Duck Enteritis Virus Research Articles

Molecular characterization and functional analysis of duck IKKα

IκB kinase α (IKKα) is a vital component of the IKK complex, which is involved in innate immune response, inflammation, cell death and proliferation. Although the functional characteristics of IKKα have been extensively studied in mammals and fish, the roles of IKKα in avian remain largely unknown. In this study, we cloned and characterized the duck IKKα (duIKKα) gene for the first time. DuIKKα encoded a protein of 757 amino acid residues and showed high sequence identities with the goose IKKα. The duIKKα was expressed in all tested tissues, and a relatively high expression of duIKKα mRNA was detected in liver and heart. Overexpression of duIKKα dramatically increased NF-κB activity and induced the expression of duck cytokines IFN-β, IL-1β, IL-6, IL-8 and RANTES in DEFs. Knockdown of duIKKα by small interfering RNA significantly decreased LPS-, poly(I:C)-, poly(dA:dT)-, duck enteritis virus (DEV)-, or duck Tembusu virus (DTMUV)-induced NF-κB activation. Moreover, duIKKα exhibited antiviral activity against DTMUV infection. These findings provide important insights into the roles of duIKKα in avian innate immunity.

Role of cholesterol in anatid herpesvirus 1 infections in vitro

Cholesterol is an essential constituent of the cell membrane that modulates several physiological events, including virus entry into the host. Duck virus enteritis (DVE) is a contagious and lethal infection that attacks several species of waterfowl. Anatid herpesvirus 1 (AnHV-1) is the causative agent of duck viral enteritis and classified under subfamily Alphaherpesvirinae. In this study, the effect of cholesterol depletion in both host cell membrane and viral envelope on the infectivity of AnHV-1 was explored. Cholesterol depletion of chicken embryo fibroblast cells (DF-1) by methyl-β-cyclodextrin (MβCD) inhibited the infectivity of AnHV-1. This inhibitory effect was moderately reversed by the exogenous replenishment of cholesterol in the cells. Furthermore, the inhibition of endogenous cholesterol synthesis by a statin drug also inhibited the infectivity of AnHV-1. Presumably, the removal of cholesterol from AnHV-1 envelope might be disrupting the viral envelope resulting in its diminished infectivity. The presence of a relatively hydrophobic cavity in MβCD can be used to extract cholesterol from the cell membrane. Loss of infectivity of the virus might be due to the effects of MβCD mediated cholesterol depletion from the cell membrane. The results implicate that the cell membrane cholesterol is vital for the infectivity of AnHV-1 in DF-1 cells, and its depletion from virion curtails the infectivity by destabilizing the envelope.

The pathogenesis of a North American H5N2 clade 2.3.4.4 group A highly pathogenic avian influenza virus in surf scoters (Melanitta perspicillata)

BackgroundAquatic waterfowl, particularly those in the order Anseriformes and Charadriiformes, are the ecological reservoir of avian influenza viruses (AIVs). Dabbling ducks play a recognized role in the maintenance and transmission of AIVs. Furthermore, the pathogenesis of highly pathogenic AIV (HPAIV) in dabbling ducks is well characterized. In contrast, the role of diving ducks in HPAIV maintenance and transmission remains unclear. In this study, the pathogenesis of a North American A/Goose/1/Guangdong/96-lineage clade 2.3.4.4 group A H5N2 HPAIV, A/Northern pintail/Washington/40964/2014, in diving sea ducks (surf scoters, Melanitta perspicillata) was characterized.ResultsIntrachoanal inoculation of surf scoters with A/Northern pintail/Washington/40964/2014 (H5N2) HPAIV induced mild transient clinical disease whilst concomitantly shedding high virus titers for up to 10 days post-inoculation (dpi), particularly from the oropharyngeal route. Virus shedding, albeit at low levels, continued to be detected up to 14 dpi. Two aged ducks that succumbed to HPAIV infection had pathological evidence for co-infection with duck enteritis virus, which was confirmed by molecular approaches. Abundant HPAIV antigen was observed in visceral and central nervous system organs and was associated with histopathological lesions.ConclusionsCollectively, surf scoters, are susceptible to HPAIV infection and excrete high titers of HPAIV from the respiratory and cloacal tracts whilst being asymptomatic. The susceptibility of diving sea ducks to H5 HPAIV highlights the need for additional research and surveillance to further understand the contribution of diving ducks to HPAIV ecology.

Research Note: Duck plague virus glycoprotein I influences cell–cell spread and final envelope acquisition

To determine the role of glycoprotein I (gI) in duck plague virus (DPV), a gI-deleted mutant (BAC-CHv-ΔgI) and a gI-revertant virus (BAC-CHv-ΔgI Rev) were constructed by using a markerless two-step Red recombination system implemented on the DPV genome cloned into a bacterial artificial chromosome (BAC). Mutants were characterized on duck embryo fibroblast (DEF) cells compared with wild-type virus. BAC-CHv-ΔgI produced viral plaques on DEF cells that were on average approximately 57.2% smaller than those produced by BAC-CHv-ΔgI Rev and wild-type virus. Electron microscopy confirmed that deleting of gI resulted in nucleocapsids accumulated around the cytoplasm vesicles and few of them could complete the final envelopment process. These results clearly indicated that DPV gI plays significant roles in viral cell-cell spread and viral final envelopment process.

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.

Construction of the recombinant duck enteritis virus delivering capsid protein VP0 of the duck hepatitis A virus

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.

Possible role of traditional medicinal plant Neem (Azadirachta indica) for the management of COVID-19 infection

Novel coronavirus disease (COVID-19) originated from Wuhan, China. Afterwards COVID-19 outbreak was declared as a Public Health Emergency of International Concern by World Health Organization (WHO). Now around two and half million peoples are suffering and several thousands are dead worldwide due to the highly infectious and deadly nature of the virus. Unfortunately, no standard medicine or vaccine is available to treat the disease. The major clinical symptoms for COVID-19 are fever, chills, cough, fatigue, respiratory symptoms, diarrhoea and shortness of breath. Clinical symptom based Indian traditional medicinal practices like Ayurveda and Siddha could be beneficial to treat and prevent the infection. Indian origin traditional medicinal plant Neem (Azadirachta indica) has been reported to have anti-viral potential against bovine herpes virus type-1, poliovirus type 1, duck plague virus, dengue virus type-2, newcastle disease virus, infectious bursal disease virus, avian influenza virus and group B coxsackievirus. Neem is widely used as Ayurvedic medicine to treat fever, cough, asthma and diarrhea which are also reported as the common clinical symptoms for COVID-19. Neem is recorded to enhance both humoral and cell mediated immune response during viral infection. Multidimensional anti-viral therapeutic potentials of Neem insist to hypothesize its probable application to control COVID-19 along with modern medicinal practices. But, series of experimental database and translational research are required to establish the hypothesis.

Duck Enteritis Virus VP16 Antagonizes IFN-β-Mediated Antiviral Innate Immunity.

Duck enteritis virus (DEV) can successfully evade the host innate immune responses and establish a lifelong latent infection in the infected host. However, the study about how DEV escapes host innate immunity is still deficient up to now. In this study, for the first time, we identified a viral protein VP16 by which DEV can obviously downregulate the production of IFN-β in duck embryo fibroblast (DEF). Our results showed that ectopic expression of VP16 decreased duck IFN-β (duIFN-β) promoter activation and significantly inhibited the mRNA transcription of IFN-β. Further study showed that VP16 can also obviously inhibit the mRNA transcription of interferon-stimulated genes (ISGs), such as myxovirus resistance protein (Mx) and interferon-induced oligoadenylate synthetase-like (OASL). Furthermore, we found that this anti-interferon activity of VP16 depended on its N-terminus (aa1-200). Coexpression analysis revealed that VP16 selectively blocked duIFN-β promoter activity at the duIRF7 level rather than duIRF1. Based on the results of coimmunoprecipitation analysis (co-IP) and indirect immunofluorescence assay (IFA), VP16 was able to bind to duck IRF7 (duIRF7) directly, but did not interact with duck IRF1 (duIRF1) in vitro.

Complete genome sequence of an isolate of duck enteritis virus from China.

Here, we present the complete genomic sequence of duck enteritis virus (DEV) strain SD, isolated in China in 2012. The virus was virulent in experimentally infected 2-month-old ducks. The DEV SD genome is 160,945 base pairs (bp) in length. The viral genome sequence, when compared to that of strain DEV CSC, which was isolated in 1962, showed three discontinuous deletions of 101bp, 48bp and 417bp within the inverted repeats. A comparison of the amino acid (aa) sequences of all ORFs of the CSC and SD isolates demonstrated an11-aa deletion, two single-aa deletions, and one single-aa deletion in LORF3, UL47, UL4, respectively. Moreover, 38 single aa variations were also detected in 24 different ORFs. These results will further advance our understanding of the genetic variations involved in evolution.

Serum enzyme activities in different varieties of Duck of Assam in healthy and duck plague out break

Serum enzyme activities in three different varieties of duck namely Pati, Khaki Campbell and Nageswari from different parts of Assam state were evaluated in during duck plague outbreak and compared with healthy ducks. A significant increase (P

The Pivotal Roles of US3 Protein in Cell-to-Cell Spread and Virion Nuclear Egress of Duck Plague Virus

The duck plague virus (DPV) US3 protein, a homolog of the herpes simplex virus-1 (HSV-1) US3 protein that is reported to be critical for viral replication, has been minimally studied. Therefore, to investigate the function of the DPV US3 protein, we used scarless Red recombination technology based on an infectious bacterial artificial chromosome (BAC) containing the DPV Chinese virulent strain (CHv) genome and successfully constructed and rescued a US3-deleted mutant and the corresponding revertant virus (BAC-CHv-ΔUS3 and BAC-CHv-ΔUS3R, respectively). For viral growth characteristics, compared to the parental and revertant viruses, the US3-deleted mutant showed an approximately 100-fold reduction in viral titers but no significant reduction in genome copies, indicating that the US3-deleted mutant exhibited decreased viral replication but not decreased viral DNA generation. In addition, the US3-deleted mutant formed viral plaques that were 33% smaller on average than those formed by the parental and revertant viruses, demonstrating that US3 protein affected the viral cell-to-cell spread of DPV. Finally, the results of electron microscopy showed that the deletion of US3 resulted in a large number of virions accumulating in the nucleus and perinuclear space, thus blocking virion nuclear egress. In this study, we found that the DPV US3 protein played pivotal roles in viral replication by promoting viral cell-to-cell spread and virion nuclear egress, which may provide some references for research on the function of the DPV US3 protein.

Alterations in serum constituents of different duck varieties of Assam in healthy and duck plague out break

Alterations of serum constituents in three different varieties of duck namely Pati, Khaki Campbell and Nageswari from different parts of the state were evaluated in healthy and duck plague outbreak. A significant reduction (P

Role of Emergency Vaccination as a Trial to Control DEV Infection in Muscovy Ducklings

Duck viral enteritis (DVE) is an important viral disease affect ducks all over the world. Vaccination is an effective way to control DVE, and an attenuated vaccine is used extensively worldwide. In this study, we attempted to control the disease by emergency vaccination. Three out of four groups (group 1, 2 and 3) of 21 days old Muscovy ducklings were infected with 0.5ml of 105.5 dELD50/ml of field duck enteritis virus (DEV) isolate. After the onset of clinical signs (6 days post infection), two out of three infected groups (group 2 and 3) were vaccinated with live attenuated vaccine using oral and S/C routes, respectively. Birds of infected- non vaccinated group (group1) showed the characteristic clinical and pathological features of the disease at the fifth day post infection that progressed throughout 21 days of the experiment, with 33.3% mortality rates. The severity and frequency of clinical and pathological findings in infected-vaccinated groups (group 2 and 3) were relatively lower than those of group1. Only 13.3% mortality rate was recorded in group 2; moreover no death was detected in group 3. The presence of DEV in the tissues collected from dead and sacrificed live ducklings at the end of experiment was confirmed by PCR. Our results showed that the use of commercial live attenuated vaccine via SC route as an emergency vaccine could ameliorate the clinical and pathological findings of the disease and considered as an effective tool to control DEV infection.

Duck plague virus gE serves essential functions during the virion final envelopment through influence capsids budding into the cytoplasmic vesicles

Duck plague virus (DPV), a member of the alphaherpesviruses subfamily, causes massive ducks death and results in a devastating hit to duck industries in China. It is of great significance for us to analyze the functions of DPV genes for controlling the outbreak of duck plague. Thus, glycoproteins E (gE) of DPV, which requires viral cell-to-cell spreading and the final envelopment in herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV), was chosen herein. The gE mutant virus BAC-CHv-ΔgE was constructed by using a markerless two-step Red recombination system implemented on the DPV genome cloned into a bacterial artificial chromosome (BAC). Viral plaques on duck embryo fibroblast (DEF) cells of BAC-CHv-ΔgE were on average approximately 60% smaller than those produced by BAC-CHv virus. Viral replication kinetics showed that BAC-CHv-ΔgE grew to lower titers than BAC-CHv virus did in DEF cells. Electron microscopy confirmed that deleting of DPV gE resulted in a large number of capsids accumulating around vesicles and very few of them could bud into vesicles. The drastic inhibition of virion formation in the absence of the DPV gE indicated that it played an important role in virion morphogenesis before the final envelopment of intracytoplasmic nucleocapsids.

High-mobility group box 1 protein (HMGB1) from Cherry Valley duck mediates signaling pathways and antiviral activity

High-mobility group box 1 protein (HMGB1) shows endogenous damage-associated molecular patterns (DAMPs) and is also an early warning protein that activates the body’s innate immune system. Here, the full-length coding sequence of HMGB1 was cloned from the spleen of Cherry Valley duck and analyzed. We find that duck HMGB1(duHMGB1) is mostly located in the nucleus of duck embryo fibroblast (DEF) cells under normal conditions but released into the cytoplasm after lipopolysaccharide (LPS) stimulation. Knocking-down or overexpressing duHMGB1 had no effect on the baseline apoptosis rate of DEF cells. However, overexpression increased weakly apoptosis after LPS activation. In addition, overexpression strongly activated the IFN-I/IRF7 signaling pathway in DEF cells and significantly increased the transcriptional level of numerous pattern recognition receptors (PRRs), pro-inflammatory cytokines (IL-6, TNF-α), IFNs and antiviral molecules (OAS, PKR, Mx) starting from 48 h post-transfection. Overexpression of duHMGB1 strongly impacted duck virus replication, either by inhibiting it from the first stage of infection for novel duck reovirus (NDRV) and at late stage for duck Tembusu virus (DTMUV) or duck plague virus (DPV), or promoting replication at early stage for DTMUV and DPV infection. Importantly, data from duHMGB1 overexpression and knockdown experiments, time-dependent DEF cells transcriptional immune responses suggest that duHMGB1 and RIG-I receptor might cooperate to promote the expression of antiviral proteins after NDRV infection, as a potential mechanism of duHMGB1-mediated antiviral activity.

Isolation and Selection of Duck Primary Cells as Pathogenic and Innate Immunologic Cell Models for Duck Plague Virus.

Duck plague virus (DPV) is a representative pathogen transmitted among aquatic animals that causes gross lesions and immune inhibition in geese and ducks. The mechanism of organ tropism and innate immune evasion of DPV has not been completely deciphered due to a lack of cell models to study the innate immune manipulation and pathogenicity of aquatic viruses. In the present study, we isolated five types of duck primary cells [duck embryo fibroblasts (DEFs), neurons, astrocytes, peripheral blood mononuclear cells (PBMCs), and monocytes/macrophages] to identify appropriate cell models for DPV, using tropism infection and innate immunologic assays. Cells responded differently to stimulation with DNA viruses or RNA virus analogs. DPV infection exhibited broad tropism, as the recombinant virulent strain (CHv-GFP) infected DEFs, neurons, astrocytes, and monocytes/macrophages, but not the PBMCs, as the expression of EGFP was negligible. The basal levels of innate immunity molecules were highest in monocytes/macrophages and lower in DEFs and astrocytes. Conversely, the titer and genomic copy number of the attenuated virus strain was higher in DEFs and astrocytes than in neurons and monocytes/macrophages. The titer and genomic copy number of the attenuated virus strain were higher compared with the virulent strain in DEFs, neurons, and astrocytes. The innate immune response was not significantly induced by either DPV strain in DEFs, neurons, or astrocytes. The virulent strain persistently infected monocytes/macrophages, but the attenuated strain did so abortively, and this was accompanied by the phenomenon of innate immune inhibition and activation by the virulent and attenuated strains, respectively. Blockage of IFNAR signaling promoted replication of the attenuated strain. Pre-activation of IFNAR signaling inhibited infection by the virulent strain. The selection assay results indicated that induction of innate immunity plays an essential role in controlling DPV infection, and monocytes/macrophages are an important cell model for further investigations. Our study provided practical methods for isolating and culturing duck primary cells, and our results will facilitate further investigations of organ tropism, innate immune responses, latent infection, and the effectiveness of antiviral drugs for treating DPV and potentially other aerial bird pathogens.

Pathodynamics of Circulating Strains of Duck Enteritis Virus: A Step Forward to Understand Its Pathogenesis.

Duck enteritis virus (DEV) causes an acute and contagious infection in duck. The present study was carried out to evaluate the pathogenicity and pathodynamics of DEV isolates from different natural outbreaks in the Assam Province of India. A total of six wild-type isolates of DEV were revived in ducklings to determine its biologic characterization. Postmortem examination of infected ducklings revealed DEV-specific gross lesions in different organs. The presence of DEV was confirmed by its genome amplification and the presence of viral antigens from collected tissue samples by indirect fluorescent antibody test. All the isolates revived in ducklings were further propagated in duck embryo fibroblast cells. Highly virulent and low virulent isolates of DEV were selected for further study based on median duck infectivity dose (DID50) and median tissue culture infectivity dose (TCID50). The highly virulent isolate of DEV had values of 102 DID50/ml and 106.33 TCID50/ml, whereas the low virulent strain had titers of 10 DID50/ml and 104.83 TCID50/ml in the cell culture. Our results showed replication of DEV in ducks with the highest and lowest viral titers in the thymus and bursa of Fabricius, respectively. In addition, microscopic analysis revealed necrosis and degeneration of submucosal esophageal glands and glandular epithelium. The study will be useful to understand the organ tropism and pathologic alteration among the virulent DEV isolates.

Protection against fowl cholera in ducks immunized with a combination vaccine containing live attenuated duck enteritis virus and recombinant outer membrane protein H of Pasteurella multocida

ABSTRACTFowl cholera is a highly contagious disease within the global duck farming industry. This study aimed at formulating and evaluating the protective efficacy of a combination vaccine containing a recombinant outer membrane protein H (rOmpH) of Pasteurella multocida strain X-73 with a live attenuated duck plague vaccine into a single dose. Four groups of ducks received different treatments and the groups were labelled as non-vaccinated, combined vaccination, duck plague vaccination and rOmpH vaccination, respectively. The combined vaccination group was comprised of live attenuated duck plague commercial vaccine with 100 µg rOmpH to a total volume of 0.5 ml/duck/intramuscular administration. All groups were challenged with avian P. multocida strain X-73 via intranasal administration. In addition, blood samples were collected monthly over a period of 6 months to determine the appropriate antibody level by indirect ELISA. The indirect ELISA results in the combination vaccine group revealed that the average levels of the serum antibody against the duck enteritis virus (0.477 ± 0.155) and fowl cholera (0.383 ± 0.100) were significantly higher than those values in the non-vaccinated control group (0.080 ± 0.027 and 0.052 ± 0.017), respectively (P < 0.05). Moreover, all vaccinated ducks were effectively protected from fowl cholera. This preliminary study indicated that a combination vaccine did not affect the antibody response in the subjects while protecting the ducks against experimental P. multocida infection. This combination vaccine should be considered part of an alternative pre-treatment strategy that could replace the monovalent vaccine.

Duplicate US1 Genes of Duck Enteritis Virus Encode a Non-essential Immediate Early Protein Localized to the Nucleus

The duplicate US1 genes of duck enteritis virus (DEV) encode a protein with a conserved Herpes_IE68 domain, which was found to be closely related to the herpes virus immediate early regulatory protein family and is highly conserved among counterparts encoded by Herpes_IE68 genes. Previous studies found the homologous proteins HSV-1 ICP22 and VZV ORF63/ORF70 to be critical for virus transcription and replication. However, little is known about the DEV ICP22 protein. In this paper, we describe the characteristics of this protein based on pharmacological experiments, real-time quantitative Polymerase Chain Reaction, Western blot, and immunofluorescence assays. We also investigate the role of the protein in DEV replication via mutation of US1. As a result, we found that the DEV ICP22 protein is a non-essential immediate early protein predominantly located in the nucleus of infected DEF cells and that DEV replication is impaired by US1 deletion. We also found that ICP22 contains a classical nuclear localization signal (NLS) at 305-312AA, and ICP22 cannot enter the nucleus by itself after mutating residue 309.

Duck enteritis virus UL21 is a late gene encoding a protein that interacts with pUL16

BackgroundpUL21 is a conserved protein of Alphaherpesvirinae that performs multiple important functions. The C-terminus of pUL21 in other members of this subfamily has RNA-binding ability; this domain contributes to pseudorabies virus (PRV) retrograde axonal transport in vitro and in vivo and participates in newly replicated viral DNA packaging and intracellular virus transport. However, knowledge regarding duck enteritis virus (DEV) pUL21 is limited.ResultsWe verified that DEV UL21 is a γ2 gene that encodes a structural protein. Moreover, we observed that pUL21 localized to the nucleus and cytoplasm. DEV pUL21 interacted with pUL16 and formed a complex in transfected human embryonic kidney (HEK) 293 T cells and DEV-infected duck embryo fibroblasts (DEFs). These results were further confirmed by CO-IP assays.ConclusionsThe DEV UL21 gene is a late gene, and pUL21 localizes to the nucleus and cytoplasm. DEV UL21 is a virion component. In addition, pUL21 can interact with pUL16. These findings provide insight into the characteristics of UL21 and the interaction between pUL21 and its binding partner pUL16. Our study enhances the understanding of DEV pUL21.