Recombinant Duck Enteritis Virus-Vectored Bivalent Vaccine Effectively Protects Against Duck Hepatitis A Virus Infection in Ducks

Duck enteritis virus (DEV) and duck hepatitis A virus (DHAV) are prevalent duck pathogens, causing significant economic losses in the duck industry annually. Using a fosmid-based rescue system, we generated two DEV recombinants, rDEV-UL26/27-P13C and rDEV-US7/8-P13C, in which the P1 and 3C genes from DHAV type 3 (DHAV-3) were inserted into the DEV genome between genes UL26 and UL27 or genes US7 and US8. We inserted a self-cleaving 2A-element between P1 and 3C, allowing the production of both proteins from a single open reading frame. P1 and 3C were simultaneously expressed in infected chicken embryo fibroblasts, with no difference in growth kinetics between cells infected with the recombinant viruses and those infected with the parent DEV. Both recombinant viruses induced neutralizing antibodies against DHAV-3 and DEV in ducks. A single dose of the recombinant viruses induced solid protection against lethal DEV challenge and completely prevented DHAV-3 infection as early as 7 days post-vaccination. These recombinant P1- and 3C-expressing DEVs provide potential bivalent vaccines against DEV and DHAV-3 infection in ducks.

Development of a Colloidal Gold Immunochromatographic Assay for Duck Enteritis Virus Detection Using Monoclonal Antibodies

Duck viral enteritis is a highly contagious and fatal disease of commercial waterfowl flocks. The disease occurs sporadically or epizootically in mainland China due to insufficient vaccinations. Early and rapid diagnosis is important for preventive intervention and the control of epizootic events in clinical settings. In this study, we generated two monoclonal antibodies (MAbs) that specifically recognized the duck enteritis virus (DEV) envelope glycoprotein B and tegument protein UL47, respectively. Using these MAbs, a colloidal gold-based immunochromatographic assay (ICA) was developed for the efficient detection of DEV antigens within 15 min. Our results showed that the detection limit of the developed ICA strip was 2.52 × 103 TCID50/mL for the virus infected cell culture suspension with no cross-reactivity with other pathogenic viruses commonly encountered in commercially raised waterfowl. Using samples from experimentally infected ducks, we demonstrated that the ICA detected the virus in cloacal swab samples on day three post-infection, demonstrating an 80% concordance with the PCR. For tissue homogenates from ducks succumbing to infection, the detection sensitivity was 100%. The efficient and specific detection by this ICA test provides a valuable, convenient, easy to use and rapid diagnostic tool for DVE under both laboratory and field conditions.

Duck enteritis virus pUL47, as a late structural protein localized in the nucleus, mainly depends on residues 40 to 50 and 768 to 777 and inhibits IFN-β signalling by interacting with STAT1

Duck enteritis virus (DEV) is a member of the Alphaherpesvirinae subfamily. The characteristics of some DEV genes have been reported. However, information regarding the DEV UL47 gene is limited. In this study, we identified the DEV UL47 gene encoding a late structural protein located in the nucleus of infected cells. We further found that two domains of DEV pUL47, amino acids (aa) 40 to 50 and 768 to 777, could function as nuclear localization sequence (NLS) to guide the nuclear localization of pUL47 and nuclear translocation of heterologous proteins, including enhanced green fluorescent protein (EGFP) and beta-galactosidase (β-Gal). Moreover, pUL47 significantly inhibited polyriboinosinic:polyribocytidylic acid [poly(I:C)]-induced interferon beta (IFN-β) production and downregulated interferon-stimulated gene (ISG) expression, such as Mx and oligoadenylate synthetase-like (OASL), by interacting with signal transducer and activator of transcription-1 (STAT1).

Molecular Characterization and Pathogenicity of an Indian Isolate of Duck Enteritis Virus Recovered From a Natural Outbreak

Background: Duck plague is a highly contagious viral disease reported in our country very often with significant economic loss. There are some bottlenecks with the currently used ‘Holland strain’ vaccine that involves cumbersome process of vaccine production in embryonated chicken eggs. With the future goal of development of an indigenous cell culture vaccine for duck plague, the present study is aimed at isolation of an Indian strain of DEV from a natural outbreak and its characterization for the seed virus purpose. Methods: Liver samples were collected from the suspected ducks died during a natural outbreak in Kerala and subjected to polymerase chain reaction (PCR) to confirm presence of viral DNA. The duck enteritis virus (DEV) was isolated by inoculation of PCR positive samples in embryonated duck eggs/ducklings and its pathogenicity was studied. Further, the DEV recovered from the infected duck embryo and duckling liver was confirmed by PCR amplification of the viral DNA polymerase gene and its sequence analysis. Result: Out of 12 liver samples tested eight (8) were found to be positive for duck plague by PCR. The DEV infected duck embryos and ducklings died showing typical signs and characteristic gross and microscopic lesions. PCR amplification of viral DNA targeting the DNA polymerase gene yielded amplicon of expected size of 446bp. The amplicon sequence showed 99-100% homology with other DEV isolates, thus confirming the new isolate as DEV, named as DEV/India/IVRI-2016 and the gene sequence has NCBI acc. no. KX511893.

Simultaneous Tracking of Capsid, Envelope Protein Localization in Living Cells Infected with Double Fluorescent Duck Enteritis Virus

Background Duck enteritis virus can cause an acute, contagious and lethal disease of many species of waterfowl. An infectious bacterial artificial chromosome clone of duck enteritis virus (DEV) vaccine strain pDEV-EF1 has been constructed in our previous study. To visually studying DEV, a double-labeled recombinant virus have been constructed. Methods based on pDEV-EF1, a recombinant mutated clone pDEV-UL35(c)CFP-gC(c)mRFP which carries a red fluorescent protein(mRFP) gene attached to the viral envelope protein gC, combined with a cyan fluorescent protein (CFP) gene fused to the viral capsid UL35 gene was constructed by two-step Red/ET recombination and the recombinant virus rDEV-UL35(c)CFP-gC(c)mRFP was rescued from chicken embryo fibroblasts (CEFs) by calcium phosphate precipitation. Then protein expression were detected by Western blot analysis, and subcellular location of gC and UL35 were observed by confocal microscopy. Viral morphogenesis were observed by transmission electron microscopy (TEM). Results The recombinant virus rDEV-UL35(c)CFP-gC(c)mRFP was rescued from chicken embryo fibroblasts (CEFs) by calcium phosphate precipitation. Western blot analysis showed UL35-CFP and gC-mRFP are both expressed in fusion forms in rDEV-UL35(c)CFP-gC(c)mRFP-infected CEFs, and subcellular location study showed gC-mRFP was mainly localized in whole cell at 36 h.p.i. and 48 h.p.i.; and then mostly migrated to cytoplasm after 60 h.p.i.; UL35-CFP was localized in nucleus in all stages of virus infection. Transmission electron microscopy indicated that viral particles at different stages of morphogenesis (A capsids, B capsids, C capsids) were observed in virus-infected cells. However,mature C capsids was less in rDEV-UL35(c)CFP-gC(c)mRFP-infected cells than rDEV-dEF1GFP and rDEV-gC(c)mRFP-infected samples. Conclusions This study has laid a foundation for visually studying localization, transportation of DEV capsid protein and envelope glycoprotein, as well as virus assembly, virion movement and virus-cell interaction.