Effects Of US3 Protein On Virulence Of Duck Plague Virus And Evaluation Of Immune Effect Of Its Deletion Strain

Duck Plague Virus（DPV）infection can cause severe disease or death of ducks,geese and other waterfowl.Its US3 gene encodes a protein kinase and is conserved in alpha herpes virus.It can phosphorylate a variety of viral and host proteins and is an important protein of DPV.In this paper,the pathogenicity and immunogenicity of US3 gene deletion and complement virus were studied in order to have a deeper understanding of the characteristics of DPV and provide basic data and reference for the prevention and control of duck plague.1.Pathogenicity assessment of US3 gene deletion strains of duck plague virusThe US3 gene complement strain was constructed by the Bacterial Artificial Chromosome（BAC）platform（p BAC-CHv）of duck plague virus already built in the laboratory using the traceless deletion technique.The bacterial artificial chromosome（BAC）strain and the complement strain of US3 gene were respectively identified and subcultured.The US3 gene deletion strain was infected with ducks and observed for 10 days.The results showed that the body temperature of the ducks infected by parental virus（DPV-CHv）and the ducks infected with reverting virus began to rise（above 42.5℃）on day 3,the weight growth stopped or slowed down,the autopsy showed widespread bleeding in all tissues and organs,and the death rate was 30%on day 4.Like the blank control ducks,the body temperature fluctuated within the normal range（40.5～42.5℃）,the weight increase trend was the same,no death occurred,and no lesions were found in all organs and tissues.The results showed that DPV-CHv-?US3 strain weakened virulence and did not cause disease in ducklings.2.Temporal and spatial distribution of US3 gene deletion virus in ducksThe 14-day-old ducks were infected with 3 strains of 10⁶ TCID₅₀ DPV-CHv-?US3,and the tissues and organs were taken at different times,respectively,and the viral load was detected by quantitative PCR.The results showed that the absent virus was the same as that of the ducks infected with parental virus and reversion virus at day 1.Viruses were detected in all tissues and organs(10^6.309.03 copies/g).There was significant difference between viral load of absent virus infected ducks(10^6.306.92 copies/g)and that of parental virus infected ducks(10^7.097.61 copies/g)（P<0.05）or extremely significant difference（P<0.01）（except spleen,bursi of Farsi,duodenum and thymus）.The viral load in spleen reached the highest level at day 5(10^7.1 copies/g in ducks infected with deletion virus and 10^9.6 copies/g in ducks infected with parental virus and reverting virus).At 9 days,the viral load of deletion-infected ducks except duodenum and rectum was significantly lower（P<0.05）or significantly lower（P<0.01）than that of parental-infected ducks(10^5.816.78 copies/g)and revirulent infected ducks(10^6.28-8.32 copies/g).3.Evaluation of immune effect of DPV-CHv-ΔUS3 strain（1）Challenge protection:14-day-old ducklings were immunized with DPV-CHv-ΔUS3 strain equivalent to 1 commercially available duck plague attenuated vaccine copy number,respectively.On the 14-th day after immunization,100 LD₅₀ of DPV-CHv was used to challenge.The results showed that the ducks immunized with DPV-CHv-ΔUS3 strain and attenuated duck plague vaccine all survived,and the protection rates were 100%;All 10 ducks in the non-immunized control group died on the 7-th day after the challenge.After the challenge,the body temperature rose above 43°C,and the ducks stopped eating and drinking,and their body weight decreased significantly.（2）The effect of immune duck on the removal of strong toxin in the body:The DPV-CHV-?US3immune ducks were attacked with DPV potent toxicity of 100 LD₅₀,tissues and organs were collected,and the DPV potent toxicity content was detected by q PCR.The results showed that:At 5-days,the number of copies was 10^7.889.86 copies/g in ducks immunized against virus deletion and 10^9.3010.26 copies/g copies/g in ducks immunized against plague vaccine,both significantly lower than that of control ducks（P<0.05）(10^10.5214.59copies/g).The virulence levels of 10-day absence virus immunized ducks and duck plague vaccine immunized ducks decreased to 10^7.038.36 copies/g and 10^7.699.43 copies/g,respectively.The results showed that US3 deletion virus could partially eliminate the strong virus in ducks after immunization,just like commercial duck plague vaccine.（3）Cloacal detoxification of immune ducks:After attacking DPV-CHV-?US3 immunized ducks with100 LD₅₀ DPV,a cloaca swab of the immunized ducks was collected,and the q PCR test results showed that,The cloacal virus titer of 3-day virus-free ducks(10^4.68 copies/100?L)was significantly lower than that of non-immunized ducks(10^5.38 copies/100?L).At day 7,all the unimmunized ducks died,and the virus titers of the ducks immune to virus deficiency and vaccine reached the peak,which were为10^5.78 copies/100?L and 10^7.42copies/100?L,respectively.At 9 d,the virus titers of virus-free and virus-free ducks decreased to 10^4.92copies/100?L and 10^6.5 copies/100?L,respectively.The results showed that US3 deletion virus can reduce the detoxification level of cloaca of immune ducks like duck plague vaccine4.Cytokine changes,antibody levels and the rule of growth and decline after US3 gene deletion virus immunization（1）Cytokine changes in immune ducks:After injecting DPV-CHV-?US3 with 100 LD₅₀ DPV,the transcription levels of cytokines IFN-α,IFN-γ,IL-2,IL-4 and IL-6 in thymus were detected by q PCR at day 5and day 10.The results showed that the cytokine transcription levels of DPV-CHv-ΔUS3 immunized ducks and duck plague vaccine immunized ducks were increased to different degrees,indicating that US3 gene deletion virus could induce the increase of some cytokines as well as duck plague vaccine.（2）The antibody level and the law of growth and decline of immune ducks:After immunizing ducks with 10^4.5 TCID₅₀ DPV-CHv-?US3,neutralizing antibodies began to be detected at day 7,the same as those immunized ducks with duck plague vaccine,and the mean values were2^2.46 and 2^2.54,respectively,with no significant difference（P>0.05）.At day 14,the numbers were 2^3.54 and 2^3.73,respectively.At day 60,the neutralizing antibody peaked(mean 2^4.56 and 2^4.52),and then began to decline.At day 95,the antibody levels were 2^4.56 and 2^4.52,respectively,which were not significantly different from those at day 14.The results showed that DPV-CHv-?US3 immune ducks and duck plague vaccine immune ducks have similar rule of antibody growth and decline,and can reach the level of antibody after duck plague vaccine immunization.In summary,this study proves that after the US3 gene deletion,the virulence of DPV is greatly weakened,and it has no pathogenicity to ducks.After immunizing ducks with the US3 deletion strain,ducks can be induced to produce immunity that is comparable to commercially available duck plague vaccines.With a comparable level of neutralizing antibody,it can resist 100 times the half-lethal dose of duck DPV virulent attack,has a good immune protection effect,and has the potential to be further developed into an attenuated vaccine.