Development Of Subunit Vaccine And DNA Vaccine Of Gyrovirus 3 And Immunoprotection Evalution

In recent years,Transmissible viral proventriculitis?TVP?has brought great harm to the poultry industry.It can occur in different breeds of laying hens and broilers and the epidemicity of the disease is very widespread.The incidence rate is generally 15%⁶0%in the poorly managed chicken farms.However,the pathogenic pathogen of transmissible viral proventriculitisis has not been conclusive.It is related to viruses such as reovirus,reticuloendotheliosis virus and adenovirus.In this case,better immune prevention measures cannot be used to prevent and control the viruses.In 2017,Li Gen identified the Gyrovirus 3?GyV3?in the proventriculus of the chickens with TVP through the high-throughput sequencing.GyV3 is the single-stranded circular DNA virus.By returning to animal experiments,it was found that GyV3 can cause the symptoms of chicken immunosuppression and proventriculus enlargement.However,there has been no any research about the vaccines of GyV3 so far.Therefore,subunit vaccine and DNA vaccine were prepared for immunogenicity research and protection of animals.GyV3 belongs to the single-stranded circular DNA virus.It consists of three open reading frames,namely capsid protein VP1?1392 bp?,skeleton protein VP2?720 bp?and apoptotic protein VP3?378 bp?.The subunit vaccine was prepared by selecting the amino acids from the 90th to the 463th of VP1 protein.The amino acids from the 90th to the 463th of VP1 protein and VP2 protein were co-expressed and connected through flexible amino acid linker?GSGGS?to prepare the subunit vaccine.The target proteins were expressed in E.coli expression system.The results of SDS-PAGE and Western blot showed that the target protein was expressed successfully.We constructed pEGFP-VP1 plasmidas by connecting VP1 with pEGFP-C1 plasmidas as DNA vaccine.The pEGFP-VP1 expression plasmid was transfected into DF1 cells to identify expression of the target protein through Western blot using the monoclonal antibody of EGFP.Animal immunogenicity tests were carried out to further verify the immunogenicity of the vaccines.Furthermore,animal protection tests were carried out to verify the animal protection effects of the vaccines.There was no significant difference in body weight between vaccine groups and control group.The antibody titers of35-days old chickens were tested.VP1_90-4630-463 subunite vaccine group reached 1:25600 and VP1_90-463-VP2 subunite vaccine group reached 1:12800;EGFP-VP1 DNA vaccine group reached 1:160.In animal protective test,the infection rate of GyV3 virus was detected one week after challenge.The animal protective rate of both subunit vaccines was as high as100%;EGFP-VP1 DNA vaccine group was 70%.The clinical symptoms,antibody titers,growth status of chickens,and histopathological changes of experimental animals showed that the immunogenicity and immunoprotection of the subunit vaccines are significantly better than the DNA vaccine.Although both subunit vaccines have good immunogenicity and 100%immunoprotection,the VP1_90-4630-463 subunit vaccine induces a higher antibody titer than the VP1_90-463-VP2 subunit vaccine,so the VP1_90-4630-463 subunit vaccine is superior to the VP1_90-46-VP2 subunit vaccine.This study is the first to carry out the research about the GyV3 vaccine,which provides a possibility for clinical prevention of GyV3 and prevention of transmissible viral proventriculitis.