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Duck hepatitis A virus type 1 (DHAV-1) disease causes significant economic losses to the duck industry. Duck enteritis virus (DEV) is frequently used as a viral vector for aquatic poultry vaccination, but no recombinan DEV expressing DHAV-1 VP0 has been developed. In this study, we established a system for rescuing the DEV C-KCE vaccine strain by transfecting cells with six fosmid DNAs. We generated a recombinant virus (rDEV-ul41VP0) by inserting the VP0 gene of DHAV-1 into the ul41 region in the DEV C-KCE genome. DHAV-1 VP0 was stably expressed in the rDEV-ul41VP0 infected cells, but did not affect the replication properties of DEV C-KCE in cells. Duck experiments showed that rDEV-ul41VP0 could provided full protection against the lethal DEV Chinese standard challenge (DEV CSC) and conferred 70% protection against DHAV-1 161/79 at 3 days postvaccination. These results indicate that rDEV-ul41VP0 rapidly induces protection against DEV CSC and DHAV-1 in ducks, and can be served as a bivalent vaccine against DEV and DHAV-1., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Duck hepatitis A virus (DHAV) causes an infectious disease that mainly affects 1- to 4-week-old ducklings, resulting in considerable loss to the duck industry. Although there have been many studies on DHAV in recent years, the effects on host infection and pathogenesis of DHAV-1 remain largely unknown. This study investigated the effects of the DHAV-1 structural protein VP3 on DHAV-1 virus adsorption and apoptosis to explore the role of VP3 in the viral life cycle. The effects of DHAV-1 VP3 and an antibody against the protein on virion adsorption was analyzed by qRT-PCR. The results showed that the virus copy number for the rabbit anti-VP3 IgG-treated group was significantly lower than that for the negative control group but higher than that for the rabbit anti-DHAV-1 IgG-treated group. This result indicates that VP3 mediates DHAV-1 virus adsorption but that it is not the only protein that involved in this process. In addition, a eukaryotic recombinant plasmid, pCAGGS/VP3, was transfected into duck embryo fibroblasts (DEFs), and the apoptotic rate was determined by DAPI staining, the TUNEL assay and flow cytometry. DAPI staining showed nucleus fragmentation and nuclear edge shifting. TUNEL assay results revealed yellow nuclei, and flow cytometry indicated a significant increase in the apoptotic rate. In addition, qRT-PCR revealed increased in the transcriptional levels of the apoptotic caspase-3, -8 and -9, with the largest increase for caspase-3, followed by caspase-9 and caspase-8. Enzyme activity analysis confirmed these results. Furthermore, the VP3 protein decreased the mitochondrial membrane potential, and the transcriptional levels of the proapoptotic factors Bak, Cyt c and Apaf-1 in the mitochondrial apoptotic pathway were significantly upregulated. These data suggest that expression of VP3 in DEFs induces apoptosis and may primarily activate caspase-3-induced apoptosis through mitochondrion-mediated intrinsic pathways. The findings provide scientific data to clarify DHAV-1 infection and pathogenesis.

Duck hepatitis A virus (DHAV) is the major pathogen of duck viral hepatitis, which has caused great economic losses to duck breeding industry. As an effective delivery tool for protein antigens, Lactococcus lactis (L. lactis) has been successfully used to stimulate mucosal and systemic immune response. In this study, a recombinant L. lactis named NZ3900-VP1 was constructed, which could express VP1 protein of DHAV type 3 (DHAV-3) by using a nisin-controlled expression (NICE) system. The animal experiment in both mice and ducklings were performed to detect the immune response and protection effect of oral vaccination by the recombinant L. lactis. The results showed that oral vaccination with L. lactis NZ3900-VP1 significantly induced specific anti-VP1 IgG antibodies and mucosal secretory immunoglobulin A (sIgA) of DHAV-3 in mice and ducklings, and cytokines including interleukin-2 (IL-2), interferon gamma (IFN-γ), interleukin-10 (IL-10) and interleukin-4 (IL-4). Notably, the ducklings vaccinated with L. lactis NZ3900-VP1 were effectively protected when facing natural infestation of DHAV-3, which indicated that the recombinant L. lactis could serve as an effective vaccine to prevent DHAV-3 infection in ducklings., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

To develop an indirect enzyme-linked immunosorbent assay(I-ELISA) method based on 3A protein of duck hepatitis A virus type 1(DHAV-1) for detection of DHAV-1 antibody, the recombinant protein 3A of DHAV-1 was expressed in E.coli and detected by Western blotting with DHAV-1 infected duck serum. A 3A-ELISA method using the expressed 3A protein as coating antigen for the detection of antibodies to DHAV-1 was developed. The optimal antigen, serum and enzyme-labeled antibody dilutions were 1:200(6.185 μg/ml), 1:20 and 1:2000, respectively. The optimal blocking buffer was 5% BSA. The cutoff value was determined to be 0.274, and the analytical sensitivity was 1:1280. There was no cross reaction between DHAV-1 infected duck serum and other common pathogenic duck serum, indicating that I-ELISA could be used to detect DHAV-1 infected duck serum. The coefficients of variation(CVs) were lower than 10%. The concordance between the I-ELISA based on the 3A subunit of DHAV-1 and that based on the whole DHAV-1 particle was 92.7%. Taken together, the 3A-ELISA method is a highly sensitive and specific test that could be used for screening for DHAV-1 infection and monitoring DHAV-1 antibody., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Duck hepatitis A virus type 1 (DHAV-1) is one of the most lethal pathogens in the duck industry. The attenuated vaccine (the CH60 strain) is cultivated through serial passage in chicken embryos and is widely used for the prevention and control of the disease. However, the specific mechanism underlying its adaptation in chicken embryos has not been fully elucidated. In this study, we first infected chicken embryo fibroblasts (CEFs) with the DHAV-1 CH60 strain. The peak of viral proliferation occurred within 36-48 h post-infection. The different DHAV-1 strains significantly induced the expression of IFNα, IFNγ, and Suppressor of cytokine signaling 3 (SOCS3) in CEFs, and we found that SOCS3 overexpression significantly promoted viral replication. Furthermore, SOCS3 overexpression significantly inhibited the expression of IFNα but promoted the expression of IFNγ. In addition, SOCS3 overexpression clearly decreased the mRNA levels of STAT1 and STAT3 in the Janus kinase (JAK)-STAT signaling pathway and inhibited the expression of the antiviral proteins MX1 and OASL. Immune-precipitation assays indicated that SOCS3 and IFNα do not physically interact. Subcellular localization of SOCS3 and IFNα revealed that SOCS3 was mainly located in the nucleus and cytoplasm, while IFNα was located only in the cytoplasm. Co-localization of these two proteins was not observed in the cytoplasm. In conclusion, the DHAV-1 CH60 strain may inhibit the expression of IFNα by increasing the SOCS3 protein and SOCS3 can in turn decrease STAT1 and STAT3 mRNA levels, thereby inhibiting the antiviral protein MX1 and ultimately promoting viral proliferation, indirectly assisting in viral adaptation in chicken embryos.

Duck virus hepatitis (DVH) caused by duck hepatitis A virus type 1 (DHAV-1) is an acute and highly contagious disease affecting young ducklings. The VP1 protein is one of the major structural proteins of DHAV-1 carries critical epitopes responsible for the induction of neutralizing antibodies. In this study, we have successfully constructed an immune phage display VHHs library against DHAV-1 with the size of 6 × 10 6 colonies. A nanobody (Nb) against VP1 protein of DHAV-1, named Nb25, was identified from the immunized phage display library. Nb25 could react with the conserved linear B-cell epitope of 174 PAPTST 179 in DHAV-1 VP1, even though Nb25 showed no neutralizing activity to DHAV-1. To the best of our knowledge, this is the first report about preparation of anti-DHAV-1 Nbs and identification of the specific conserved linear B-cell epitope of DHAV-1 with Nb, which will facilitate the serologic diagnosis of DHAV-1 infection., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The avian adeno-associated virus (AAAV) has been proved to be an efficient gene transfer vector for human gene therapy and vaccine research. In this experiment, an AAAV-based vaccine was evaluated for the development of a vaccine against duck hepatitis a virus type 1 (DHAV-1). The major capsid VP1 gene was amplified and subcloned into pFBGFP containing the inverted terminal repeats of AAAV, and then the recombinant baculovirus rBac-VP1 was generated. The recombinant AAAV expressing the VP1 protein (rAAAV-VP1) was produced by co-infecting Sf9 cells with rBac-VP1 and the other 2 baculoviruses containing AAAV functional genes and structural genes respectively, and confirmed by electron microscopy, Western blotting and immunofluorescence assays. Quantitative real-time PCR revealed that the titer of rAAAV-VP1 was about 9 × 1012 VG/mL. Immunogenicity was studied in ducklings. One day ducklings were injected intramuscularly once with rAAAV-VP1. Serum from rAAAV-VP1-vaccinated ducklings showed a systemic immune response evidenced by VP1-specific enzyme-linked immunosorbent assay and virus neutralization test. Furthermore, all ducklings inoculated with rAAAV-VP1 were protected against DHAV-1 challenge. The data of quantitative real-time RT-PCR from livers of challenged ducklings also showed that the level of virus copies in rAAAV-VP1 group was significantly lower than that of the PBS group. Collectively, these results demonstrate that the AAAV-based vaccine is a potential vaccine candidate for the control of duck viral hepatitis.

The avian adeno-associated virus (AAAV) is a replication-defective nonpathogenic virus that has been proved to be useful as a viral vector in gene delivery. In this study, the feasibility of AAAV for transgenic expression of duck hepatitis A virus (DHAV) VP3 structural protein and its ability to induce protective immunity in ducklings was assessed. The recombinant AAAV (rAAAV-VP3) expressing the VP3 protein was prepared by co-infection of Sf9 cells with recombinant baculovirus (rBac-VP3) containing VP3 gene flanked by inverted terminal repeats (ITRs) of AAAV and the other two recombinant baculovirus expressing AAAV functional and structural genes, respectively. The generation of rAAAV-VP3 was demonstrated by electron microscopy, immunofluorescence assay, and western blot analysis. One day old ducklings were inoculated with rAAAV-VP3 or commercial attenuated vaccine and then challenged with DHAV-1 strain SH two weeks post vaccination. Anti-DHAV-1 antibodies were detected in all vaccinated groups by ELISA, and the titers between the rAAAV-VP3 group and the attenuated vaccine group were not statistically significant. Real time RT-PCR analysis showed that the virus copy numbers in the livers of the PBS control group were significantly higher than that of the rAAAV-VP3 and attenuated vaccine groups. In conclusion, we demonstrated that the VP3 expression mediated by rAAAV in ducklings could induce protective immunity against DHAV challenge, and this could be a candidate vaccine for the control of duck viral hepatitis. Keywords: avian adeno-associated virus; duck hepatitis A virus; VP3 gene; immunogenicity.

Duck hepatitis A virus type 1 (DHAV-1) is one of the most common and lethal pathogens in young ducklings. Live-attenuated DHAV vaccine (CH60 strain) developed by passaging in chicken embryos provided effective immune protection for ducklings. However, the accurate mechanism for such adaption in chicken embryos is not fully revealed. Here, we utilize RNA-sequencing to perform global transcriptional analysis of DHAV-1-innoculated embryonated livers along with histopathological and ultrastructural analysis. This study revealed that infection with DHAV-1 strain CH60 is associated with enhanced type I and II interferon responses, activated innate immune responses, elevated levels of suppressor of cytokine signaling 1 and 3 ( SOCS1 and SOCS3 ) accompanied with abnormalities in multiple metabolic pathways. Excessive inflammatory and innate immune responses induced by the CH60 strain are related to severe liver damage. Our study presents a comprehensive characterization of the transcriptome of chicken embryos infected with DHAV-CH60 and provides insight for in-depth exploration of viral adaption and virus-host interactions.

To construct phage antibody library for VP3 protein of duck hepatitis virus type 1(DHAV-1) and pan the specific single-chain variable fragment antibody (scFv), total RNA was extracted from the protein VP3- immunized mice spleen., vp3 gene encoding VP3 protein was amplified from the genome of DHAV-1 by RT-PCR method for the following recombinant pET-VP3 construction, immunogenic VP3 expression and purification, and combined with SOE-PCR method to complete the assembly of scFv. The scFv gene was cloned into pCANTAB5E vector for phage antibody library construction. Finally, the library for anti-VP3 scFv was screened by four rounds of adsorption-elution-enrichment with the purified VP3 protein. The characters of binding ability, specificity and neutralization of soluble antibodies expressed were evaluated by ELISA. The results showed 7 VP3-specific scFvs have been screened and identified with high both sensitivity and specificity for binding DHAV-1. To our knowledge, this is the first report for VP3-specific scFv of DHAV-1 and potentially promising application used in prevention and treatment of duck viral hepatitis., (Copyright © 2018. Published by Elsevier B.V.)

Duck viral hepatitis type I is a rapidly spreading infection lethal in young ducklings, caused by the duck hepatitis A virus (DHAV). Vaccination of breeder ducks is a common practice to control DHAV. However, maintaining proper maternal antibody levels in large flocks is difficult. Therefore, a simple vaccination strategy that can induces stable high antibody levels through mass vaccination is desirable. We evaluated a DHAV vaccination strategy for breeder ducks involving oral administration under field conditions, and examined the kinetics of antibody response in the ducks and their progeny. The strategy included a primary intramuscular vaccination, followed by secondary and tertiary oral vaccinations. Five weeks after the primary vaccination, virus-neutralizing antibody titers increased by 8.4 ± 1.3 log 2 . The titers remained stable at around 9.0 ± 1.1 log 2 for up to 36 weeks. None of the progeny died when challenged with virulent DHAV at 1, 7 or 14 days of age. The transfer percentage of antibodies from the breeder ducks to their progeny was 12.8 ± 3.0%. When antibody levels of the progeny were measured from the day of hatching to 20 days of age, the levels steadily declined, reaching a mean titer of 0 log 2 at 20 days. The half-life of the maternally derived antibodies against DHAV was 3.4 ± 1.1 days. Our vaccination strategy might be effective in breeder ducks because it can be easily applied and induced strong immunity. Moreover, our results might provide a foundation for the mechanistic study of maternally derived antibodies in passive protection., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Duck hepatitis A virus type 1 (DHAV-1) is one of the most harmful pathogens in the duck industry. The infection of adult ducks with DHAV-1 was previously shown to result in transient cytokine storms in their kidneys. To understand how DHAV-1 infection impacts the host liver, we conducted animal experiments with the virulent CH DHAV-1 strain and the attenuated CH60 commercial vaccine strain. Visual observation and standard hematoxylin and eosin staining were performed to detect pathological damage in the liver, and viral copy numbers and cytokine expression in the liver were evaluated by quantitative PCR. The CH strain (10 8.4 copies/mg) had higher viral titers than the CH60 strain (10 4.9 copies/mg) in the liver and caused ecchymotic hemorrhaging on the liver surface. Additionally, livers from ducklings inoculated with the CH strain were significantly infiltrated by numerous red blood cells, accompanied by severe cytokine storms, but similar signs were not observed in the livers of ducklings inoculated with the CH60 strain. In conclusion, the severe cytokine storm caused by the CH strain apparently induces hemorrhagic lesions in the liver, which might be a key factor in the rapid death of ducklings.

Duck virus hepatitis caused by duck hepatitis A virus (DHAV) is an acute and contagious disease. To better understand the pathogenic mechanism of DHAV-3 in ducklings, an infection experiment was performed. Our results showed that typical symptoms were observed in the infected ducklings. DHAV-3 could infect many tissues, leading to pathological lesions, especially on the livers and spleen, and the host immune responses are activated in infection. Real-time quantitative PCR demonstrated that expression of many innate immune-related genes was mostly up-regulated in the livers and spleen, and antiviral innate immune response was established, but not sufficient to restrict the virus replication of lethal dose. Many major pattern recognition receptors (PRRs) (RIG-1, MDA5, and TLR7) are involved in the host immune response to DHAV-3, and the expression of interferon (IFNα, IFNβ and IFNγ) and antiviral proteins (MX, OAS and PKR) are also up-regulated in the liver and spleen. The expression of most cytokines (IL-1β, IL-2 and IL-6) was also up-regulated to different degrees and was various; the expression of IL-2 increased most significantly in liver. Our data provide a foundation for further study of the pathogenicity of duck virus hepatitis and extend our understanding of the immune responses of ducklings to DHAV-3 infection., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Ferritin heavy polypeptide 1 (FTH1) plays a pivotal role in response to viral infections. FTH1 expression is modulated by various pathogens, but the regulatory mechanisms are unknown. We firstly construct duck hepatitis virus 1 (DHV-1) infection model, including morbid ducklings, non-morbid ducklings and control ducklings. Then the mRNA expression of duck FTH1 (duFTH1) was measured mRNA expression of duck FTH1 (duFTH1) in the liver and spleen after duck hepatitis virus 1 (DHV-1) infection using quantitative polymerase chain reaction (qPCR) and found that duFTH1 mRNA was down-regulated significantly in morbid ducklings (liver, P < 0.01; spleen, P < 0.05) compared with the control ducklings. We also found that duFTH1 expression was significantly higher in the spleen (P < 0.01) and liver (P < 0.05) of non-morbid ducklings than in morbid ducklings. Moreover, DNA methylation of the duFTH1 promoter was examined by bisulfite sequencing (BSP) and we found that the duFTH1 promoter was hypomethylated, the relative methylation was only 5.9% and 2.0% in the morbid ducklings and non-morbid ducklings, respectively. The promoter contained a -55 C/T mutation in 75% of non-morbid ducklings, and this polymorphism affected promoter activity. Further analysis suggested that this mutation altered the binding site of the transcription factor NRF1. Binding of NRF1 to the FTH1 promoter was confirmed by electrophoretic mobility shift assay (EMSA) analysis. Thus, our findings revealed the NRF1 was a negative regulator, and lossed of binding of NRF1 to duFTH1 promoter due to -55C/T mutation enhances duFTH1 expression in non-morbid ducks, which provided molecular insights into the effect of duFTH1 expression via promoter polymorphisms, but not DNA methylation, in response to DHV-1 challenge., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Context: The flavonoid prescription baicalin-linarin-icariin-notoginsenoside R1 (BLIN) has a curative effect on duck virus hepatitis (DVH) caused by duck hepatitis A virus type 1 (DHAV-1). However, the mechanism of this curative effect is not understood., Objective: This study investigates the mechanism of the curative effect of BLIN on DVH caused by DHAV-1. We analyzed the anti-DHAV-1 reproduction mechanism and immuno-regulatory effect of BLIN., Materials and Methods: The anti-DHAV-1 reproduction effects of BLIN at 20, 10, 5 and 2.5 μg/mL in vitro, as well as the influence of BLIN at 20 μg/mL on DHAV-1 adsorption, replication and release were tested using the qRT-PCR method. The promotion abilities of BLIN at 20, 10, 5 and 2.5 μg/mL on T- and B-lymphocyte proliferation were investigated by the MTT method. IL-2 and IFN-γ levels and total anti-DHAV-1 antibody secretion after treatment with DHAV-1 for 4, 8 and 54 h were determined by ELISA., Results: BLIN showed a dose-dependent DHAV-1 reproduction inhibitory effect. The inhibitory effect was highest at 20 μg/mL, where DHAV-1 adsorption and release were significantly lower. Meanwhile, BLIN at 5 μg/mL significantly increased T and B lymphocyte proliferation. BLIN stimulated total anti-DHAV-1 antibody secretion in ducklings at the dosage of 4 mg per duckling, but did not stimulate IL-2 and IFN-γ secretion significantly., Conclusions: BLIN inhibits DHAV-1 reproduction by suppressing its adsorption and release. Additionally, BLIN promoted the duckling antiviral response.

Duck hepatitis A subtype 1 virus (DHAV-1) infection causes high mortality in ducklings, resulting in significant losses to duck industries. VP3 is a structural protein of DHAV-1. However, B-cell epitopes on VP3 have not been investigated. To stimulate VP3 antibody response, eukaryotic expression plasmid pCI-neo-VP3 was constructed and used as DNA immunogen to prepare mAbs. Western blot showed that 25.5 kDa VP3 could be detected by mAbs in duck embryo fibroblast (DEF) cells transfected with pCI-neo-VP3. Immunofluorescence assay showed that mAbs could specifically bind to DEF cells infected with DHAV-1. DAPI staining indicated that VP3 localizes to the cytoplasm and nucleus of DHAV-1 infected DEF. With neutralizing mAb 3B7, minimal epitope PSNI was mapped. Sequence alignment indicated that 205 PSNI 208 is highly conserved among DHAV-1, but different from those of DHAV-2 and DHAV-3. Epitope peptide reacted specifically with DHAV-1-positive duck sera by dot blotting, revealing PSNI is DHAV-1 type-specific epitope and the importance of these amino acids in antibody-epitope binding reactivity. These findings provided useful information for understanding the antigenicity of VP3 and might be valuable in the development of epitope-based vaccine or diagnostic kit for DHAV-1 infection and provide insights for understanding the pathogenesis of DHAV-1.

Because of their better immunogenicity and the improved protection they afford, live attenuated vaccines derived from serial passaging in an abnormal host are widely used to protect humans or animals from certain pathogens. Here, we used a virulent and a chicken embryo-attenuated duck hepatitis A virus genotype 1 to compare the different regulated immune responses induced by viruses with differing virulence. In this study, the attenuated strains had lower protein expression levels than the virulent strains as identified by immunohistochemistry. This may be caused by apparent codon usage bias selected during passage. Furthermore, lower translation efficiency led to decreased viral replication, which is highly dependent on non-structural viral protein expression. Although the two strains had differing levels of virulence, both could induce strong innate immune responses and robust Tc or Th cell populations during the early stages of the immune response. However, due to fixed single nucleotide polymorphisms (SNPs) selected by passage, the virulent and attenuated strains may induce differing immune responses, with stronger Tc cell immunity induced by the attenuated strain in the spleen and thymus and stronger Tc cell immunity induced by the virulent strain in the liver, lung, bursa of Fabricius and Harderian gland. Four immune related genes (RIG-1, MDA5, IFN-β, and IL-6) were highly differentially expressed in the Harderian gland, bursa of Fabricius and thymus. This study has provided further information about differences in virus-host interactions between duck hepatitis A viruses of differing virulence.

Duck hepatitis A virus 1 (DHAV-1) is the principal pathogen that causes duck viral hepatitis (DHV), a highly fatal infectious disease in ducklings. Given the importance of the humoral immune response in the clearance of DHAV-1, indirect enzyme-linked immunosorbent assays (I-ELISAs) to detect immune indices, including IgG, IgM and IgA1, were developed and evaluated in this study. The optimal concentrations of coating-antigen were 1.79μg/ml, 2.23μg/ml and 2.23μg/ml for IgG, IgM and IgA1, respectively. Meanwhile, the optimal dilutions of sera were 1:80, 1:40 and 1:40, respectively; and of the conjugates were 1:300, 1:1800 and 1:800, respectively. Based on these conditions, three linear regression equations, y=1.363+1.954x (r 2 =0.983), y=1.141+2.228x (r 2 =0.970) and y=1.103+1.559x (r 2 =0.995) were derived for IgG, IgM and IgA1, respectively. Analytical sensitivities of the new methods were 1:2560, 1:1280 and 1:640 for IgG, IgM and IgA1, respectively. The concordances between the I-ELISAs and serum-neutralization were 95.2% for IgG and IgA1, and 75% for IgM. Although there was a weak cross-reaction with DHAV-3 positive serum for the IgG and IgA1 tests, it didn't affect the ability to detect DHAV-1 specific antibodies. Thus, these new I-ELISAs were shown to be potentially convenient methods to survey the status of humoral immune response to DHAV-1., (Copyright © 2016 Elsevier B.V. All rights reserved.)

An indirect enzyme-linked immunosorbent assay (I-ELISA) based on the recombinant VP3 protein of duck hepatitis A virus type 1 (DHAV-1) was developed and evaluated in this study. The optimal antigen, serum and enzyme-labeled antibody dilutions were 1:160 (0.94μg), 1:160 and 1:2000, respectively. The optimal blocking buffer was 1% gelatin. The cutoff value was determined to be 0.332, and the analytical sensitivity was 1:1280 (OD450-630=0.37). The results of the specificity evaluation showed that no cross-reactivity existed between DHAV-1 antiserum and other common duck-sensitive pathogens, except for duck hepatitis A virus type 3 (DHAV-3), suggesting that this could be a common approach for the simultaneous detection of DHAV-1 and DHAV-3 antibodies. The coefficients of variation (CVs) for all of the tested samples were lower than 10%. The concordance between the I-ELISA based on the VP3 subunit of DHAV-1 and that based on the whole DHAV-1 particle was 96%. These results indicate that the VP3-based I-ELISA method has high sensitivity, specificity, and repeatability and is as effective as the DHAV-1-based I-ELISA method for sero-surveillance. Thus, it may be a convenient and novel method for DHAV antibody detection and epidemiological surveillance of DHAV prevalence., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Duck virus hepatitis (DVH), mainly caused by duck hepatitis A virus (DHAV), is a severe disease threaten to duck industry and has worldwide distribution. As the major structural protein, the VP1 protein of DHAV is able to induce neutralizing antibody in ducks. In this study, a monoclonal antibody (mAb) 4F8 against the intact DHAV-1 particles was used to identify the possible epitope in the three serotypes of DHAV. The mAb 4F8 had weak neutralizing activities to both DHAV-1 and DHAV-3, and reacted with the conserved linear B-cell epitopes of (75)GEIILT(80) in DHAV-1 VP1 and (75)GEVILT(80) in DHAV-3 VP1 protein, respectively, while not with DHAV-2 VP1. This was the first report about identification of the common conserved neutralizing linear B-cell epitope of DHAV-1 and DHAV-3, which will facilitate understanding of the antigenic structure of VP1 and the serologic diagnosis of DHAV infection., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Bush Sophora Root polysaccharide (BSRPS) and its sulfate, sulfated Bush Sophora Root polysaccharide (sBSRPS), possess the antiviral activities against duck hepatitis A virus. However their antiviral mechanisms are still not clear. This paper reported their immuno-enhancing roles in the therapeutic effects for duck virus hepatitis (DVH). The effects of BSRPS and sBSRPS on stimulating lymphocyte proliferation were investigated by MTT methods. After that, ducklings were challenged with DHAV and treated with BSRPS and sBSRPS. Meanwhile, the total antibody (Ab), cytokines including interferon gamma (IFN-γ), hepatocyte growth factor (HGF), interleukin (IL)-2, IL-6 and IL-8 were determined by enzyme-linked immuno sorbent assay methods. The results showed that BSRPS owned a fine hepatoprotective effect with stable HGF producing ability. Sulfated modification was able to increase the proliferation rates of B and T lymphocytes and the secretions of total Ab, IFN-γ and IL-2, as comparison with those of BSRPS group. In summary, both of them exhibited immuno-enhancing effects on the therapeutic effects for DVH, and the capacity of sBSRPS was stronger than that of BSRPS., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Objective: To simultaneously detect antibodies against Duck hepatitis A type 1 (DHAV-1) and type 3 (DHAV-3) viruses, we developed an indirect enzyme-linked immunosobent assay (ELISA) with bacterially expressed recombinant viral protein as antigen in Escherichia coli., Methods: We amplified the full-length VP3 gene of DHAV-1 and the full-length VP1 gene of DHAV-3 through reverse transcription-polymerase chain reaction (RT-PCR) and then cloned them into pET-32a expression vector, designated as pET-1VP3-3VP1. The fusion protein DHAV-1VP3-3VP1 expressed correctly and was subsequently used to develop an indirect ELISA assay., Results: DHAV-1VP3-3VP1 fusion protein expressed in BL21 (DE3) cells following induction by Isopropyl-beta-D-1-thiogalactopyranoside (IPTG). The expressed protein was very antigenic and reactive to virus-specific antibodies in western blot assay. The optimal working concentration for coating antigen was 1.0 microg per well and the working concentration of serum samples was 1:200 dilution and the cut-off value that distinguished the positive from negative serum samples was OD650 > OR = 0.38., Conclusion: The ELISA method based on the prokaryotic expression of VP3 (DHAV-1) and VP1 proteins (DHAV-3) can be used effectively for the clinical detection antibodies against DHAV-1 and DHAV-3.

The interferon-induced proteins with tetratricopeptide repeats (IFITs) protein family mediates antiviral effects by inhibiting translation initiation, cell proliferation, and migration in the interferon (IFN) dependent innate immune system. Several members of this family, including IFIT1, IFIT2, IFIT3 and IFIT5, have been heavily studied in mammals. Avian species contain only one family member, IFIT5, and little is known about the role of this protein in birds. In this study, duck IFIT5 (duIFIT5) full-length mRNA was cloned by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of the cDNA ends (RACE). Based on the sequence obtained, we performed a series of bioinformatics analyses, and found that duIFIT5 was most similar to homologs in other avian species. Also, duIFIT5 contained eight conserved TPR motifs and two conserved multi-domains (TPR&#95;11 and TPR&#95;12). Finally, we used duck hepatitis virus type 1 (DHV-1) and polyriboinosinicpolyribocytidylic acid (poly (I:C)) as a pathogen or a pathogen-associated molecular pattern induction to infect three-day-old domestic ducklings. The liver and spleen were collected to detect the change in duIFIT5 transcript level upon infection by quantitative real-time PCR (qRT-PCR). DuIFIT5 expression rapidly increased after DHV-1 infection and maintained a high level, while the transcripts of duIFIT5 peaked at 8h after poly (I:C) infection and then returned to normal. Taken together, these results provide a greater understanding of avian IFIT5.

Background: The VP1 protein of duck hepatitis A virus (DHAV) is a major structural protein that induces neutralizing antibodies in ducks; however, B-cell epitopes on the VP1 protein of duck hepatitis A genotype 1 virus (DHAV-1) have not been characterized., Methods and Results: To characterize B-cell epitopes on VP1, we used the monoclonal antibody (mAb) 2D10 against Escherichia coli-expressed VP1 of DHAV-1. In vitro, mAb 2D10 neutralized DHAV-1 virus. By using an array of overlapping 12-mer peptides, we found that mAb 2D10 recognized phages displaying peptides with the consensus motif LPAPTS. Sequence alignment showed that the epitope 173LPAPTS178 is highly conserved among the DHAV-1 genotypes. Moreover, the six amino acid peptide LPAPTS was proven to be the minimal unit of the epitope with maximal binding activity to mAb 2D10. DHAV-1-positive duck serum reacted with the epitope in dot blotting assay, revealing the importance of the six amino acids of the epitope for antibody-epitope binding. Competitive inhibition assays of mAb 2D10 binding to synthetic LPAPTS peptides and truncated VP1 protein fragments, detected by Western blotting, also verify that LPAPTS was the VP1 epitope., Conclusions and Significance: We identified LPAPTS as a VP1-specific linear B-cell epitope recognized by the neutralizing mAb 2D10. Our findings have potential applications in the development of diagnostic techniques and epitope-based marker vaccines against DHAV-1.

Previous studies of duck hepatitis A virus infection have focused only on the pathogenicity and host response of one strain. Here, we show that the virulent SH strain and the attenuated FC64 strain induced varied pathogenicity, apoptosis and immune responses in the livers of 1-day-old ducklings. SH infection caused apoptosis and visible lesions in the liver; serum aspartate aminotransferase, alanine transaminase, alkaline phosphatase, γ-glutamyltransferase and total bilirubin activities were markedly upregulated; and ducklings died at 36 h post-infection (p.i.). However, FC64 infection did not induce significant symptoms or impair liver function, and all of the infected ducklings remained healthy. In addition, both virus strains replicated well in the liver, spleen and intestine, whilst the SH strain replicated more efficiently than FC64. IFN-γ, IL-2, inducible nitric oxide synthase and nitric oxide were strongly induced by SH infection, and may be associated with the pathogenicity of the SH strain. IFN-α, IFN-β, IFN-stimulated transmembrane protein 1, IFN-stimulated gene 12, 2',5'-oligoadenylate synthetase-like and IL-6 were moderately induced by SH infection at 24 h p.i., and dramatically induced by FC64 infection at 36 h p.i. The intensive induction of cytokines by FC64 may be involved in restriction of virus replication and stimulation of adaptive immune responses. Ducklings inoculated with FC64 produced high levels of antiviral antibodies within 45 days p.i. The low virulence and strong immune response of FC64 rendered this strain a good vaccine candidate, as confirmed by a protective assay in this study., (© 2014 The Authors.)

The VP1 protein of duck hepatitis type 1 virus (DHV-1), one of the major structural proteins, is able to induce neutralizing antibody in ducks, but a monoclonal antibody (mAb) against VP1 protein has never been characterized. Four hybridoma cell lines secreting anti DHV-1A VP1 mAbs were prepared and designated 2D9, 2D10, 5F7, and 3E8. Immunoglobulin subclass tests differentiated them as IgG1 (2D9 and 2D10) and IgG2b (5F7 and 3E8). Dot blot and western blotting assays showed that mAbs reacted with His-VP1 protein in a conformation-independent manner. Competitive binding assays indicated that mAbs delineated three epitopes, namely A, B and C, of VP1. Immunofluorescence assays indicated that mAbs could specifically bind to duck embryo fibroblast (DEF) cells infected with DHV-1A. mAbs 2D9, 2D10, and 5F7 had universal reactivity to heterologous DHV-1As tested in an antigen-capture ELISA, suggesting that they are highly conserved among DHV-1As. An antigen-capture ELISA could detect DHV-1A protein VP1 with a clear difference in absorbance values between the liver samples of DHV-1A- and mock-infected birds, indicating that the mAb capture ELISA is a useful method for the detection of DHV-1A infections., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Duck hepatitis virus (DHV) affects 1-week-old but not 3-week-old ducks, and it causes a more severe disease in the younger ducks. These differences may be partially due to the host response to DHV infection. In order to understand this difference, we characterized the pathobiology of and innate immune response to DHV infection in 1-day-old (1D) and 3-week-old (3 W) ducks. Viral RNA was detected in duck livers at 24, 36 and 72 h after inoculation with DHV at a dose of 10(3) LD50. Virus-induced pathology ranged from no clinical signs to severe disease and death, and it was more severe in the 1D ducks. Infection with DHV induced up-regulation of gene expression of Toll-like receptor (TLR)-7, TLR3, retinoic-acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA-5), interleukin (IL)-6, interferon (IFN)-α, interferon-induced transmembrane protein 1 (IFITM1), interferon-stimulated gene 12 (ISG12), and 2'-5' oligoadenylate synthetase-like gene (OASL) in the livers of 3 W ducks. Of these, IL-6, OASL and ISG12 mRNA levels were more than 100-fold higher in infected 3 W ducks than in mock-infected ducks of the same age. These genes were induced much less in infected 1D ducklings. We present evidence that a lower level of viral replication in the hepatocytes of 3 W ducks, whose basal level of cytokines is higher than that in 1D ducklings, may be related to the strong innate immunity induced. From our data, we conclude that duck age plays an important role in the pathogenicity of and innate immune responses to DHV.

The VP1 gene of duck hepatitis virus type 1 (DHV-1) strain VJ09 was amplified by reverse transcription PCR from the liver of a duckling with clinical symptoms of viral hepatitis. The resulting VP1 cDNA was 720 bp in length and encoded a 240-amino-acid protein. In VP1 gene-based phylogenetic analysis, the VJ09 strain grouped with DHV-1 genotype C. The VP1 gene was inserted into the expression vector pPICZαA and expressed in Pichia pastoris. The expressed VP1 protein was purified and identified by western blot analysis. To evaluate the recombinant VP1's immunogenic potential in ducklings, the antibodies raised in the immunized ducklings were titrated by ELISA, and lymphocyte proliferation and virus neutralization assays were performed. The results show that the recombinant VP1 protein induced a significant immune response in ducklings and this could be a candidate for the development of a subunit vaccine against DHV-1 genotype C.

VP1 protein, the capsid protein of duck hepatitis A virus (DHAV), contains critical epitopes for inducing a protective immune response. Due to its low-level expression in Escherichia coli (E. coli), the function of this protein is poorly characterized. In this study, a codon-optimized VP1 gene was chemically synthesized in terms of the codon usage bias in E. coli and subcloned into pET32a (+) to increase its expression. The recombinant VP1 fusion protein was purified from inclusion body by Ni(2+) affinity chromatography His-Bind Resin and used to raise the rabbit anti-DHAV-VP1 polyclonal antibody. The expression of the codon-optimized VP1 gene in E. coli was significantly increased when compared to the wild-type VP1 gene, having an at least 17-fold increase. Western blot analysis showed that the recombinant protein was recognized by the rabbit anti-DHAV polyclonal antibody. Western blot also demonstrated that the rabbit anti-DHAV-VP1 polyclonal antibody could recognize the purified VP1 fusion protein specifically, and in the indirect immunofluorescent assays (IFA), the antibody was able to probe the VP1 protein in DHAV-1 infected cells. In conclusion, codon optimization increased dramatically DHAV VP1 expression in E. coli and the His-tagged VP1 fusion protein showed good antigenicity and immunogenicity., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A suicidal DNA vaccine based on a Semliki Forest virus (SFV) replicon was evaluated for the development of a vaccine against duck hepatitis virus type 1 (DHV-1). The VP1 gene of DHV-1 was cloned and inserted into pSCA1, an SFV DNA-based replicon vector. The resultant plasmid, pSCA/VP1, was transfected into BHK-21 cells and the antigenicity of the expressed protein was confirmed using an indirect immunofluorescence and western blot assay. Immunogenicity was studied in ducklings. Ducklings were injected intramuscularly two times with pSCA/VP1 at 14 days intervals. Anti-DHV-1 antibodies were detected by ELISA, the lymphocyte proliferation response was also tested by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide method and neutralizing antibodies were measured by microneutralization tests. Our results showed that DHV-1-specific antibodies, neutralizing antibodies and lymphocyte proliferation were well induced in ducklings. Furthermore, all the ducklings were protected against challenge with wild DHV-1. In conclusion, we demonstrate that the suicidal DNA vaccine is a promising vaccine candidate facilitating the prevention of duck hepatitis caused by DHV-1., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Duck hepatitis A virus genotype C (DHAV-C), recognized recently, is one of the pathogens causing fatal duck viral hepatitis in ducklings, especially in Asia. To demonstrate the pathogenesis of the DHAV-C isolate, 3-day-old specific pathogen free ducklings were inoculated subcutaneously with a DHAV-C isolate and the clinical signs were observed. Virus distribution, histological and apoptotic morphological changes of various tissues were examined at different times post inoculation. The serial, characteristic changes included haemorrhage and swelling of the liver. Apoptotic cells and virus antigen staining were found in all of the tissues examined. Where more virus antigen staining was detected, there were more severe histopathological and apoptotic changes. The amount of virus antigen and the histological and apoptotic morphological changes agreed with each other and became increasingly severe with length of time after infection. Apoptotic cells were ubiquitously distributed, especially among lymphocytes, macrophages and monocytes in immune organs such as the bursa of Fabricius, thymus and spleen, and in liver, kidney and cerebral cells. Necrosis was also observed within 72 h post inoculation in all organs examined, except the cerebrum, and was characterized by cell swelling and collapsed plasma membrane. These results suggest that the recent outbreak of disease caused by DHAV-C virus is pantropic, causing apoptosis and necrosis of different organs. The apoptosis and necrosis caused by the DHAV-C field strain in this study is associated with pathogenesis and DHAV-C-induced lesions.

Duck hepatitis virus type 1 (DHV-1) causes a highly contagious disease in ducklings and is often associated with liver necrosis, hemorrhages, and high mortality. In the current study, the expression levels of gene transcripts encoding proinflammatory cytokines and the virus were measured by quantitative reverse-transcription PCR in duck livers after infection with a DHV-1 JX isolate obtained from natural cases in Hubei Province, China. In addition, sera IL-1β, IL-6, and alanine aminotransferase levels were quantified. Liver histopathology was examined following DHV-1 infection. The ducklings died within 1 to 2 d postinfection (d.p.i.) because of typical liver degeneration, hemorrhage, necrosis, and bile-duct epithelial cell proliferation. Transcripts of the cytokines IFN-α, IL-6, TNF-α, and IL-10 decreased by 0.5 d.p.i. and then gradually increased at 1 d.p.i. Similarly, DHV-1 JX 3D gene levels in the liver sharply increased at 1 d.p.i. and then maintained a high level. In contrast, liver TNF-α and IL-1β transcripts showed no increased expression of the cytokine gene postinfection and significantly decreased compared with the expression at 0.25 d.p.i., only the expression of IFN-α transcripts increased 128-fold by 1 d.p.i. Changes in the serum IL-6 level remained relatively stable postinfection and not significantly different compared with that of the control (P > 0.05), whereas serum levels of IL-1β significantly decreased at 0.5 d.p.i. and increased from 1 d.p.i. onwards (P < 0.05). Serum alanine aminotransferase levels significantly increased 2 d.p.i. compared with that of the control group (P < 0.01), which seemed to keep with the number of dead ducks. The cytokines exhibited a biphasic pattern following DHV-1 JX infection. Taken together, the data indicated that duckling liver inflammatory responses were produced following experimental DHV-1 JX infection involving multiple cytokines.

Three novel Anas platyrhynchos avian β-defensins (Apl&#95;AvBDs), Apl&#95;AvBD4, 7 and 12, were identified successfully and characterized in tissues from Peking ducks in the present study. The cDNA fragment of Apl&#95;AvBD4 contained 171 bp, and encoded 56 amino acids. The complete nucleotide sequences of Apl&#95;AvBD7 and 12 contained 204 bp and 198 bp open reading frames, which encoded 67 and 65 amino acids, respectively. Both recombinant and synthetic forms of the three Apl&#95;AvBDs showed antibacterial activity against most of the bacteria investigated, including Gram-negative and Gram-positive bacteria, except for Salmonella choleraesuis. In addition, the antibacterial activity of all the three Apl&#95;AvBDs decreased significantly in 150 mM NaCl. Significant antiviral activity of the three Apl&#95;AvBDs was shown against duck hepatitis virus (DHV). However, none of the Apl&#95;AvBDs showed significant hemolytic activity. Additionally, the expressions of the three Apl&#95;AvBDs in response to DHV infection was highly variable, and significant upregulation of Apl&#95;AvBD7 in liver was found in response to infection at different time points. Expression of Apl&#95;AvBD4 in thymus, and of Apl&#95;AvBD7 in bone marrow was induced in a time-dependent fashion by DHV infection. In contrast, expression of Apl&#95;AvBD12 was found to be significantly decreased, and was hard to detect in cecal tonsil, spleen, bursa of Fabricius, and thymus of ducks at some time points after DHV infection. The present results demonstrate that Apl&#95;AvBDs play vital roles in the immune response of ducks against bacterial and viral pathogens., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The VP1-encoding gene of the duck hepatitis type 1 virus (DHV-1) HP-1 strain was cloned and expressed in Escherichiacoli. The open reading frame (ORF) of VP1 comprised 714 bp and encoded 238 amino acids, with a predicated molecular mass of 26.5 kDa. The expressed VP1 fusion protein in E. coli was detected by Western blotting with duck anti-DHV-1 polyclonal serum. A VP1-ELISA using the expressed VP1 protein as a coating antigen for the detection of antibodies to DHV-1 in ducks was developed. In comparison with the virus neutralization test, the specificity and sensitivity of the VP1-ELISA was 92.5% and 96.7%. Comparative analysis between Western blots and the VP1-ELISA showed that the concordance between the two methods was 86%. The VP1-ELISA did not react with the anti-sera to other duck viral pathogens, implying that this protein is specific for the recognition of duck anti-DHV-1 antibodies. Taken together, the VP1-ELISA is a highly sensitive and specific test that could be used for screening for DHV-1 infection and monitoring antibody titres against DHV-1., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Objective: We developed an indirect enzyme-linked immunosorbent assay (ELISA) by the recombinant VP1 protein of duck hepatitis virus (DHV) expressed in Escherichia coli to detect antibodies against DHV., Methods: The VP1 gene of DHV was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into pMD18-T and pET-32a vectors to get a prokaryotic expression vector pET-32a-VP1. DHV VP1 gene was expressed and analyzed. A method of enzyme-linked immunosorbent assay (ELISA) was developed and studied., Results: We obtained the recombinant plasmid pET-32a-VP1 with correct sequence and orientation. DHV VP1 gene was expressed in E. coli BL21 (DE3) at a high level and had good immunoreactivity by SDS-PAGE and western blot. The optimum working concentration of antigen was 5.0 microg in 100 microL per well, the working concentration of serum samples was 1 : 100 dilution and the critical value was OD450 > or = 0.302 for the ELISA assay. The rate of coincidence of ELISA and virus neutralization test (VN) was 97.5% by detecting 80 serum samples. The method was specific, sensitive and could be applied for antibody detection of maternal antibody and the rule of antibody growth and decline in immunizing ducklings., Conclusion: The ELISA method developed by the purified recombinant protein could be used to detect antibodies against DHV.

Duck hepatitis strains 90D and 04G were determined to be antigenically unrelated to type 1 duck hepatitis virus (DHV-1) by in vitro cross-neutralization assay. The genome sequences of 90D and 04G revealed that both strains of the new serotype DHV (N-DHV) possessed a typical picornavirus genome organization apart from the unique possession of three in-tandem 2A genes present in DHV-1. The 2A1, 2A2, and 2A3 proteins represented an aphthovirus-like 2A protein, AIG1-like protein, and human parechovirus-like 2A protein, respectively. The N-DHV genome displayed unique features, compared to the DHV-1 genome. The 366 nt 3'UTR of N-DHV, the largest determined thus far among picornaviruses, was 52 nt longer than DHV-1. The pairwise percent identity of the nucleic acid and amino acid sequences at 1D region of N-DHV and DHV-1 were only 69.1-69.7 and 70.1-70.5%, respectively. Finally, phylogenetic and evolutionary analysis of N-DHV revealed that the N-DHV and DHV-1 belong to two different clusters of a novel genus in the Picornaviridae family.

Sera from wild geese were tested for antibodies to selected viral pathogens at a resting site for wild waterfowl in Germany. Serum samples from both bean geese (Anser fabalis) and white-fronted geese (Anser albifrons) collected in October 1991 were examined using serological methods licensed for routine diagnosis in domestic poultry. Of 130 sera tested, antibodies to several infectious agents were found including Newcastle disease virus (45%), goose parvovirus (48%), avian reovirus (29%), and avian adenovirus or egg drop syndrome 76 virus (6%). Antibodies against duck hepatitis virus were not detected. Differences in seroprevalences were not detected between the two geese species. While role and significance of wild geese in the epidemiology of avian diseases remains to be determined, it is possible that they could be of some importance as reservoirs and carriers of certain viral diseases of domestic poultry.

The most important virus-induced diseases associated with heavy losses in the domestic goose are Derzsy's disease which is caused by a goose parvovirus and duck plague (duck viral enteritis) which is caused by an avian herpesvirus. Both diseases still occur but can be prevented by timely vaccinations. Antibodies against Influenza A viruses of the subtypes H1, H5, and H7 as well as against avian paramyxoviruses of the serogroups 4, 6, and 8, respectively, were not detected in any of the examined sera. However, antibodies against paramyxovirus type 1 were detected in sera of one source. Haemagglutination inhibition or neutralizing antibodies against avian adenoviruses (EDS76 virus and goose adenovirus of the serotypes 1, 2, and 3) were quite often detected. Based on the present knowledge their pathogenic potential is minor. Neutralizing antibodies against a reovirus originating from Muscovy ducks and against a chicken reovirus (strain U Con S 1133) were quite frequently detected. In 35 of 564 examined geese sera hepatitis B virus was found.

To define the residues involved in duck hepatitis B virus (DHBV) neutralization at the amino acid level, we have used a procedure combining monoclonal antibodies (MAbs) and overlapping octapeptides (Pepscan). Two neutralizing MAbs (SD20 and 900), specific for the pre-S protein were shown to reduce DHBV infectivity in vivo by 75 and 90%, respectively, while complete protection of ducklings was achieved with a polyclonal antiserum raised against the bacterially expressed first 131 amino acids of the DHBV pre-S region (DHBpre-S). Using fusion polypeptides, the binding sites of these MAbs were localized between aa 77 and 100 on pre-S protein. We have used octapeptides spanning the pre-S sequence from aa 64 to 115 for fine mapping of these epitopes. Within the sequence scanned, the polyclonal anti-DHBpre-S antiserum recognized a region exclusively limited to the residues E82-K95, suggesting immunodominance of this region in the sequence aa 64-115. The epitope recognized by Mab 900 was mapped within the same region, whereas the epitope recognized by Mab SD20 was localized downstream from this region. To define the amino acids essential for binding to the highly neutralizing Mab 900, we have used single amino acid replacement and demonstrated that two residues Q87 and W88 were important for antibody recognition.

A duck hepatitis virus (DHV), isolated from ducks on a farm in Virginia in 1963, was shown to be only partially related to DHV type I (DHV-I) in cross-neutralization and in cross-protection tests. The virus, named DHV-Ia, apparently is a serologic variant of DHV-I; both viruses are serologically distinct from DHV type III. Pathologic responses to DHV-Ia were similar to those described for DHV-I infection.

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibody to duck hepatitis virus (DHV) is described. The results of ELISA were compared with those of an agar gel diffusion precipitin (AGDP) test and a serum-neutralization (SN) test. The specificity of the ELISA was in accordance with the specificity of the AGDP and SN tests, but there was a difference in sensitivity. The positive detection rates of ELISA, SN test, and AGDP test for 93 clinical samples were 68.8%, 68.8%, and 18.8%, respectively. A positive/negative (P/N) value larger than or equal to 2.1 plus an absorbance value larger than or equal to 0.4 was used as a comprehensive positive standard for the ELISA. This eliminated false-positive reactions. The results showed that the ELISA was a rapid, sensitive, and accurate method for detecting antibody to DHV.

Ducks were induced to develop high-level duck hepatitis virus (DHV)-neutralizing antibodies by inculation with a chicken-embryo-adapted DHV via subcutaneous, intramuscular, and intratracheal routes. Administration of the DHV orally in a gelatin capsule failed to stimulate immune response in the ducks. Contact controls of these ducks also remained negative for anti-DHV antibodies. These observations indicated that the DHV administered orally, in gelatin capsule, failed to infect the ducks. None of numerous duck anti-DHV immune sera, with virus-neutralizing activity in the range of 1.8 to 5.57 log10 median- embryo-infective-dose (EID50) neutralization index, developed precipitin lines against a variety of DHV preparations tested in low- and high-ionic-strength agar. The results suggest that the agar-gel immunodiffusion test is unsuitable for serologic testing of duck sera for anti-DHV antibody activity. Virus-neutralizing activity was revealed in both immunoglobulin M (IgM) and IgG classes of sera of actively immunized ducks. Immunodiffusion tests of Sephadex G-200 fractions of 1-day-old duckling sera with monospecific rabbit anti-duck IgM (DIgM) serum failed to detect DIgM. These results demonstrated that the IgM is not being transferred from the dam to the newly hatched ducklings. Seven- and 14-day-old ducks had DIgM in their sera. However, this IgM had no DHV-neutralizing activity, indicating that it was newly developed by the ducklings, which had no active DHV immune response, not having been exposed to DHV.

Neutralising antibodies were present in duckling serum four days after vaccination of two-day-old ducklings with live duck hepatitis virus vaccines. The antibodies were found in the macroglobulin and 7S fractions by Sephadex G200 chromatography, had gamma or beta 2 mobility in immunoelectrophoresis and reacted with an antiserum to duck immunoglobulins.

Duck hepatitis, a highly fatal disease of ducklings, must be controlled in successful commercial duck enterprises. Several procedures which have been effective in controlling the disease are 1) vaccination of adult breeders with passive transfer of antibodies through the yolk to the duckling, 2) administration of antisera to ducklings at the onset of signs of disease, and 3) parenteral vaccination of ducklings with attenuated DH virus after the loss of passive immunity. However, in each of these procedures, some losses usually occur and the administration of antiserum or vaccine to 2- to 3-week-old ducklings is costly and involves physical stress. Experimental studies of oral immunization in ducklings from immunized and nonimmunized parents were conducted to determine the feasibility of a convenient control program with little stress to the ducklings. Ducklings immunized with attenuated live duck hepatitis virus by intramuscular method, oral instillation, and through the drinking water were challenged with virulent duck hepatitis virus either intramuscularly or orally. Intramuscular, oral instillation, and drinking water application all provided adequate protection on challenge with virulent virus. Rise in neutralizing antibody was detected following immunization especially in sera of ducklings from nonimmunized parents. On the basis of successful experimental results, over 350,000 ducklings from immunized parents on a commercial farm have been sucessfully vaccinated by the drinking water method.

An assay to determine 50 per cent neutralisation end points of duck anti-duck hepatitis virus sera was developed using a plaque assay to quantify residual virus. The optimal conditions were determined. Ducklings produce a serological response within four days of vaccination and the response reaches a maximum within nine days.

Egg-attenuated duck hepatitis type (Rispens H55) was exhaustively tested as a potential vaccine under controlled conditions in ducklings fully susceptible to the disease at day 2 after hatching. Data are presented which indicate that this vaccine fulfils essential criteria of efficacy in terms of (a) the optimal age at which successful vaccination is practicable (day 2 or earlier), (b) the rapidity of onset of immunity (in 48 to 72 hours), (c) the high level of immunity induced (88.0 to 94.0 per cent), (d) the persistence of this degree of immunity in the individual bird throughout the period when it would otherwise be at risk (until the end of the fourth week of life) and (e) the consistency of the effects of the vaccine in successive groups of ducklings hatched over a four year period. Employed as a vaccine. H55 was completely innocuous to the vaccinated ducklings under laboratory conditions.

The in vitro isolation, propagation, and characterization of duck hepatitis virus Type III (DHV-III), is described. This virus, which is serologically distinct from the classical (Type I) DHV, replicated in liver and kidney cell cultures of duck origin. Replication was limited in chicken and quail kidney and duck embryo fibroblast cultures. It did not replicate in a variety of other cell cultures of avian or mammalian origin. The virus was grown successfully in embryonating eggs of ducks, but not of chickens. DHV-III passed through a 50-nm membrane filter, was stable at pH 3.0 and resisted treatment with 5% chloroform. Virus growth was not inhibited by treatment with 5-iodo-2-deoxyuridine. Electron-microscope examination revealed crystalline arrays in the cytoplasm; virus particles had cubic symmetry, and were about 30 nm in diameter. By these properties, this virus can be classified as a member of the picornavirus group.

The immune response to duck hepatitis B virus (DHBV) had not been elucidated. An assay was therefore established to detect the presence of antibody to DHB surface antigen (anti-DHBs) in serum of experimentally inoculated and naturally infected ducks. Anti-DHBs in serum was detected by indirect RIA from the percentage inhibition of binding of rabbit anti-DHBs to purified DHBsAg. Specificity was confirmed by positive and negative controls, infected and noninfected sera, and a mouse monoclonal antibody to DHB core antigen (anti-DHBc). Serum and liver samples were tested for DHBV DNA by dot-blot hybridization assay. Adult ducks repeatedly inoculated with DHBV remained non-viraemic but developed anti-DHBs. This antibody activity neutralized the infectivity of DHBV, which was experimentally inoculated into 1-day-old ducklings. In naturally infected flocks anti-DHBs was detected in a proportion of noninfected adult ducks as well as 1-day-old hatchlings. Anti-DHBs activity in hatchlings neutralized the infectivity of experimentally inoculated DHBV. Pekin ducks can therefore mount a neutralizing antibody response to DHBV, and immunity may be transferred in ovo from dam to off-spring.