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.

[Establishment of a method to detect duck hepatitis B virus covalently closed circular DNA based on rolling circle amplification].

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.

Antiviral activity of methyl helicterate isolated from Helicteres angustifolia (Sterculiaceae) against hepatitis B virus.

The anti-HBV effect of methyl helicterate (MH), a triterpenoid isolated from the Chinese herb Helicteres angustifolia, was explored both in vitro and in vivo. In the HBV-transfected cell line HepG2.2.15, the secretion of HBsAg/HBeAg, the levels of HBV DNA and cccDNA, and the amount of viral RNA were significantly decreased after treatment with MH for 144h. In addition, MH had no inhibitory effect on the mitochondrial DNA content. In DHBV-infected ducklings, MH significantly reduced the serum DHBV DNA, liver total viral DNA, and cccDNA levels. Furthermore, analysis of the liver pathological changes confirmed the hepatoprotective effect of MH. These results indicate that MH efficiently inhibits HBV replication both in vitro and in vivo and that MH may be a major bioactive ingredient in H. angustifolia.

[Cloning and sequence analysis of the DHBV genome of the brown ducks in Guilin region and establishment of the quantitative method for detecting DHBV].

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.

Development and application of a universal Taqman real-time PCR for quantitation of duck hepatitis B virus DNA.

To develop a quantitative assay for universal detection of duck hepatitis B virus (DHBV) DNA, a Taqman real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) assay was developed using primers and probes based on genomic sequences located at nucleotide 241-414 of the DHBV Core region which possesses the highest homology among the 44 DHBV genomes available in Genbank. The DHBV Core gene cloned in pGEM-T was used to generate DHBV DNA standard. The assay had a lowest detection limit of 10(3) copies/ml and a good linear standard curve (Y=-3.989X+49.086, r(2)=0.9993) over a wide range of input DHBV DNA (10(3) to 10(10) copies/ml). The standard deviation of intra- and inter-assay was 0.01-0.06 and 0.05-0.16, respectively, and the coefficient of variation was 1.3-1.8%. The specificity of the assay was validated using duck hepatitis virus type 1, hepatitis B virus, and E. coli DNA. Comparison of ABI 7300 and Bio-Rad iQ5 PCR instruments yielded highly consistent results. The assay showed a positive rate of 63.8% (51/80) DHBV DNA in peripheral blood and liver tissue from ducks from Xi'an, China. The FQ-PCR developed is highly sensitive, specific, reproducible and versatile, and may be used to universally detect DHBV DNA of different DHBV strains.

[Research on the gene structure of duck hepatitis B virus and its encoding proteins].

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.

[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.

Identification of natural recombination in duck hepatitis B virus.

Due to its high similarity to human hepatitis B virus (HBV), duck HBV (DHBV) is often used as an important model for HBV research. While inter-genotypic recombination of HBV is common, it has not been reported with DHBV. In this study, 32 non-redundant DHBV complete genomes were analyzed using phylogenetic methods and classified into two clusters, corresponding to the 'Chinese' and 'Western country' branches previously reported based on geographical distribution. One 'Chinese' branch strain was isolated in Australia and three 'Western country' branch strains were isolated in China, suggesting cross-geographical distribution of both branches. Recombination analyses of the 32 DHBV genomes identified two possible inter-genotypic recombination events with high confidence value. These recombination events occurred between the lineages represented, respectively, by the Chinese isolate GD3 (AY536371, 'Chinese' branch) and the American isolate DHBV16 (K01834, 'Western country' branch), giving rise to two Chinese recombinant isolates CH4 (EU429324) and CH6 (EU429326). The identification of inter-genotypic recombination among circulating DHBV isolates suggests the usefulness of DHBV as a model for studying the mechanism of HBV recombination.

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.

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.

Assembly and budding of a hepatitis B virus is mediated by a novel type of intracellular vesicles.

UNLABELLED: Formation of enveloped viruses involves assembly and budding at cellular membranes. In this study, we elucidated the morphogenesis of hepadnaviruses on the ultrastructural and biochemical level using duck hepatitis B virus (DHBV) as a model system. Formation of virus progeny initiates at the endoplasmic reticulum (ER) and is conserved both in vitro and in vivo. The morphogenesis proceeds via membrane-surrounded vesicles containing both virions and subviral particles, indicating a common morphogenetic pathway. The virus particle-containing vesicles (VCVs) are generated and maintained by reorganization of endomembranes accompanied by a striking disorganization of the rough ER (rER). VCVs are novel organelles with unique identity and properties of ER, intermediate compartment, endosomes, and multivesicular bodies. VCVs are dynamic structures whose size and shape are regulated by both membrane fusion and fission. CONCLUSION: Our data indicate a strong reorganization of endomembranes during DHBV infection, resulting in the biogenesis of novel organelles serving as multifunctional platforms for assembly and budding of virus progeny.

LC-MS characterization of efficacy substances in serum of experimental animals treated with Sophora flavescens extracts.

Anti-DHBV (duck hepatitis B virus) activity was found in the aqueous extracts of Sophora flavescens Ait. in vivo. Liquid chromatography/electrospray ionization ion trap mass spectrometry was applied to characterize the components in duck serum after oral administration of S. flavescens extract. Oxymatrine (1), sophoranol (2), sophoridine (3) and matrine (4) were identified in the serum. Further research on the four compounds was evaluated for their antiviral activity against HBV (hepatitis B virus) in cell culture. The results suggested that oxymatrine, sophoranol and matrine were the efficacy substances for anti-HBV activity in aqueous extracts of S. flavescens Ait.

Enzymatic treatment of duck hepatitis B virus: topology of the surface proteins for virions and noninfectious subviral particles.

The large surface antigen L of duck hepatitis B virus exhibits a mixed topology with the preS domains of the protein alternatively exposed to the particles' interior or exterior. After separating virions from subviral particles (SVPs), we compared their L topologies and showed that both particle types exhibit the same amount of L with the following differences: 1--preS of intact virions was enzymatically digested with chymotrypsin, whereas in SVPs only half of preS was accessible, 2--phosphorylation of L at S118 was completely removed by phosphatase treatment only in virions, 3--iodine-125 labeling disclosed a higher ratio of exposed preS to S domains in virions compared to SVPs. These data point towards different surface architectures of virions and SVPs. Because the preS domain acts in binding to a cellular receptor of hepatocytes, our findings implicate the exclusion of SVPs as competitors for the receptor binding and entry of virions.

The effect of surgical immunomodulation on liver inflammation and clearance of DHBV 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.

Rise in gamma interferon expression during resolution of duck hepatitis B virus infection.

Gamma interferon (IFN-gamma) expression plays a crucial role in the control of mammalian hepatitis B virus (HBV) infection. However, the role of duck INF-gamma (DuIFN-gamma) in the outcome of duck HBV (DHBV) infection, a reference model for hepadnavirus replication studies, has not yet been investigated. This work explored the dynamics of DuIFN-gamma expression in liver and peripheral blood mononuclear cells (PBMCs) during resolution of DHBV infection in adolescent ducks in relation to serum and liver markers of virus replication, histological changes and humoral response induction. DHBV infection of 3-week-old ducks resulted in transient expression of intrahepatic preS protein (days 3-14) and mild histological changes. Low-level viraemia was detected only during the first 10 days of infection and was accompanied by early anti-preS antibody response induction. Importantly, a strong increase in intrahepatic DuIFN-gamma RNA was detected by real-time RT-PCR at days 6-14, which coincided with a sharp decrease in both viral DNA and preS protein in the liver. Interestingly, liver DuIFN-gamma expression remained augmented to the end of the follow-up period (day 66) and correlated with portal lymphocyte infiltration and persistence of trace quantities of intrahepatic DHBV DNA in animals that had apparently completely resolved the infection. Moreover, in infected ducks, a moderate increase was detected in the levels of DuIFN-gamma in PBMCs (days 12-14), which coincided with the peak in liver DuIFN-gamma RNA levels. These data reveal that increased DuIFN-gamma expression in liver and PBMCs is concomitant with viral clearance, characterizing the resolution of infection, and provide new insights into the host-virus interactions that control DHBV infection.

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.

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.