Qihui Luo

Complete Genome Sequence of Riemerella anatipestifer Reference Strain

Riemerella anatipestifer is an infectious pathogen causing serositis in ducks. We had the genome of the R. anatipestifer reference strain ATCC 11845 sequenced. The completed draft genome consists of one circular chromosome with 2,164,087 bp. There are 2,101 genes in the draft, and its GC content is 35.01%.

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.

Expression and characterization of the UL31 protein from duck enteritis virus.

BackgroundPrevious 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.ResultsThe 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.ConclusionIn 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.

Identification and characterization of duck plague virus glycoprotein C gene and gene product

BackgroundViral envelope proteins have been proposed to play significant roles in the process of viral infection.ResultsIn this study, an envelope protein gene, gC (NCBI GenBank accession no. EU076811), was expressed and characterized from duck plague virus (DPV), a member of the family herpesviridae. The gene encodes a protein of 432 amino acids with a predicted molecular mass of 45 kDa. Sequence comparisons, multiple alignments and phylogenetic analysis showed that DPV gC has several features common to other identified herpesvirus gC, and was genetically close to the gallid herpervirus.Antibodies raised in rabbits against the pET32a-gC recombinant protein expressed in Escherichia coli BL21 (DE3) recognized a 45-KDa DPV-specific protein from infected duck embryo fibroblast (DEF) cells. Transcriptional and expression analysis, using real-time fluorescent quantitative PCR (FQ-PCR) and Western blot detection, revealed that the transcripts encoding DPV gC and the protein itself appeared late during infection of DEF cells. Immunofluorescence localization further demonstrated that the gC protein exhibited substantial cytoplasm fluorescence in DPV-infected DEF cells.ConclusionsIn this work, the DPV gC protein was successfully expressed in a prokaryotic expression system, and we presented the basic properties of the DPV gC product for the first time. These properties of the gC protein provided a prerequisite for further functional analysis of this gene.

Immunohistochemical detection and localization of new type gosling viral enteritis virus in paraformaldehyde-fixed paraffin-embedded tissue.

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 48 h PI, in the liver, kidney, pancreas and myocardium from 72 h, and in the cerebrum and cerebellum from 96 h, while it was hardly detected in the respiratory organs at any time. The positive staining reaction could be detected in NGVEV-infected goslings until 600 h 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 72 h 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.

Detection of anatid herpesvirus 1 gC gene by TaqMan™ fluorescent quantitative real-time PCR with specific primers and probe

BackgroundAnatid herpesvirus 1 (AHV-1) is known for the difficulty of monitoring and controlling, because it has a long period of asymptomatic carrier state in waterfowls. Furthermore, as a significant essential agent for viral attachment, release, stability and virulence, gC (UL44) gene and its protein product (glycoprotein C) may play a key role in the epidemiological screening. The objectives of this study were to rapidly, sensitively, quantitatively detect gC gene of AHV-1 and provide the underlying basis for further investigating pcDNA3.1-gC DNA vaccine in infected ducks by TaqMan™ fluorescent quantitative real-time PCR assay (FQ-PCR) with pcDNA3.1-gC plasmid.ResultsThe repeatable and reproducible quantitative assay was established by the standard curve with a wide dynamic range (eight logarithmic units of concentration) and very good correlation values (1.000). This protocol was able to detect as little as 1.0 × 101 DNA copies per reaction and it was highly specific to AHV-1. The TaqMan™ FQ-PCR assay successfully detected the gC gene in tissue samples from pcDNA3.1-gC and AHV-1 attenuated vaccine (AHV-1 Cha) strain inoculated ducks respectively.ConclusionsThe assay offers an attractive method for the detection of AHV-1, the investigation of distribution pattern of AHV-1 in vivo and molecular epidemiological screening. Meanwhile, this method could expedite related AHV-1 and gC DNA vaccine research.

His6-Tagged UL35 Protein of Duck Plague Virus: Expression, Purification, and Production of Polyclonal Antibody

Objective: Duck plague virus (DPV), the causative agent of duck plague (DP), is an alphaherpesvirus that causes an acute, febrile, contagious, and septic disease of waterfowl. UL35 protein (VP26) is a major capsid protein encoded by the UL35 gene, which is located in the unique long (UL) region of the DPV genome. To investigate the specific roles of VP26, the UL35 gene was amplified from the DPV DNA by polymerase chain reaction (PCR) and subcloned into pET-32a(+). Methods: The resultant prokaryotic expression vector, pET-32a(+)/UL35, includes an amino-terminal His6 as a fusion partner. Escherichia coli BL21 (DE3) competent cells were transformed with the construct and protein expression was subsequently induced by the addition of isopropyl-β-D-thiogalactopyranoside to the culture medium. Protein lysates were submitted to SDS-PAGE to evaluate recombinant protein expression. Results: The band that corresponded to the predicted protein size (33 kDa) was observed on the SDS-PAGE gel. The recombinant His6-tagged VP26 fusion protein was expressed at a high level in an insoluble form (inclusion bodies) and constituted about 24% of the total cellular protein. Then, the fusion protein was purified to near homogeneity using single-step immobilized metal affinity chromatography on a nickel-nitrilotriacetic acid affinity resin, yielding about 620 mg per liter culture. After purification, New Zealand white rabbits were immunized with purified His6-tagged VP26 in order to raise polyclonal antibody against this recombinant protein. Using the resultant sera, Western blot analysis showed that the recombinant protein was recognized by the polyclonal antibody. Conclusion: Thus, the polyclonal antibody prepared here may serve as a valuable tool to study the functional involvement of VP26 in the DPV life cycle.

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

BackgroundA 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.ResultsAfter 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.ConclusionsThis 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.

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.

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

BackgroundThe 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.ResultsAccording 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.ConclusionsIn 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.