The Duck Cell Virus GC Gene Mediated Host Cell Adsorption Characteristics And GC~-/TK~-double Gene Deletion Strain Construction

Duck plague can cause acute, thermal and septic shock of ducks, goose, swans and other anseriformes. Due to the high morbidity and mortality, duck plague is currently one of the most serious infections disease in the waterfowl industry. We examined the roles of DPV gC in the adsorption during duck plague virus infection and hope to build DPV gC~-/TK~- mutant strains and its revertant using Red recombination. The contents of our research and results are as follows:1. Role of duck plague virus glycoprotein C in viral adsorption:its interactions with cell surface heparan sulfateTo determine the role of gC in DPV adsorption to DEF cells, we comparied the adsorption ability of both the wide-type virus (DPV-CHv) and the mutant (DPV-△gC-EGFP), and confirmed the role of in the interaction between DPV and HS.Firsty, DNA copy numbers of both DPV-△gC-EGFP and DPV-CHv were determined using RT-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 results showed that DNA copy number for the wild-type DPV-CHv was much higher than DPV-AgC-EGFP within 90 min adsorption, especial the significantly higher viral copies was observed at 60 min adsorption, and both the wild-type and mutant viral strains is no significant from 90 min afterwards. After allowing adsorption for 1h, we found that (i) the DNA copy number in the culture increased in a time dependent manner, and (ii) the DPV-CHv DNA copy number was significantly higher than DPV-△gC-EGFP at 74 h. The results indicated the proliferation of virion varies directly with the adsorption of DPV which were co-determined by gC and other genes.Then, to explore the interactions between DPV and cell surface HS, the effects of exogenous heparin on viral infectivity and adsorption were determined. Furthermore, to examine whether removal of heparin-like moieties from the cell surface would prevent adsorption and entry of viruses, we pretreated DEF cells with heparinase prior to exposure to the wild-type or truncated virus. These experiments revealed that gC-truncated virus and wild-type DPV-CHv were not sensitive to heparin and heparinase treatment.Next, to examine interactions between the glycoproteins and heparin, these interactions were mimicked using recombinant DPV glycoproteins DPV-gB, DPV-gC, or DPV-gE proteins and affinity chromatography. These results showed that the E. coli-expressed DPV-gB could bind to heparin, while the DPV-gC and DPV-gE could not bind to heparin. Therefore, we concluded that gC of DPV may not be able to specifically bind to HS moieties expressed on the cell surface.Overall, this study demonstrated that the gC of DPV can mediate viral adsorption in an HS-independent manner, and gC had no effect on the total quantity of viral adsorption. 2. Construction of gC~-/TK~- mutant strains of DPV and the growth characteristicsThe gC and TK gene were non-essential of herpes virus replication and related to viral virulence. And based on the constructed rescued BAC recombinant DPV virus platform above with the TK gene deletion, gC deletion was generated by Red recombination to construct gC~-/TK~- mutant strains, named CHv-BAC-GAgC. And its gC revertant was generated, which was known as DPV CHv-BAC-G△gCR. Then DPV CHv-BAC-GAgC and DPV CHv-BAC-G△gCR were identified by PCR, sequencing and RFLP analysis, and the rescued the recombinant virus were identified by indirect immunofluorescence (IFA). Meanwhile, we examined the growth characteristic research of DPV CHv-BAC-GAgC and DPV CHv-BAC-G△gCR. Viral infection in DEF was performed as follows, then overlaid with 0.5% methyl cellulose and stained with 0.5% crystal violet to visualize the plaques for observing plaque size. The results showed that the rescued mutant strains and its revertant can generate same CPE as the parental virus did, but the plaque size of CHv-BAC-GAgC was less than that others. The same viral DNA copy number of DPV CHv-BAC-G, DPV CHv-BAC-GAgC, DPV CHv-BAC-G△gCR were added to cell monolayers, and we evaluated the virus growth according to the perspective of viruses proliferation at different post-infection. The results indicated that (i) the DNA copy number in cell lysates increased in a time dependent manner, and reached peak at 36 h post-infection, as well as decreased from 36 h afterwards and were no significant, and (ii) the DNA copy number in the culture supernatants also increased in a time dependent manner, and reached peak at 120h post-infection. The same time post-infection of viral DNA copy numbers was no significant. So the one step growth curve revealed that the constructed mutant strains and its revertant can exhibit same propagation cycle in infected cells, it may be attributed to affect viral release of gC gene.