Yufei Guo

Development and evaluation of an antigen-capture ELISA for detection of the UL24 antigen of the duck enteritis virus, based on a polyclonal antibody against the UL24 expression protein

Abstract An antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) method was developed for the efficient detection of the UL24 antigen of the duck enteritis virus (DEV) using polyclonal antibodies. Ducks and rabbits were immunized, respectively, with expressed UL24 recombinant protein. The IgG antibodies against UL24 from ducks and rabbits were purified and used as the capture antibodies. The specificity of the optimized AC-ELISA was evaluated by use of DEV, duck hepatitis virus (DHV), duck hepatitis B virus (DHBV), gosling plague virus (GPV), Riemerella anatipestifer (R.A.), Escherichia coli (E. coli), Pasteurella multocida (P.M.) and Salmonella Enteritidis (S.E.). Only DEV specimens yielded a specific and strong signal. The limit of the sensitivity of this method for the detection of DEV was 46ng/100μl. Compared with PCR and virus isolation, the rate of agreement for the detection of experimentally infected sera was 100%. A comparative test used on clinical specimens between the neutralization test and the AC-ELISA showed that the proportions of true positives and true negatives by the AC-ELISA were 0.90 and 0.67 respectively. These results indicated that the AC-ELISA approach is rapid, sensitive, and reliable for specific detection of DEV antigen.

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

BackgroundAnatid 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.ResultsThe detection limit of the assay was 1 × 101 standard 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.ConclusionThe 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.

Complete nucleotide sequence of the duck plague virus gE gene

Duck plague (DP), which is caused by duck plague virus (DPV), is a disease that severely affects various types of waterfowl (ducks, geese, and swans). DPV infection causes significant economic losses to the commercial duck industry because of the consequent high mortality and low egg production rates [7]. DPV has been classified as belonging to the subfamily Alphaherpesvirinae of the family Herpesviridae based on the report of the Eighth International Committee on Taxonomy of Viruses (ICTV), but it has not been grouped into any genus [4]. Currently, there is little information on the molecular characteristics of DPV, and very few DPV genomic sequences have been published. Here, we report the complete sequence of the newly isolated gE gene of DPV.

Purification of anatid herpesvirus 1 particles by tangential-flow ultrafiltration and sucrose gradient ultracentrifugation.

Anatid herpesvirus 1 (AHV-1) infection causes substantial economic losses to the world-wide waterfowl production. However, little is known about the efficient method used to study the purification of AHV-1 and the negative staining morphology of the purified virus particles. This lack of knowledge is one of the important factors that have affected the progress of research studies on AHV-1 molecular virology to such an extent that they are lagging far behind those on other members of the same family Herpesviridae. Therefore, an efficient method for purifying AHV-1 from cell-culture medium has been developed. Abundant AHV-1 particles, whose morphological features match those of herpesvirus, were obtained by using the following procedures: (1) conventional differential centrifugation for removal of debris after cell disruption, (2) tangential-flow ultrafiltration coupled with sucrose density gradient ultracentrifugation for isolation of the virus, and (3) conventional differential ultracentrifugation for virus concentration. The purified AHV-1 particles were subjected to transmission electron microscopy (TEM), infectivity and recovery tests, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting assay, and agar gel diffusion test (AGDT). The results of examinations revealed that purified AHV-1 particles were free of visible contamination or degradation. The purified AHV-1 particles were biologically active and were successful in initiating infection upon inoculation into susceptible duck embryo fibroblast. The procedures are reliable technically and feasible for purification of large volumes of viruses.

Identification and characterization of the duck enteritis virus UL51 gene

Compared to the UL51 gene of other alphaherpesviruses, the duck enteritis virus (DEV) UL51 gene contains ten conserved motifs and has a close evolutionary relationship with members of the genus Mardivirus. The DEV UL51 gene product was identified using a rabbit polyclonal antiserum raised against a 6-His-UL51 fusion protein expressed in Escherichia coli as a 34-kDa protein. Western blotting and RT-(real time) PCR analysis of DEV-infected cells showed that the protein was produced at the late stage of infection and that its production was highly dependent on viral DNA synthesis, suggesting that the gene should be classified as γ2 class. Analysis of extracellular virions revealed that the protein was a component of extracellular mature DEV virions. Indirect immunofluorescence studies localized most of the protein to the juxtanuclear region. These results will provide a basis for further functional analysis of the gene.

Characterization of subcellular localization of duck enteritis virus UL51 protein

BackgroundKnowledge of the subcellular localization of a protein can provide useful insights about its function. While the subcellular localization of many alphaherpesvirus UL51 proteins has been well characterized, little is known about where duck enteritis virus (DEV) UL51 protein (pUL51) is targeted to. Thus, in this study, we investigated the subcellular localization and distribution of DEV pUL51 by computer aided analysis, as well as indirect immunofluorescence (IIF) and transmission immunoelectron microscopy (TIEM) approaches in DEV-infected cells.ResultsThe 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.ConclusionIn 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.

Development of a fluorescent quantitative real-time polymerase chain reaction assay for the detection of Goose parvovirus in vivo

BackgroundGoose parvovirus (GPV) is a Dependovirus associated with latent infection and mortality in geese. Currently, it severely affects geese production worldwide. The objective of this study was to develop a fluorescent quantitative real-time polymerase chain reaction (PCR) (FQ-PCR) assay for fast and accurate quantification of GPV DNA in infected goslings, which can aid in the understanding of the regular distribution pattern and the nosogenesis of GPV in vivo.ResultsThe detection limit of the assay was 2.8 × 101 standard DNA copies, with a sensitivity of 3 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 intraassay and interassay coefficients of variation.ConclusionThe high sensitivity, specificity, simplicity, and reproducibility of the GPV fluorogenic PCR assay, combined with a high throughput, make this method suitable for a broad spectrum of GPV etiology-related applications.

Cloning, Expression, Purification and Characterization of UL24 Partial Protein of Duck Enteritis Virus

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° 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 (OD450nm = 2.5) on day 28 after immunization, while with mean titers of 1:10,240 (OD450nm = 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.

Anatid herpesvirus 1 CH virulent strain induces syncytium and apoptosis in duck embryo fibroblast cultures.

Abstract Anatid herpesvirus 1 (AHV-1) CH virulent strain was first isolated from an infected duck and it was found that this virus strain could induce cytopathic effect (CPE) in duck embryo fibroblast (DEF). Following AHV-1 infection, DEF showed morphological changes such as cell rounding, improved refractivity and detachment from the culture surface. However, its pathological characteristics were not adequately known. Related studies were performed and the results showed that syncytium formation could be observed as the other type of CPE in AHV-1 infection. Hematoxylin-eosin staining and 4’, 6-diamidino-2-phenylindole (DAPI) staining of infected DEF were each used to visualize the shape and distribution of chromatin within nuclei and nuclear fragmentation was observed. Chromatin condensation and margination, as well as formation of apoptotic bodies were observed by transmission electron microscopy (TEM). DNA ladder formation was detected in AHV-1 infected cells and apoptosis of the infected DEF was also detected by flow cytometry analysis of Annexin V-FITC/PI staining method. Therefore, it was suggested that AHV-1 virulent strain can induce syncytium and apoptosis in DEF. Syncytium formation and apoptosis observed in this study may contribute to the elucidation of AHV-1 pathogenesis.

Replication kinetics of duck virus enteritis vaccine virus in ducklings immunized by the mucosal or systemic route using real-time quantitative PCR.

A chicken embryo-adapted duck enteritis virus (DEV) strain is the most widely used vaccine against duck virus enteritis (DVE) infection. The kinetics of attenuated DEV vaccine was examined in tissues of ducklings vaccinated by the mucosal or systemic route at 20 days of age and sampled regularly up to 60 days post-vaccination (p.v.). Significant numbers of virus genomes in the lymphoid and other parenchymatous organs were first detected at 60 min p.v., and subsequently rose to peak levels during 90 min to 1 day p.v. independent of the route of vaccine administration. The peak level of vaccine virus in the individual parenchymatous organs of subcutaneously immunized ducklings was significantly higher than that of orally or nasally immunized ducklings. The route of vaccine administration had significant effect on the initial tissue distribution of vaccine virus in respiratory and digestive tracts. Vaccine viruses spread to digestive tract and trachea tissues by mucosal route, i.e. oral and nasal administration, early than that by subcutaneous route. The rapid early increase of vaccine virus levels in all samples examined followed by a steady decline from 90 min to 6 days p.v. The real-time PCR analysis of a variety of tissues is significant for further investigation of the mechanism of vaccinal protection, and the optimization of vaccination regimes.