RNA-seq analysis of chlorogenic acid intervention in duck embryo fibroblasts infected with duck plague virus.

INTRODUCTION: Chlorogenic acid, the primary active component in Chinese medicines like honeysuckle, exhibits anti-inflammatory and antiviral effects. It has been demonstrated that chlorogenic acid effectively prevents and treats Duck enteritis virus (DEV) infection. This study aims to further elucidate the mechanism by which chlorogenic acid prevents DEV infection. METHODS: Duck embryo fibroblast (DEF) cells were pre-treated with chlorogenic acid before being infected with DEV. Cell samples were collected at different time points for transcriptomic sequencing, while qPCR was used to detect the proliferation of DEV. Additionally, 30-day-old ducks were treated with chlorogenic acid, and their lymphoid organs were harvested for histopathological sections to observe pathological damage. The proliferation of DEV in the lymphoid organs was also detected using qPCR Based on the transcriptomic sequencing results, NF-κB1 gene was silenced by RNAi technology to analyze the effect of NF-κB1 gene on DEV proliferation. RESULTS: Compared to the viral infection group, DEF cells in the chlorogenic acid intervention group exhibited significantly reduced DEV load (P < 0.05). Transcriptomic sequencing results suggested that chlorogenic acid inhibited DEV proliferation in DEF cells by regulating NF-κB signaling pathway. The results of RNAi silencing suggested that in the three treatment groups, compared with the DEV experimental group, there was no significant difference in the effect of pre-transfection after transfection on DEV proliferation, while both the pre-transfection after transfection and the simultaneous transfection group showed significant inhibition on DEV proliferation Furthermore, compared to the virus infection group, ducks in the chlorogenic acid intervention group showed significantly decreased DEV load in their lymphoid organs (P < 0.05), along with alleviated pathological damage such as nuclear pyretosis and nuclear fragmentation. CONCLUSIONS: Chlorogenic acid effectively inhibits DEV proliferation in DEF and duck lymphatic organs, mitigates viral-induced pathological damage, and provides a theoretical basis for screening targeted drugs against DEV.

Duck STING mediates antiviral autophagy directing the interferon signaling pathway to inhibit duck plague virus infection.

Migratory birds are important vectors for virus transmission, how migratory birds recognize viruses and viruses are sustained in birds is still enigmatic. As an animal model for waterfowl among migratory birds, studying and dissecting the antiviral immunity and viral evasion in duck cells may pave a path to deciphering these puzzles. Here, we studied the mechanism of antiviral autophagy mediated by duck STING in DEF cells. The results collaborated that duck STING could significantly enhance LC3B-II/I turnover, LC3B-EGFP puncta formation, and mCherry/EGFP ratio, indicating that duck STING could induce autophagy. The autophagy induced by duck STING is not affected by shRNA knockdown of ATG5 expression, deletion of the C-terminal tail of STING, or TBK1 inhibitor BX795 treatment, indicating that duck STING activated non-classical selective autophagy is independent of interaction with TBK1, TBK1 phosphorylation, and interferon (IFN) signaling. The STING R235A mutant and Sar1A/B kinase mutant abolished duck STING induced autophagy, suggesting binding with cGAMP and COPII complex mediated transport are the critical prerequisite. Duck STING interacted with LC3B through LIR motifs to induce autophagy, the LIR 4/7 motif mutants of duck STING abolished the interaction with LC3B, and neither activated autophagy nor IFN expression, indicating that duck STING associates with LC3B directed autophagy and dictated innate immunity activation. Finally, we found that duck STING mediated autophagy significantly inhibited duck plague virus (DPV) infection via ubiquitously degraded viral proteins. Our study may shed light on one scenario about the control and evasion of diseases transmitted by migratory birds.

Marek's disease virus prolongs survival of primary chicken B-cells by inducing a senescence-like phenotype.

Marek's disease virus (MDV) is an alphaherpesvirus that causes immunosuppression and deadly lymphoma in chickens. Lymphoid organs play a central role in MDV infection in animals. B-cells in the bursa of Fabricius facilitate high levels of MDV replication and contribute to dissemination at early stages of infection. Several studies investigated host responses in bursal tissue of MDV-infected chickens; however, the cellular responses specifically in bursal B-cells has never been investigated. We took advantage of our recently established in vitro infection system to decipher the cellular responses of bursal B-cells to infection with a very virulent MDV strain. Here, we demonstrate that MDV infection extends the survival of bursal B-cells in culture. Microarray analyses revealed that most cytokine/cytokine-receptor-, cell cycle- and apoptosis-associated genes are significantly down-regulated in these cells. Further functional assays validated these strong effects of MDV infections on cell cycle progression and thus, B-cell proliferation. In addition, we confirmed that MDV infections protect B-cells from apoptosis and trigger an accumulation of the autophagy marker Lc3-II. Taken together, our data indicate that MDV-infected bursal B-cells show hallmarks of a senescence-like phenotype, leading to a prolonged B-cell survival. This study provides an in-depth analysis of bursal B-cell responses to MDV infection and important insights into how the virus extends the survival of these cells.

Duck plague virus tegument protein vp22 plays a key role in the secondary envelopment and cell-to-cell spread.

Duck plague virus (DPV) is one of the major infectious and fatal diseases of geese, ducks, and other wild waterfowl. The DPV UL49 gene product VP22 is one of the most abundant tegument proteins. However, the role of the DPV VP22 is enigmatic to be clarified. In this study, we found deletion of the UL49 gene resulted in reduced viral growth curve and smaller plaque size in duck embryo fibroblast (DEF) cells, confirming that DPV VP22 is required for efficient viral growth in vitro. In addition, deletion of the UL49 gene inhibited the secondary envelopment of the virus, the release of viral particles, and the spread of viruses between cells. Our study signified the importance of VP22 for DPV secondary envelopment, release, cell-to-cell spread, and accumulation of viral RNA. These findings provide a basis for further study of the function of VP22 in DPV or other herpesviruses.

Differential expression profile and in-silico functional analysis of long noncoding RNA and mRNA in duck embryo fibroblasts infected with duck plague virus.

BACKGROUND: Duck plague virus (DPV), belonging to herpesviruses, is a linear double-stranded DNA virus. There are many reports about the outbreak of the duck plague in a variety of countries, which caused huge economic losses. Recently, increasing reports revealed that multiple long non-coding RNAs (lncRNAs) can possess great potential in the regulation of host antiviral immune response. Furthermore, it remains to be determined which specific molecular mechanisms are responsible for the DPV-host interaction in host immunity. Here, lncRNAs and mRNAs in DPV infected duck embryonic fibroblast (DEF) cells were identified by high-throughput RNA-sequencing (RNA-seq). And we predicted target genes of differentially expressed genes (DEGs) and formed a complex regulatory network depending on in-silico analysis and prediction. RESULT: RNA-seq analysis results showed that 2921 lncRNAs were found at 30 h post-infection (hpi). In our study, 218 DE lncRNAs and 2840 DE mRNAs were obtained in DEF after DPV infection. Among these DEGs and target genes, some have been authenticated as immune-related molecules, such as a Macrophage mannose receptor (MR), Anas platyrhynchos toll-like receptor 2 (TLR2), leukocyte differentiation antigen, interleukin family, and their related regulatory factors. Furthermore, according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis, we found that the target genes may have important effects on biological development, biosynthesis, signal transduction, cell biological regulation, and cell process. Also, we obtained, the potential targeting relationship existing in DEF cells between host lncRNAs and DPV-encoded miRNAs by software. CONCLUSIONS: This study revealed not only expression changes, but also the possible biological regulatory relationship of lncRNAs and mRNAs in DPV infected DEF cells. Together, these data and analyses provide additional insight into the role of lncRNAs and mRNAs in the host's immune response to DPV infection.

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.

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.

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.

Why the evolution of vaccine resistance is less of a concern than the evolution of drug resistance.

Vaccines and antimicrobial drugs both impose strong selection for resistance. Yet only drug resistance is a major challenge for 21st century medicine. Why is drug resistance ubiquitous and not vaccine resistance? Part of the answer is that vaccine resistance is far less likely to evolve than drug resistance. But what happens when vaccine resistance does evolve? We review six putative cases. We find that in contrast to drug resistance, vaccine resistance is harder to detect and harder to confirm and that the mechanistic basis is less well understood. Nevertheless, in the cases we examined, the pronounced health benefits associated with vaccination have largely been sustained. Thus, we contend that vaccine resistance is less of a concern than drug resistance because it is less likely to evolve and when it does, it is less harmful to human and animal health and well-being. Studies of pathogen strains that evolve the capacity to replicate and transmit from vaccinated hosts will enhance our ability to develop next-generation vaccines that minimize the risk of harmful pathogen evolution.

Marek's Disease Virus-Encoded MicroRNA 155 Ortholog Critical for the Induction of Lymphomas Is Not Essential for the Proliferation of Transformed Cell Lines.

MicroRNAs (miRNAs) are small noncoding RNAs with profound regulatory roles in many areas of biology, including cancer. MicroRNA 155 (miR-155), one of the extensively studied multifunctional miRNAs, is important in several human malignancies such as diffuse large B cell lymphoma and chronic lymphocytic leukemia. Moreover, miR-155 orthologs KSHV-miR-K12-11 and MDV-miR-M4, encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) and Marek's disease virus (MDV), respectively, are also involved in oncogenesis. In MDV-induced T-cell lymphomas and in lymphoblastoid cell lines derived from them, MDV-miR-M4 is highly expressed. Using excellent disease models of infection in natural avian hosts, we showed previously that MDV-miR-M4 is critical for the induction of T-cell lymphomas as mutant viruses with precise deletions were significantly compromised in their oncogenicity. However, those studies did not elucidate whether continued expression of MDV-miR-M4 is essential for maintaining the transformed phenotype of tumor cells. Here using an in situ CRISPR/Cas9 editing approach, we deleted MDV-miR-M4 from the MDV-induced lymphoma-derived lymphoblastoid cell line MDCC-HP8. Precise deletion of MDV-miR-M4 was confirmed by PCR, sequencing, quantitative reverse transcription-PCR (qRT-PCR), and functional analysis. Continued proliferation of the MDV-miR-M4-deleted cell lines demonstrated that MDV-miR-M4 expression is not essential for maintaining the transformed phenotype, despite its initial critical role in the induction of lymphomas. Ability to examine the direct role of oncogenic miRNAs in situ in tumor cell lines is valuable in delineating distinct determinants and pathways associated with the induction or maintenance of transformation in cancer cells and will also contribute significantly to gaining further insights into the biology of oncogenic herpesviruses.IMPORTANCE Marek's disease virus (MDV) is an alphaherpesvirus associated with Marek's disease (MD), a highly contagious neoplastic disease of chickens. MD serves as an excellent model for studying virus-induced T-cell lymphomas in the natural chicken hosts. Among the limited set of genes associated with MD oncogenicity, MDV-miR-M4, a highly expressed viral ortholog of the oncogenic miR-155, has received extensive attention due to its direct role in the induction of lymphomas. Using a targeted CRISPR-Cas9-based gene editing approach in MDV-transformed lymphoblastoid cell lines, we show that MDV-miR-M4, despite its critical role in the induction of tumors, is not essential for maintaining the transformed phenotype and continuous proliferation. As far as we know, this was the first study in which precise editing of an oncogenic miRNA was carried out in situ in MD lymphoma-derived cell lines to demonstrate that it is not essential in maintaining the transformed phenotype.

Replication of Marek's Disease Virus Is Dependent on Synthesis of De Novo Fatty Acid and Prostaglandin E2.

Marek's disease virus (MDV) causes deadly lymphoma and induces an imbalance of the lipid metabolism in infected chickens. Here, we discovered that MDV activates the fatty acid synthesis (FAS) pathway in primary chicken embryo fibroblasts (CEFs). In addition, MDV-infected cells contained high levels of fatty acids and showed increased numbers of lipid droplets (LDs). Chemical inhibitors of the FAS pathway (TOFA and C75) reduced MDV titers by approximately 30-fold. Addition of the downstream metabolites, including malonyl-coenzyme A and palmitic acid, completely restored the inhibitory effects of the FAS inhibitors. Furthermore, we could demonstrate that MDV infection activates the COX-2/prostaglandin E2 (PGE2) pathway, as evident by increased levels of arachidonic acid, COX-2 expression, and PGE2 synthesis. Inhibition of the COX-2/PGE2 pathway by chemical inhibitors or knockdown of COX2 using short hairpin RNA reduced MDV titers, suggesting that COX-2 promotes virus replication. Exogenous PGE2 completely restored the inhibition of the COX-2/PGE2 pathway in MDV replication. Unexpectedly, exogenous PGE2 also partially rescued the inhibitory effects of FAS inhibitors on MDV replication, suggesting that there is a link between these two pathways in MDV infection. Taken together, our data demonstrate that the FAS and COX-2/PGE2 pathways play an important role in the replication of this deadly pathogen.IMPORTANCE Disturbances of the lipid metabolism in chickens infected with MDV contribute to the pathogenesis of disease. However, the role of lipid metabolism in MDV replication remained unknown. Here, we demonstrate that MDV infection activates FAS and induces LD formation. Moreover, our results demonstrate that MDV replication is highly dependent on the FAS pathway and the downstream metabolites. Finally, our results reveal that MDV also activates the COX-2/PGE2 pathway, which supports MDV replication by activating PGE2/EP2 and PGE2/EP4 signaling pathways.

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.

Genetic characterization of a Marek's disease virus strain isolated in Japan.

BACKGROUND: Marek's disease virus (MDV) causes malignant lymphomas in chickens (Marek's disease, MD). MD is currently controlled by vaccination; however, MDV strains have a tendency to develop increased virulence. Distinct diversity and point mutations are present in the Meq proteins, the oncoproteins of MDV, suggesting that changes in protein function induced by amino acid substitutions might affect MDV virulence. We previously reported that recent MDV isolates in Japan display distinct mutations in Meq proteins from those observed in traditional MDV isolates in Japan, but similar to those in MDV strains isolated from other countries. METHODS: To further investigate the genetic characteristics in Japanese field strains, we sequenced the whole genome of an MDV strain that was successfully isolated from a chicken with MD in Japan. A phylogenetic analysis of the meq gene was also performed. RESULTS: Phylogenetic analysis revealed that the Meq proteins in most of the Japanese isolates were similar to those of Chinese and European strains, and the genomic sequence of the Japanese strain was classified into the Eurasian cluster. Comparison of coding region sequences among the Japanese strain and MDV strains from other countries revealed that the genetic characteristics of the Japanese strain were similar to those of Chinese and European strains. CONCLUSIONS: The MDV strains distributed in Asian and European countries including Japan seem to be genetically closer to each other than to MDV strains from North America. These findings indicate that the genetic diversities of MDV strains that emerged may have been dependent on the different vaccination-based control approaches.

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).

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.

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 101 bp, 48 bp and 417 bp 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.

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.

Fowl 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.

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

BACKGROUND: pUL21 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. RESULTS: We 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. CONCLUSIONS: The 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.

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

BACKGROUND: Aquatic 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. RESULTS: Intrachoanal 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. CONCLUSIONS: Collectively, 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.

Antiviral effect of baicalin on Marek's disease virus in CEF cells.

BACKGROUND: Baicalin, the main metabolic component of Scutellaria baicalensis Georgi, has various pharmacological properties including anti-inflammatory, anti-oxidant, anti-apoptotic, anti-bactericidal and anti-viral. The purpose of this study was to investigate the anti-Marek's disease virus (MDV) activities of baicalin in CEF cells. RESULTS: Here, we showed that baicalin could inhibit viral mRNA, protein levels and overall plaque formation in a time-dependent manner. We also found that baicalin could consistently inhibit MDV replication and directly affect the virus infectivity. Moreover, baicalin treatment has no effect on expression level of antiviral cytokine and inflammatory cytokines in MDV infected CEFs. CONCLUSIONS: These results demonstrate that baicalin could be a potential drug against MDV infection.