b>CMV-driven expression vector encoding APOBEC3G-HA and replication competent 1.3 fold over-length HBV were constructed. There was a dose dependent decrease in the levels of intracellular core-associated viral (HBV and DHBV) DNA and extracellular production of HBsAg and HBeAg. Levels of intracellular core-associated viral RNA were also decreased, but the expression of HBcAg remained almost unchanged.</p><p><b>CONCLUSION</b>APOBEC3G suppresses HBV and DHBV replication and also suppresses HBsAg and HBeAg expression.</p>


Subject(s)
APOBEC-3G Deaminase , Cytidine Deaminase , Genetics , Hep G2 Cells , Hepatitis B Surface Antigens , Metabolism , Hepatitis B Virus, Duck , Physiology , Hepatitis B e Antigens , Metabolism , Hepatitis B virus , Physiology , Humans , RNA, Messenger , Genetics , Virus Replication
14.
Article in Chinese | WPRIM | ID: wpr-353235

ABSTRACT

<p><b>OBJECTIVE</b>To study the inhibitory effect of binding peptides on duck hepatitis B virus (DHBV) replication in duck hepatocytes.</p><p><b>METHODS</b>Specific binding peptides to duck hepatitis B virus polymerase (DHBVP) were screened by phage display technology (PDT), then were sequenced and synthesized. Binding peptides were added into primary culture of duck hepatocytes infected with DHBV in vitro. DHBV-DNA in the cytoplasm, cell nucleus and medium supernatant was assayed over time.</p><p><b>RESULTS</b>Seven binding peptides were obtained after 3-round screening by PDT. Duck primary hepatocytes infected by DHBV were treated with above obtained binding peptides. The DHBV-DNA levels in medium supernatant and cytoplasm of duck hepatocytes treated with synthesized peptides (the 3rd and the 6th peptide) were significantly lower than those of control cells (P<0.05).</p><p><b>CONCLUSION</b>Specific binding peptides to DHBVP could inhibit the replication of DHBV.</p>


Subject(s)
Animals , Cells, Cultured , Ducks , Hepadnaviridae Infections , Virology , Hepatitis B Virus, Duck , Genetics , Hepatitis, Viral, Animal , Virology , Hepatocytes , Virology , Peptide Nucleic Acids , Pharmacology , RNA-Directed DNA Polymerase , Metabolism , Virus Replication
15.
Article in Chinese | WPRIM | ID: wpr-353234

ABSTRACT

<p><b>OBJECTIVE</b>To explore the inhibitory effect of combination of lamivudine with thymosin alpha1 (Talpha1) on the replication of duck hepatitis B virus (DHBV).</p><p><b>METHODS</b>Peking ducks of 1 d old were challenged with DHBV-positive serum and used as a duck hepatitis B model. After treated with lamivudine for three months, the ducks were randomly grouped and treated with or without Talpha1 for 8 d. Serum DHBV titrate was observed by semi-quantitative PCR, and inflammation and degeneration of hepatocytes were observed by pathology examination.</p><p><b>RESULTS</b>The serum DHBV titrate was significantly reduced (4483.2+/-5193.4 compared with 9351.8+/-5059.6) after lamivudine treatment, and it was reduced more significantly(1692.2+/-589.2) after combination treatment with Talpha1. Lamivudine reduced the degeneration degree of hepatocytes (3.2+/-0.8 compared with 4.6+/-0.5) and the inflammation degree of liver (6.2+/-3.3 compared with 8.6+/-2.8). The combination treatment with Talpha1 increased liver inflammation degree (9.0+/-5.2).</p><p><b>CONCLUSION</b>Both Talpha1 and lamivudine may reduce the replication of DHBV in Peking ducks and combination treatment may have the better anti-virus effect and enhance immune response in liver.</p>


Subject(s)
Animals , Animals, Newborn , Antiviral Agents , Therapeutic Uses , Cells, Cultured , Drug Therapy, Combination , Ducks , Hepadnaviridae Infections , Drug Therapy , Hepatitis B Virus, Duck , Genetics , Hepatitis, Viral, Animal , Drug Therapy , Hepatocytes , Virology , Lamivudine , Therapeutic Uses , Thymosin , Therapeutic Uses , Virus Replication
16.
Article in Chinese | WPRIM | ID: wpr-282360

ABSTRACT

<p><b>OBJECTIVE</b>To discuss the possibility of hepatitis B virus (HBV) transmission through dental handpieces.</p><p><b>METHODS</b>Investigation was carried on methods for disinfecting and sterilizing dental handpieces and the condition of HBsAg contamination on dental handpieces before and after disinfection and sterilization by randomly sampling all special stomatological hospitals and dental clinics in a same city and 10 dental departments from the third, second and first class hospitals. The possibility of HBV transmission through dental handpieces was probed by investigating whether ducks can be infected by bath liquid of dental handpieces contaminated by DHBV, while in such bath liquid, DHBV can not be detected by serum dot hybridization.</p><p><b>RESULTS</b>From 2001 to 2004, in methods to disposing dental handpieces, the use of autoclave was remarkably increased while of the disinfectant wipe, immersion and other methods was remarkably decreased. The positive rate of HBsAg from dental handpieces in practice was 1.65%. It was evident that the bath liquid of dental handpieces contaminated by DHBV can conduct infection in vivo test of duck, while DHBV can not be detected in such bath liquid by serum dot hybridization, it is proved that the negative result of HBsAg in non-sterilized dental handpieces can not eliminate the possibility of HBV transmission through dental handpieces.</p><p><b>CONCLUSION</b>There might exist the possibility of HBV transmission through dental handpieces however, the autoclaves might kill the virus contaminating on dental handpieces.</p>


Subject(s)
Animals , DNA, Viral , Blood , Dental Instruments , Virology , Ducks , Virology , Equipment Contamination , Hepatitis B , Hepatitis B Virus, Duck , Genetics , Sterilization , Methods , Reference Standards
17.
Chinese Journal of Hepatology ; (12): 341-343, 2003.
Article in Chinese | WPRIM | ID: wpr-305948

ABSTRACT

<p><b>OBJECTIVE</b>To clone and analyze duck hepatitis B virus genome from Chongqing brown duck.</p><p><b>METHODS</b>Duck hepatitis B virus (DHBV) DNA extracted from a Chongqing brown duck was amplified by PCR and cloned into PGEM-T vector using T-A clone method. The sequence of this DHBV genome was analyzed with some softwares after identified.</p><p><b>RESULTS</b>The duck hepatitis B virus genome from Chongqing brown duck (DHBVcq), which was 3 024 nucleotides long, contained three ORFs whose onset and end nucleotides were in accord with those of HPUGA, encoding P, PreC/C and PreS/S protein respectively. Comparison of this strain with other DHBV reported in GenBank showed that the homology of DHBVcq and M32990 got the highest score of 94.9% at nucleotide level, while DHBVcq and DHBVCG got the least (89.8%). Most of the conserved regulation nucleotides and amino acids sequence found in other DHBV were also identified in DHBVcq. The epsilon region of DHBVcq, which was important for encapsidation of pgRNA and synthesis of minus-strand DNA, differed from that of most other DHBV strains, forming a stem-loop conformation with a three- nucleotides upper stem rather than a common nine-nucleotides one in free status.</p><p><b>CONCLUSION</b>The successful clone and analysis of DHBVcq provide further studies with helpful information.</p>


Subject(s)
Animals , Cloning, Molecular , DNA, Viral , Chemistry , Genetics , Ducks , Hepatitis B Virus, Duck , Classification , Genetics , Hepatitis Virus, Duck , Genetics , Open Reading Frames , Polymerase Chain Reaction , Sequence Analysis, DNA , Sequence Homology
18.
Article in Chinese | WPRIM | ID: wpr-281863

ABSTRACT

<p><b>BACKGROUND</b>To determine the feasibility of inhibition of duck hepatitis B virus (DHBV) DNA replication by antisense phosphorothioate oligodeoxynucleotides corresponding to DHBV transcription region.</p><p><b>METHODS</b>The authors designed three antisense phosphorothioate oligodeoxynucleotides which correspond to DHBV PreS1,PreS2 and S antigen gene promotors respectively. The DNA replication level was detected with Southern blot method and cpm calculation.</p><p><b>RESULTS</b>Primary duck hepatocyte culture was treated with 1.5 micromol/L antisense oligodeoxynucleotides in vitro, all the antisense fragments caused a firm inhibition of viral DNA replication and the inhibition rates were 61.5%, 69.3% and 62.4%, respectively. In vivo, the animals were treated with 10 microgram/g PreS1 antigen gene promotor antisense oligodeoxynucleotides per day for 6 days and a very strong inhibition rate of 87.9% was obtained.</p><p><b>CONCLUSIONS</b>The results demonstrated the potential clinical application of antisense phosphorothioate oligodeoxynucleotides in clinics.</p>


Subject(s)
Animals , DNA Replication , DNA, Viral , Ducks , Hepadnaviridae Infections , Virology , Hepatitis B Surface Antigens , Blood , Hepatitis B Virus, Duck , Genetics , Physiology , Hepatitis, Viral, Animal , Virology , Oligodeoxyribonucleotides, Antisense , Pharmacology , Protein Precursors , Blood , Virus Replication
19.
Article in English | IMSEAR | ID: sea-125271

ABSTRACT

Duck hepatitis B virus (DHBV) infected carrier ducks serve as a useful model for testing anti-hepadnavirus drugs. This needs a well characterised duck hepatitis B virus strain. We cloned and sequenced the complete genome of duck hepatitis B virus strain of Indian origin. It was 3021 nucleotides in length and had all the recognisable open reading frames (Polymerase: 20-2530 nucletides, Surface: 693-1787 nucleotides and Core: 2518-412). Using an inoculum of 50 microliters serum containing 1 x 10(11) virus particles/ml, we could infect 80% of one day old ducklings and develop a duck colony.


Subject(s)
Animals , Antiviral Agents/pharmacology , Base Sequence , Ducks , Genome, Viral , Hepadnaviridae Infections/drug therapy , Hepatitis B Virus, Duck/drug effects , Molecular Sequence Data
20.
Indian J Pathol Microbiol ; 1994 Jan; 37(1): 53-8
Article in English | IMSEAR | ID: sea-73282

ABSTRACT

A DNA molecular hybridization technique employing Duck Hepatitis B Virus (DHBV) DNA of 3.0 kilobase pairs as a probe was used to screen for the presence of DHBV DNA in blood samples, collected from 90 apparently healthy Indian country ducks. Six out of 90 ducks showed positivity for DHBV DNA in serum (5.4%) and only 4 out of 6 DHBV DNA positive ducks answered in Counter Immuno Electrophoresis (CIEP) using specific antibody against DHBV surface antigen raised in Guinea pig. The results indicate the pilot observation that (a) DHBV carrier status exists to a tune of 5.4% among apparently healthy Indian country ducks also and (b) DHBV probe can be employed as a sensitive and reliable assay for DHBV DNA detection in DHBV infected ducks.


Subject(s)
Animals , DNA, Viral/blood , Ducks/microbiology , Hepatitis B Virus, Duck/genetics , India , Nucleic Acid Hybridization
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