Duck Enteritis Virus Replication Research Articles

Duck Enteritis Virus Inhibits the cGAS-STING DNA-Sensing Pathway To Evade the Innate Immune Response.

Cyclic GMP-AMP synthase (cGAS), a key DNA sensor, detects cytosolic viral DNA and activates the adaptor protein stimulator of interferon genes (STING) to initiate interferon (IFN) production and host innate antiviral responses. Duck enteritis virus (DEV) is a duck alphaherpesvirus that causes an acute and contagious disease with high mortality in waterfowl. In the present study, we found that DEV inhibits host innate immune responses during the late phase of viral infection. Furthermore, we screened DEV proteins for their ability to inhibit the cGAS-STING DNA-sensing pathway and identified multiple viral proteins, including UL41, US3, UL28, UL53, and UL24, which block IFN-β activation through this pathway. The DEV tegument protein UL41, which exhibited the strongest inhibitory effect, selectively downregulated the expression of interferon regulatory factor 7 (IRF7) by reducing its mRNA accumulation, thereby inhibiting the DNA-sensing pathway. Ectopic expression of UL41 markedly reduced viral DNA-triggered IFN-β production and promoted viral replication, whereas deficiency of UL41 in the context of DEV infection increased the IFN-β response to DEV and suppressed viral replication. In addition, ectopic expression of IRF7 inhibited the replication of the UL41-deficient virus, whereas IRF7 knockdown facilitated its replication. This study is the first report identifying multiple viral proteins encoded by a duck DNA virus, which inhibit the cGAS-STING DNA-sensing pathway. These findings expand our knowledge of DNA sensing in ducks and reveal a mechanism through which DEV antagonizes the host innate immune response. IMPORTANCE Duck enteritis virus (DEV) is a duck alphaherpesvirus that causes an acute and contagious disease with high mortality, resulting in substantial economic losses in the commercial waterfowl industry. The evasion of DNA-sensing pathway-mediated antiviral innate immunity is essential for the persistent infection and replication of many DNA viruses. However, the mechanisms used by DEV to modulate the DNA-sensing pathway remain poorly understood. In the present study, we found that DEV encodes multiple viral proteins to inhibit the cGAS-STING DNA-sensing pathway. The DEV tegument protein UL41 selectively diminished the accumulation of interferon regulatory factor 7 (IRF7) mRNA, thereby inhibiting the DNA-sensing pathway. Loss of UL41 potently enhanced the IFN-β response to DEV and impaired viral replication in ducks. These findings provide insights into the host-virus interaction during DEV infection and help develop new live attenuated vaccines against DEV.

Duck Enteritis Virus Protein Kinase US3 Inhibits DNA Sensing Signaling by Phosphorylating Interferon Regulatory Factor 7.

The cytosolic DNA sensing pathway mediates innate immune defense against infection by many DNA viruses; however, viruses have evolved multiple strategies to evade the host immune response. Duck enteritis virus (DEV) causes an acute and contagious disease with high mortality in waterfowl. The mechanisms employed by DEV to block the DNA sensing pathway are not well understood. Here, we sought to investigate the role of DEV US3, a serine/threonine protein kinase, in the inhibition of DNA sensing. We found that ectopic expression of DEV US3 significantly inhibited the production of IFN-β and expression of interferon-stimulated genes induced by interferon-stimulatory DNA and poly(dA-dT). US3 also inhibited viral DNA-triggered IFN-β activation and promoted DEV replication in duck embryo fibroblasts, while knockdown of US3 during DEV infection enhances the IFN-β response and suppresses viral replication. US3 inhibited the DNA-sensing signaling pathway by targeting interferon regulatory factor 7 (IRF7), and the kinase activity of US3 was indispensable for its inhibitory function. Furthermore, we found that US3 interacts with the activation domain of IRF7, phosphorylating IRF7, blocking its dimerization and nuclear translocation, and finally leading to the inhibition of IFN-β production. These findings expand our knowledge on DNA sensing in ducks and reveal a novel mechanism whereby DEV evades host antiviral immunity. IMPORTANCE Duck enteritis virus (DEV) is a duck alphaherpesvirus that causes an acute and contagious disease with high mortality, resulting in substantial economic losses in the commercial waterfowl industry. The evasion of DNA-sensing pathway-mediated antiviral innate immunity is essential for the persistent infection and replication for many DNA viruses. However, the strategies used by DEV to block the DNA-sensing pathway are not well understood. In this study, DEV US3 protein kinase was demonstrated to inhibit the DNA-sensing signaling via binding to the activation domain of interferon regulatory factor 7 (IRF7), which induced the hyperphosphorylation of IRF7 and abolished IRF7 dimerization and nuclear translocation. Our findings provide insights into how duck herpesviral kinase counteracts host antiviral innate immunity to ensure viral replication and spread.

Two nuclear localization signals regulate intracellular localization of the duck enteritis virus UL13 protein

Duck enteritis virus (DEV) multifunctional tegument protein UL13 is predicted to be a conserved herpesvirus protein kinase; however, little is known about its subcellular localization signal. In this study, through transfection of 2 predicted nuclear signals of DEV UL13 fused to enhanced green fluorescent protein, 2 bipartite nuclear localization signals (NLS) were identified. We found that ivermectin blocked the NLS-mediated nuclear import of DEV UL13, showing that the nuclear localization signal of DEV UL13 is a classical importin α- and β-dependent process. We constructed a DEV UL13 mutant strain in which the NLS of DEV UL13 was deleted to explore whether deletion of the NLS affects viral replication. Amino acids 4 to 7 and 90 to 96 were predicted to be NLSs, further proving that nuclear import occurs via a classical importin α- and β-dependent process. We also found that the NLS of pUL13 had no effect on DEV replication in cell culture. Our study enhances the understanding of DEV pUL13. Taken together, these results provide significant information regarding the biological function of pUL13 during DEV infection.

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.

Downregulation of microRNA-30a-5p contributes to the replication of duck enteritis virus by regulating Beclin-1-mediated autophagy

BackgroundMicroRNAs (miRNAs) is increasingly recognized as an important element in regulating virus-host interactions. Our previous results showed that cellular miR-30a-5p was significantly downregulated after duck enteritis virus (DEV) infection cell. However, whehter or not the miR-30a-5p is involved in DEV infection has not been known.MethodsQuantitative reverse-transcription PCR (qRT-PCR) was used to measure the expression levels of miRNAs(miR-30a-5p) and Beclin-1 mRNA. The miR-30a-5p - Beclin-1 target interactions were determined by Dual luciferase reporter assay (DLRA). Western blotting was utilized to analyze Beclin-1-mediated duck embryo fibroblast (DEF) cells autophagy activity. DEV titers were estimated by the median tissue culture infective dose (TCID50).ResultsThe miR-30a-5p was significantly downregulated and the Beclin-1 mRNA was significantly upregulated in DEV-infected DEF cells. DLRA confirmed that miR-30a-5p directly targeted the 3′- UTR of the Beclin-1 gene. Overexpression of miR-30a-5p significantly reduced the expression level of Beclin-1protein (p < 0.05), leading to the decrease of Beclin-1-mediated autophagy activity, which ultimately suppressed DEV replication (P < 0.05). Whereas transfection of miR-30a-5p inhibitor increased Beclin-1-mediated autophagy and triggered DEV replication during the whole process of DEV infection (P < 0.01).ConclusionsThis study shows that miR-30a-5p can inhibit DEV replication through reducing autophagy by targeting Beclin-1. These findings suggest a new insight into virus-host interaction during DEV infection and provide a potential new antiviral therapeutic strategy against DEV infection.

US10 Protein Is Crucial but not Indispensable for Duck Enteritis Virus Infection in Vitro

To investigate the function of the duck enteritis virus (DEV) tegument protein US10, we generated US10 deletion and revertant mutants (ΔUS10 and US10FRT) via two-step RED recombination based on an infectious BAC clone of DEV CHv-BAC-G (BAC-G). In multistep growth kinetic analyses, ΔUS10 showed an approximately 100-fold reduction in viral titer, while the genome copies decreased only 4-fold compared to those of BAC-G. In one-step growth kinetic analyses, there were no significant differences in genome copies among BAC-G, ΔUS10 and US10FRT, but ΔUS10 still showed a 5- to 20-fold reduction in viral titer, and the replication defect of ΔUS10 was partially reversed by infection of US10-expressing cells. The transcription levels of Mx, OASL, IL-4, IL-6 and IL-10 in ΔUS10-infected duck embryo fibroblasts (DEFs) were significantly upregulated, while TLR3 was downregulated compared with those in BAC-G-infected DEFs. Taken together, these data indicated that US10 is vital for DEV replication and is associated with transcription of some immunity genes.

Impaired Cellular Energy Metabolism Contributes to Duck-Enteritis-Virus-Induced Autophagy via the AMPK-TSC2-MTOR Signaling Pathway.

Duck enteritis virus (DEV) is a large, complex double-stranded DNA virus that induces duck embryo fibroblast (DEF) cells autophagy, which is beneficial to its own replication, but the mechanism has not been described. In this study, we showed that impaired cell energy metabolism is involved in DEV-induced autophagy, whereby ATP synthesis is disrupted in cells after DEV infection, which causes metabolic stress and activation of autophagy. Methyl pyruvate (MP) inhibited conversion of LC3I to LC3II and accumulation of GFP-LC3, which could reverse the energy loss caused by DEV infection. Inhibition of DEV replication by MP confirmed the above view. We found that infection with DEV activated the metabolic regulator 5′ AMP-activated kinase (AMPK) and inhibited activity of mechanistic target of rapamycin (mTOR). In the cases where AMPK expression was inhibited, the LC3-I conversion to LC3-II ratio was decreased, as was GFP-LC3 point and DEV replication; in addition, inhibition of p-mTOR showed a reverse trend. We also found that tuberous sclerosis (TSC) 2 was a key mediator between AMPK and mTOR through activation by phosphorylation. siRNA targeting TSC2 was transfected to reverse the inhibition of mTOR, and down-regulate autophagy level and DEV replication, but AMPK expression was not changed, while siRNA targeting AMPK inhibited activation of TSC2. In conclusion, our findings indicate that energy metabolism in cell damage induced by DEV contributes to autophagy via the AMPK–TSC2–MTOR signaling pathway, which provides a new perspective for DEV and host interactions.

The duck enteritis virus early protein, UL13, found in both nucleus and cytoplasm, influences viral replication in cell culture

The UL13 protein of the duck enteritis virus (DEV), predicted to encode a Ser/Thr protein kinase, belongs to the family of conserved herpesvirus protein kinases (CHPK), which plays an important role in herpesvirus proliferation. In this study, truncated UL13 was expressed as a fusion protein of approximately 44kDa using a prokaryotic expression system, and this protein was used to generate a specific anti-UL13 antibody. This antibody detected UL13 starting at 4h post infection in duck embryonic fibroblast cells and identified UL13 to be present in both the cytoplasm and the nucleus. UL13 RNA was found to be transcribed starting at 2h post infection, and the synthesis of the UL13 mRNA was found to be sensitive to the protein synthesis inhibitor cycloheximide (CHX) and tolerant of the DNA polymerase inhibitor ganciclovir (GCV). Its nuclear location and status as an early gene suggested that DEV UL13 might play important roles in DEV replication, which was confirmed by comparing the proliferation of a UL13-knockout mutant virus, a revertant virus, and the parent virus in cell culture. The specific mechanisms of UL13 in viral replication need to be further studied.

Preliminary study of the UL55 gene based on infectious Chinese virulent duck enteritis virus bacterial artificial chromosome clone

BackgroundLethal Duck Enteritis Virus (DEV) infection can cause high morbidity and mortality of many species of waterfowl within the order Anseriformes. However, little is known about the function of viral genes including the conserved UL55 gene among alpha herpes virus due to the obstacles in maintenance and manipulation of DEV genome in host cells.MethodsIn this paper, we constructed an infectious bacteria artificial chromosome (BAC) clone of the lethal clinical isolate duck enteritis virus Chinese virulent strain (DEV CHv) by inserting a transfer vector containing BAC mini-F sequence and selection marker EGFP into UL23 gene using homologous recombination. UL55 deletion and its revertant mutant were generated by two-step RED recombination in E. coli on basis of rescued recombinant virus. The function of UL55 gene in DEV replication and its effect on distribution of UL26.5 protein were carried out by growth characteristics and co-localization analysis.ResultsThe complete genome of DEV CHv can be stably maintained in E. coli as a BAC clone and reconstituted again in DEF cells. The generated UL55 deletion mutant based on DEV CHv-BAC-G displayed similar growth curves, plaque morphology and virus titer of its parental virus in infected Duck Embryo Fibroblast (DEF) cells. Immunofluorescence assay indicated that the loss of UL55 gene do not affect the distribution of UL26.5 protein in intracellular. These data also suggest infectious BAC clone of DEV CHv will facilitate the gene function studies of DEV genome.ConclusionsWe have successfully developed an infectious BAC clone of lethal clinical isolate DEV CHv for the first time. The generated UL55 gene mutant based on that demonstrated this platform would be a very useful tool for functional study of DEV genes. We found the least known DEV UL55 is dispensable for virus replication and UL26.5 distribution, and it could be a very promise candidate locus for developing bivalent vaccine. Experiment are now in progress for testifying the possibility of UL55 gene locus as an exogenous gene insertion site for developing DEV vectored vaccine.

Autophagy activated by duck enteritis virus infection positively affects its replication

Duck enteritis virus (DEV) is an acute, septic, sexually transmitted disease that occurs in ducks, geese and other poultry. Autophagy is an evolutionarily ancient pathway that is important in many viral infections. Despite extensive study, the interplay between DEV and autophagy of host cells is not clearly understood. In this study, we found that DEV infection triggers autophagy in duck embryo fibroblast (DEF) cells, as demonstrated by the appearance of autophagosome-like double- or single-membrane vesicles in the cytoplasm of host cells and the number of GFP-LC3 dots. In addition, increased conversion of the autophagy marker protein LC3-I and LC3-II and decreased p62/SQSTM1 indicated complete autophagy flux. Heat-inactivated DEV infection did not induce autophagy, suggesting that the trigger of autophagy in DEF cells depended on DEV replication. When autophagy was pharmacologically inhibited by LY294002 or wortmannin, DEV replication decreased. The DEV offspring yield decreased when small interference RNA was used to interfere with autophagy related to the genes Beclin-1 and ATG5. In contrast, after treating DEF cells with rapamycin, an inducer of autophagy, DEV replication increased. These results indicated that DEV infection induced autophagy in DEF cells and autophagy facilitated DEV replication.

Antiviral effect of resveratrol in ducklings infected with virulent duck enteritis virus

Duck enteritis virus (DEV) is a double-stranded DNA virus belonging to the alphaherpesvirinae subfamily of the herpesviridae. Although vaccines were wildly used in controlling this disease, some infection could still not be prevented and led to significant economic losses as a result of mortality and decreased egg production. However, there is no antiviral drug against DEV. Resveratrol was identified to exert its antiviral activity by inhibiting the DEV replication in preliminary investigations. In the present study, we confirmed that resveratrol significantly reduced the mortality of ducklings which infected with a virulent strain of DEV. With resveratrol treatment, the survival rate increased by almost 80% at 8 days post infection (dpi). Pathological symptoms of ducklings caused by DEV were also relieved by resveratrol. The virus load in blood and tissues were effectively depressed when compared with the untreated group. In the assay of immune cytokines, the resveratrol exerted a dual-regulation effect. These results suggest that resveratrol is expected to be a new alternative control measure for DEV infection.

Identification of the Nuclear Localization Signal Region of Duck Enteritis Virus UL14 and Its Interaction with VP16

Object: Duck enteritis virus (DEV) is a member of the Alphaherpesvirinae viruses. VP16 and pUL14 are both predicted to be tegument proteins of DEV. Methods: An indirect immunofluorescence assay (IFA) was performed for preliminary analysis of the colocalization of pUL14 and VP16, which detected their subcellular localization in duck embryo fibroblasts (DEFs) during virus replication. The coexpression of pUL14 and VP16 was detected in transfected DEFs. A bimolecular fluorescence complementation (BiFC) assay was used to confirm a direct interaction between pUL14 and VP16. To better characterize the nuclear localization domain of pUL14, we designed a series of deletion mutants and transfected them with VP16. Results: Our IFA findings indicated that pUL14 binds to VP16 in the cytoplasm and that pUL14 leads VP16 import into the nucleus during DEV replication. The BiFC assay revealed the presence of pUL14 and VP16 complexes. Furthermore, 1-98 amino acid (aa) at the N-terminus of pUL14 played a role in the nuclear localization signal (NLS) region and promoted translocation of VP16 into the nucleus to complete the virus life cycle. Conclusions: Our findings indicated that pUL14 could transport VP16 into the nucleus and that the N-terminal 1-98 aa may contain the NLS domain of pUL14.