Background: Previous studies indicate that the UL31 protein and its homology play similar roles in nuclear egress of all herpesviruses. However, there is no report on the UL31 gene product of DEV. In this study, we expressed and presented the basic properties of the DEV UL31 product. Results: The entire ORF of the UL31 was cloned into pET 32a (+) prokaryotic expression vector. Escherichia coli BL21(DE3) competent cells were transformed with the construct followed by the induction of protein expression by the addition of IPTG. Band corresponding to the predicted sizes (55 kDa) was produced on the SDS-PAGE. Over expressed 6×His-UL31 fusion protein was purified by nickel affinity chromatography. The DEV UL31 gene product has been identified by using a rabbit polyclonal antiserum raised against the purified protein. A protein of approximate 35 kDa that reacted with the antiserum was detected in immunoblots of DEV-infected cellular lysates, suggesting that the 35 kDa protein was the primary translation product of the UL31 gene. RT-PCR analyses revealed that the UL31 gene was transcribed most abundantly during the late phase of replication. Subsequently, Immunofluorescence analysis revealed that the protein was widespread speckled structures in the nuclei of infected cells. Western blotting of purified virion preparations showed that UL31 was a component of intracellular virions but was absent from mature extracellular virions. Finally, an Immunofluorescence assay was established to study the distribution of the UL31 antigen in tissues of artificially DEV infected ducks. The results showed that the UL31 antigen was primarily located in the cells of digestive organs and immunological organs. Conclusion: In this work, we present the basic properties of the DEV UL31 product. The results indicate that DEV UL31 shares many similarities with its HSV or PRV homolog UL31 and suggest that functional cross-complementation is possible between members of the Alpha herpesvirus subfamily. Furthermore, in vivo experiments with ducks infected with UL31-defective isolates of DEV will also be of importance in order to assess the possible role of the UL31 protein in viral pathogenesis. These properties of the UL31 protein provide a prerequisite for further functional analysis of this gene.

Highly pathogenic avian influenza virus (HPAIV) is a highly contagious disease which is a zoonotic pathogen of significant economic and public health concern. The outbreaks caused by HPAIV H5N1 of Asian origin have caused animal and human disease and mortality in several countries of Southeast Asia, such as Bangladesh, Cambodia, China, India, Indonesia, Laos, Myanmar, Thailand and Viet Nam. For the first time since 1961, this HPAIV has also caused extensive mortality in wild birds and has sparked debate of the role wild birds have played in the spread of this virus. Other than confirmed mortality events, little is known of this virus in wild birds. There is no report on the seroprevalence of avian influenza H5 infection in wild migratory birds in Yunnan Province. In this study we examined live wild birds in Yunnan Province for H5 specific antibody to better understand the occurrence of this disease in free living birds.

Background: In previous studies, the expression and localization characteristics of duck plague virus (DPV) gE protein have been described in cultured cells, but the properties of DPV gE protein have not been reported in vivo. Immunofluorescence analysis had been used for the detection of virus antigen, but there was no report on the use of this technique for the detection of DPV gE. In this study, we investigated the distribution of DPV gE protein on DPV-infected ducks using polyclonal antibody raised against the recombinant His-gE fusion protein by indirect immunofluorescence assay (IFA). Results: The recombinant gE protein was highly immunogenicity by ELISA, and the gE was used as an antigen for the preparation of polyclonal antibody, which could be used the first antibody for further experiment to study the distribution of DPV gE protein in DPV-infected tissues by indirect immunofluorescence assay. DPV gE protein were distributed in the immune organs (thymus, bursa of fabricius (BF), Harders glands, spleen), the digestive organs (liver, duodenum, jejunum, ileum), and the other parenchymatous organs (kidney, myocardium, cerebrum, and lung) of DPV-infected ducks, but the positive immunofluorescence signal was not seen in the muscle and pancreas. The lymphocytes, reticulum cells, macrophages, epithelial cells, and hepatocytes served as the principal site for the localization of DPV gE antigen. Moreover, the intensity of fluorescence increased sharply from 12 to 216 h post-infection (p.i.). Conclusions: In this work, the immunogenicity of the recombinant gE protein was analyzed by ELISA, and we presented the distribution properties of DPV gE antigen in infected ducks for the first time, which may be useful for understanding the pathogenesis of DPV. These properties of the gE protein provided the prerequisite for further functional analysis.

Background: Duck plague virus (DPV) is the causative agent of Duck Plague (DP) that causes significant morbidity and mortality throughout duck-producing areas of the world. The diagnosis of DP currently relies on the use of live or inactivated whole DPV virion as antigens in ELISA, but it is too laborious and expensive for routine application, and it is still difficult to get purified DPV virion with current technology.Results: In this study, we describe the expression and purification of a recombinant Thymidine Kinase (TK) protein which makes antigen in an in-house developed, optimized and standardized ELISA. The specificity of the optimized TK-ELISA was evaluated by antisera against Duck Plague Virus (DPV), Duck Hepatitis B Virus (DHBV), Duck Hepatitis Virus (DHV),Riemerella Anatipestifer(R. A),Escherichia coli(E. coli) andSalmonella anatum(S. anatum). Only antisera against DPV yielded a specific and strong signal. In order to determine the sensitivity of the TK-ELISA, a panel of diluted sera was tested, and the minimum detection limit of 1:2560 (OD450 nm = 0.401) was obtained according to the endpoint cut-off (0.2438). The repeatability and reproducibility under the experimental conditions demonstrates a low variability (P > 0.05). The suspected sera samples (n = 30) were determined by TK-ELISA and the positive rate is 90% (27/30), and the TK-ELISA showed 83.33% (22+3/30) coincidence rate with the Serum Neutralization Test (SNT) and 90% (24+3/30) coincidence rate with the whole DPV virion based-ELISA (DPV-ELISA). When defining the dynamics of antibody response to attenuated live DPV vaccine, the maximum antibodies is reached after 4 weeks.Conclusions: The results suggest that the TK-ELISA provides high specificity, sensitivity, repeatability and reproducibility for detection of anti-DPV antibodies in duck sera, and has the potential to be much simpler than DPV-ELISA and SNT for the sera epidemiological investigation.

The gE protein of duck plague virus is the important membrane glycoprotein, its protein characterization has not been reported. In this study, we expressed and presented the characterization of the DPV gE product. According to the sequence of the gE gene, a pair of primers were designed, and the DNA product with 1490bp in size was amplified by using the polymerase chain reaction (PCR). The PCR product was cloned into pMD18-T vector, and subcloned into pET32a(+), generating the recombinant plasmid pET32a/DPV-gE. SDS-PAGE analysis showed that the fusion pET32a/DPV-gE protein was highly expressed after induction by 0.2 mM IPTG at 30°C for 4.5 h in Rosseta host cells. Over expressed 6×His-gE fusion protein was purified by nickel affinity chromatography, and used to immunize the rabbits for the preparation of polyclonal antibody. The result of the intracellular localization revealed that the gE protein was appeared to be in the cytoplasm region. The real time PCR, RT-PCR analysis and Western blotting revealed that the gE gene was produced most abundantly during the late phase of replication in DPV-infected cells. In this work, the DPV gE protein was successfully expressed in a prokaryotic expression system, and we presented the basic properties of the DPV gE product for the first time. These properties of the gE protein provided a prerequisite for further functional analysis of this gene.

Previous studies have indicated that the VP19c protein and its homology play similar roles in capsid assembly of all Alphaherpesvirus subfamily. However, there is no report on the VP19c protein of duck enteritis virus (DEV). In this study, we expressed the DEV VP19c protein and presented its antigenic properties. Moreover, we developed polyclonal antibody against the VP19c protein and characterized it. A recombinant VP19c (rVP19c) and N-terminal were expressed in Escherichia coli (E.coli) and purified by Ni2+-affinity chromatography. The antigenic properties of the recombinant protein were determined by Western blot and indirect enzyme-linked immunosorbent assay (ELISA). Furthermore, the polyclonal antibodies against the purified recombinant proteins were produced and the titer of polyclonal antibody was determined by ELISA analysis. Finally, the antibody was used to recognize the VP19c in the cells infected with DEV in the immunofluorescence assay. The N-terminally His-tagged rVP19c and rVP19c(N) were produced as inclusion bodies in E. coli strain BL21 (DE3) with molecular weight of about 66 and 46 kDa. Then the proteins were purified to reach the level of homogeneity. Western blot and ELISA analysis that the rVP19c seems to be structurally and antigenically very similar to native VP19c and the N-terminus of VP19c may contain most antigenic linear-epitopes. Furthermore, ELISA analysis demonstrated that the titer of polyclonal antibody was approximately 1:12800, and in the immunofluorescence assay, the antibody was able to recognize the VP19c in the cells infected with DEV. To our knowledge, this was the first report on basic properties of DEV VP19c protein. In the present study, we obtained a high-level expression of the recombinant VP19c protein as well as high titers of rabbit polyclonal antibody against to VP19c protein. The anti-rVP19c serum was able to detect the VP19c protein in DEV infected cells and the VP19c protein targeted to the nucleus as distinct punctate speckles. This specific polyclonal antibody provides a good tool for further studying structural and functional characterization of DEV VP19c.