The Role Of Fiber Proteins In The Infection Of Serotype 4 Fowl Adenovirus And Their Application In Diagnostics

Since 2015,the large-scale outbreak of hepatitis-hydropericardium syndrome(HHS)caused by highly pathogenic serotype 4 fowl adenovirus(FAdV-4)has occurred in many provinces in China,and also outbreaked globally.The mortality caused by FAdV-4 rangs from 30%-80%,which has resulted in huge economic losses to the global poultry industry.However,little is known about the infection mechanism of highly pathogenic FAdV-4,and there is no commercial serological technique for the detection of the specific antibody against FAdV-4.In this study,through targeting the surface proteins Fiber-1 and Fiber-2 of FAdV-4,the mechanism that Fiber-1 directly mediated FAdV-4 infection through its shaft+knob domain was identified,the immune protection effect of knob domain of Fiber-1 was evaluated,the protein 14-33ε interacted with Fiber-1 was identified,the fusion proteins of GST-Fiber-1 and GSTFiber-2 were expressed and purified,and the indirect ELIS As and competitive ELISA for specific detection of antibody against FAdV-4 were developed.All above results and findings provided the targets and technical supports for the effective immune prevention and control of FAdV-4 infection1 Shaft-knob domain of Fiber-1 directly mediated the infection of FAdV-4In order to identify which Fiber directly mediates for the infection of FAdV-4 among Fiber-1 and Fiber-2,in this study,on the basis of eukaryotic and prokaryotic expression of Fiber-1 and Fiber-2,the superinfection resistance analysis and recombinant protein interfering assay were carried out We found that Fiber-1,but not Fiber-2,was the key factor for directly triggering the infection of FAdV-4.Superinfection resistance analysis of Fiber-1 trunctions further revealed that Shaftknob domain of fiber-1 was required for FAdV-4 infection.In addition,the mouse polyclonal antibody against the recombinant protein His-Knob showed a good neutralizing activity against FAdV-4 infection in vitro.Further,the animal experimemt revealed that His-Knob as an antigen could provide efficient protection against the lethal challenge of FAdV-4 in SPF chickens.The chickens vaccinated with His-Knob did not showe any clinical symptoms and pathological changes,and also effectively block viruses shedding.All above results not only proved that Fiber-1 and its Shaft-knob domains played the important role in mediating FAdV-4 infection,but also demonstrated that the recombinant protein His-Knob could be used as a subunit vaccine candidate for prevention and control of FAdV-4 infection.2 Fiber-1 of FAdV-4 efficiently interacted with host protein 14-3-3εFiber-1 could directly mediate FAdV-4 infection through its Shaft-knob domain,demonstrating that Fiber-1 is the significant viral protein in binding to the cellular receptors while FAdV-4 infecting host cells.Recently,the CAR was identified as cellular receptor for FAdV-4 infection via Fiber-1.In order to further identify the cellular proteins interacting with Fiber-land explore their roles in FAdV-4 infection,in this study,mAb 3B5 specific to Fiber-1 was used to co-immunoprecipitate LMH cell lysates transfected with pcDNA3.1-F1 or infected FAdV-4,and then the silver stain,mass spectrum and western blot analysis were carried out.The result showed that Fiber-1 of FAdV-4 could effectively interact with the host cellular protein 14-3-3ε.And the polyclonal antibody against the recombinant protein His-14-3-3ε which prepared by immunizing mice with prokaryotic expression protein could effectively recognize the endogenous 14-3-3ε protein in LMH cells.14-3-3ε belongs to the 14-33 protein family,which not only plays significant roles in cell signal transduction,but also is responsible for the activity of protein kinases and protein phosphorylases.Although the over-expression of 14-3-3ε-Flag in FAdV-4-sensitive LMH cells had no effect on FAdV-4 replication,the discovery of the interaction between Fiber-1 and the host protein 14-3-3ε and the preparation of polyclonal antibody against His-14-3-3εprotein could provide a new host target and the biomaterial for further exploring the role and mechanism of Fiber-1 in the infection and pathogenesis of FAdV-4.3 Establishment of an indirect ELISA for detection of antibody against FAdV-4To develop the rapid serological diagnostic techniques for the detection of the antibody against FAdV-4,in this study,two indirect ELISA(iELISA)methods for specific detection of antibody against FAdV-4 were established using two purified recombinant proteins GST-Fiber-1 and GST-Fiber-2 as the coating antigens,and all parameters of the iELISAs were optimized.Specificity assay showed that the iELISAs could only react with the positive sera against FAdV-4 and FAdV-10,but could not react with the positive sera against the other pathogens including FAdV-1,FAdV-2,FAdV-3,FAdV-5,FAdV-6,FAdV-7,FAdV-8b,FAdV-9,FAdV-11,ALV-J,IBV,NDV,ARV,EDS and H9N2.Stability analysis showed that the intra-assay/interassay variation coefficient were both less than 10%.Moreover,the iELISAs were able to be effectively applied in detecting antibodies against FAdV-4 from chickens either infected with FAdV-4 or immunized with FAdV-4 vaccine.In comparison with Biocheck commercial detection kit,the two iELISAs showed the better sensitivity for detection of the antibody against FAdV-4.All these demonstrate that the two iELISAs based on GST-Fiber-1 and GST-Fiber-2 generated here have the good application value in the detection of antbody against FAdV-4 for diagnostics of clinical FAdV-4 infection and monitoring the efficacy of FAdV-4 vaccines.4 Establishment of a cELISA for detection of antibody against FAdV-4To develop a more convenient and rapid serological diagnostic technique for the detection of the antibody against FAdV-4.In this study,a competitive ELISA method(cELISA)for the detection of antibody against FAdV-4 was established by using the purified protein GST-Fiber-2 and HRP-labeled monoclonal antibody 3C2 specific to Fiber-2 respectively used as coating antigen and secondary antibody,and all parameters of the cELISA were optimized.And the percent of inhibition rate(PI)was determined as the judgement standard of the cELISA,Sample with the PI of ≥31%was judged to be positive.Specificity test showed that the iELISAs could only react with the positive sera against FAdV-4 and FAdV-10,but could not react with the positive sera against the other pathogens including FAdV-1,FAdV-2,FAdV-3,FAdV5,FAdV-6,FAdV-7,FAdV-8b,FAdV-11,ALV-J,IBV,NDV,ARV,EDS and H9N2.Stability analysis showed that the intra-assay/inter-assay variation coefficient were both less than 10%.In comparison with Bio-check commercial detection kit,the cELISA showed the better specificity in detection.The establishment of this cELISA with the good specificity and stability for specific detection of antibody ananist FAdV4 not only provides the rapid technique for the detection of FAdV-4 infection in different poultry species,but also can be used for the immunological monitoring of vaccination.