Construction Of US10 Deletion Strain Of Duck Plague Virus And Evaluation Of It's Immune Effect

Duck plague is a common infectious disease in the goosewings,mainly infecting ducks and geese.The pathogen is duck plague virus(DPV),which belongs to the herpesvirus family?-herpesvirus subfamily.It has caused serious economic losses to the duck and goose industry around the world.The sequencing analysis of the whole genome showed that the strong strain of US10 gene of DPV weak strain lacked a sequence,which suggested that US10 gene might be related to the virulence of the virus,but there was no experimental study.In this paper,two spotted duck plague virus US10-deleted strains have been spot-purified and BAC elements were removed respectively,We obtained the DPV-USGFP-deficient strains DPV-EGFP-?US10 and DPV-?US10 with the green fluorescent reporter gene and no reporter gene,and measured the virulence and immune effect of the two deleted viruses.The results of the experiment are as follows:1.Construction and genetic stability of duck plague virus US10 deletion strain with green fluorescent reporter gene and no reporter geneThe DPV US10 deletion virus constructed and preserved in the laboratory was repeatedly spotted and purified to obtain the US10 deletion virus DPV-EGFP-?US10 with a green fluorescent reporter gene;the BPV element was removed from the DPV US10 deletion virus constructed in the laboratory and stored DPV-BAC-?US10.The missing UL23 gene was replenished to obtain the DPV US10 deletion virus DPV-?US10 without reporter gene.Two strains of US10-deleted virus were successively passaged on duck embryo fibroblast(DEF)for 25 generations and 10 generations on 14-day-old ducklings.US10response status,virus titration and comparison were determined by PCR and sequencing,The pathogenic condition of susceptible animals determines the genetic stability of the missing virus.Results:The two US10-deficient viruses DPV-EGFP-?US10 and DPV-?US10 were stably passaged on DEF cells and ducks,the virus US10 did not recover,and the virus copy number and cell culture of every passages generation liver tissues,the virus copy number was stable at about 10^8.5copies/mL and 10⁸copies/g,respectively.There was no death in virus infection ducks of each generation,and no pathological changes were observed in necropsy.It shows that the missing virus is stable during the passaging process,no back mutation occurs,and the pathogenicity of susceptible animals has not changed significantly,that is,both strains of US10 deleted virus have good genetic stability.2.Safety of DPV US10 deficient strain to ducksIn order to investigate whether the deletion of the US10 gene has an effect on the virulence of DPV,two DPV US10-deficient viruses,DPV-EGFP-?US10,DPV-?US10,and the CHv-50 were injected intramuscularly with 14-day-old ducklings at 10⁶and 10⁷TCID₅₀/mouse,respectively.Set a blank control and observe continuously for 10 days.Results:The ducks in the parental virus group had their body temperature increased to above 43°C on the second day,and the ducks in the 10⁶and 10⁷TCID₅₀groups also died;while the ducks in the two different doses of the US10-deficient group and the blank control group had normal body temperature(40.5?42.5?)without death.It shows that the deletion of US10 gene can reduce the virulence of DPV,and the two DPV US10-deficient virus strains are not pathogenic to ducks.3.The immune protection effect of duck plague virus US10 deletion strain on ducksTwo DPV US10-deficient viruses,DPV-EGFP-?US10 and DPV-?US10,were immunized with 14-day-old ducklings with a single dose of the same commercial attenuated vaccine(10^7.7copies or 10⁵TCID₅₀),Blood was collected at 14 days after immunization to detect the production of DPV-specific ELISA antibodies and neutralizing antibodies;Challenged with 100 LD₅₀CHv virulent 14 days after immunization,observed 10days,recorded body temperature and mortality.Results:The temperature of ducks in the blank control group increased on the 4th day and all died on the 7th day;the body temperature of the commercial attenuated vaccine group fluctuated within the normal range without death;the DPV-EGFP-?US10 and DPV-?US10 group ducks As with the commercial attenuated vaccine group,there was no increase in body temperature or death.DPV-specific ELISA antibodies and neutralizing antibodies were detected in duck serum of two strains of DPV US10-deficient virus,which were significantly or significantly different from the blank group.In summary:the duck plague virus US10 gene is a non-essential gene related to virus virulence;the duck plague virus US10 deleted strain is safe for ducks,and the deleted strain can stimulate ducks to produce DPV-specific antibodies and thus have a good challenge against CHv The protective effect has the potential as a genetic engineering vaccine.