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In this study, apoptosis was induced by new type gosling viral enteritis virus (NGVEV) in experimentally infected goslings is reported in detail for the first time. After 3-day-old goslings were orally inoculated with a NGVEV-CN strain suspension, the time course of NGVEV effects on apoptotic morphological changes of the internal tissues was evaluated. These changes were observed by histological analysis with light microscopy and ultrastructural analysis with transmission electron microscopy. DNA fragmentation was assessed with a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and DNA ladder analysis. A series of characteristic apoptotic morphological changes including chromatin condensation and margination, cytoplasmic shrinkage, plasma membrane blebbing, and formation of apoptotic bodies were noted. Apoptosis was readily observed in the lymphoid and gastrointestinal organs, and sporadically occurred in other organs after 3 days post-infection (PI). The presence and quantity of TUNEL-positive cells increased with infection time until 9 days PI. DNA extracted from the NGVEV-infected gosling cells displayed characteristic 180~200 bp ladders. Apoptotic cells were ubiquitously distributed, especially among lymphocytes, macrophages, monocytes, and epithelial and intestinal cells. Necrosis was subsequently detected during the late NGVEV-infection phase, which was characterized by cell swelling, plasma membrane collapse, and rapidly lysis. Our results suggested that apoptosis may play an important role in the pathogenesis of NGVE disease.

Background: Previous studies have indicated that the UL16 protein and its homologs from herpesvirus were conserved and played similar roles in viral DNA packaging, virion assembly, budding, and egress. However, there was no report on the UL16 gene product of duck enteritis virus (DEV). In this study, we analyzed the amino acid sequence of UL16 using bioinformatics tools and expressed in Escherichia coli Rosetta (DE3) induced by isopropy1-β-D-thiogalactopyranoside (IPTG). The recombinant protein was produced, purified using a Ni-NTA column and used to generate the polyclonal antibody against UL16. The intracellular distribution of the DEV UL16 product was carried out using indirect immunofluorescence assay., Results: 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., Conclusions: 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.

Knowledge of the intracellular location of a protein can provide useful insights into its function. Bioinformatic studies have predicted that the DEV pUL38 mainly targets the cytoplasm and nucleus. In this study, we obtained anti-pUL38 polyclonal sera. These antibodies were functional in western blotting and immunofluorescence in DEV-infected duck embryo fibroblasts (DEFs). pUL38 was expressed as a 51-kDa protein from 8 h post-infection onward, initially showing a diffuse distribution throughout the cytoplasm, and later in the nucleus. Furthermore, pUL38 was found in purified virus. These results provide the first evidence of the kinetics of expression and intracellular localization of DEV pUL38.

The UL24 gene of duck enteritis virus (DEV) is conserved across herpesviruses, but its protein characterization has not been reported. We expressed the UL24 gene in Escherichia coli BL21 from a recombinant plasmid pET32a/DEV-UL24 and used the resulting protein to raise antiserum. This antiserum recognized a 38-kDa protein in lysates from infected cells. SDS-PAGE analysis showed that the UL24 partial protein was highly expressed after induction by 0.4 mM IPTG at 30 degrees for 6 h. The results of purification revealed that expression protein was more purified using the method of electrophoresis than that of chromatography, but the yield was lower. In immunogenicity analysis, the protein could significantly elicit a specific antibody response in immunized ducklings when compared with the control groups, and the titers against expression protein reached the peak 1:5,120 (OD(450nm) = 2.5) on day 28 after immunization, while with mean titers of 1:10,240 (OD(450nm) = 3.37) in DEV commercial attenuated vaccine strain immunized duckling groups. It showed that expression protein is immunogenic in laboratory ducklings. On the basis of subcellular location, UL24 appeared to be predominantly nuclear membrane-associated, especially at later times in infection, and provided a good tool to further study the biofunctions of UL24 protein., (Copyright 2009 S. Karger AG, Basel.)

The article presents a study on the expression and distribution of tegument proteins from duck enteritis virus UL51 protein (UL51p) in experimentally infected ducks. For the study, researchers analyzed the DEV CHv strain from China. They used an immunoperoxidase staining approach in order to find the protein in paraffin-embedded tissues. They found that several DEV UL51p were distributed in the bursa of Fabricius, thymus, spleen, liver, esophagus, small intestine and large intestine of the ducks. They also foun dthat the protein can be expressed in the cytoplasm of different cell types.

To establish an ideal method for the detailed definition of the tropism of the goose adenovirus new-type gosling viral enteritis virus (NGVEV) in conventional paraformaldehyde-fixed paraffin-embedded gosling tissue sections, an indirect immunofluorescence assay was established and optimized in this study for the first time. Three-day-old goslings orally inoculated with the CN strain of NGVEV were killed from 0.5 h to 30 days post-infection (p.i.) at intervals, and organs were collected and prepared for immunofluorescence assay and ultrastructural observation. NGVEV antigens could be detected in the gastrointestinal tract and lymphoid organs as early as 24 h p.i. The lymphoid organs (bursa of Fabricius, thymus, Harderian gland and spleen), the digestive organs (proventriculus, gizzard, duodenum, jejunum, ileum, caecum, rectum and liver), kidney, myocardium and brain of NGVEV-infected goslings showed extensive evidence of presence of viral antigens. Lymphocytes, macrophages, mesenchymes, endothelial cells, epithelia, superficial and crypt mucosal cells, glandular cells, and fibrocytes served as the principal site for NGVEV localization. The number of positive cells and intensity of fluorescence increased until 15 days p.i. Ultrastructural observation showed that NGVEV particles could be seen from 3 days p.i. onwards. [ABSTRACT FROM AUTHOR]

Abstract: Duck virus enteritis (DVE) is an acute and contagious herpes virus infection of duck, geese and swans with high morbidity and mortality. The development of specific mucosal immune system against duck enteritis virus (DEV) infection for ducks has been hindered by a lack of knowledge concerning the purification of immunoglobulin A (IgA) of duck. In the present work, the method for purification of duck immunoglobulin A was developed, and the induction of intestinal mucosal immune responses against DEV was studied by orally infected ducklings with virulent DEV. The results showed that a continuous increased DEV DNA levels were observed in blood and various organs examined of orally infected ducklings throughout the infection, which was accompanied by the development of infection in ducklings from mild progressed to severe pathological lesions. Furthermore, a marked increased level of DEV-specific IgA and IgG antibodies in bile, serum and the intestinal tract, as well as the density of IgA+ cells in intestine were detected between 1 and 12days p.i., followed by a drastic reduction of the antibody levels and the density of IgA+ cells at 15days p.i. The results indicate that the DVE infection can stimulate both IgA-dominated antibody immune responses in the intestinal tract, and IgG-dominated antibody systemic immunity in the serum of ducklings orally inoculated with virulent DEV. The severe lesions of the villus epithelial cells and the lymphoid organs can suppress the intestinal mucosal immune responses. [Copyright &y& Elsevier]

Abstract: To determine the distribution and localization of new type gosling viral enteritis virus (NGVEV) in paraformaldehyde-fixed paraffin-embedded tissues of experimentally infected goslings, for the first time, an immunohistochemical (IHC) staining method was reported. Anti-NGVEV polyclonal serum was obtained from the rabbits immunized with purified NGVEV antigen, which was extracted by caprylic-ammonium sulphate method and purified through High-Q columns anion exchange chromatography. Three-day-old NGVEV-free goslings were orally inoculated with NGVEV-CN strain suspension as infection group and phosphate buffered saline solution (PBS) as control group, respectively. The tissues were collected at sequential time points between 0.5 and 720h post inoculation (PI), and prepared for IHC staining and ultra-structural observation. The positive immunoreactivity could be readily detected in the lymphoid and gastrointestinal organs of infected goslings as early as 48h PI, in the liver, kidney, pancreas and myocardium from 72h, and in the cerebrum and cerebellum from 96h, while it was hardly detected in the respiratory organs at any time. The positive staining reaction could be detected in NGVEV-infected goslings until 600h PI, and no positive staining cell could be observed in the controls. The highest levels of viral antigen were found in the bursa of Fabricius (BF), thymus, proventriculus, gizzard and intestine tract, moreover, the liver, kidney, spleen, myocardium and pancreas were intensively and widely stained. The target cells had a ubiquitous distribution, especially included the epithelial cells, endothelial cells, superficial and crypt mucosal cells, glandular cells, fibrocytes, macrophages and lymphocytes, which served as the principal sites for antigen localization. The ultra-structural observation by transmission electron microscope (TEM) further indicated that NGVEV particles could be widely detected in the lymphoid and digestive organs of infected goslings from 72h PI onwards. This work may be useful not only for offering a possibility of routine diagnosis of NGVE, but also for better understanding of the pathogenesis of the disease. [Copyright &y& Elsevier]