Identification Of The Cellular Attachment Receptor Of Egg Drop Syndrome Virus Based On Structure Determination Of The EDSV Fiber Protein And The Immunogenicity Of The Protein

Egg Drop Syndrome is caused by Egg Drop Syndrome Virus(EDSV),which primarily causes a sudden severe drop in egg production as well as the production of shell-less,thin-shelled,discolored,or misshapen eggs in apparently healthy laying birds.EDSV was first reported in Holland in 1976,and then spread throughout the world.Besides,EDSV could infect10-day-old Pekin ducklings and caused severe acute respiratory symptoms.In the late 1980 s,EDSV infection appeared in China in broiler breeders,and then many strains were isolated in many areas.Fiber protein is an important structural protein of EDSV,which was involved in virus assembly,virus-receptor binding,and virus immunogenicity.The protein consists of three domains:a N-terminal tail,the long shaft with repetitive triple ?-spiral motif,and a C-terminal globular knob domain,which usually recognizes the primary cellular receptor.In the present study,based on the structural determination of EDSV fiber protein,we have investigated the mechanism of EDSV-host interaction,and the immunogenicity of the recombinant fiber protein.Firstly,we determined the crystal structure of the fiber protein to reveal the structural basis for the function of the EDSV fiber protein.The coding sequence for the predicted EDSV fiber knob domain and several adjacent amino acids was cloned into the prokaryotic expression vector.The recombinant plasmid was transformed into E.coli BL21(DE3)cells.Protein expression was induced with IPTG and the expression level reached about 70%.Subsequently,purification and characterization of the fiber protein were performed using affinity chromatography,size exclusion chromatography(SEC)and dynamic light scattering(DLS).It was shown that the purified fiber protein was homogenous trimer with strong hemagglutination.The protein was then crystallized and data sets were collected and processed.The atomic coordinates and structure factors of the EDSV fiber protein have been deposited in the Protein Data Bank(PDB code 6ITX).Based on the structural analysis,we aimed at the investigation of the mechanism of EDSV infection and the immunogenicity of the recombinant fiber protein.EDSV is the only member of the avian group ? Aviadenovirus within the family Adenoviridae.Adenovirus infections are usually initiated by the interaction of their fiber knobs with the primary receptors.The structural fold of EDSV fiber knob is more similar to CELO long fiber knob,compared with human adenovirus fiber knob.Based on previous studies of CELO,we proposed that the two avian adenoviruses probably bind with the same cellular receptor coxsackievirus and adenovirus receptor(CAR)in a unique fashion.In the present study,we demonstrated that knockdown of the CAR remarkably impaired virus infection in the EDSV permissive DEF cells.And the EDSV fiber knob was shown to specifically interact with DEF cells by flow cytometry analysis.Furthermore,confocal,co-immunoprecipitation,and pull-down analysis validated that the fiber knob specifically recognized and interacted with CAR.In inhibition assays,soluble fiber knob or CAR protein significantly inhibited EDSV attachment in a dose-dependent manner.Moreover,overexpression of CAR resulted in an increase in the binding of EDSV virions to both permissive and nonpermissive cells.Therefore,these results indicate that CAR is a cellular attachment receptor for EDSV.Inactivated EDSV vaccines produced in embryonated duck eggs or cell culture systems have been widely used for the prophylaxis of EDS.However,recombinant subunit vaccines that are efficacious and inexpensive are a desirable alternative.The crystal structure of the recombinant fiber protein reveals that the protein forms a trimer,which is close to the natural conformation of the antigen.To detect the immunogenicity of the EDSV fiber protein,the protein was used to immunize SPF chickens.The results of neutralization test and hemagglutination inhibition assays(HI)indicated that the protein induced high level of humoral immune responses,and the titer of antibodies could maintain until 20 weeks after first immunization.Furthermore,we detected the IL-2,IL-4,IFN-? secretion and lymphocyte proliferation of chickens,and the results indicated that the chickens produced cellular immune responses.Finally,the copies of virus gene in liver were detected by RT-PCR after the challenge of EDSV,revealing that the fiber protein vaccine effectively reduced the virus infection.In conclusion,the recombinant fiber protein produced as described here has the potential to be an EDSV vaccine candidate.