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The early phase after hepatitis B virus infection could play a crucial role in clearance and/or persistence of the virus, particularly in neonates. This work compared the early phase of duck hepatitis B virus infection in 1-day-old (D1) and 28-day-old (D28) ducks to determine whether differences in viral or host innate immune response can be related to the difference in outcome. In the first phase, almost immediately after inoculation, virus was taken up by components of the reticulo-endothelial systems, particularly liver-specific macrophages, Kupffer cells. Very early after infection, the induction of alpha interferon by infected hepatocytes occurred and was rapidly reinforced by recruitment of effector lymphocytes, which directly or indirectly caused apoptosis, eliminating infected hepatocytes, as was seen in mature birds. In addition, a lack of lymphocytic infiltration of the liver was found in D1 ducks, which supports the suggestion that the innate immune network is less effective in D1 ducks. Taken together, these results suggest that failure of the co-ordinated innate immune response rather than a defect in induced antiviral cell-mediated immunity may be the key factor which makes baby ducks vulnerable to persistence of hepadnavirus infection.

The key to developing a therapeutic vaccine for chronic hepadnavirus infection lies in the characteristics of the host-immune response which leads to clearance of acute infection. Groups of 28-day-old ducks which had been surgically bursectomized (n = 10) or thymectomized (n = 13) on the day of hatch or were untreated (n = 21) were inoculated with 10(9) viral genome equivalents (vge) DHBV, then bled twice a week, and euthanased 40 days later. Serum and liver were tested for DHBV DNA and total leukocytes and peripheral blood mononuclear cells (PBMCs) counted. Liver and spleen sections were either stained with hematoxylin and eosin, and graded for inflammation or stained with peroxidase-labeled anti-human CD3 antibody and examined for T lymphocyte distribution. PBMC counts were similar in all groups. DHBV infection combined with bursectomy increased significantly, while thymectomy decreased significantly the total leukocyte count. The spleen and liver bursectomy increased T lymphocyte number while B cells were decreased. Converse changes were observed in thymectomized ducks. Histological evidence of hepatitis was present in infected control and bursectomized ducks but not in the uninfected control or infected thymectomized ducks. In control animals, DHBV challenge caused viremia in 17 and persistent infection in 11 (56%). Fewer thymectomized ducks (3/13, 23%) and significantly more (100%) bursectomized ducks remained persistently infected (P < 0.001). Unexpectedly, bursectomy led to persistence of infection while clearance of infection occurred normally in thymectomized ducks despite decreased T lymphocyte numbers. This suggests that clearance requires T and B lymphocyte collaboration., ((c) 2006 Wiley-Liss, Inc.)

Many viruses including HIV, hepatitis C and hepatitis B, have an outer lipid envelope which maintains inserted viral peptides in the "correct" functional conformation and orientation. Disruption of the lipid envelope by most solvents destroys infectivity and often results in a loss of antigenicity. This communication outlines a novel approach to viral inactivation by specific solvent delipidation which modifies the whole virion rendering it non-infective, but antigenic. Duck hepatitis B virus (DHBV) was delipidated using a diisopropylether (DIPE) and butanol mixture and residual infectivity tested by inoculation into day-old ducks. Delipidation completely inactivated the DHBV (p < 0.001). Delipidated DHBV was then used to vaccinate ducks. Three doses of delipidated DHBV induced anti-DHBs antibody production and prevented high dose challenge infection in five out of six ducks. In comparison, five of six ducks vaccinated with undelipidated DHBV and four of four ducks vaccinated with glutaraldehyde inactivated DHBV were unprotected (p < 0.05). Although this solvent system completely inactivated DHBV, viral antigens were retained in an appropriate form to induce immunity. Delipidation of enveloped viruses with specific organic solvents has potential as the basis for development of vaccines.

Duck hepatitis is a convenient model of hepatitis B virus (HBV) infection, but the lack of immunological reagents hampers investigation of pathogenesis and vaccine development. The aim of this study was to define T-cell epitopes in the surface peptide recognized by vaccinated immune birds. Blastogenesis assays were used to test the proliferative response of spleen mononuclear cells to synthetic peptides spanning the pre-S/S region in 22 naïve and 13 immunized and challenged immune ducks. Roughly > or = 50% of the immune ducks responded to five immunodominant peptides eliciting a statistically greater proliferative response than in naïve birds. Fewer ducks responded to an additional six peptides. No statistically significant difference could be shown for the response to 11 peptides between the immune ducks and the naïve ducks. There was no clustering of the immunodominant peptides which were located throughout the surface antigen at sites of major swings in hydrophobicity. A number of peptides which induce lymphoblastogenesis in vaccinated immune ducks have been identified. Their role in spontaneous recovery from duck hepatitis B infection merits investigation.

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.

Purpose: Nosocomial transmission of viral hepatitis and retrovirus infection has been reported. The expected risk is greatest for the hepatitis B virus (HBV). The duck HBV (DHBV) has similar biologic and structural characteristics to HBV and has been adopted as a suitable model for disinfectant testing., Methods: Angioscopic examination of the external jugular vein was performed on DHBV-infected ducks. After use, the instrument was air dried for 3 minutes. Samples were obtained by flushing the channel with 5 mL of phosphate buffered saline solution. The samples were collected immediately after drying (control), after flushing with 5 mL of water, after glutaraldehyde disinfection for 5, 10, and 20 minutes, and after ethylene oxide gas sterilization. Angioscopes were either precleaned or uncleaned before disinfection/sterilization. Residual infectivity was assessed with inoculation of samples into the peritoneal cavity of day-old ducks (n = 231)., Results: DNA analysis results of liver samples showed that all 38 control ducks became infected. The frequency of DHBV infection was reduced to 93% (14 of 15) by flushing the angioscope with 5 mL of sterile water. No transmission occurred after the use of any of the properly precleaned and disinfected/sterilized angioscopes. However, after the use of the uncleaned angioscopes, the transmission rate was 90% (9 of 10) and 70% (7 of 10) after 5 and 10 minutes of contact time, respectively, in 2% glutaraldehyde. Even after the recommended 20 minutes of contact time, there was still 6% (2 of 35) transmission. After ethylene oxide sterilization, two of the recipient ducklings (2 of 35) were infected with DHBV., Conclusion: There was no disease transmission after reuse of disposable angioscopes adequately cleaned before disinfection or sterilization. However, if the angioscopes are inadequately cleaned, DHBV can survive despite glutaraldehyde disinfection or ethylene oxide sterilization. This contrasts with previous in vitro and in vivo data with solid surgical instruments. It is postulated that the presence of a narrow lumen or residual protein shielding within the lumen may compromise effective inactivation of hepadnaviruses on angioscopes, with the potential risk for patient-to-patient transmission.

Duck hepatitis B virus (DHBV) has been a useful model for hepadnavirus infection. There have been few studies on immunity to DHBV and none describing the cell-mediated immune response by acute and chronically infected ducks. A duck hepatitis B antigen-specific blastogenesis assay was used to measure DHBV antigen-specific responses of duck peripheral blood (PBMC) and splenic mononuclear cells (SMCs) from uninfected control ducks, ducks acutely or chronically infected with DHBV, and ducks immune to DHBV. A comparison of the group mean responses by PBMC to DHBV surface antigen (DHBsAg) found that the immune group was significantly different to the other three groups (controls or unexposed, P < 0.0001; acutely infected, P< 0.01; chronically infected, P < 0.01). The responses to DHBsAg by PBMC of the acute group (P< 0.01) were significantly different also to that of the unexposed group. For DHBV core antigen (DHBcAg), significant differences in the responses were found between immune ducks and unexposed (P < 0.0005) and acutely infected (P < 0.05) groups. The SMC showed a significant difference between unexposed ducks and immune ducks (P< 0.05) in the group mean responses to DHBsAg. The responses to DHBcAg were significantly different between the immune group and the acute (P < 0.01) and unexposed (P < 0.01) groups. The group mean of unexposed ducks was also significantly different to that of acutely infected ducks (P < 0.01). This study indicates that the cellular immune response in immune animals differs from acutely and chronically infected ducks. Further studies of these differences may provide some explanations for the differing outcomes of DHBV infection.

Human hepatitis B virus (HBV) is an important cause of nosocomial infections and can be transmitted by contaminated instruments. However, tests of the efficacy of sterilization of materials and equipment contaminated by HBV are difficult to perform because the virus cannot be cultured in the laboratory. In this study, we aimed to evaluate the capability of a low temperature, hydrogen peroxide gas plasma sterilizer (Sterrad, Advanced Sterilization Products, Irvine California,) to inactivate duck hepatitis B virus (DHBV). In laboratory efficacy studies using DHBV dried on to glass filter carriers and exposed to one-half of the hydrogen peroxide gas plasma sterilization process, there was a 10(7) or greater decrease in the viral titer, with no infectivity detected on the carriers after treatment. In-use studies were performed using a laparoscope that was experimentally contaminated with DHBV to mimic the possible transmission of infection between successive patients. Following exposure to the hydrogen peroxide gas plasma sterilization process no transmission of DHBV infection from the laparoscope occurred despite obvious visual soiling with blood (N = 8) while the transmission rate for the unprocessed laparoscope (positive control) was 100% (26/26), and that for instruments after a water wash was 63% (7/11). In conclusion the hydrogen gas plasma sterilization process completely inactivates DHBV a representative of the hepadna group of viruses.

An antigen-specific lymphoblastogenesis assay for duck hepatitis B surface antigen (DHBsAg) and duck hepatitis B core antigen (DHBcAg) was developed using mononuclear cells from the peripheral blood (PBMC) or spleens (SMC) of immune ducks. Optimal culture conditions for the assay were determined by testing a number of variables, including antigen concentration, cell numbers/well, and the day of harvest. The specificity of the assay was assessed. The assay used 10% pooled duck serum supplement, and 8 x 10(5) cells/well for PBMC or 5 x 10(5) cells/well for SMC. The optimum antigen concentration ranged from 0.01 to 0.1 microgram/ml for both DHBsAg and DHBcAg. Maximum antigen-specific blastogenesis occurred between 4 to 7 days after establishment of the culture. The use of PHA (10 micrograms/ml) mitogenesis could predict the optimal cell numbers/well for antigen-specific blastogenesis. The assay demonstrated specific responses by immune ducks compared with those of unexposed ducklings and adult ducks (for DHBsAg P < 0.001; DHBcAg P < 0.05). For immune ducks, PBMC from all 8 ducks responded to DHBsAg, however, cells from only 4 of 7 immune ducks, responded to DHBcAg. Splenic mononuclear cells from all immune ducks responded to either DHBsAg or DHBcAg or both antigens.

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.

The effects of extracts of five Australian Phyllanthus species (P. hirtellus, P. gunnii, P. gasstroemii, P. similis and P. tenellus), other plant extracts and the antiviral drug foscarnet on duck hepatitis B virus (DHBV) endogenous DNA polymerase (DNAp) activity were compared. All 5 Phyllanthus species caused 50% inhibition at concentrations of dry weight between 350-800 micrograms/ml, which is comparable with the effect described for P. amarus on the DNAp of human and woodchuck hepatitis B viruses. Incubation of P. hirtellus with 100 ID50 DHBV neutralized infection. However, neither P. gasstroemi extract, given by intraperitoneal injection (i.p.) at a dose of 20 mg/kg 3 times per week to ducklings early in the incubation period, or P. hirtellus extract, given to established DHBV carrier ducklings, prevented or eliminated infection.

The efficacy of three proprietary glutaraldehyde disinfectants and their component bases was assessed using the duck hepatitis B virus (DHBV) model. Inactivation of infectivity of undiluted serum containing 10(6.8) ID50/ml DHBV was assessed after a mixture with an equal volume of disinfectant had stood at room temperature for 10 min. A dried spill of infectious serum was simulated using sterile filter paper disks, saturated with serum containing DHBV, dried and then exposed to test disinfectant for 10 min. Residual infectivity, and hence the reduction in virus titre, was determined by inoculation of dilutions of the treated samples into 1-day-old ducklings. A greater than 3 log10 reduction in virus titre could be demonstrated for the disinfectants as well as for some of their component bases. Disinfectant activity varied according to the method of viral presentation but a reduction of exposure time from 10 to 2.5 min did not diminish activity. The experimental protocol permits a comparative and quantitative assessment of the efficacy of both established and new disinfectants.

The therapeutic efficacy of antiviral agents for postexposure prophylaxis to hepadnavirus infection has been studied using acyclovir and foscarnet in the duck hepatitis B virus (DHBV) model. A total of 112 Pekin-Aylesbury ducks were inoculated with DHBV at 11 days post-hatch. Three days later, groups of these birds were injected intraperitoneally twice daily for 10 days with acyclovir (25 mg/kg) or foscarnet (250 mg/kg) or phosphate-buffered saline. Serum samples were taken before, during, and up to 4 weeks post-treatment and were analysed for DHBV DNA by dot hybridization. Liver tissue obtained at sacrifice was examined for viral DNA and for histological changes. At completion of treatment with acyclovir, 21 of 22 ducks were not viremic, compared with 6 of 26 control birds (P less than 0.001). Four weeks after withdrawal of acyclovir, 12 of 20 ducks remained nonviremic, compared with 2 of 23 controls (P less than 0.01). In liver tissue, viral DNA was detected in 10 of 19 treated ducks, compared with 21/24 controls (P less than 0.01). Histological changes of hepatitis were present in more of the control birds than in the treated group. The results with foscarnet treatment were similar, although a smaller inoculum of DHBV was used and fewer control birds became infected. The administration of antiviral agents soon after exposure prevented productive infection in approximately 50% of birds. Therefore, the use of a safe antiviral agent such as acyclovir, which can be given orally, should be considered in post-exposure prophylaxis against human hepatitis B virus (HBV) infection.