Pathogenicity Evaluation Of Duck Plague Virus LORF5 Gene-deletion Strain

Duck plague?DP?is one of the most serious infectious diseases in the duck industry.Duck plague virus?DPV?the pathogen of DP was classified to the family Herpesviridae subfamily Alphaherpesvirinae.LORF5 gene is one of the unique genes of Avian herpesvirus,and its gene function has not been reported in DPV.In this thesis,to investigate the effect of LORF5 gene on the pathogenicity of DPV CHv,the LORF5 gene-deletion virus was constructed and its proliferation curve was determined in vitro and in vivo,and the pathogenicity was evaluated in vivo.The main research contents and results are as follows:1.Construction of LORF5 gene deletion recombinant virus and determination its proliferation curve in vitroBased on DPV CHv-BAC-G,the DPV CHv-BAC-?LORF5 clone strain was constructed by Red homologous recombination,and the UL23 gene?BAC element insertion site?was replenished on the strain.After rescue and plaque purification,The BAC component was removed and LORF5gene-deletion virus DPV CHv-?LORF5 was constructed.DPV CHv,DPV CHv-?LORF5,DPV CHv-BAC-G and DPV CHv-BAC-?LORF5 viruses with MOI=0.01 were inoculated into DEF cells,respectively,and 24 h,48 h,72 h and 96 h after infection,the viral genome copies number were determined.The results showed that LORF5 gene-deleted viruses showed similar proliferation patterns within 96 h after infection with the parental virus.Compared with the parental virus,the virus content of the LORF5 gene-deletion virus was slightly lower than that of the parental virus at each time point,but there was no significant difference,indicating that the deletion of LORF5 gene did not significantly affect the proliferation of the virus on DEF cells.2.Pathogenicity evaluation of DPV CHv-?LORF5 and its proliferation in liverDPV CHv-?LORF5 virus and parental virus DPV CHv were inoculated into 14-day-old ducklings at 10³,10⁴,10⁵,10⁶,10⁷ TCID₅₀/dose,respectively,and a mock group was established at the same time.The study continues to observe for one week.The results showed that the high-dose challenge group(10⁵,10⁶ and 10⁷ TCID₅₀)of the parent virus showed high fever and death;the liver was found to have hepatic hemorrhage and congestion,intestinal bleeding,thymus atrophy and severe bleeding,bursal atrophy bleeding,Which were characteristic pathological changes of duck plague.However,DPV CHv-?LORF5 virus challenge groups except for the thymic hemorrhage observed in the high-dose challenge group(10⁶ and 10⁷ TCID₅₀),the pathological changes of other tissues organs,high fever and death did not be observed.Liver histological examination of DPV CHv 10⁷ TCID₅₀ challenge group showed severe steatosis,necrosis and hemorrhage,however DPV CHv-?LORF5 10⁷ TCID₅₀0 challenge group only slightly steatosis and hemorrhage.These data showed that loss of LORF5 reduces the pathogenicity of DPV virus to ducklings.14-day-old ducklings were injected with DPV CHv and DPV CHv-?LORF5 at a dose of 10⁷TCID₅₀,and the virus genome copies were quantified in duck livers at 1 d,2 d,3 d,5 d and 7 d after infection.The results showed that DPV CHv-?LORF5 and DPV CHv viruses showed similar proliferation patterns in the infected duck liver within 5 d after challenge,and maintained a certain virus titer.There was no significant differences in the virus content at each time point.After 5 d,the titer of the parental virus continued to rise,while the titer of the LORF5-deficient virus decreased,indicating that LOFR5 deletion does not significantly affect the DPV proliferation within 5 d after viral infection in liver of ducklings,but it will affect the DPV sustained proliferation in the liver of ducklings.In summary,the results of this study indicate that the LORF5 gene is non-essential for DPV replication,and its deletion can reduce the pathogenicity of the DPV virus to ducklings,but it does not significantly affect the virus proliferation in DEF and ducklings liver within 5 d after viral infection.