Duck hepatitis B virus (DHBV) shares many basic characteristics with hepatitis B virus (HBV) and is an attractive model for vaccine development.  In this study, DHBV DNA vaccines were designed to express envelope and capsid fusion proteins to enhance the breadth of immune response in ducks.  Attenuated Salmonella typhimurium (SL7207) was used as a carrier and adjuvant to boost the magnitude of immune response.  Based on this strategy, novel DNA vaccines (SL7207-pVAX1-LC and SL7207-pVAX1-SC) were generated.  Growth kinetics, genetic stabilities and relative transcription levels of the L, S and C genes introduced by these vaccine strains were measured before inoculation to guarantee safety and efficacy.  The relative transcript levels of the CD4 and CD8 T genes and the antibody levels (IgY) in ducks receiving the vaccines were higher than those in single gene delivered groups.  Additionally, the copy number of covalently closed circular DNA in hepatocytes after DHBV challenge also provided evidence that our fusion vaccines could enhance the protective efficiency against DHBV infection in ducks.

    The capsid (Cap) protein, which is the only structural protein of duck circovirus (DuCV), is the most important antigen for the development of vaccines against DuCV and the virus’s serological diagnostic methods. In order to use yeast expression system to produce a large quantities of DuCV Cap protein which is close to its natural form to display the antigen peptides perfectly, the Cap gene was optimized into the codon-optimized capsid (Opt-Cap) gene towards the preference of yeast firstly. Then, the genes of Cap and Opt-Cap were separately cloned into pPIC9K plasmid and transformed into Picha pastoris GS115. The strains that displayed the phenotype of Mut+ and contained multiple inserts of expression cassette were selected from those colonies. After the induction expression, the secretory type of Cap protein, which was about 43 kDa, was best expressed under 0.5% (v/v) methanol and sorbitol induction. Compared with the Cap gene, the expression level of Opt-Cap gene was much higher. What’s more, the purified Cap protein had a good reactivity to its specific polyclone antibody and DuCV-positive serum, and it was able to self-assemble into virus-like particles (VLPs). These VLPs, with a diameter of 15–20 nm and without a nucleic acid structure, showed a high level of similarity to DuCV particles in size and shape. All of the results demonstrated that, based on the codon-optimization, it is suitable to use the P. pastoris expression system to produce DuCV VLPs on a large scale. It is the first time that a large amounts of DuCV VLPs were produced successfully in P. pastoris, which might be particularly useful for the further studies of serological diagnosis and vaccines of DuCV.

Many mammalian herpes viruses utilize heparan sulfate (HS) moieties present on cell surface proteoglycans as receptors for cell entry, and this process also requires viral glycoprotein C (gC) homologues.  However, our understanding of the role of gC in facilitating attachment of other alpha-herpes viruses such as the duck plague virus (DPV) remains preliminary.  To study the role of gC during DPV infection, we used a gC-deleted mutant virus (DPV-ΔgC-EGFP).  Examination of the viral copy number by real-time PCR, as well as time course studies of viral adsorption and proliferation revealed that gC was involved in the viral binding to the cell surface.  The affinity of viral glycoproteins (gB-DPV, gC-DPV, and gE-DPV) to HS was assessed using a prokaryotic expression system and HiTrap^TM Heparin HP column chromatography.  In addition, to confirm that gC played a role in the interaction between DPV and HS, viruses were treated with the HS analogue heparin and host cells were treated with its inhibitors heparinase prior to exposure to DPV-ΔgC-EGFP or wild-type strain Chinese virulent duck plague virus (DPV-CHv).  The effects of heparin and heparinase on virus infectivity demonstrated that function of gC on viral adsorption is independent of interactions between gC and heparin sulfate on cell surface.  All in all, this study demonstrated that the gC of DPV can mediate viral adsorption in an HS-independent manner, which distinguish it from the gC of some other alpha-herpes viruses.  Future studies will be required to identify the receptors involved in gC protein binding to cells.  This work provides us a foundation for further studies of examining the roles of gC in the adsorption during duck plague virus infection.

    CD8, a glycoprotein on the surface of T cells, is involved in the defense against viral infection and plays significant roles in antigen presentation and in the antiviral immune response. CD8 is composed of two chains. Of these, the CD8α chain was chosen for the detection because it involved in both the CD8αα homodimer and the CD8αβ heterodimer. Here, we established a double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) for specific detection of goose CD8α (goCD8α). The results showed that the optimal coated antibody and antigen dilutions were 1:50 (the antibody titer was 1:12 800) and 1:32 (0.3 ng mL^–1), respectively, while the optimal capture antibody and horseradish peroxidase (HRP)-labelled goat anti-rabbit IgG dilutions were 1:50 (the antibody titer was 1:51 200) and 1:4 000 (the antibody titer was 1:5 000), respectively. The optimal blocking buffer was 5% bovine serum albumin (BSA). The best incubating condition was overnight at 4°C, the best blocking time was 120 min and the best anti-capture antibody working time was 150 min. In addition, the minimum dose detectable by DAS-ELISA was 5×10–3 ng mL^–1. Most importantly, goCD8α expression levels in goose spleen mononuclear cells (MNCs) post-Goose parvoviruse (GPV) infection were found to be significantly up-regulated using the DAS-ELISA method, which was consistent with previous results obtained using real-time quantitative PCR. In conclusion, the DAS-ELISA method reported here is a novel, specific technique for the clinical detection of goCD8α.