Comparative genomic analysis of duck enteritis virus strains.
ABSTRACT: The icosahedral virion of duck enteritis virus (DEV) is roughly spherical and approximately 150 nm in diameter. Here, we describe the genomic features of DEV CHv together with a draft genome sequence and its annotation, highlighting the homogeneity and heterogeneity of this genome in comparison with its reference genomes.
Project description:The Chinese virulent (CHv) strain of duck enteritis virus (DEV) has a genome of approximately 162,175 nucleotides with a GC content of 44.89%. Here we report the complete genomic sequence and annotation of DEV CHv, which offer an effective platform for providing authentic research experiences to novice scientists. In addition, knowledge of this virus will extend our general knowledge of DEV and will be useful for further studies of the mechanisms of virus replication and pathogenesis.
Project description:Lethal 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.In 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.The 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.We 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.
Project description:Duck enteritis virus (DEV) belongs to the family Herpesviridae and is an important epornitic agent that causes economic losses in the waterfowl industry. The Chinese virulent (CHv) and attenuate vaccines (VAC) are two different pathogenic DEV strains. MicroRNAs (miRNAs) are a class of non-coding RNAs that regulate gene expression in viral infection. Nonetheless, there is little information on virulent duck enteritis virus (DEV)-encoded miRNAs.Using high-throughput sequencing, we identified 39 mature viral miRNAs from CHv-infected duck embryo fibroblasts cells. Compared with the reported 33 VAC-encoded miRNAs, only 13 miRNA sequences and 22 "seed sequences" of miRNA were identical, and 8 novel viral miRNAs were detected and confirmed by stem-loop RT-qPCR in this study. Using RNAhybrid and PITA software, 38 CHv-encoded miRNAs were predicted to target 41 viral genes and formed a complex regulatory network. Dual luciferase reporter assay (DLRA) confirmed that viral dev-miR-D8-3p can directly target the 3'-UTR of CHv US1 gene (p < 0.05). Gene Ontology analysis on host target genes of viral miRNAs were mainly involved in biological regulation, cellular and metabolic processes. In addition, 598 novel duck-encoded miRNAs were detected in this study. Thirty-eight host miRNAs showed significant differential expression after CHv infection: 13 miRNAs were up-regulated, and 25 miRNAs were down-regulated, which may affect viral replication in the host cell.These data suggested that CHv encoded a different set of microRNAs and formed a unique regulatory network compared with VAC. This is the first report of DEF miRNAs expression profile and an analysis of these miRNAs regulatory mechanisms during DEV infection. These data provide a basis for further exploring miRNA regulatory roles in the pathogenesis of DEV infection and contribute to the understanding of the CHv-host interaction at the miRNA level.
Project description:BACKGROUND: The function and kinetics of some herpsvirus UL16 gene have been reported. But there was no any report of duck enteritis virus (DEV) UL16 gene. FINDINGS: The kinetics of DEV UL16 gene was examined in DEV CHv infected duck embryo fibroblasts (DEFs) by establishment of real-time quantitative reverse transcription polymerase chain reaction assay (qRT-PCR) and western-blotting. In this study, UL16 mRNA was transcript at a low level from 0-18 h post-infection (p.i), and peaked at 36 h p.i. It can't be detected in the presence of acyclovir (ACV). Besides, western-blotting analysis showed that UL16 gene expressed as an apparent 40-KDa in DEV infected cell lysate from 12 h p.i, and rose to peak level at 48 h p.i consistent with the qRT-PCR result. CONCLUSIONS: These results provided the first evidence of the kinetics of DEV UL16 gene. DEV UL16 gene was a late gene and dependent on viral DNA synthesis.
Project description:To investigate the function of the duck enteritis virus (DEV) tegument protein US10, we generated US10 deletion and revertant mutants (?US10 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, ?US10 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, ?US10 and US10FRT, but ?US10 still showed a 5- to 20-fold reduction in viral titer, and the replication defect of ?US10 was partially reversed by infection of US10-expressing cells. The transcription levels of Mx, OASL, IL-4, IL-6 and IL-10 in ?US10-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.
Project description:BACKGROUND:There is little information regarding the duck enteritis virus (DEV) US10 gene and its molecular characterization. METHODS:Duck enteritis virus US10 was amplified and cloned into the recombinant vector pET32a(+). The recombinant US10 protein was expressed in Escherichia coli BL21 cells and used to immunize rabbits for the preparation of polyclonal antibodies. The harvested rabbit antiserum against DEV US10 was detected and analyzed by agar immunodiffusion. Using this antibody, western blotting and indirect immunofluorescence analysis were used to analyze the expression level and subcellular localization of US10 in infected cells at different time points. Quantitative reverse-transcription PCR (qRT-PCR) and pharmacological inhibition tests were used to ascertain the kinetic class of the US10 gene. A mass spectrometry-based strategy was used to identify US10 in purified DEV virions and quantify its abundance. RESULTS:The recombinant pET32a(+)/US10 protein was expressed as inclusion bodies, purified by gradient urea washing, and used to prepare specific antibodies. The results of qRT-PCR, western blotting, and pharmacological inhibition tests revealed that US10 is mainly transcribed in the late stage of viral replication. However, the presence of the DNA polymerase inhibitor ganciclovir and the protein synthesis inhibitor cycloheximide blocked transcription. Therefore, US10 is a ?2 (true late) gene. Indirect immunofluorescence analysis showed that US10 proteins were initially diffusely distributed throughout the cytoplasm, but with the passage of time, they gradually relocated to a perinuclear region. The US10 protein was detected in purified DEV virions by mass spectrometry, but was not detected by western blotting, indicating that DEV US10 is a minor virion protein. CONCLUSIONS:The DEV US10 gene is a ?2 gene and the US10 protein is localized in the perinuclear region. DEV US10 is a virion component.
Project description: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 ?2 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.
Project description:Duck enteritis virus (DEV) belongs to the subfamily Alphaherpesvirinae, and information on the DEV UL41 gene is limited.The DEV UL41 gene was cloned into the pET32a(+) vector and expressed in a prokaryotic expression system. Antiserum was raised against a bacterially expressed UL41-His fusion protein for further experiments. Transcription was quantified and UL41 protein expression levels were determined in DEV-infected cells at different time points by RT-qPCR and western blotting, respectively. DEV virions were purified by sucrose gradient centrifugation and analyzed by mass spectrometry to identify protein content. We confirmed the DEV UL41 gene kinetic class using a pharmacological test. IFA was used to analyze the intracellular localization of pUL41.The recombinant expression plasmid, pET-32a(+)-UL41, which highly expresses a 76.0 kDa fusion protein, was constructed and expressed in E. coli BL21 (DE3) after induction with 0.2 mM IPTG at 30 °C for 10 h, generating a specific mouse anti-UL41 protein polyclonal antibody. RT-qPCR and western blot analyses revealed that the UL41 transcript number peaked at 36 hpi, and peak protein expression occurred at 48 hpi. The pharmacological test showed that UL41 was a γ2 gene. Mass spectrometry analysis showed that pUL41 was a virion component. IFA results revealed that pUL41 was localized throughout DEV-infected cells but only localized to the cytoplasm of transfected cells. DEV pUL47 translocated pUL41 to the nuclei of DEF cells; this translocation was dependent on predicted pUL47 NLS signals (40-50 aa and 768-777 aa).DEV UL41 is a γ2 gene that encodes a virion structural protein, pUL41 localizes throughout DEV-infected cells but only localizes to the cytoplasm of transfected cells. pUL41 cannot autonomously localize to the nucleus, as this nuclear localization is dependent on predicted DEV pUL47 NLS signals (40-50 aa and 768-777 aa).
Project description:Duck is susceptible to many pathogens, such as duck hepatitis virus, duck enteritis virus (DEV), duck tembusu virus, H5N1 highly pathogenic avian influenza virus (HPAIV) in particular. With the significant role of duck in the evolution of H5N1 HPAIV, control and eradication of H5N1 HPAIV in duck through vaccine immunization is considered an effective method in minimizing the threat of a pandemic outbreak. Consequently, a practical strategy to construct a vaccine against these pathogens should be determined. In this study, the DEV was examined as a candidate vaccine vector to deliver the hemagglutinin (HA) gene of H5N1, and its potential as a polyvalent vaccine was evaluated. A modified mini-F vector was inserted into the gB and UL26 gene junction of the attenuated DEV vaccine strain C-KCE genome to generate an infectious bacterial artificial chromosome (BAC) of C-KCE (vBAC-C-KCE). The HA gene of A/duck/Hubei/xn/2007 (H5N1) was inserted into the C-KCE genome via the mating-assisted genetically integrated cloning (MAGIC) to generate the recombinant vector pBAC-C-KCE-HA. A bivalent vaccine C-KCE-HA was developed by eliminating the BAC backbone. Ducks immunized with C-KCE-HA induced both the cross-reactive antibodies and T cell response against H5. Moreover, C-KCE-HA-immunized ducks provided rapid and long-lasting protection against homologous and heterologous HPAIV H5N1 and DEV clinical signs, death, and primary viral replication. In conclusion, our BAC-C-KCE is a promising platform for developing a polyvalent live attenuated vaccine.
Project description:Here, we present the complete genome sequence of an attenuated duck enteritis virus (DEV) obtained by serial chicken embryo passage. Compared with a virulent DEV, there is a serial deletion in unique long open reading frame 11 (LORF11) and unique long region 2 (UL2). This study will aid in further exploration of the molecular pathogenesis of DEV.