Development of TaqMan® MGB fluorescent real-time PCR assay for the detection of anatid herpesvirus 1

<p>Abstract</p> <p>Background</p> <p>Anatid herpesvirus 1 (AHV-1) is an alphaherpesvirus associated with latent infection and mortality in ducks and geese and is currently affecting the world-wide waterfowl production severely. Here we describe a fluorescent quantitative real-time PCR (FQ-PCR) method developed for fast measurement of AHV-1 DNA based on TaqMan MGB technology.</p> <p>Results</p> <p>The detection limit of the assay was 1 × 10¹standard DNA copies, with a sensitivity of 2 logs higher than that of the conventional gel-based PCR assay targeting the same gene. The real-time PCR was reproducible, as shown by satisfactory low intra-assay and inter-assay coefficients of variation.</p> <p>Conclusion</p> <p>The high sensitivity, specificity, simplicity and reproducibility of the AHV-1 fluorogenic PCR assay, combined with its wide dynamic range and high throughput, make this method suitable for a broad spectrum of AHV-1 etiologically related application.</p

Expression and characterization of the UL31 protein from duck enteritis virus

<p>Abstract</p> <p>Background</p> <p>Previous studies indicate that the UL31 protein and its homology play similar roles in nuclear egress of all <it>herpesviruses</it>. 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.</p> <p>Results</p> <p>The entire ORF of the UL31 was cloned into pET 32a (+) prokaryotic expression vector. <it>Escherichia coli </it>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.</p> <p>Conclusion</p> <p>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 <it>Alpha</it>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.</p

Induction of immune responses in ducks with a DNA vaccine encoding duck plague virus glycoprotein C

<p>Abstract</p> <p>Background</p> <p>A DNA vaccine expressing glycoprotein C (gC) of duck plague virus (DPV) was evaluated for inducing immunity in ducks. The plasmid encoding gC of DPV was administered via intramuscular (IM) injection and gene gun bombardment.</p> <p>Results</p> <p>After immunization by both routes virus-specific serum antibody and T-cell responses developed. Vaccination of ducks by IM injection induced a stronger humoral, but weaker cell-mediated immune response. In contrast, a better cell-mediated immune response was achieved by using a gene gun to deliver DNA-coated gold beads to the epidermis with as little as 6 μg of DNA.</p> <p>Conclusions</p> <p>This demonstrated that both routes of DNA inoculation can be used for eliciting virus-specific immune responses. Although DNA vaccine containing DPV gC is effective in both intramuscular injection and gene gun bombardment, the latter could induce significantly higher cell-mediated responses against DPV.</p

Characterization of the duck enteritis virus UL55 protein

<p>Abstract</p> <p>Background</p> <p>Characteration of the newly identified duck enteritis virus UL55 gene product has not been reported yet. Knowledge of the protein UL55 can provide useful insights about its function.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

Cloning, expression and characterization of gE protein of Duck plague virus

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

Immunofluorescence Analysis of Duck plague virus gE protein on DPV-infected ducks

<p>Abstract</p> <p>Background</p> <p>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).</p> <p>Results</p> <p>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.).</p> <p>Conclusions</p> <p>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.</p

Expression and intracellular localization of duck enteritis virus pUL38 protein

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

Expressing gK gene of duck enteritis virus guided by bioinformatics and its applied prospect in diagnosis

<p>Abstract</p> <p>Background</p> <p>Duck viral enteritis, which is caused by duck enteritis virus (DEV), causes significant economic losses in domestic and wild waterfowls because of the high mortality and low egg production rates. With the purpose of eliminating this disease and decreasing economic loss in the commercial duck industry, researching on glycoprotein K (gK) of DEV may be a new kind of method for preventing and curing this disease. Because glycoproteins project from the virus envelope as spikes and are directly involved in the host immune system and elicitation of the host immune responses, and also play an important role in mediating infection of target cells, the entry into cell for free virus and the maturation or egress of virus. The gK is one of the major envelope glycoproteins of DEV. However, little information correlated with gK is known, such as antigenic and functional characterization.</p> <p>Results</p> <p>Bioinformatic predictions revealed that the expression of the full-length gK gene (<it>fgK</it>) in a prokaryotic system is difficult because of the presence of suboptimal exon and transmembrane domains at the C-terminal. In this study, we found that the <it>fgK </it>gene might not be expressed in a prokaryotic system in accordance with the bioinformatic predictions. Further, we successfully used bioinformatics tools to guide the prokaryotic expression of the <it>gK </it>gene by designing a novel truncated <it>gK </it>gene (<it>tgK</it>). These findings indicated that bioinformatics provides theoretical data for target gene expression and saves time for our research. The recombinant tgK protein (tgK) was expressed and purified by immobilized metal affinity chromatography (IMAC). Western blotting and indirect enzyme-linked immunosorbent assay (ELISA) showed that the tgK possessed antigenic characteristics similar to native DEV-gK.</p> <p>Conclusions</p> <p>In this work, the DEV-<it>tgK </it>was expressed successfully in prokaryotic system for the first time, which will provide usefull information for prokaryotic expression of alphaherpesvirus gK homologs, and the recombinant truncated gK possessed antigenic characteristics similar to native DEV gK. Because of the good reactionogenicity, specificity and sensitivity, the purified tgK could be useful for developing a sensitive serum diagnostic kit to monitor DEV outbreaks.</p

Characterization of duck enteritis virus UL53 gene and glycoprotein K

<p>Abstract</p> <p>Background</p> <p>Most of the previous research work had focused on the epidemiology and prevention of duck enteritis virus (DEV). Whilst with the development of protocols in molecular biology, nowadays more and more information about the genes of DEV was reported. But little information about DEV UL53 gene and glycoprotein K(gK) was known except our reported data.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

Expression and characterization of recombinant VP19c protein and N-terminal from duck enteritis virus

<p>Abstract</p> <p>Background</p> <p>Previous studies have indicated that the VP19c protein and its homology play similar roles in capsid assembly of all <it>Alphaherpesvirus </it>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.</p> <p>Methods</p> <p>A recombinant VP19c (rVP19c) and N-terminal were expressed in <it>Escherichia coli </it>(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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p