Distribution Characteristics of DNA Vaccine Encoded with Glycoprotein C from Duck Plague Virus in Ducks and Preliminary Study of the Biological Characteristics of GC-deleted DPV and Ge-deleted DPV

As one virus of genus Mardivirus, subfamily Alphaherpesvirinae, family Herpesviridae, order Herpesvirales, duck plague virus (DPV), a pan-tropic systemic infection virus, is the cause of an acute contagious disease-duck plague-of ducks, geese, and swans, which is characterized by vascular damage, tissue hemorrhages, digestive mucosal eruptions, lesions of lymphoid organs, and degenerative changes in parenchymatous organs. The disease is an important epidemic affecting waterfowl breeding industryAt present, the prevention and control of duck plague is mainly depended on both live attenuated and inactivated vaccines. Inactivated vaccines are relative safety, but give only short-term immunity, and need larger doses and higher costs. Although high immunogenicity and longer term of immunoprotection do live attenuated vaccines possess, they still have some problem need to solve, including the latent dangers of persistence infection and virulence reversion, and the difficulty in distinguishing vaccinated birds from infected ones. Therefore, it has important practical significance to develop more safe and effective genetically engineered vaccines.Bioinformatic analysis showed that glycoprotein C of duck plague virus is highly conserved among different strains. And it was proved by prokaryotic expression that glycoprotein C had high immunogenicity. Accordingly, gC gene was chosed as the protective antigen gene and subcloned into the eukaryotic expression vector pcDNA3.1(+) in this study. The results of restriction endonuclease digestion and indirect immunofluorescence assay confirmed that the gC gene was inserted correctly and expressed in COS-7cells. In order to obtain a large number of the eukaryotic expression plasmid, the condition of high-cell-density fermentation was studied. Obtained from maxipreparation and purification, plasmid was analyzed by spectrophotometer, restriction analysis and agarose gel electrophoresis. It was showed that plasmid contained little impurity and the supercoiled structure up to90%.In order to clarify the effect of immunological adjuvant and immune pathways, complexes of gC gene vaccine with lipid or chitosan were inoculated into ducks by intramuscular, oral and intranasal. At the same time, gC gene vaccine was inoculated into ducks with the gene gun bombardment and intramuscular injection of different doses. And then, the tissue distribution characteristics of gC gene vaccine was analyzed by quantitative real-time PCR based TaqManTM probe. The results showed that1) the liposomes and chitosan can effectively improve the tissue distribution of gC gene vaccine in ducks; 2) the gene gun bombardment has the highest transfection efficiency;3) oral vaccination benefits the distribution of gene vaccine in the digestive tract and bursa of Fabricius;4) intranasal immunization can improve the distribution of gene vaccine in respiratory tract and brain;5) intramuscular injection mediate more rapid plasmid distribution in duck tissues;6) intramuscular and gene gun bombardment are dose-related.CHv strain has the largest genome among three strains of duck plague virus which genomes have been sequenced completely. As a virulent strain, CHV strain was choosed to more effectively reveal the role of the gene in duck plague virus by the recombinant studies. In the present study, gC gene was chosed as target gene. Firstly, two fragments flanking gC gene about1.3kb were amplified and subcloned into pUC19. Then, a DPV transfer vector pUC-AgC-EGFP was constructed, which contained an EGFP reporter. In order to effectively control the insertion direction of the EGFP gene to express against gC promoter, multiple cloning sites between Xho I and Xba I pEGFP-C1were first deleted. And then, a1579bp fragment, which contained the sequences of Human cytomegalovirus (CMV), immediate early promoter and SV40early mRNA polyadenylation signal, was amplified and clonee into T vector. PCR fragment, Co-transfected pUC-△gC-EGFP with DPV CHv in DEF, a recombinant DPV, designated as DPV-AgC-EGFP, was successfully obtained after plague purification which combined with green fluorescent under inverted fluorescence microscope.DPV-AgC-EGFP was examined by continuous passage30or more, PCR, sequencing and biological characteristics in vitro and in vivo. It was shown that EGFP gene can be stably inherited and correctly expressed. The results of PCR and sequencing showed that the gC gene sequence of DPV-AgC-EGFP in line with expectations. Compared with DPV parental strain, the total virus titer of DPV-AgC-EGFP decreased by approximately50times, and the supernatant virus titer was mostly affected and decreased40times. Additionally, the DPV-AgC-EGFP mainly passed through the cell in MEM and it is difficult to find free the DPV-AgC-EGFP virus particles under transmission electron microscope. All above results showed that the DPV gC played a role in virus assembly. In addition, the fact that DEF infected with DPV-AgC-EGFP DEF formed multinucleated giant cells showed that gc maybe inhibit cell fusion. Furthermore, the results in vivo showed DPV-AgC-EGFP reduced the pathogenicity, induced neutralizing antibodies and protected completely vaccinated ducks from challenge of DPV virulent. Therefore, the gC-deleted DPV can be developed to an effective genetically engineered vaccine for duck plague.The similarity of gE gene between different DPV strains Duck Plague virus was99%. A gE-based ELISA method has been established for its good immunogenicity and reactivity. Named as DPV-AgE-EGFP, a gE-deleted DPV, which can express EGFP, was constructed since the gE gene of alphaherpesvirus was an important virulence gene. The results of PCR, sequencing and immunofluorescence assay showed that the gE gene of DPV-△gE-EGFP was replaced by EGFP gene and cannot be expressed no longer. The results of continuous passage showed that the DPV-△gE-EGFP was able to be stably inherited and express EGFP protein. Compared with the DPV parental strain, the virus titer of DPV-AgE-EGFP reduced by about9times, the plaque formed by DPV-AgE-EGFP also decreased, and it was shown by one step growth curves that the proliferation of DPV-AgE-EGFP slowed down in early infection. These results indicated that the deletion of gE impacted the ability of DPV through cell-to-cell spread. Moreover, The results obtained of in vivo studies revealed that the gE gene play an important role in virulence of DPV because there is no abnormal phenomenon in clinical symptoms except slightly increasing in temperature in ducks vaccinated with DPV-△gE-EGFP. Although the neutralizing antibodies induced by DPV-△gE-EGFP were significantly lower than that induced by DPV-AgC-EGFP, the gE-deleted mutant induced completely protection in inoculated ducks from manifestation of clinical symptoms after viral challenge. The results demonstrate that the gE-deletion mutant is good candidate for DPV marker vaccine.