Optimizing environmental safety and cell-killing potential of oncolytic Newcastle Disease virus with modifications of the V, F and HN genes.

Newcastle Disease Virus (NDV) is an avian RNA virus, which was shown to be effective and safe for use in oncolytic viral therapy for several tumour malignancies. The presence of a multi basic cleavage site (MBCS) in the fusion protein improved its oncolytic efficacy in vitro and in vivo. However, NDV with a MBCS can be virulent in poultry. We aimed to develop an NDV with a MBCS but with reduced virulence for poultry while remaining effective in killing human tumour cells. To this end, the open reading frame of the V protein, an avian specific type I interferon antagonist, was disrupted by introducing multiple mutations. NDV with a mutated V gene was attenuated in avian cells and chicken and duck eggs. Although this virus still killed tumour cells, the efficacy was reduced compared to the virulent NDV. Introduction of various mutations in the fusion (F) and hemagglutinin-neuraminidase (HN) genes slightly improved this efficacy. Taken together, these data demonstrated that NDV with a MBCS but with abrogation of the V protein ORF and mutations in the F and HN genes can be safe for evaluation in oncolytic viral therapy.

Effect of Maternal Marginal Zinc Deficiency on Development, Redox Status, and Gene Expression Related to Oxidation and Apoptosis in an Avian Embryo Model.

Maternal severe zinc (Zn) deficiency resulted in growth retardation and high mortality during embryonic development in human. Therefore, this study is aimed at evaluating the effect of maternal marginal Zn deficiency on the development and redox status to avoid severe Zn deficiency using an avian model. A total of 324 laying duck breeders at 214 days old were randomly allotted into 3 dietary Zn levels with 6 replicates of 18 ducks per replicate. The birds were fed experimental diets including 3 dietary supplemental Zn levels of 0 mg/kg (maternal Zn-deficient group, 29.2 mg Zn/kg diet), 60 mg/kg (maternal Zn-adequate group), and 120 mg/kg (maternal Zn-high group) for 6 weeks. Dietary Zn levels had on effect on egg production and fertility (P > 0.05), whereas dietary Zn deficiency decreased breeder plasma Zn concentration and erythrocytic alkaline phosphatase activity at week 6 and inhibited erythrocytic 5'-nucleotidase (5'-NT) activity at weeks 2, 4, and 6 (P < 0.05), indicating that marginal Zn-deficient status occurred after Zn depletion. Maternal marginal Zn deficiency increased embryonic mortality and contents of superoxide anion radical, MDA, and PPC and reduced MT content and CuZnSOD activity in duck embryonic livers on E29. The MDA content was positively correlated with embryonic mortality. Maternal marginal Zn deficiency increased BCL2-associated X protein and Caspase-9 mRNA expressions as well as decreased B-cell lymphoma-2 and MT1 mRNA and signal AKT1 and ERK1 protein expressions (P < 0.05). Breeder plasma Zn concentration and erythrocytic 5'-NT activities at week 6 were positively correlated with GSH-Px activity and GPx, MT1, and BCL2 mRNA expressions in embryonic livers on E29. In conclusion, erythrocytic 5'-NT activity could be more rapid and reliable to monitor marginal Zn-deficient status. Marginal Zn deficiency impaired hatchability and antioxidant defense system and then induced oxidative damage and apoptosis in the embryonic liver, contributing to the greater loss of duck embryonic death.

Antioxidant activities and protective effects of duck embryo peptides against H2O2-induced oxidative damage in HepG2 cells.

Previous work showed that peptides from duck eggs incubated for 15 D presented high total antioxidant activities. Here, this work explore the antioxidant activities of different segments, ZT1 (≤3 KD), ZT2 (≤10 KD), and ZT3 (≤30 KD), derived from duck embryo peptides and their protective effects against H2O2-induced oxidative damage in HepG2 cells. Peptides present no cytotoxicity to HepG2 cells. Moreover, ZT1 exhibits a higher ability to scavenge several radicals as well as stronger inhibition of H2O2-induced oxidative stress than ZT2 and ZT3. The activities of catalase and glutathione peroxidase as well as total superoxide dismutase increase in a concentration-dependent manner. Peptides are isolated from ZT1 and then subjected to LC-MS/MS to identify their sequences, followed by functional annotation, bioinformatics prediction, and hot-spot motif recognition. As a result, 413 potential functional peptides are identified, with some compounds exhibiting more than 1 function. This work will help for exploring bioactive substances in duck embryo eggs and enhance the utilization value of duck or other poultry eggs.

Anti-angiogenic activity of Gracilaria coronopifolia J.G. Agardh extract by lowering the levels of trace metals (iron, zinc and copper) in duck chorioallantoic membrane and in vitro activation of AMP-kinase.

AMP-activated protein kinase (AMPK) is an intracellular energy sensor important in metabolic regulation, cell growth, and survival. However, the specific role of AMPK signaling pathway in the inhibition of angiogenesis remains unclear. The study highlights the activity on AMP activated protein kinase signaling pathways of a marine algae, Gracilaria coronopifolia, and its effects on angiogenesis. It was found that the most potent extract, GCD, inhibited angiogenesis significantly in the duck chorioallantoic membrane assay and also activated the enzyme AMP-kinase, in vitro. The dichloromethane extract was found most active in inhibiting angiogenesis in the duck chorioallantoic membrane (IC50 = 1.21 µg/mL) followed by GCH (IC50 = 3.08 µg/mL) (p = 0.479) and GCM (IC50 = 8.93 µg/mL) (p = 0.042). Benferroni post hoc analysis revealed that there was no significant difference between the percent inhibitions of GCH and GCM extracts (p = 0.479). Consequently, angiogenic inhibition caused lowering of iron, zinc, and copper levels in the duck CAM. Thin layer chromatography and gas chromatography-mass spectrometry revealed the components of each extracts. Notably, this is the first report on the kinase activity of a red algae G. coronopifolia extracts and a colorimetric-based quantification of angiogenesis based on metal content of CAM. Our data also suggest a novel therapeutic approach for inhibiting angiogenesis through the AMPK pathway.

Transcriptome reveals B lymphocyte apoptosis in duck embryonic bursa of Fabricius mediated by mitochondrial and Fas signaling pathways.

As a central immune organ unique to birds, the bursa of Fabricius (BF) provides a proper microenvironment for B-cell development. The bursal B-cells undergo rapid proliferation and differentiation at the embryonic stages, but 95% of them undergo apoptosis after hatching. Few studies have focused on the cause of bursal B-cells apoptosis at the embryonic stages in birds. To explore the cause, we compared the transcriptional profiles of three characteristic embryonic stages in duck, including embryonic day 14 (ED14), 22 (ED22) and 1 day after hatching (D1). Our results showed that the apoptotic B-cells were first observed at ED22 while there were no apoptotic B-cells at ED14. By performing enrichment analysis for DEGs and qRT-PCR, our results demonstrated that both mitochondrial and Fas signaling pathways mediated bursal B-cell apoptosis during the duck embryonic development. Further, protein-protein interactions (PPIs) and KEGG enrichment analysis together showed that BMP4, FoxO1 and IGF-1 may regulate bursal B-cells apoptosis. In addition, the DEGs showed two stage-specific expression patterns. By analyzing the genes of two expression patterns, the results indicated that B-cell false differentiation may be one of the reasons of apoptosis in the duck embryonic BF. Overall, these data demonstrated that from ED14-ED22, apoptosis of bursal B-cells was mediated by mitochondrial and Fas signaling pathways and could be regulated by BMP4, FoxO1 and IGF-1 in duck. One of the primary causes of bursal B-cell apoptosis may be false differentiation in B-cells.

Cytotoxicity of various chemicals and mycotoxins in fresh primary duck embryonic fibroblasts: a comparison to HepG2 cells.

To screen cost-effectively the overall toxicity of a sample, particularly in the case of food and feed ingredient quality control, a sensitive bioassay is necessary. With the wide variety of cytotoxicity assays, performance comparison between assays using different cells has become of interest. Fresh primary duck embryonic fibroblasts (DEF) were hypothesized to be a sensitive tool for in vitro cytotoxicity screening; cell viability of DEF in response to various cytotoxins was determined and compared with response of HepG2 cells. The IC50 values by the alamar blue assay in the DEF cells had a high correlation (R(2) = 0.96) with those obtained in HepG2 cells. Within the same toxin, primary DEF yielded significantly lower IC50 values than that obtained from HepG2 cells using the MTT and alamar blue assay. Additionally, primary DEF responded to all mycotoxins tested using the alamar blue assay, while HepG2 was less sensitive, particularly at short exposure times. The estimated IC50 for aflatoxin B1 , fumonisins B1 and deoxynivalenol in DEF after 72 h incubation were 3.69, 4.19 and 1.26 µg ml(-1) , respectively. Results from the current study suggest that primary DEF are more sensitive to cytotoxins and mycotoxins compared to HepG2, and thus may have great potential as an effective tool for cytotoxicity assessment. The question remains whether in vitro IC50 values can accurately predict in vivo toxicity; however, the current study accentuates the need for further attention to identify sensitive cell models for in vitro cytotoxicity screening and subsequent exploration of species-specific prediction models for in vivo toxicity. Copyright © 2016 John Wiley & Sons, Ltd.

Cadmium induces the differentiation of duck embryonic bone marrow cells into osteoclasts in vitro.

This study aimed to determine the in vitro effect of cadmium on the differentiation of duck embryonic bone marrow cells into osteoclasts. Bone marrow cells were harvested from 23-day old Gaoyou duck embryos and were cultured with either 50 nmol/L cadmium alone or different cadmium concentrations (0, 5, 10, 20 and 50 nmol/L) in combination with macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL). Tartrate-resistant acid phosphatase (TRAP) staining, pit formation assay with bovine cortical bone slices, and co-staining with tetramethyl rhodamine isothiocyanate (TRITC)-conjugated phalloidin and Hoechst 33258 were performed to determine the number of TRAP-positive cells and bone resorption activity. Cadmium at a concentration ⩾ 10 nmol/L in the presence of M-CSF and RANKL significantly increased in a concentration-dependent manner both the number of TRAP-positive cells (35-160%) and bone resorption activity (36-261%) (P<0.05). High cadmium concentrations in the presence of M-CSF and RANKL markedly promoted the formation of filamentous (F)-actin rings in differentiated osteoclasts. In conclusion, cadmium promotes in vitro the differentiation of duck embryonic osteoclasts in the presence of M-CSF and RANKL.

Influence of osteoprotegerin on differentiation, activation, and apoptosis of Gaoyou duck embryo osteoclasts in vitro.

ABSTRACT The aim of this study was to determine the influence of osteoprotegerin (OPG) on the differentiation, activation, and apoptosis of Gaoyou duck embryo osteoclasts cultured in vitro. Bone marrow cells were harvested from 23-d-old Gaoyou duck embryos and cultured in the presence of different concentrations of OPG (group A: no added factors, group B: 30 ng/mL of OPG, and group C: 100 ng/mL of OPG). Tartrate-resistant acid phosphatase (TRAP) staining, pit formation assay, and co-staining with tetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin and Hoechst 33258 were all performed to determine the number of TRAP-positive cells, bone resorption activity, and the level of apoptosis, respectively. The number of TRAP-positive cells and the net expansion of pit formations area peaked on d 7 of culture in all 3 groups. The number of osteoclasts and the total volume of pit formations in OPG-treated groups were significantly lower compared with group A (P < 0.05). At each time point, the net expansion of pit formations area correlated with the number of TRAP-positive cells. The OPG inhibited the de novo formation of filamentous (F)-actin rings and promoted the disruption of existing F-actin rings in mature osteoclasts. In addition, OPG induced apoptosis in mature osteoclasts, as demonstrated by morphological changes in the nuclei. In osteoclast precursors, OPG inhibited differentiation and downregulated the formation of F-actin rings. In mature osteoclasts, OPG suppressed activation and enhanced the development of apoptosis, observed as a decrease in the number of TRAP-positive cells, the disruption of F-actin rings and morphological changes of the nuclei.

Developmental changes in cell proliferation and apoptosis in the normal duck thymus.

Cell proliferation and apoptosis in the normal duck thymus during embryonic and post-embryonic development were studied. The flow cytometry assay shows that the level of G(0)/G(1) thymic cell population and the proportion of apoptotic cells increased with age, while the levels of S phase, G(2) + M phase and the proliferating index decreased with age. Proliferation cell nuclear antigen (PCNA) was mainly detected in the nuclei of lymphocytes. The number of PCNA-positive cells in the cortex and medulla significantly decreased with age. Transferase-mediated dUTP nick-end labelling (TUNEL) reaction stained apoptotic bodies in the cytoplasm of macrophages and free apoptotic bodies or nuclei with condensed chromatin in lymphocytes. The number of TUNEL-positive cells in the cortex and medulla markedly increased with age. The amount of proliferation and apoptotic cells in the thymic cortex was higher than that in the medulla. The balance between proliferation and apoptosis in the duck thymus may account for the process of thymic development and involution.

Embryotoxicity of weathered crude oil from the Gulf of Mexico in mallard ducks (Anas platyrhynchos).

Weathered crude oil in the Gulf of Mexico can result from oil spills such as the Deepwater Horizon incident that occurred on April 20, 2010 or from natural seeps. Adult waterbirds of the Gulf Coast region may become exposed to weathered crude oil while foraging, wading, or resting, and residues can then be transferred to nests, eggs, and hatchlings. Although the toxicity of many types of crude oil to avian embryos has been thoroughly studied, the effects of weathered crude oil on developing avian embryos are not well characterized. The objective of the present study was to examine embryotoxicity of weathered crude oil collected from the Gulf of Mexico in June 2010 using mallard ducks (Anas platyrhynchos) as a model species. Weathered crude oil was applied to fertilized mallard duck eggs by paintbrush in masses ranging from 0.1 to 99.9 mg on day 3 of incubation. Mortality occurred as early as day 7 and the conservatively derived median lethal application of weathered crude oil was 30.8 mg/egg (0.5 mg/g egg) or 30.7 µl/egg (0.5 µl/g egg). Body mass, liver and spleen mass, crown-rump and bill lengths, and frequency of deformities were not significantly different among hatchlings from oiled and control eggs. In comparison to published reports of fresh crude oil embryotoxicity, weathered crude oil was considerably less toxic. We conclude that avian toxicity varies according to the degree of crude oil weathering and the stage of embryonic development at the time of exposure. Results indicate bird eggs exposed to weathered crude oil from the Gulf of Mexico during summer 2010 may have had reduced hatching success.

Coordinated and sequential activation of neutral and acidic DNases during interdigital cell death in the embryonic limb.

Interdigital tissue regression during embryonic development is one of the most representative model systems of morphogenetic cell death, but the degenerative cascade accounting for this process awaits clarification. Although the canonical apoptotic caspase pathway appears to be activated in the interdigital mesenchyme committed to die, neither genetic nor chemical blockage of caspases or their downstream effectors, is sufficient to prevent cell death. Hence, alternative and/or complementary dying pathways must also be responsible for this degenerative process. In this work we have chosen to study the endonucleases during the regression of the interdigital tissue of avian embryos to gain insights into the molecular mechanisms accounting for programmed cell death in this system. We show that caspase activated DNase, which is a neutral DNase associated with the caspase apoptotic pathway, appears to be the main endonuclease only at an initial phase of interdigit regression. However at peak stages of the degenerative process, the acidic DNases L-DNase II and lysosomal DNase IIB become predominant in the system and markers for cell autophagy become moderately up-regulated. Consistent with the activation of acidic endonucleases we observed that microenvironmental pH value in the interdigits decreased to levels only appropriate for acidic enzymes. Furthermore, we found that overexpression of lysosomal DNase IIB in embryonic limb mesoderm promoted cell death, which was also accompanied by up-regulation and activation of L-DNase II. Up-regulation of acidic DNases was maintained in interdigits explanted to culture dishes, where the participation of exogenous professional phagocytes of hematopoietic origin is avoided. Finally, and consistent with all our findings, up-regulation of acidic DNases was much reduced in the webbed interdigits of duck embryos, characterized by a rudimentary interdigital degenerative process. We conclude that the regression of the interdigital tissue involves a coordinated and sequential activation of the caspase and lysosomal degenerative molecular cascades.

[Preliminary study on apoptosis of DEF cells induced by new type gosling viral enteritis virus (NGVEV) infection].

The characteristics changes of apoptosis of Duck Embryo Fibroblasts (DEF) cells induced by New type gosling viral enteritis virus, NGVEV) were observed by means of HE staining, electron microscopy and Annexin V-FITC/PI fluorescent staining. During 24-48 h post infection (pi), the difference of morphological change between infected DEF cells and the mock infected cells was invisible. At 72 h pi, the nuclear chromatin was getting condensed through HE staining; apoptotic morphological change such as abnormal shape of the nucleus, condensation of the cytoplasm and chromatin were observed under electron microscope; and the early apoptotic cells (Annexin V-FITC positive and PI negative) were detected under fluorescence microscope. At 96-120 h pi, by means of HE staining and electron microscopy, the advanced morphological change of apoptosis such as formation of different kinds of apoptotic bodies, and shrink of the DEF cells and nucleus were detected; under fluorescence microscope the different stages of the apoptotic DEF can be easily distinguished: early apoptotic cells (Annexin V-FITC postive and pi negative), advanced or late apoptotic cells (both Annexin V-FITC and PI positive), necrosis cells or dead cells (Annexin V-FITC negative and PI positive). This investigation shows that NGVEV might induce apoptosis and form characteristic apoptotic morphological changes in the DEF cells. NGVEV inducement of apoptosis may be an important mechanism of efficient dissemination of virus progeny.

Morphologic observations of new type gosling viral enteritis virus (NGVEV) virulent isolate in infected duck embryo fibroblasts.

The morphogenesis of the new type gosling viral enteritis virus (NGVEV) and the characteristic ultrastructural changes in the duck embryo fibroblasts (DEFs) were investigated by ultrathin sectioning and transmission electron microscopy after monolayer DEFs were experimentally infected with a virulent NGVEV strain. The investigation demonstrated that typical NGVEV particles were round, with a diameter ranging from 75 nm to 90 nm and that they were present in both the nucleus and cytoplasm of the infected DEFs. The mature virions contained nucleocapsids and nucleic acids. The virion penetrated the DEF, replicated, and matured in the nucleus, and they were finally released into the extracellular space via budding and disruption of the cytoplasmic membrane. With the appearance of progeny NGVEV, certain virus-related structures that were densely electron stained, which were circular, U-shaped, or irregular in appearance, could be observed in the cytoplasm of the infected DEFs. In this research, we first detected three types of intracytoplasmic inclusion bodies during the NGVEV infection, which always contained a number of NGVEV particles. Furthermore, we detected that NGVEV could induce apoptosis in DEFs, which had not been reported previously. The morphologic changes of apoptosis included shrinking of the apoptotic cells, chromatin condensation and margination, appearance of vacuoles on the cytoplasmic membrane, and the formation of apoptotic bodies. The mitochondria were ultracondensed and aggregated into compact clusters during apoptosis.

Survey of enterocyte morphology and tight junction formation in the small intestine of avian embryos.

The developing villus in the small intestine is covered by a single cell layer epithelium where tight junctions are present between the individual enterocytes. As the incubation period proceeds, the epithelium is expanding both in area as well as replenishing epithelial cells. The formation of tight junctions was evaluated in the small intestinal segments of the chicken, duck, and turkey in the final days of incubation and the first few days posthatch. The percentage of enterocyte membrane involved in tight junctions decreased as day of hatch approached followed by an increase in the 3 d after hatch. The rapid increase of epithelial cell proliferation at day of hatch may affect the percentage of cell membrane involved in tight junctions by having enterocytes of various sizes. The microvillus length changes throughout the incubation and posthatch period with differences between the crypt and villus tip. The microvillus length on the villus tip increases after hatch, whereas the crypt microvillus length remains static. Across the intestinal segments, the microvillus length is longest on the villus tip and shortest on the crypt at day of hatch in all 3 species. The observations made in cellular structure, mitochondria and nucleus location, and lipid droplets are similar to reports by other researchers, but this is the first report of those observations in both duck and turkey. The tight junctions appear to be ensconced by day of hatch with little change in cell perimeter in the next 24 h. Potential for embryonic enterocytes exist with the appearance of a goblet cell originating along the basolateral membrane and extruding the enterocytes at day of hatch.

A comparative study on the immunogenicity, safety and tolerance of purified duck embryo vaccine (PDEV) manufactured in India (Vaxirab) and Switzerland (Lyssavac-N): a randomized simulated post-exposure study in healthy volunteers.

Purified duck embryo vaccine (PDEV, Vaxirab) for rabies prophylaxis is now indigenously manufactured in India under technology transfer from Berna Biotech who made the original PDEV (Lyssavac). In the present study we have compared the two vaccines in terms of safety, immunogenicity and tolerance. The study was conducted in 220 adult healthy volunteers. It was observed that both vaccines produced neutralizing antibody titers (as determined by rapid fluorescent focus inhibition test, RFFIT) more than 0.5 IU/mL (minimum level for seroconversion) on all days tested but the titers on days 90 and 180 were significantly higher with Lyssavac. The adverse reactions produced were slightly more with Lysssavac but both vaccines were well tolerated. In conclusion, the indigenously produced PDEV (Vaxirab) was found to be equally safe and immunogenic as the original PDEV (Lyssavac) manufactured at Switzerland.

Polymorphisms in the repeat long regions of oncogenic and attenuated pathotypes of Marek's disease virus 1.

The nucleotide sequences of the terminal repeat long (TR(L)) and internal repeat long regions (IR(L)) in the genomes of 13 strains of Marek's disease virus type 1 (MDV-1) were determined and represent the largest collection of sequencing data from a contiguous region (12.8 kb) in the serotype 1 genomes. The collection of strains used in this study has been well characterized with respect to their virulence and contains members of each pathotype (4 attenuated, 1 mildly virulent, 3 virulent, 2 very virulent and 3 very virulent plus). It has previously been reported that two loci (meq and RLORF4) in the RL regions are likely to encode virulence factors based on comparative genomic studies involving vaccine and virulent strains. Additional studies using knockout mutants have provided stronger evidence that indeed RLORF4 and meq or the overlapping genes 23 kD and RLORF6 are involved in virulence. In this report, we provide evidence that additional open reading frames (ORFs) in the RL regions differ significantly between the extremes of the pathotypes (attenuated vs. nonattenuated). A deletion of 10 base pairs has been identified in RLORF12 from two attenuated strains CVI988 BP-5, p48 and RM-1, p40; and the lower virulence strain JM/102W. A deletion of 40 bp was also identified in RLORF4 of the attenuated strain R2/23, passage 106. A 177 bp insertion within the meq loci has been identified in most of the attenuated strains examined. Interestingly, R2/23 did not contain this insertion but instead truncated proteins are predicted for the three overlapping ORFs (meq, 23 kD and RLORF6) due to a frameshift mutation. Single nucleotide polymorphisms (SNPs), which loosely partition between attenuated and nonattenuated strains, have been identified in the ORFs encoding RLORF12, RLORF8, meq, 23 kD, RLORF6, RLORF4, RLORF3 and ICP0 and three previously unidentified short ORFs: MHLS, MLHG and MPSG. Although no single nucleotide polymorphism in the RL regions could predict virulence, their overall contribution to virulence can now be examined in defined mutants containing additional insertions or deletions in ORFs, suspected of encoding virulence factors, identified by this research.

Intraembryonic avian leukosis virus subgroup C (ALV-C) inoculation producing wasting disease in ducks soon after hatching.

We have studied the pathogenic changes in Khaki Campbell ducks injected in mid embryogenesis with ALV subgroup C virus td daPR-C derived from a molecular clone. The employed duck flock was shown to be highly genetically homogeneous and was controlled for the absence of current infections. Clear symptoms of wasting disease, which appeared since one week post hatching, represented the early consequence of the virus infection. They were manifested by decreased body weight, including clear involution of thymic tissue and pronounced anaemia. Microscopically, thymuses of infected animals displayed lymphatic depletion, clearly visible in the lobular cortex. Similarly, in the bursa Fabricii follicles, a marked reduction of the cortical layer and a decrease in folicullar centres was revealed. A decrease in the antibody response correlated with bursa Fabricii atrophy. The clear signs of anaemia were confirmed by haematological measurements, red blood cell count, haematocrit value and haemoglobin included. On the basis of these and additional observations we propose that inoculation of duck embryos provides a suitable model for analysis of the wasting disease produced by ALV-C.

Development of viral disinfectant assays for duck hepatitis B virus using cell culture/PCR.

Human hepatitis B virus (HBV) is a worldwide public health problem with chronic carriers at risk for developing cirrhosis and hepatocellular carcinoma. Accidental nosocomial infections from inadequately disinfected equipment or exposure to blood and body fluids from patients are major routes. To solve such problems, disinfectants to inactivate HBV must be validated. Duck hepatitis B virus (DHBV) is accepted as a surrogate for HBV, due to their similar sensitivities to disinfectants and its safety. Ducklings are used for disinfectant efficacy assays; however, the same virus titer is obtained using duck embryonic hepatocytes. Viral titration in disinfectant efficacy assay is conducted using Southern hybridization of infected duck serum. However, this test requires radioisotopes. Therefore, disinfectant assessment protocols were developed using duck embryonic hepatocytes with polymerase chain reaction (PCR) or nested PCR. The ease of handling, lowered cost and enhanced sensitivity make PCR desirable. Chicken embryonic hepatocytes were applied to DHBV disinfectant efficacy assay. Results were consistent and could be used under certain conditions. The virucidal activities of two quaternary ammonium chloride disinfectants, n-alkyl dimethyl benzyl ammonium chloride and alkyl dimethyl benzyl ammonium chloride (10C-12C) were compared and effective concentrations were 1200 and 1800 ppm, respectively. Efficacies of these disinfectants were validated using real-time quantitative PCR. Results confirmed that the efficacy of n-alkyl dimethyl benzyl ammonium chloride was higher than alkyl dimethyl benzyl ammonium chloride (10C-12C). This assay was useful for rapid discrimination of killing potentials of disinfectants. In conclusion, these assays can be applied to other viruses that are unable to cause CPE in cell cultures and broadened the utility of DHBV as animal model for HBV.

Inhibition of duck hepatitis B virus replication by intrahepatic expression of an antiviral resistance gene.

Resistance genes coding for inhibitors of hepadnaviral replication, such as ribozymes, antisense RNA, and dominant negative mutants have been shown to be effective in transfected hepatoma cells. In vivo studies, however, are not available to date. Here we expanded the use of the duck hepatitis B virus (DHBV) model for studying antiviral resistance genes in vivo. Animals were experimentally infected by intravenous injection of DHBV-positive serum in ovo. The use of recombinant human adenovirus type 5 and avian adenovirus CELO for gene transfer was evaluated. Adenovirus type 5 transduced more than 95% and CELO less than 1% of embryonic hepatocytes in vivo. Adenovirus type 5 interfered with DHBV replication (viral cross-talk), but this effect was moderate and did not preclude analysis of specific antiviral effects. Thus adenoviral transfer of a dominant negative mutant prior to DHBV infection (intracellular immunization) yielded 100-fold suppression of viral replication compared to the green fluorescent protein marker gene. Neither gene was toxic. These data demonstrate that a prototype anthepadnaviral resistance gene is functional in vivo. Duck embryos represent a useful model for evaluating gene therapeutic strategies in vivo without the need for large scale preparations of gene delivery vehicles.