The early host innate immune response to duck hepatitis B virus infection.

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.

[Dynamic detection of duck hepatitis B virus cccDNA in serum of ducks with liver injury].

OBJECTIVE: To confirm whether DHBV cccDNA could be detected in serum of DHBV-infected ducks after liver injury. METHODS: Twenty four ducks with persistent DHBV infection were divided into 4 groups with the following treatments: A, D-galactosamine (D-GalN, 2.2 g/kg) and lipopolysaccharide (LPS, 100 µg/kg); B, 10 mg/kg of carbon tetrachloride (CCl4) every 12 h twice following D-GalN and LPS; C, 15 mg/kg of CCl4 every 12 h twice following D-GalN and LPS; D, normal saline as normal control (NC). At 0 h, 24 h, 36 h and 48 h post-treatment, sera were collected from each duck for determination of serum DHBV load, DHBV cccDNA and alanine aminotransferase (ALT). And ducks were eventually sacrificed to obtain liver specimens for pathological assessment of liver lesions and determination of intrahepatic total DHBV DNA and DHBV cccDNA. RESULTS: (1) No obvious pathological change was observed in the liver of ducks from NC group while the indices of liver injury were significantly different between Groups A, B and C; (2) DHBV cccDNA was undetectable in the sera of ducks from NC and A group at all time points. In contrast, DHBV cccDNA, varying from 3.17 × 10(3) copies/ml to 1.72 × 10(4) copies/ml, was detected in the sera of 2 ducks from Group B and 3 ducks from Group C at 36 h post-treatment. The occurrence of DHBV cccDNA in serum was significantly correlated with the degree of liver injury while no significant association with serum ALT level and DHBV load as well as with the level of intrahepatic total DHBV DNA and DHBV cccDNA was observed. CONCLUSION: DHBV cccDNA may be detected in the serum when the liver of duck is seriously damaged. The incidence of DHBV cccDNA occurrence in the serum is significantly associated with the severity of liver injury.

Simple and visible detection of duck hepatitis B virus in ducks and geese using loop-mediated isothermal amplification.

In this study, loop-mediated isothermal amplification (LAMP) was used to establish a rapid, specific, and visual detection method for duck hepatitis B virus (DHBV). The design and synthesis of 4 specific LAMP primers were based on the conserved gene region of the DHBV genome, and the optimum temperature and time of the LAMP reaction were 63°C and 50 min, respectively. The LAMP assay was confirmed to be specific for DHBV detection and had the same sensitivity as the quantitative PCR assay. A visual detection method for rapid determination of results was developed using a color indicator containing phenol red and cresol red. A color change was produced based on a pH change in the reaction system, indicating a positive reaction. For the detection of samples from ducks and geese, the LAMP method has the advantages of simplicity, high sensitivity and specificity, good visibility, and low cost. Moreover, it is more practical and convenient than PCR-related assays for the clinical detection of DHBV.

Viral load in 1-day-old ducklings acutely infected with duck hepatitis B virus by different doses and routes of inoculation.

In order to define clearly the conditions leading to the outcome of acute duck hepatitis B virus (DHBV) infection, 1-day-old Pekin ducklings were infected with DHBV by different routes and given different doses of inoculum. Groups of 24 ducklings were inoculated either intravenously via the vena cruralis, or intraperitoneally with pooled serum containing either 1.6 x 10(7) or 1.6 x 10(4) DHBV genomes. One control duck from each group was inoculated with an equal volume of normal duck serum. A sensitive and reproducible real-time polymerase chain reaction assay based on TaqMan technology was developed for the detection and quantitation of DHBV DNA in the serum and liver. DHBAg was observed in the hepatocytes by immunohistochemistry. Histological changes in the liver tissue were also observed. The results demonstrate that ducklings at each time point and in all groups developed detectable viraemia. In each group, DHBV DNA in the liver was at a lower level than in serum and the peak DNA titre was found in serum earlier than in the liver. In the low-dose groups it was always at a lower level than in the high-dose groups. The DHBV replication levels appeared to be directly related to the number of DHBAg-positive hepatocytes. The variation trends of DHBAg-positive hepatocytes were similar in the high-dose groups. Histological changes were associated with liver viral DNA levels. We suggest that this dose and route of inoculation can be used as a model to study acute DHBV infections.

Molecular characterization of duck hepatitis B virus isolated from Hubei brown ducks.

The objective of this study was to characterize the genome structure of duck hepatitis B virus (DHBV) isolated from Hubei brown ducks. The natural carrier rate of DHBV in adult ducks from Hubei area was investigated and the DHBV DNA-positive serum screened out. The complete genome of a DHBV strain was amplified by polymerase chain reaction (PCR) and cloned into T vector and sequenced. The results showed that the carrier rate of DHBV in Hubei brown ducks was 10 %. This strain (GenBank accession number DQ276978) had a genome of 3024 nucleotides with three overlapping open reading frames encoding the surface, core and polymerase proteins respectively. Comparison of the strain with 17 DHBV strains registered in GenBank revealed a homology from 89.3 % to 93.5 % at the nucleotide level. The sequences of the structural and functional domains of these proteins were highly conserved. The strain was found to share more signature amino acids in the polymerase genes with the "Chinese" DHBV strains than those of the "Western" country strains. This finding was also corroborated by a phylogenetic tree analysis. Therefore, the DQ276978 might belong to a subtype of the Chinese DHBV strains.

Alternative methods for validation of cell culture infection with duck hepatitis B virus.

Hepatitis B virus (HBV) is an important virus used in disinfection procedures for blood spillage. However, validation of HBV inactivation is difficult, since there are no feasible infectivity assays. In some countries, the duck HBV (DHBV) is recognized as a suitable model for testing antiviral activity of chemical biocides against HBV. Currently, DHBV-infected ducks are required for preparation of the test virus as well as eggs from DHBV-free flocks for testing DHBV infectivity. To improve the practicality of the system, we suggested to use commercially available embryonated duck eggs for preparation of DHBV-susceptible hepatocyte cultures and to exclude infected hepatocytes by pre-screening with qualitative detection of DHBV DNA using polymerase chain reaction (PCR). A standardized DHBV test virus was prepared from the DHBV DNA-transfected hepatoma cell line D2, which contained 10(11)DHBV DNA molecules per mL detected by light cycler real-time PCR. Infection of cell cultures was most efficient 4 days after plating. The best identification of infected cultures was possible 6 days after infection with immunofluorescence using an antiserum against DHBV surface antigen.

[The possibility of hepatitis B virus transmission through dental handpieces].

OBJECTIVE: To discuss the possibility of hepatitis B virus (HBV) transmission through dental handpieces. METHODS: 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. RESULTS: 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. CONCLUSION: There might exist the possibility of HBV transmission through dental handpieces however, the autoclaves might kill the virus contaminating on dental handpieces.

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.

An in vitro system for the enzymological analysis of avian hepatitis B virus replication and inhibition in core particles.

A detailed analysis of the hepatitis B virus (HBV) replication reaction is important both in understanding viral biology and in developing effective antiviral drugs. This can best be achieved by studying the viral reverse transcriptase (RT) in its natural context, encapsidated within viral core particles in a multiprotein complex, rather than as an isolated enzyme. In order to facilitate a precise enzymological analysis of the avian HBV-RT reaction and its inhibition within replicating cores, a scheme for the purification and analysis of intracellular core particles derived from infected liver tissue has been devised, optimized and evaluated. The purification scheme itself is simple and rapid, and results in preparations with a 25-fold increase in endogenous polymerase activity that persists for over 5 h under assay conditions. In order to assess the suitability of these preparations for mechanistic studies, a thorough evaluation of purity was undertaken, revealing predominantly pure viral protein and nucleic acid, free of contaminating cellular polymerases and phosphatase activities that potently degrade nucleotides and antiviral drugs. Parameters governing optimal polymerase activity have been determined, and an assay for DHBV-RT activity has been developed which offers the highest purity and specific polymerase activity currently available to study hepadnaviral replication and inhibition.

Immuno disc assay for screening duck hepatitis B surface antigen in serum, liver tissue and cultured hepatocytes.

An immuno disc assay (IDA) for semi-quantitative analysis of the surface antigen (DHBsAg) of duck hepatitis B virus (DHBV) is described. Unpurified antigen preparations were adsorbed onto punched-out nitrocellulose membrane discs. Rabbit antiserum raised against serum-derived gradient-purified DHBsAg was used for detecting the antigen. Cross-reacting antibodies in the rabbit antiserum were removed using normal duck serum and normal duck hepatocytes. The sensitivity of the IDA was compared with that of the Western blot analysis and was observed to be of the same order, but differed slightly for DHBsAg in liver and sera. In contrast to Western blot analysis, antigen specificity for the IDA included the S-protein. Immunodetection was carried out in microtitre plates, but the procedure was accelerated by attaching the antigen-adsorbed discs to an adhesive plate sealer. The IDA was exemplified for measuring DHBsAg in duck serum, duck liver homogenates and viral protein synthesis in cultures of DHBV-infected hepatocytes.

Inactivation of duck hepatitis B virus by a hydrogen peroxide gas plasma sterilization system: laboratory and 'in use' testing.

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.

Establishment and assessment of two methods for quantitative detection of serum duck hepatitis B virus DNA.

AIM: To establish and assess the methods for quantitative detection of serum duck hepatitis B virus (DHBV) DNA by quantitative membrane hybridization using DHBV DNA probe labeled directly with alkaline phosphatase and fluorescence quantitative PCR (qPCR). METHODS: Probes of DHBV DNA labeled directly with alkaline phosphatase and chemiluminescent substrate CDP-star were used in this assay. DHBV DNA was detected by autoradiography, and then scanned by DNA dot-blot. In addition, three primers derived from DHBV DNA S gene were designed. Semi-nested primer was labeled by AmpliSensor. Standard curve of the positive standards of DHBV DNA was established after asymmetric preamplification, semi-nested amplification and on-line detection. Results from 100 samples detected separately by alkaline phosphatase direct-labeled DHBV DNA probe with dot-blot hybridization and digoxigenin-labeled DHBV DNA probe hybridization. Seventy samples of duck serum were tested by fluorescent qPCR and digoxigenin-labeled DHBV DNA probe in dot-blot hybridization assay and the correlation of results was analysed. RESULTS: Sensitivity of alkaline phosphatase direct-labeled DHBV DNA probe was 10 pg. The coincidence was 100% compared with digoxigenin-labeled DHBV DNA probe assay. After 30 cycles, amplification products showed two bands of about 180 bp and 70 bp by 20 g/L agarose gel electrophoresis. Concentration of amplification products was in direct proportion to the initial concentration of positive standards. The detection index was in direct proportion to the quantity of amplification products accumulated in the current cycle. The initial concentration of positive standards was in inverse proportion to the number of cycles needed for enough quantities of amplification products. Correlation coefficient of the results was (0.97, P<0.01) between fluorescent qPCR and dot-blot hybridization. CONCLUSION: Alkaline phosphatase direct-labeled DHBV DNA probe in dot-blot hybridization and fluorescent qPCR can be used as valuable means to quantify DHBV DNA in serum.

A teratoma in a duck infected congenitally with duck hepatitis B virus.

A teratoma was diagnosed in an 8-month-old pekin duck based on the presence of tissue derived from embryonic ectoderm, mesoderm, and endoderm in the neoplasm. The neoplasm was examined for the presence of duck hepatitis B virus, because the duck was congenitally infected with the virus, a member of the hepadnavirus family that is associated with hepatic neoplasms in hepadnavirus-infected mammals. Persistent infection occurred in the liver, but no evidence of viral infection was found in the neoplasm.

Ultrastructural study of hepatitis B virus in biliary epithelial cells of duck liver.

Liver specimens from the Shanghai Ma ducks congenitally infected with duck hepatitis B virus (DHBV) were examined by immunohistochemical technique and electron microscopy. All the 12 serum DHBV positive ducks showed varying degrees of positive straining in hepatocytes. In 8 of the 12 ducks, DHBV antigen was discovered in the cytoplasm of biliary epithelial cells. Conventional electron microscopy revealed two kinds of virus particles in the biliary epithelial cells: 1. the incomplete virus, 40-50 nm in diameter and spherical in shape with an outer membrane, located mainly in the dilated cisternae of rough endoplasmic reticulum of the cells in large amounts; 2. the complete virions, 55-60 nm in diameter, were spherical with an outer membrane and located mainly in the cytoplasmic vesicles in small amount. We believe the particles found in the biliary epithelial cells in this study were DHBV particles. It is most likely that the infection and replication of DHBV not only take place in the liver cells but also in the biliary epithelial cells.

Characterisation of the Indian strain of duck hepatitis B virus (DHBV) and development of a carrier duck colony for antiviral drug testing.

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.

[Duck hepatitis B virus (DHBV) and viral DNA in duck hepatocellular carcinoma and liver tissue].

The hybridization test for duck hepatitis B virus DNA was performed in 34 ducks with hepatoma from Qidong, Jiangsu province. Among the 34 hepatoma ducks, 18 were positive for DHBV DNA in the serum and 27 were positive in the tumor and/or liver tissue. Tissue sections were stained with Victoria blue nuclear fast red for detecting DHBV surface antigen. Victoria blue positive cells were found in 11 tumors and 15 paratumorous regions of 23 ducks with hepatocellular carcinoma. Although paratumorous regions were positively stained in 3 of 8 ducks with cholangiocarcinoma, all were negative within their tumors. All paratumorous regions and 2 tumor regions of 3 ducks with hepatocellular-cholangiocarcinoma were positive for Victoria blue. The results suggest that duck hepatocellular carcinoma be closely related to DHBV infection.

Dot blot hybridization assay for the detection of duck hepatitis B virus DNA among healthy Indian country ducks.

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.

[A study of duck hepatitis B virus infection in Chinese ducklings].

Intraperitoneal inoculation of duck hepatitis B virus in three different dosages (9 x 10(7), 1.8 x 10(8), 9 x 10(8) DHBV particles) into 3 to 21 day-old Chinese ducklings provided from a DHBV free flock produced a persistent infection up to 93.3% in 60 animals. The serum and liver specimens of these ducklings were examined by DNA dot blot hybridization on the 30th day after inoculation. The results showed that: (1) examination of viral DNA in liver was more sensitive and reliable than estimation of the DNA in serum for detecting DHBV infection in inoculated ducklings; (2) the liver DHBV DNA level did not coincide with the degree of liver hepatitis induced; (3) 21-day-old Chinese ducklings were also susceptible to DHBV infection, the infection rate of this group was 100% (10/10).

[Ultrastructural study of duck hepatitis B virus in biliary epithelial cells of duck liver].

Liver specimens from Shanghai Ma ducks infected with duck hepatitis B virus (DHBV) were examined by immunohistochemical technique and electron microscopy. The results showed that DHBV and DHBV antigen were found not only in the hepatocytes but also in the biliary epithelial cells of infected ducks. Electron microscopy also revealed that incomplete spherical particles, 40-50 nm in diameter, were present in the dilated cisternae of rough endoplasmic reticulum (RER), and complete spherical virions, 55-65 nm in diameter, existed in the cytoplasmic vesicles and cytoplasm in small amounts. The results of the present study seem to confirm the possibility of infection and replication of DHBV not only in the liver cells but also in the biliary epithelial cells of ducks.

[The effects of Ara-A on viral markers in duck hepatitis B virus carrier ducks].

Duck hepatitis B virus (DHBV) carrier ducks of one week old were injected with Ara-A (adenine arabinoside) of different dose including 2.5 (11 ducks), 5.0 (11), 10.0 (10) and 20.0 (10) mg/kg for 14 days. This antiviral effect showed dose-dependence up to 5.0 mg/kg and this dose seemed effective to obtain significant antiviral effect. Viral DNA and DNA polymerase activity were reduced significantly from the 1st week after starting the administration of Ara-A. This antiviral effect was maintained even at the 1st week after discontinuation of the drug. These findings were quite similar to those observed in HBV carriers. With the increasing necessity of Ara-A treatment in patients who will not respond to interferon therapy, DHBV seemed a suitable model for the investigation of the dose and antiviral effect of Ara-A treatment in humans.