Effects of US3 protein kinase activity on localization of UL31/UL34 protein and nucleocapsids egress of duck plague virus.

Duck plague virus (DPV) is a pathogen causing duck plague and has caused huge economic losses in poultry industry. In our previous report, US3 gene deletion from DPV genome seriously impaired virus replication. In this study, we constructed a US3 kinase-inactive mutant (US3K213A) to further explore the function of US3 protein (pUS3) in DPV. Our results showed that the loss of pUS3 kinase activity caused lower viral titers, smaller plaque sizes and a blockage of capsids nuclear egress including primary enveloped virion (PEV) accumulation compared to the parental virus infection. It indicates that the effects of DPV pUS3 on viral propagation depended on its kinase activity. In addition, we conducted electron microscopy analysis to show the outer nuclear membrane (ONM) evaginations and the nuclear envelope (NE) deep invagination in US3K213A-infected cells. Finally, an irregular distribution of pUL31/pUL34 in the NE in △US3- and US3K213A-infected cells and an interaction of pUS3 and pUL31 were found, which suggests that pUS3 potentially targets pUL31 and regulates the localization of pUL31/pUL34 to promote nucleocapsids egress through its kinase activity.

Adaptation and characterization of Anatid herpesvirus 1 in different permissible cell lines.

Duck viral enteritis is an acute, contagious infection of Anatidae family members. The disease is caused by Anatid herpesvirus 1 (AnHV-1). The infection of AnHV-1 is controlled by vaccination to the flock with chick embryo adapted attenuated vaccine in developed countries. However, its economic impact in developing countries is substantial and there is a need to understand the cell culture spectrum of the virus to produce its vaccine on a mass scale. In the present study, the permissivity of AnHV-1 for different cells was analyzed. The AnHV-1 showed enhanced replication following its serial passage in CEF, DF-1, Vero, MDCK, and QT-35 cells. The characteristic cytopathic effect (CPE) of rounding and clumping of cells were observed in CEF, DF-1, Vero, and QT-35 cell lines. The infectivity and viral replication were highest in CEF, DF-1, Vero, and QT-35 cells. In contrast, the results suggested that MDCK cells are less permissive for AnHV-1 infection with negligible CPE and reduced viral replication. Heterologous cell culture systems other than chicken embryo fibroblasts to adapted live vaccine viruses will provide a system devoid of other avian infectious agents. Moreover, it can be used for the propagation and cultivation of AnHV-1 vaccine strain for developing cell culture-based vaccines with high titer and could be an economical alternative for the existing options.

Gga-miR-181a modulates ANP32A expression and inhibits MDCC-MSB-1 cell.

Marek's disease (MD), a highly contagious T cell lymphoid neoplasia disease of chickens, causes huge economic losses to the poultry industry. It is the only one tumor disease which can be prevented by vaccine in chickens; therefore, MD is considered to be an excellent model to study the pathogenesis of virus-induced cancer. Recently, abundant evidences have verified that miRNAs are regulators in the process of neoplastic transformation. In our previous study on miRNome analysis of MDV-induced lymphoma in chicken, we found that gga-miR-181a was downregulated drastically in MDV-infected spleens. To further investigate the role of gga-miR-181a in MDV-induced lymphomagenesis, we performed cell migration assay, and the results suggested that gga-miR-181a suppressed the migration of MDV-transformed lymphoid cell (MSB-1). Subsequently, luciferase reporter gene assay revealed that acidic nuclear phosphoprotein 32A (ANP32A) was a functional target gene of gga-miR181a. Real-time PCR and western blot assay showed that the mRNA and protein levels of ANP32A were downregulated in gga-miR-181a mimic group at 48-h and 96-h post-transfection, respectively, indicating that ANP32A was modulated by gga-miR-181a. All the results suggested that gga-miR-181a was an inhibitor in MSB-1 cell migration. ANP32A was a direct target gene of gga-miR-181a and they were implicated in MD lymphoma tumorigenesis.

UL28 and UL33 homologs of Marek's disease virus terminase complex involved in the regulation of cleavage and packaging of viral DNA are indispensable for replication in cultured cells.

Processing and packaging of herpesvirus genomic DNA is regulated by a packaging-associated terminase complex comprising of viral proteins pUL15, pUL28 and pUL33. Marek's disease virus (MDV) homologs UL28 and UL33 showed conserved functional features with high sequence identity with the corresponding Herpes simplex virus 1 (HSV-1) homologs. As part of the investigations into the role of the UL28 and UL33 homologs of oncogenic MDV for DNA packaging and replication in cultured cells, we generated MDV mutant clones deficient in UL28 or UL33 of full-length MDV genomes. Transfection of UL28- or UL33-deleted BAC DNA into chicken embryo fibroblast (CEF) did not result either in the production of visible virus plaques, or detectable single cell infection after passaging onto fresh CEF cells. However, typical MDV plaques were detectable in CEF transfected with the DNA of revertant mutants where the deleted genes were precisely reinserted. Moreover, the replication defect of the UL28-deficient mutant was completely restored when fragment encoding the full UL28 gene was co-transfected into CEF cells. Viruses recovered from the revertant construct, as well as by the UL28 co-transfection, showed replication ability comparable with parental virus. Furthermore, the transmission electron microscopy study indicated that immature capsids were assembled without the UL28 expression, but with the loss of infectivity. Importantly, predicted three-dimensional structures of UL28 between MDV and HSV-1 suggests conserved function in virus replication. For the first time, these results revealed that both UL28 and UL33 are essential for MDV replication through regulating DNA cleavage and packaging.

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-ß 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 (ß-Gal). Moreover, pUL47 significantly inhibited polyriboinosinic:polyribocytidylic acid [poly(I:C)]-induced interferon beta (IFN-ß) 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).

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-ß-cyclodextrin (MßCD) 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 MßCD can be used to extract cholesterol from the cell membrane. Loss of infectivity of the virus might be due to the effects of MßCD 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.

Abrogation of Marek's disease virus replication using CRISPR/Cas9.

Marek's disease virus (MDV) is a highly cell-associated alphaherpesvirus that causes deadly lymphomas in chickens. While vaccination protects against clinical symptoms, MDV field strains can still circulate in vaccinated flocks and continuously evolve towards greater virulence. MDV vaccines do not provide sterilizing immunity, allowing the virus to overcome vaccine protection, and has increased the need for more potent vaccines or alternative interventions. In this study, we addressed if the CRISPR/Cas9 system can protect cells from MDV replication. We first screened a number of guide RNAs (gRNAs) targeting essential MDV genes for their ability to prevent virus replication. Single gRNAs significantly inhibited virus replication, but could result in the emergence of escape mutants. Strikingly, combining two or more gRNAs completely abrogated virus replication and no escape mutants were observed upon serial passaging. Our study provides the first proof-of-concept, demonstrating that the CRISPR/Cas9 system can be efficiently used to block MDV replication. The presented findings lay the foundation for future research to completely protect chickens from this deadly pathogen.

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.

Patodinámica de las cepas circulantes del virus de la enteritis del pato: Un paso adelante para comprender su patogenia. El virus de la enteritis del pato (DEV) causa una infección aguda y contagiosa en el pato. El presente estudio se llevó a cabo para evaluar la patogenicidad y la patodinámica de los aislamientos del virus de la enteritis del pato de diferentes brotes naturales en la provincia de Assam en la India. Se replicaron un total de seis aislamientos del virus de la enteritis del pato de tipo silvestre en patitos para su caracterización biológica. El examen post mortem de los patitos infectados reveló lesiones macroscópicas específicas de la enteritis viral en diferentes órganos. La presencia del virus de la enteritis viral de pato fue confirmada por su amplificación del genoma y por la presencia de antígenos virales mediante la prueba indirecta de anticuerpos fluorescentes con muestras de tejido recolectadas. Todos los aislamientos replicados en patitos se propagaron adicionalmente en células de fibroblastos de embriones de pato. Se seleccionaron aislamientos del virus de la enteritis del pato altamente virulentos y poco virulentos para un estudio adicional basado en la dosis de infectividad en el pato (DID50) y la dosis de infectividad de cultivo de tejidos (TCID50). El aislado altamente virulento del virus de la enteritis del pato mostró valores de 102 DID50/ml y 106.33 TCID50/ml, mientras que la cepa virulenta baja tenía títulos de 10 DID50/ml y 104.83 TCID50/ml en cultivo celular. Nuestros resultados mostraron la replicación del virus de la enteritis viral en patos con los títulos virales más altos y más bajos en el timo y en la bolsa de Fabricio, respectivamente. Además, el análisis microscópico reveló necrosis y degeneración de las glándulas esofágicas submucosas y del epitelio glandular. El estudio será útil para comprender el tropismo de los órganos y la alteración patológica entre los aislados virulentos del virus de la enteritis viral del pato.

Duck Enteritis Virus VP16 Antagonizes IFN-ß-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-ß in duck embryo fibroblast (DEF). Our results showed that ectopic expression of VP16 decreased duck IFN-ß (duIFN-ß) promoter activation and significantly inhibited the mRNA transcription of IFN-ß. 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-ß 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.

Pervasive Differential Splicing in Marek's Disease Virus can Discriminate CVI-988 Vaccine Strain from RB-1B Very Virulent Strain in Chicken Embryonic Fibroblasts.

Marek's disease is a major scourge challenging poultry health worldwide. It is caused by the highly contagious Marek's disease virus (MDV), an alphaherpesvirus. Here, we showed that, similar to other members of its Herpesviridae family, MDV also presents a complex landscape of splicing events, most of which are uncharacterised and/or not annotated. Quite strikingly, and although the biological relevance of this fact is unknown, we found that a number of viral splicing isoforms are strain-specific, despite the close sequence similarity of the strains considered: very virulent RB-1B and vaccine CVI-988. We validated our findings by devising an assay that discriminated infections caused by the two strains in chicken embryonic fibroblasts on the basis of the presence of some RNA species. To our knowledge, this study is the first to accomplish such a result, emphasizing how relevant a comprehensive picture of the viral transcriptome is to fully understand viral pathogenesis.

Glutaminolysis and Glycolysis Are Essential for Optimal Replication of Marek's Disease Virus.

Viruses may hijack glycolysis, glutaminolysis, or fatty acid ß-oxidation of host cells to provide the energy and macromolecules required for efficient viral replication. Marek's disease virus (MDV) causes a deadly lymphoproliferative disease in chickens and modulates metabolism of host cells. Metabolic analysis of MDV-infected chicken embryonic fibroblasts (CEFs) identified elevated levels of metabolites involved in glutamine catabolism, such as glutamic acid, alanine, glycine, pyrimidine, and creatine. In addition, our results demonstrate that glutamine uptake is elevated by MDV-infected cells in vitro Although glutamine, but not glucose, deprivation significantly reduced cell viability in MDV-infected cells, both glutamine and glucose were required for virus replication and spread. In the presence of minimum glutamine requirements based on optimal cell viability, virus replication was partially rescued by the addition of the tricarboxylic acid (TCA) cycle intermediate, α-ketoglutarate, suggesting that exogenous glutamine is an essential carbon source for the TCA cycle to generate energy and macromolecules required for virus replication. Surprisingly, the inhibition of carnitine palmitoyltransferase 1a (CPT1a), which is elevated in MDV-infected cells, by chemical (etomoxir) or physiological (malonyl-CoA) inhibitors, did not reduce MDV replication, indicating that MDV replication does not require fatty acid ß-oxidation. Taken together, our results demonstrate that MDV infection activates anaplerotic substrate from glucose to glutamine to provide energy and macromolecules required for MDV replication, and optimal MDV replication occurs when the cells do not depend on mitochondrial ß-oxidation.IMPORTANCE Viruses can manipulate host cellular metabolism to provide energy and essential biosynthetic requirements for efficient replication. Marek's disease virus (MDV), an avian alphaherpesvirus, causes a deadly lymphoma in chickens and hijacks host cell metabolism. This study provides evidence for the importance of glycolysis and glutaminolysis, but not fatty acid ß-oxidation, as an essential energy source for the replication and spread of MDV. Moreover, it suggests that in MDV infection, as in many tumor cells, glutamine is used for generation of energetic and biosynthetic requirements of the MDV infection, while glucose is used biosynthetically.

Galectin-1 induces immune response and antiviral ability in Cherry Valley ducks after duck plague virus infection.

Galectin-1, as a typical animal galactose-binding protein, it is found on the cell surface and in the extracellular matrix. Cloning the full-length coding sequence of galectin-1 from the spleens of Cherry Valley ducks revealed that the coding sequence of duck galectin-1 (duGal-1) comprises 405 bp, encoding 134 amino acids. Homologic analysis revealed its amino acid sequence is most identical to that of Anas platyrhynchos (98.8%) followed by Gallus gallus. Quantitative real-time PCR analysis indicated that duGal-1 mRNA is broadly expressed in healthy Cherry Valley duck tissues, primarily in the heart and trachea but minimally in the lung and skin. Meanwhile, the duGal-1 expression is slightly upregulated in the infected liver and spleen. Furthermore, the expression levels of ISGs (Mx, PKR, OAS) and some cytokines such as IFN-α, IL-1ß, IL-2, are up-regulated to varying degrees after overexpression the duGal-1, In contrast, Knockdown of duGal-1 found that the expression levels of ISGs and some inflammatory cytokines were down-regulated. Antiviral assay showed that duGal-1 could inhibit viral replications early during infection. This is the first study of the cloning, tissue distribution, and antiviral immune responses of duGal-1, and findings imply it is involved in the early stages of antiviral innate immune responses to duck plague virus infections in ducks.

Duck RIG-I restricts duck enteritis virus infection.

Retinoic acid-inducible gene I (RIG-I) is a nucleic acid sensor that plays a key role in host antiviral defenses. Duck viral enteritis (DEV) is a DNA virus that causes significant economic losses to the poultry industry worldwide. Although RIG-I is known to be involved in a common antiviral signaling pathway triggered by RNA viruses, its role in DEV infection remains unclear. In this study, we demonstrated that DEV infection increased the expression levels of interferon ß (IFN-ß) and RIG-I in ducks both in vivo and in vitro. Furthermore, overexpression of duck RIG-I significantly upregulated the expression of interferon-stimulated genes, including myxovirus resistance protein (Mx), Interferon-induced oligodenylate synthetase-like (OASL) and IFN-ß. We therefore used overexpression and knockdown methods to determine if RIG-I affected DEV infection in ducks. Viral infection was inhibited by RIG-I, and enhanced by knockdown of RIG-I expression using small interfering RNA. RIG-I overexpression also activated signal transducer and activator of transcription 1 (STAT1), as a member of the JAK-STAT family. The combined results following STAT1 knockdown and RIG-I overexpression suggested that the antiviral activity of RIG-I was STAT1-dependent. Overall, these findings indicate that RIG-I effectively restricts DEV replication and may play a vital role in the host immune response to DEV infection in ducks.

Marek's Disease Virus Disables the ATR-Chk1 Pathway by Activating STAT3.

Oncogenic virus replication often leads to genomic instability, causing DNA damage and inducing the DNA damage response (DDR) pathway. The DDR pathway is a cellular pathway that senses DNA damage and regulates the cell cycle to maintain genomic stability. Therefore, the DDR pathway is critical for the viral lifecycle and tumorigenesis. Marek's disease virus (MDV), an alphaherpesvirus that causes lymphoma in chickens, has been shown to induce DNA damage in infected cells. However, the interaction between MDV and the host DDR is unclear. In this study, we observed that MDV infection causes DNA strand breakage in chicken fibroblast (CEF) cells along with an increase in the DNA damage markers p53 and p21. Interestingly, we showed that phosphorylation of STAT3 was increased during MDV infection, concomitantly with a decrease of Chk1 phosphorylation. In addition, we found that MDV infection was enhanced by VE-821, an ATR-specific inhibitor, but attenuated by hydroxyurea, an ATR activator. Moreover, inhibition of STAT3 phosphorylation by Stattic eliminates the ability of MDV to inhibit Chk1 phosphorylation. Finally, we showed that MDV replication was decreased by Stattic treatment. Taken together, these results suggest that MDV disables the ATR-Chk1 pathway through STAT3 activation to benefit its replication.IMPORTANCE MDV is used as a biomedical model to study virus-induced lymphoma due to the similar genomic structures and physiological characteristics of MDV and human herpesviruses. Upon infection, MDV induces DNA damage, which may activate the DDR pathway. The DDR pathway has a dual impact on viruses because it manipulates repair and recombination factors to facilitate viral replication and also initiates antiviral action by regulating other signaling pathways. Many DNA viruses evolve to manipulate the DDR pathway to promote virus replication. In this study, we identified a mechanism used by MDV to inhibit ATR-Chk1 pathways. ATR is a cellular kinase that responds to broken single-stranded DNA, which has been less studied in MDV infection. Our results suggest that MDV infection activates STAT3 to disable the ATR-Chk1 pathway, which is conducive to viral replication. This finding provides new insight into the role of STAT3 in interrupting the ATR-Chk1 pathway during MDV replication.

Molecular and pathological characterization of duck enteritis virus in Egypt.

In winter 2016, a fatal disease outbreak suspected to be duck virus enteritis (DVE) stroke over a million ducklings in 10 white Pekin and Muscovy ducks flocks in Dakahlia and Gharbia Governorates, Egypt, causing heavy economic losses. The disease quickly killed 20%-60% of affected farms. The clinical signs were inappetence, ataxia, crowding in corners, partially closed eye lids and blue beaks. Post mortem examination revealed white necrotic foci in liver, mottled spleen and sometimes cecal core. A total of 10 intestines, livers and spleens samples were collected from diseased flocks. Each sample was pooled randomly from eight to ten ducklings. Polymerase chain reaction (PCR) and histopathological examination were utilized for DEV identification in collected samples. Nucleotides sequences of the amplified DNA polymerase gene were compared with the other DEVs available on GeneBank. Also, existence of co-infection with Salmonella spp. was verified via PCR. DEV nucleic acid was detected by PCR in 8 of 10 collected samples (80%) with positive amplification of polymerase gene. Histopathological examination revealed eosinophilic and basophilic intranuclear inclusion bodies in enterocytes. In some infected enterocytes, intranuclear and intracytoplasmic inclusions were observed in the same cell. Respectively, eosinophilic intranuclear inclusion bodies found in hepatocytes and reticular cells of liver and spleen of diseased ducklings. Four of the 10 collected samples showed positive results for Salmonella spp. infection that may be involved in enhancing infection with DEV. The identified DEVs revealed close genetic relationship with DEVs detected previously in India and China indicating potential transmission of the virus from there that crucially needs further work for better understanding of virus origin. In conclusion, our study revealed infection of duckling farms with DEV and Salmonella that necessitate the implementation of restricted early preventive and control measures for both diseases to decrease the expected economic losses.

Co-localization of and interaction between duck enteritis virus glycoprotein H and L.

BACKGROUND: Duck Enteritis Virus (DEV), belonging to the α-herpesvirus subfamily, is a linear double-stranded DNA virus. Glycoprotein H and L (gH and gL), encoded by UL22 and UL1, are conserved in the family of herpesviruses. They play important roles as gH/gL dimers during viral entry into host cells through cell-cell fusion. The interaction between gH and gL has been confirmed in several human herpesviruses, such as Herpes Simplex Virus (HSV), Epstein-Barr virus (EBV) and Human Cytomegalovirus (HCMV). In this paper, we studied the interaction between DEV gH and gL. RESULTS: Recombinant plasmids pEGFP-N-gH and pDsRED-N-gL were constructed successfully. Expressions of both DEV gH and gL were observed after incubation of COS-7 cells transfected with pEGFP-N-gH and pDsRED-N-gL plasmids after 12 h, respectively. Also, the co-localization of a proportion of the gH and gL was detected in the cytoplasm of COS-7 cells after co-transfection for 24 h. Then, pCMV-Flag-gL and pCMV-Myc-gH recombinant plasmids were constructed and co-transfected into COS-7 cells. It was showed that both gH and gL were tested with positive results through co-immunoprecipitation and Western-blotting. CONCLUSIONS: Our results demonstrated not only the co-localization of DEV gH and gL in COS-7 cells, but also the interaction between them. It will provide an insight for the further studies in terms of protein-protein interaction in DEV.

Duck enteritis virus activates CaMKKß-AMPK to trigger autophagy in duck embryo fibroblast cells via increased cytosolic calcium.

BACKGROUND: The results of our previous study showed that impaired cellular energy metabolism contributes to duck enteritis virus-induced autophagy via the 5`-adenosine monophosphate-activated protein kinase (AMPK)/tuberous sclerosis complex 2/mammalian target of rapamycin pathway in duck embryo fibroblast (DEF) cells. However, it remains unknown whether any other underlying mechanisms of AMPK activation are involved in autophagy induction. METHODS: The activity of CaMKKß and AMPK in DEF cells infected with DEV were evaluated.The Effect of inhibitory activity of CaMKKß on DEV-induced autophagy was investigated. In addtion to, the cytosolic calcium level in DEF cells infected with DEV were evaluated.The Effect of inhibitory cytosolic calcium level on DEV-induced autophagy was investigated. RESULTS: In this study, duck enteritis virus (DEV) infection activated CaMKKß and its substrate molecule AMPK at 36, 48, and 60 h post-infection (hpi). STO-609, a CaMKKß inhibitor, or CaMKKß siRNA significantly inhibited the activation of DEV to AMPK, LC3I to LC3II transformation, and GFP-LC3 puncta distribution. In addition, inhibition of CaMKKß activity also significantly reduced progeny DEV titer and gB protein expression. Besides, cytosolic calcium (Ca2+) was higher in DEV-infected cells than mock controls at 36, 48, and 60 hpi, respectively. Treatment of DEV-infected cells with 1,2-Bis (2-aminophenoxy) ethane-N, N, N', N-tetraacetic acid (BAPTA-AM) significantly reduced intracellular Ca2+ ion concentrations, as well as CaMKKß and AMPK activities, and subsequent autophagy, in addition to viral protein synthesis and viral titer. CONCLUSIONS: These results showed that elevated [Ca2+]cyto-mediated activation of CaMKKß managed the activation of AMPK, which then positively regulated autophagy, thereby providing further insight into DEV-host interactions.

Synergistic Viral Replication of Marek's Disease Virus and Avian Leukosis Virus Subgroup J is Responsible for the Enhanced Pathogenicity in the Superinfection of Chickens.

Superinfection of Marek's disease virus (MDV) and avian leukosis virus subgroup J (ALV-J) causes lethal neoplasia and death in chickens. However, whether there is synergism between the two viruses in viral replication and pathogenicity has remained elusive. In this study, we found that the superinfection of MDV and ALV-J increased the viral replication of the two viruses in RNA and protein level, and synergistically promoted the expression of IL-10, IL-6, and TGF-ß in chicken embryo fibroblasts (CEF). Moreover, MDV and ALV-J protein expression in dual-infected cells detected by confocal laser scanning microscope appeared earlier in the cytoplasm and the nucleus, and caused more severe cytopathy than single infection, suggesting that synergistically increased MDV and ALV-J viral-protein biosynthesis is responsible for the severe cytopathy. In vivo, compared to the single virus infected chickens, the mortality and tumor formation rates increased significantly in MDV and ALV-J dual-infected chickens. Viral loads of MDV and ALV-J in tissues of dual-infected chickens were significantly higher than those of single-infected chickens. Histopathology observation showed that more severe inflammation and tumor cells metastases were present in dual-infected chickens. In the present study, we concluded that synergistic viral replication of MDV and ALV-J is responsible for the enhanced pathogenicity in superinfection of chickens.

Duck plague virus Glycoprotein J is functional but slightly impaired in viral replication and cell-to-cell spread.

To analyse the function of the duck plague virus (DPV) glycoprotein J homologue (gJ), two different mutated viruses, a gJ deleted mutant ΔgJ and a gJR rescue mutant gJR with US5 restored were generated. All recombinant viruses were constructed by using two-step of RED recombination system implemented on the duck plague virus Chinese virulent strain (DPV CHv) genome cloned into a bacterial artificial chromosome. DPV-mutants were characterized on non-complementing DEF cells compared with parental virus. Viral replication kinetics of intracellular and extracellular viruses revealed that the ΔgJ virus produce a 10-fold reduction of viral titers than the gJR and parental virus, which especially the production of extracellular infectivity was affected. In addition, the ΔgJ virus produced viral plaques on DEF cells that was on average approximately 11% smaller than those produced by the gJR and parental viruses. Electron microscopy confirmed that although DPV CHv without gJ could efficiently carry out viral replication, virion assembly and envelopment within infected cells, the ΔgJ virus produced and accumulated high levels of anuclear particles in the nuclear and cytoplasm. These results show that the gJ slightly impaired in viral replication, virion assembly and cell-to-cell spread, and is not essential in virion envelopment.

Epstein-Barr virus-encoded RNAs (EBERs) complement the loss of Herpesvirus telomerase RNA (vTR) in virus-induced tumor formation.

Marek's disease virus (MDV) is an alphaherpesvirus that causes fatal lymphomas in chickens and is used as a natural virus-host model for herpesvirus-induced tumorigenesis. MDV encodes a telomerase RNA subunit (vTR) that is crucial for efficient MDV-induced lymphoma formation; however, the mechanism is not completely understood. Similarly, Epstein Barr-virus (EBV) encodes two RNAs (EBER-1 and EBER-2) that are highly expressed in EBV-induced tumor cells, however their role in tumorigenesis remains unclear. Intriguingly, vTR and EBER-1 have interaction partners in common that are highly conserved in humans and chickens. Therefore, we investigated if EBER-1 and/or EBER-2 can complement the loss of vTR in MDV-induced tumor formation. We first deleted vTR (v∆vTR) and replaced it by either EBER-1 or EBER-2 in the very virulent RB-1B strain. Insertion of either EBER-1 or EBER-2 did not affect MDV replication and their expression levels were comparable to vTR in wild type virus. Intriguingly, EBER-2 restored tumor formation of MDV that lacks vTR. EBER-1 partially restored MDV oncogenicity, while tumor formation was severely impaired in chickens infected with v∆vTR. Our data provides the first evidence that EBERs possess tumor-promoting properties in vivo using this natural model for herpesvirus-tumorigenesis.