| Duck tembusu virus disease is an emerging infectious diseases which can lead to a severe drop in egg production of the laying ducks and growth retardation.Meat-type duck and laying duck can be infected with the virus.The incidence of virus disease in duck tembusu group can reach 100%,the mortality rate was 5%~15%.This disease has caused a serious economic loss to the poultry industry in China.Currently,the prevention of DTMUV mainly rely on the inactivated vaccine and attenuated vaccine.However,there are many defects in the inactivated vaccine and attenuated vaccine,such as short duration,serious side effects,and virulence return.Therefore,it is necessary to develop some safe,efficient and vaccinated animals vaccines against DTMUV.It has been proved that DTMUV E protein can cause structural protein protective neutralizing antibodies,which play an important role in the process of the virus in the host cell invasion.In this study,the recombinant adenovirus expressing DTMUV E protein was constructed,and its immunogenicity was analyzed in ducklings.The details of this study are as follows:Firstly,DTMUV E gene was amplified with specific primers from DTMUV AH-F10strain(GenBank Accession No.:KM102539).Subsequently,E gene was cloned into shuttle vector pacAd-CMV K-NpA,after characterized with PCR,restrict enzymes and sequencing,the recombinant shuttle plasmids were linearized with the Pac I restrict enzyme,and was contransfected to the AAV-293 cells combinant with the linearized backbone vector pacAd5 9.2-100.The positive rAd was packaged in AAV-293 cells,named rAd-E.rAd-E.The titer of rAd-E was(TCID50)was 10-6.17/0.1mL.The results of IFA and Western blotting analysis revealed that the recombinant adenoviruses could express DTMUV E protein in cells,and the expressed proteins had good reactionogenicity.The 7-day-old healthy ducklings were randomly divided into 4 group.Experiment group,duckings were immunized with rAd-E(108 TCID50).Positive control group,ducklings were inoculated with the killed vaccine(DTMUV AH-F10 strain).Ducklings were inoculated with wtAd(108TCID50).Ducklings were immunized with 0.5mL PBS.The specific antibodies and neutralizing antibodies against DTMUV were evaluated using an indirect enzyme-linked immunosorbent assay(ELISA)and a microneutralization test,respectively.Cell-mediated immune responses were also examined using a lymphocyte proliferation assay.The results showed that specific antibodies could be detected from 1week post primary immunization,the antibody level was significantly higher than that ofGroup 3 and Group4,but lower than Group 2;The highest titers of neutralizing antibody reached 1:16,lower than that of Group 2.Lymphocyte proliferation test showed that the stimulation index of Group 1 was higher than that of inactivated vaccine group,and there was significant difference between the two groups(P < 0.05).The expression of IL-4 and IFN-γ in Group 1 ducklings were also higher than that of inactivated vaccine group.These results showed that the recombinant adenovirus could induced strong cellular immune response.All ducklings were challenged with 0.5mL(ELD50,10-2.38/0.1mL)wild DTMUV AH-TL15 strain via intramuscular injection.The incidence and mortality were recorded,and the duckings were dissected.Protective test and HE staining showed that the protection rate of inactivated vaccine was100%,the protection of recombinant adenovirus rate reached 80%.The tissue samples of ducklings immunized with inactivated vaccine and recombinant adenovirus group have no obvious lesions,vascular cuff,neuronophagia phenomena,and neuronal degeneration within the brains in PBS group and wt Ad group.In summary,the recombinant adenovirus rAd-E can induce ducklings specific humoral immune response and cellular immune response in ducklings,and could protect experimental ducklings from wild DTMUV attack.These results provide a good basis for further developing new vaccines against DTMUV infection. |
