The Development And Immune Efficacy Evaluation Of DTMUV Attenuated Vaccine

Duck Tambusu virus disease caused by Duck Tembusu virus?DTMUV?is an acute,potent infectious disease characterized by decreased egg production rate and hemorrhagic ovary inflammation.The Spotted Duck was most easily to infect DTMUV,followed by the Cherry Valley Pekin Duck,the Muscovy Duck and Goose.In recently years,DTMUV has spread to the major duck-farming region in China,causing significant economic losses in the duck industry.It is imperative to develop an effective duck tabutsu vaccine.In this study,the virulent strain ZJSBL01 stored in laboratory were identified by electron microscopy.It was found that the virus particles were spherical and enveloped under the electron microscope.The size of the virus particles was about 50 nm.The morphology was consistent with the report.The ZJSBL01 strain was serially passaged on DF-1 cells for 50 passages,followed by 50 passages on DF-1 cells and 50 passages on BHK-21 cells induced by 5-FU to obtain different generations of virus strains.After purification of the ZJSBL01 strain with different generations of plaques,the proliferation was studied on BHK-21 cells.The results showed that the maximum virus titer was reached at 48 h after the virus inoculation in different generations of ZJSBL01 strains.The whole genome sequencing of the ZJSBL01 strain was performed from different generations.The results showed that compared with the parental strain,12 of the F100generation nucleotide mutations were routinely passaged,resulting in 9 amino acid mutations;the induced mutations were passaged in the F50-50 generation.There were 71nucleotide mutation sites,which resulted in 20 amino acid mutations.The immune-associated protein of Duck Tambusu virus is mainly structural protein E protein.Compared with the parental virus,the F100 generation E protein of the duck has 2 amino acid mutations,and the E protein of the F50-50 generation has 5 amino acid mutations.It was shown that the nucleotide and amino acid mutation rates of the induced mutations were higher.In order to determine the safety and immunogenicity of the ZJSBL01 strain in different generations,the animal studies in the juvenile duck were performed in this study.The results showed that the F50-40 generation has higher safety and immunity protection and can be used as vaccine candidate strain.Further study on the genetic stability and safety of the candidate strains of the F50-40 vaccine revealed that the F50-40 was stable in DF-1cells,and that it was not virulent to susceptible ducks.Ducks have good safety and the egg production rate of ducks after inoculation remains stable.At the same time,the immunopotency of candidate strains of F50-40 vaccine was found to have good immunogenicity for egg-laying ducks and egg-laying ducks,and the egg production rate of ZJSBL01 strains remained stable after challenge.Finally,the best immunization dose was tested for different duck species.The results showed that the optimal immunization dose for immune protection of Cherry Valley Duck was 5×10⁴ TCID₅₀/only,for ducks 3×10⁴ TCID₅₀/only,and for Muscovy Ducks 5×10⁴TCID₅₀/only.Compared with Muscovy ducks,ducklings and Cherry Valley ducks have a longer duration of immune protection at optimal doses.Efficacy evaluation of F50-40 with different immunizing doses and attenuated vaccines on the market was conducted on laying ducks.The results showed that after the ZJSBL01 strain was challenged,the egg production rate in the different immunization dose groups of F50-40 remained stable.The egg production rate decreased slightly,and the optimum immunization dose for F50-40 in laying ducks was determined to be 5×10⁴ TCID₅₀/only.In summary,this study first through the generation of weak strains of ZJSBL01 in DF-1 and BHK-21 cells.The whole gene sequencing of different generations of ZJSBL01strain revealed that there were nucleotide and amino acid mutations in E protein and further animal experiments.To evaluate the safety and immunogenicity of different generations of ZJSBL01 strains,and to select vaccine candidate strain F50-40 successfully.After studying the genetic stability,safety,and immune efficacy of F50-40,it was determined that the F50-40 pairs were different.The optimal immunization dose for ducks was compared with the attenuated duck vaccine in the market.At the same immunization dose,F50-40 had better immunity protection and determined the optimal immunity for laying ducks.The dose was 5 x 10⁴ TCID₅₀/only.