| Fowl adenovirus(FAdV)belongs to the family Adenoviridea and genus Aviadenovirus.According to characteristics of genotype and sera cross-neutralization assay,FAdVs can be divided into 5 species(FAdV-A～E)and 12 serotypes(FAdV-1～8a and 8b～11).Since the prevalence of hepatitis-hydropericardium syndrome(HHS)in 2015,more and more cases caused by infection of fowl adenovirus have been reported.Inclusion body hepatitis(IBH)caused by serotype 8b fowl adenovirus(FAdV-8b)is gradually prevalent in China.Typical clinical symptoms of IBH are hemorrhagic spots and necrotic foci on the liver surface,and chickens with IBH have an acute morbidity and mortality rate of 2-10%.IBH mainly affects chickens aged 3-5 weeks,and causes huge economic losses to the poultry industry.However,the infection and pathogenic mechanism of IBH caused by FAdV-8b are still unclear,and there is no commercial specific serological test and commercial vaccines in China.In this study,we targeted the Fiber which is an important protein on the surface of fowl adenovirus.On the basis of cloning and expressing the fiber of FAdV-8b,the indirect ELISA technique for detection of antibody against FAdV-8b was established,and the specific monoclonal antibody against the Fiber protein of FAdV-8b was generated.The recombinant FAdV-4 virus FA4-F8b expressing the Fiber protein of FAdV-8b was developed by CRISPR-Cas9 gene editing technology,and the FA4-F8b can protect both FAdV-4 and FAdV-8b.In addition,the Fiber-1 of FAdV-4 can effectively inhibit the infection and replication of FAdV-8b in susceptible cells.1.Establishment of Fiber-based indirect ELISA for detection of antibody against FAdV-8bIn order to establish an indirect ELISA for detection of the specific antibodies against FAdV-8b,the fiber of FAdV-8b was cloned and expressed.After obtaining the fusion expression product GST-F of Fiber protein and GST,the purified recombinant protein GSTF was used as coating antigen to establish an indirect ELISA method for detection of antibody against FAdV-8b by exploring the conditions of coating concentration,dilution rate of primary antibody,diluent solution of primary antibody,incubation time of primary antibody,diluent solution of secondary antibody,incubation time of secondary antibody and chromogenic time.Specificity test showed that the ELISA only reacted with the positive sera of FAdV-7/8,but had no reaction with other sera detected.Compared with the commercial ELISA kit and IFA method,the ELISA generated here was more sensitive than the IFAd,and had a high coincidence rate(about 94%)with commercial ELISA kit for the detection of antibody against FAdV.The feasibility of the ELISA for detecting the sera of the infected chicken and sera of the clinical immunized chicken demonstrated that the indirect ELISA established here could not only be used for the diagnosis of infection of FAdV-7/8,but also be applied for monitoring the antibody level of the immunized chickens.2.Preparation and characterization of monoclonal antibodies against the Fiber of FAdV-8bIn order to generate monoclonal antibodies(mAbs)against Fiber of FAdV-8b,in this study,the purified recombinant protein GST-F was used as an immunogen to immunize mice.Finally,we generated two novel monoclonal antibodies against Fiber protein of FAdV-8,named as 4D9 and 5F10.Although they had no neutralizing activity against the infection of FAdV-8,mAbs 4D9 and 5F10 had good reactive characteristics of ELISA,IFA,Western blot and IP.Both mAbs 4D9 and 5F10 can recognize Fiber protein expressed in LMH cells transfected with the plasmid or infected with FAdV-8b.The cross-reactivity of mAbs showed that mAbs 4D9 had high specificity for FAdV-8b,while 5710 had certain cross-reactivity with FAdV-7,but no cross-reactivity with other serotype fowl adenoviruses.Epitope maping using Fiber protein truncations revealed that mAbs 4D9 and 5F10 recognized 205-216aa and 129-140aa of Fiber,respectively.Further analysis of the epitopes recognized by mAbs showed that the epitope recognized by 5F10 was conserved in FAdV-7,8a and 8b,while the epitope recognized by 4D9 was only conserved in FAdV8b.The development of mAbs against the Fiber of FAdV-8b,the related characteristics and the identification of their epitopes lay the foundation for further exploring the role of Fiber in infection of FAdV-8b,identifying interaction proteins with Fiber and developing serological detection technology for FAdV-8b.3.Generation and characteristics of recombinant FAdV-4 virus expressing Fiber of FAdV-8bIn order to prevent and control the infection of FAdV-4 and FAdV-8b at the same time,in this study,a novel recombinant FAdV-4 virus FA4-F8b expressing Fiber protein of FAdV8b was constructed by using CRISPR-Cas9 gene-editing technology,homologous recombination technology and the recombinant FAdV-4 expressing EGFP constructed previously by our group.Virus replication assay in vitro showed that FA4-F8b could effectively and stably replicate in LMH cells,and the virus titer could reach 108 TCID50/ml.Infection study in vivo showed that FA4-F8b was highly pathogenic to SPF chickens,similar to wild-type FAdV-4,with a mortality rate of 80%.However,the inactivated FA4F8b not only effectively induced high titer of neutralizing antibodies against FAdV-4 and FAdV-8b,but also provided effective protection against FAdV-4 and FAdV-8b.All these demonstrate that the recombinant virus FA4-F8b expressing Fiber protein of FAdV-8b generated here can be used as an inactivated vaccine candidate for simultaneous prevention and control of FAdV-4 and FAdV-8b,but also proves that FAdV-4 can be used as a vaccine vector for expressing exogenous genes to construct polyvalent vaccines or multiplevaccines.4.Fiber-1 of FAdV-4 can effectively inhibit the infection of FAdV-8bDue to the previous results of superinfection resistance test,Hexon,Penton and Fiber of FAdV-8b had no significant inhibition on infection of FAdV-8b in LMH cells.In order to further explore whether FAdV-4 and FAdV-8b have a common cell receptor to mediate infection,in this study,we used Fiber-1 protein of FAdV-4 to explore the infection and replication of FAdV-8b in LMH cells through superinfection resistance assay and protein interference test in vitro.The results showed that Fiber-1 of FAdV-4 could effectively inhibit FAdV-8b infection and replication in LMH cells.Using different domains of Fiber-1 further revealed that the Shaft-Knob domain of Fiber-1 was essential for inhibiting infection and replication of FAdV-8b in LMH cells.Due to the important role of Fiber-1 and its Shaft-Knob domain in mediating infection of FAdV-4 and interaction with cell receptors,it is suggested that common cell receptors for FAdV-8b and FAdV-4 might mediate replication and infection of FAdV-8b.Subsequently,coxsackie and adenovirus receptor(CAR)gene knockout cell lines were infected with FAdV-8b,and the results showed that CAR gene knockout could influence the infection of FAdV-8b,indicating that CAR might be the cell receptor of FAdV-8b.All these findings provide novel insights into further exploring the infection mechanism of FAdV-8b and new targets for preventing and controlling for both FAdV-4 and FAdV-8b. |
PDF Full Text Request