Construction And Immune Effect Evaluation Of Duck Plague Virus LORF4-deletion Strain

Duck plague virus(DPV)can cause acute,febrile,and septic infectious diseases of anseriformes such as ducks,geese,and cygnoides.In this paper,a DPV LORF4 gene deletion virus was constructed,and its virulence changes were observed and its immune effect was evaluated.The main research contents and results are as follows:1.Construction of DPV-CHv?LORF4Using the Bacterial Artificial Chromosome(BAC)platform of the DPV CHv strain constructed in this laboratory and the Red homologous recombination technology,infectious clones(DPV-CHv-BAC-?LORF4)with the purpose of deleting the LORF4 gene have been constructed.And then another infectious clone(DPV-CHv-?LORF4)for the purpose of removing BAC functional elements mini-F have been constructed.Finally,the infectious clone was used to transfect and rescue the DPV(DPV-CHv?LORF4)that only deleted the LORF4 gene.The results of replication kinetics showed that DPV-CHv?LORF4 had similar proliferation rules with its parent strain CHv-50.Both viruses showed a gradual proliferation trend at 24?72 h,and there was no significant differences in virus titer in the supernatant or cells.This indicates that the deletion of the LORF4 gene will not affect the proliferation of DPV in host cell,and the LORF4 gene is a non-essential gene for DPV replication.2.Safety evaluation of DPV-CHv?LORF4DPV-CHv?LORF4 with TCID₅₀of 10⁴,10⁵,10⁶,10⁷were used to inoculate 14-day-old ducklings and set the CHv-50(parent strain)control.The results showed that the ducklings infected with CHv-50(parent strain)showed high fever symptoms and death.The mortality and temperature of the ducklings were positively correlated with the inoculated virus titer.The higher titer of the inoculated virus,the greater number of duckling deaths and the faster time to cause high fever.However,DPV-CHv?LORF4 did not changed the temperature of ducklings and did not caused the death of ducklings.The results indicate that the deletion of LORF4 gene significantly reduces the virulence of DPV in ducks.The results of continuous passage in duck embryo fibroblasts and ducklings indicate that the DPV-CHv?LORF4 deletion virus will not restore the LORF4 gene that has been deleted during continuous passaging.During the passage,the viral load in the duckling's liver is stable and the weak virulence is stable.The deletion of LORF4 gene has good genetic stability.3.Immune effect evaluation and antibody production of DPV-CHv?LORF4Refer to a dose of attenuated DPV vaccine(10^7.7copies each,ie 10⁵TCID₅₀),14-day-old ducklings were immunized with the DPV-CHv?LORF4:(1)100 LD₅₀DPV virulent strain was inoculated at 7 d after immunization.Observe continuously for 10 days after inoculation.The results showed that no ducks died in the DPV-CHv?LORF4 group and the vaccine group,and all of the blank controls died within 7 days after inoculation.This shows that immunizing DPV-CHv?LORF4 can provide 100%protection against DPV virulent strain,like the commercial attenuated vaccine;(2)The results of neutralizing antibody levels showed that the DPV-CHv?LORF4 can produce neutralizing antibodies against DPV on the 7th day after immunization,the antibody level gradually increased within 28 d after immunization.There was no significant difference between the DPV-CHv?LORF4 and attenuated DPV vaccine with the level of neutralizing antibody.Therefore,LORF4 gene is a non-essential gene for DPV,and the DPV with the deletion of LORF4 gene has significantly reduced virulence to ducks.The DPV-CHv?LORF4 can induce the body to produce neutralizing antibodies and provide protection against DPV virulent strain.