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Characterization of subcellular localization of duck enteritis virus UL51 protein
Chanjuan Shen, Yufei Guo, Anchun Cheng, Mingshu Wang, Yi Zhou, Dan Lin, Hongyi Xin, Na Zhang
Virology Journal , 2009, DOI: 10.1186/1743-422x-6-92
Abstract: The DEV UL51 gene product was identified as an approximate 34 kDa protein in DEV-infected cells analyzed by western blotting. Computer aided analysis suggested that DEV pUL51 is not targeted to the mitochondrial, extra-cellular or nucleus, but be targeted to the cytoplasmic in host cells, more specifically, palmitoylation of the pUL51 through the N-terminal cysteine at position 9 makes membrane association and Golgi localization possible. Using IIF analysis, we found that DEV pUL51 was first detected in a juxtanuclear region of DEV-infected cells at 9 h postinfection (p.i.), and then was detected widely distributed in the cytoplasm and especially was stronger in the juxtanuclear region from 12 to 60 h p.i. TIEM analysis revealed that DEV pUL51 was mainly associated with cytoplasmic virions and also with some membranous structure near the pUL51-specific immuno-labeling intracellular virion in the cytoplasmic vesicles; moreover, the pUL51 efficiently accumulated in the Golgi apparatus at first, and then was sent to the plasma membrane from the Golgi by some unknown mechanism.In this work, we described the basic characteristics of pUL51 subcellular localization and distribution for the first time. From these results, we concluded that palmitoylation at the N-terminal cysteine, which is conserved in all alphaherpesvirus UL51 homologs, is required for its membrane association and Golgi localization, and the pUL51 mainly localized to the juxtanuclear region of DEV-infected cells, as well seemed to be incorporated into mature virions as a component of the tegument. The research will provide useful clues for DEV pUL51 functional analysis, and will be usefull for further understanding the localization properties of alphaherpesvirus UL51 homologs.Duck enteritis virus (DEV) is a member of the subfamily Alphaherpesvirinae, and an important pathogen of waterfowl (ducks, geese, and swans), causing an acute contagious viral disease that result in substantial economic losses [1-3].
Expression and characterization of duck enteritis virus gI gene
Lijuan Li, Anchun Cheng, Mingshu Wang, Jun Xiang, Xiaoyuan Yang, Shunchuan Zhang, Dekang Zhu, Renyong Jia, Qihui Luo, Yi Zhou, Zhengli Chen, Xiaoyue Chen
Virology Journal , 2011, DOI: 10.1186/1743-422x-8-241
Abstract: The special 1221-bp fragment containing complete open reading frame(ORF) of duck enteritis virus(DEV) gI gene was extracted from plasmid pMD18-T-gI, and then cloned into prokaryotic expression vector pET-32a(+), resulting in pET-32a(+)-gI. After being confirmed by PCR, restriction endonuclease digestion and sequencing, pET-32a(+)-gI was transformed into E.coli BL21(DE3) competent cells for overexpression. DEV gI gene was successfully expressed by the addition of isopropyl-β-D-thiogalactopyranoside(IPTG). SDS-PAGE showed that the recombinant protein His6-tagged gI molecular weight was about 61 kDa. Subsequently, the expressed product was applied to generate specific antibody against gI protein. The specificity of the rabbit immuneserum was confirmed by its ability to react with the recombinant protein His6-tagged gI. In addition, real time-PCR was used to determine the the levels of the mRNA transcripts of gI gene, the results showed that the DEV gI gene was transcribed most abundantly during the late phase of infection. Furthermore, indirect immunofluorescence(IIF) was established to study the gI protein expression and localization in DEV-infected duck embryo fibroblasts (DEFs), the results confirmed that the protein was expressed and located in the cytoplasm of the infected cells, intensively.The recombinant prokaryotic expression vector of DEV gI gene was constructed successfully. The gI protein was successfully expressed by E.coli BL21(DE3) and maintained its antigenicity very well. The basic information of the transcription and intracellular localization of gI gene were presented, that would be helpful to assess the possible role of DEV gI gene. The research will provide useful clues for further functional analysis of DEV gI gene.Duck virus enteritis(DVE), also called duck plague, is an acute and contagious herpesvirus infection of waterfowls such as ducks, geese, and swans with high morbidity and mortality[1]. The causative agent of DVE is duck enteritis virus
Characterization of duck enteritis virus UL53 gene and glycoprotein K
Shunchuan Zhang, Jun Xiang, Anchun Cheng, Mingshu Wang, Ying Wu, Xiaoyuan Yang, Dekang Zhu, Renyong Jia, Qihui Luo, Zhengli Chen, Xiaoyue Chen
Virology Journal , 2011, DOI: 10.1186/1743-422x-8-235
Abstract: In our paper, the fluorescent quantitative real-time PCR(FQ-RT-PCR) assay and nucleic acid inhibition test were used to study the transcription characteristic of the DEV UL53 gene. Except detecting the mRNA of DEV UL53 gene, the product gK encoded by UL53 gene was detected through the expression kinetics of UL53 gene by the purified rabbit anti-UL53 protein polyclonal antibodies. Western-blotting and indirect immunofluorescence assays were used to detect gK. From the results of these experiments, the UL53 gene and gK were respectively identified as a late gene and a really late protein. On the other hand, the indirect immunofluorescence assay provided another information that the intracellular localization of DEV gK was mainly distributed in cytoplasm.By way of conclusions, we conceded that DEV UL53 gene is a really late gene, which is coincident with properties of UL53 homologs from other herpesvirus, such as ILTV(Infectious Laryngotracheitis virus) and HSV-1(Herpes simplex virus type 1). The properties of intracellular localization about gK protein provided a foundation for further functional analysis and further studies will be focused on constructing of the UL53 gene DEV mutant.Duck enteritis virus (DEV) is an alphaherpesvirinae that causes an acute, contagious and highly lethal disease in all ages of birds from the order Anseriformes (ducks, geese, and swans) [1-4]. DEV leads to heavy economic losses to the commercial duck industry due to its high mortality rate and decreased duck egg production [1].Whilst most of the previous research work had focused on the epidemiology and prevention of this disease [5,6]. With the development of protocols in molecular biology, nowadays more and more information about the genes of DEV was reported, such as UL5 [7], gC [8-10], UL24 [11-13], UL31 [14,15], UL35 [16,17], UL46 [18], UL38 [19], gE [20], UL51 [21], TK gene [22] and so on. While no information about DEV UL53 gene was known except our reported data [23,24], UL53 gene
Characterization of the duck enteritis virus UL55 protein
Ying Wu, Anchun Cheng, Mingshu Wang, Shunchuan Zhang, Dekang Zhu, Renyong Jia, Qihui Luo, Zhengli Chen, Xiaoyue Chen
Virology Journal , 2011, DOI: 10.1186/1743-422x-8-256
Abstract: The newly identified duck enteritis virus UL55 gene was about 561 bp, it was amplified and digested for construction of a recombinant plasmid pET32a(+)/UL55 for expression in Escherichia coli. SDS-PAGE analysis revealed the recombinant protein UL55(pUL55) was overexpressed in Escherichia coli BL21 host cells after induction by 0.2 mM IPTG at 37°C for 4 h and aggregated as inclusion bodies. The denatured protein about 40 KDa named pUL55 was purified by washing five times, and used to immune rabbits for preparation of polyclonal antibody. The prepared polyclonal antibody against pUL55 was detected and determined by Agar immundiffusion and Neutralization test. The results of Wstern blotting assay and intracellular analysis revealed that pUL55 was expressed most abundantly during the late phase of replication and mainly distributed in cytoplasm in duck enteritis virus infected cells.In this study, the duck enteritis virus UL55 protein was successfully expressed in prokaryotic expression system. Besides, we have prepared the polyclonal antibody against recombinant prtein UL55, and characterized some properties of the duck enteritis virus UL55 protein for the first time. The research will be useful for further functional analysis of this gene.Duck enteritis virus (DEV), alternatively known as Duck plague virus (DPV), is a fatal pathogen of the family Anatidae of the order anseriformes[1], leading to an acute, febrile, contagious, and septic disease to waterfowls of all ages. The resulting disease designated as duck virus enteritis (DVE) has caused serious losses in commercial duck production in domestic and wild waterfowl since it was firstly discovered in Netherlands[2]. To our knowledge, DEV has been clustered to the subfamily of alphaherpesvirinae according to the report of the Eighth International Committee on Taxonomy of Viruses (ICTV)[3]. However, it has not been classified to any genus yet.The genome of DEV is composed of a linear, double stranded DNA. In recent ye
Comparative analysis of the genes UL1 through UL7 of the duck enteritis virus and other herpesviruses of the subfamily Alphaherpesvirinae
Li, Huixin;Liu, Shengwang;Han, Zongxi;Shao, Yuhao;Chen, Shuhong;Kong, Xiangang;
Genetics and Molecular Biology , 2009, DOI: 10.1590/S1415-47572009005000003
Abstract: keywords : duck enteritis virus; ul1-ul7 genes; phylogenetic analysis.
Inhibitory Effect of Resveratrol against Duck Enteritis Virus In Vitro  [PDF]
Jiao Xu, Zhongqiong Yin, Li Li, Anchun Cheng, Renyong Jia, Xu Song, Hongke Lu, Shujun Dai, Cheng Lv, Xiaoxia Liang, Changliang He, Ling Zhao, Gang Su, Gang Ye, Fei Shi
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0065213
Abstract: Duck viral enteritis (DVE) is an acute, contagious herpesvirus infection of ducks, geese, and swans of all ages and species. This disease has been responsible for significant economic losses in domestic and wild waterfowl as a result of mortality, and decreased egg production. Resveratrol is a naturally occurring phytoalexin in specific plants and exhibits inhibitory activity against many kinds of virus. In this paper, resveratrol was found to inhibit duck enteritis virus (DEV) replication in a dose-dependent manner, with a 50% inhibition concentration of 3.85 μg/mL. The inhibition in virus multiplication in the presence of resveratrol was not attributed to direct inactivation or inhibition of virus attachment to the host cells, but to the inhibition of viral multiplication in host cells. The assay of the time of addition limited the drug effect during the first 8 h of infection. This conclusion was supported by the ultrastructure images of the early stage of DEV infection, which showed that the replication of virus nucleic acid and the formation of the capsid in the cell nucleus were suppressed. In the indirect immunofluorescence assay, proteins expression in DEV infected duck embryo fibroblasts (DEFs) within 24 h post-infection (p.i.) was also effectively suppressed by resveratrol. In summary, the resveratrol has a good activity against DEV infection in vitro, which could be attributed to that fact that several essential immediate early viral proteins for virus replication were impacted by resveratrol.
Replication kinetics of duck enteritis virus UL16 gene in vitro  [cached]
He Qin,Cheng Anchun,Wang Mingshu,Xiang Jun
Virology Journal , 2012, DOI: 10.1186/1743-422x-9-281
Abstract: 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.
Expression and characterization of UL16 gene from duck enteritis virus
Qin He, Qiao Yang, Anchun Cheng, Mingshu Wang, Jun Xiang, Dekang Zhu, Renyong Jia, Qihui Luo, Zhengli Chen, Yi Zhou, Xiaoyue Chen
Virology Journal , 2011, DOI: 10.1186/1743-422x-8-413
Abstract: In our study, UL16 gene of DEV was composed of 1089 nucleotides, which encoded 362 amino acids. Multiple sequence alignment suggested that the UL16 gene was highly conserved in herpesvirus family. The UL16 gene was cloned into a pET prokaryotic expression vector and transformed into Escherichia coli Rossetta (DE3) induced by IPTG. A 60kDa fusion protein band corresponding to the predicted size was produced on the SDS-PAGE, purified using a Ni-NTA column. Anti-UL16 polyclonal sera was prepared by immunizing rabbits, and reacted with a band in the IPTG induced cell lysates with an apparent molecular mass of 60 kDa. In vivo expression of the UL16 protein in DEV infected duck embryo fibroblast cells (DEFs) was localized mostly around perinuclear cytoplasmic area and in cytosol using indirect immunofluorescence assay.The UL16 gene of DEV was successfully cloned, expressed and detected in DEV infected DEFs for the first time. The UL16 protein localized mostly around perinuclear cytoplasmic area and in cytosol in DEV infected DEFs. DEV UL16 shared high similarity with UL16 family members, indicating that DEV UL16 many has similar function with its homologs. All these results may provide some insight for further research about full characterizations and functions of the DEV UL16.Duck viral enteritis (DVE), an acute and contagious disease, is highly lethal in all ages of birds from the order Anseriformes (ducks, geese, and swans). This disease is characterized by vascular lesions and tissue hemorrhage, as well as gastrointestinal, lymphatic, and nervous impairments [1-3]. Duck enteritis virus (DEV) is the causative agent for DVE and was first recorded in Holland in 1923 [4], more outbreaks were reported in the North America [5], Canada [6], France [7] and China [8] et al.According to the Eighth International Committee on Taxonomy of Viruses (ICTV), DEV (anatid herpesvirus I) is a member of subfamily Alphaherpesvirinae of the family Herpesviridae but not assigned to any genus
Production, purification and characterization of polyclonal antibody against the truncated gK of the duck enteritis virus
Shunchuan Zhang, Jun Xiang, Anchun Cheng, Mingshu Wang, Xin Li, Lijuan Li, Xiwen Chen, Dekang Zhu, Qihui Luo, Xiaoyue Chen
Virology Journal , 2010, DOI: 10.1186/1743-422x-7-241
Abstract: Duck virus enteritis (DVE) is an acute, contagious herpesvirus infection of ducks, geese, and swans, characterized by vascular damage, tissue hemorrhages, digestive mucosal eruptions, lesions of lymphoid organs, and degenerative changes in parenchymatous organs [1-5]. The causative agent of DVE is duck enteritis virus(DEV), composing of a linear, double-stranded DNA genome with 64.3% guanine-plus-cytosine content, which is higher than any other reported avian herpesvirus in the Alpha-herpesvirinae subfamily[6]. In duck-producing areas of the world where the diseases has been reported, DEV has produced significant economic losses in domestic and wild waterfowl due to mortality, condemnations, and decreased egg production[7].With the purpose of decreasing economic losses in the commercial duck industry, studying gK of DEV may be a new method for preferably preventing and curing this disease. Because glycoproteins are the major antigens recognized by the infected host's immune system and play an important role in mediating target cell infection, cellular entry of free viruses, and the maturation or egress of the virus [8,9]. Glycoprotein K is one of the major glycoproteins encoded by the DEV-gK gene, which is located in the unique long region of the DEV genome. Additionally, gK is capable of inducing a protective immune response in vivo and is responsible for viral binding to the cellular receptor [10,11].Although the disease has been reported in 1926, there was little information known about the functions of DEV-gK. To investigate the functions and characteristics of gK gene as well as gK, the full-length gK gene (fgK) and truncated gK gene (tgK) expression plasmid were constructed[11], only the tgK expressed efficiently in prokaryotic system (Figure 1, lane4). The recombinant tgK protein was purified by immobilized metal affinity chromatography (IMAC) and showed in (Figure 1, lane5).Then, the purified tgK was used to produce polyclonal antibody. Preimmune serum was c
Dynamic changes of apoptosis in duck embryo fibroblasts induced by new type Gosling viral enteritis virus

Shun Chen,Anchun Cheng,Mingshu Wang,Xiaoyue Chen,

自然科学进展 , 2008,
Abstract: The monolayer duck embryo fibroblast (DEF) cells were experimentally infected with new type Gosling viral enteritis virus (NGVEV) and the dynamic changes of apoptosis were detected at different time points after NGVEV infection by using transmission electron microscopy (TEM), DNA agarose gel electrophoresis and Annexin V-FITC/PI stained fluorescence-activated cell sorter (FACS).The result shows that NGVEV can induce infected cells undergo apoptosis and change regularly. A series of characteristic apoptotic morphological changes including shrink of the cells, chromatin condensation and margination, as well as formation of apoptotic bodies were observed by TEM. The typical ladder pattern of DNA fragmentation was demonstrated by agarose gel electrophoresis. And using flow cytometry analysis of Annexin V-FITC/PI staining, the dead, viable, apoptotic and necrotic cells could be analysis quantitatively.
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