Primary Duck Hepatocyte Culture and Duck Hepatitis B Virus Infection Model.

Duck hepatitis B virus (DHBV) is an avian member of the hepatotropic DNA viruses, or hepadnaviridae. It shares with the human hepatitis B virus (HBV) a similar genomic organization and replication strategy via reverse transcription, but is simpler than HBV in lacking the X gene and in expressing just two coterminal envelope proteins: Large (L) and small (S). DHBV has been extensively used as a convenient and valuable animal model for study of the hepadnaviral life cycle, and for drug screening in vitro but also in vivo. Ducks and primary duck hepatocytes (PDHs) are inexpensive, easily accessible, and readily infected with DHBV. The high levels of genome replication and protein expression in duck liver and PDHs also facilitate monitoring of viral life cycle using conventional molecular biology techniques such as Southern blot for replicative DNA and covalently closed circular DNA (cccDNA), Northern blot for viral RNAs, and Western blot for viral proteins.

Discovery and Genomic Characterization of a Novel Hepadnavirus from Asymptomatic Anadromous Alewife (Alosa pseudoharengus).

The alewife (Alosa pseudoharengus) is an anadromous herring that inhabits waters of northeastern North America. This prey species is a critical forage for piscivorous birds, mammals, and fishes in estuarine and oceanic ecosystems. During a discovery project tailored to identify potentially emerging pathogens of this species, we obtained the full genome of a novel hepadnavirus (ApHBV) from clinically normal alewives collected from the Maurice River, Great Egg Harbor River, and Delaware River in New Jersey, USA during 2015-2018. This previously undescribed hepadnavirus contained a circular DNA genome of 3146 nucleotides. Phylogenetic analysis of the polymerase protein placed this virus in the clade of metahepadnaviruses (family: Hepadnaviridae; genus: Metahepadnavirus). There was no evidence of pathology in the internal organs of infected fish and virions were not observed in liver tissues by electron microscopy. We developed a Taqman-based quantitative (qPCR) assay and screened 182 individuals collected between 2015 and 2018 and detected additional qPCR positives (n = 6). An additional complete genome was obtained in 2018 and it has 99.4% genome nucleotide identity to the first virus. Single-nucleotide polymorphisms were observed between the two genomes, including 7/9 and 12/8 synonymous vs nonsynonymous mutations across the polymerase and surface proteins, respectively. While there was no evidence that this virus was associated with disease in this species, alewives are migratory interjurisdictional fishes of management concern. Identification of microbial agents using de novo sequencing and other advanced technologies is a critical aspect of understanding disease ecology for informed population management.

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.

Adoption of Euvax-B vaccine in Nineveh

Hepatitis B infection is one of the most important health problems around the world. The high mortality rate of the hepatitis B encouraged research that led to the finding of an effective vaccine against it. The aim of the present study was to find out the use of the Euvax-B vaccine in sectors of Nineveh province. According to the results obtained in this study, in the next five years, the vaccination coverage for the second and third doses needs to improve(AU)

La infección por hepatitis B es uno de los más importantes problemas de salud del mundo. La alta tasa de mortalidad de la hepatitis B impulsó las investigaciones que llevaron a encontrar una vacuna eficaz contra la misma. El objetivo del presente estudio fue conocer el uso de la vacuna Euvax-B en sectores de la provincia de Nínive. De acuerdo con los resultados obtenidos, en los próximos cinco años, se debe incrementar la cobertura de inmunización de la segunda y tercera dosis de la vacuna(AU)

Establishment of a method to detect duck hepatitis B virus covalently closed circular DNA based on rolling circle amplification / 病毒学报

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.

Cloning and sequence analysis of the DHBV genome of the brown ducks in Guilin region and establishment of the quantitative method for detecting DHBV / 病毒学报

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.

Research on the gene structure of duck hepatitis B virus and its encoding proteins / 病毒学报

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.

Inhibitory effect of total saponins isolated from Taraphochlamys affinis on duck hepatitis B virus replication / 中国中药杂志

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.

Effects of hyperin on the cccDNA of duck hepatitis B virus and its immunological regulation / 药学学报

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.

Establishment of an in vivo model for duck hepatitis B virus infection using Hubei duckling / 中华实验和临床病毒学杂志

<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>

Experimental study on effect of Shenling Yigan Granule in antagonizing duck hepatitis B virus / 中国中西医结合杂志

<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>

Cáncer de hígado y virus de las hepatitis B y C / Liver cancer and hepatitis B and C virus

Uno de los tumores hepáticos mas frecuentes, y de mayor malignidad es el carcinoma hepatocelular (CHC), el mismo que se encuentra estrechamente relacionado con el virus de la hepatitis B y C. Según estudios realizados, se ha demostrado que hay mayor incidencia de CHC en los portadores crónicos del HBsAg, aunque el mecanismo por el cual el virus de la hepatitis B puede desarrollar el CHC no está totalmente esclarecido; no obstante existe una relación directa con los procesos necroinflamatorios crónicos que el virus puede inducir. Es importante señalar también la incidencia del CHC por VHC ya que según estudios realizados utilizando sistemas ELISA, se demostró la presencia y replicación del virus en tejido hepático tumoral, confirmando de esta forma la asociación causal entre la infección por el VHC y CHC.En ensayos realizados en Taiwan e Italia, se observó que en pacientes en los que se detectaron simultáneamente ambos marcadores virales (HBsAg y el antiVHC), el riesgo de desarrollar CHC fue mayor, en comparación a aquellos que presentaron en forma aislada, la infección por uno de los 2 virus.Los mecanismos por los cuales la presencia de los virus de las hepatitis B y C promueven el desarrollo del CHC, no esta esclarecido totalmente; pero en la revisión de la literatura se plantean hipótesis importantes en cuanto al efecto oncogénico directo que pueden tener, así como los mecanismos indirectos que actúan por un incremento en la tasa de recambio de hepatocitos o por la producción de radicales libres.Estas son las razones por las cuales la Agencia Internacional para la investigación sobre el cáncer de la OMS se ha pronunciado sobre considerarlos, en especial al VHC, como agente carcinógeno en humanos.

One of the most common liver tumors, and of greatest malignancy, is the hepatocellular carcinoma (HCC), which is closely linked to Hepatitis B and C viruses.According to some studies, it has been demonstrated that the incidence of HC is highest among chronic carriers of HBAgs, although the mechanism by which the virus can lead to HCC is not well identified, thus there is a direct relationship among the chronic necroinflammatory processes that this virus can induce.It is also important to mention the incidence of HCC due to HCV, because there is studies in which the presence and replication of the virus in a normal liver tissue has been demonstrated by Elisa procedures, therefore confirming the causative association among virus infection and HCC.In some trials conducted in Taiwan and Italy, it was observed that the patients in which both viral markers (HBAgs and antiHCV) were detected simmoultaneously, the risk of developing HCC was higher, in comparison to those who presented isolated infection by one of the 2 viruses.The mechanisms by which the presence of both viruses, hepatitis B and C can promote the development of HCC is not totally determined, but the review of the literature suggests important hypothesis concerning a direct oncogenic effect, as well as indirect mechanisms, involving an increase in the exchange rate of hepatocites or the production of free radicals.These are the reasons for which the International Agency for the investigation of cancer of the WHO has pronounced into consideration, specially the HCV as a carcinogenic agent in humans.

Combination of lamivudine with thymosin alpha1 in treatment of duck hepatitis B / 浙江大学学报·医学版

<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>

Inhibition of binding peptides on replication of duck hepatitis B virus / 浙江大学学报·医学版

<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>

Inhibition of duck hepatitis B virus DNA replication by antisense phosphorothioate oligodeoxynucleotides in vitro and in vivo / 中华实验和临床病毒学杂志

<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>