Research Of The Molecular Mechanism Of Fiber-2 On The Pathogenicity Of Serotype 4 Fowl Adenovirus

Serotype 4 fowl adenovirus(FAdV-4)is a non-enveloped virus with double stranded DNA genome,which belongs to the family Adenoviridea and genus Aviadenovirus.Since the outbreak of Hydropericardium syndrome(HPS)at Pakistan in 1987,FAdV-4 has spread to neighbouring countries gradually.Since 2015,the HPS caused by the highly pathogenic FAdV-4 has broken out in most areas of China mainland.The chickens infected with FAdV-4 showed hydropericardium,hepatitis and nephritis,with morbidity and mortality ranging from 30%～80%,causing great economic losses to the poultry industry.However,little is known about the pathogenesis of FAdV-4,and no licensed vaccines is available for FAdV-4.In Zhang’s study,Fiber-2 and hexon were identified to be closely related with the virulence of the highly pathogenic FAdV-4,However,the molecular mechanism for the pathogenesis of Fiber-2 remains unclear.In this study,we tried to explore the pathogenesis and molecular mechanism of FAdV-4 by targeting the virulent factor,Fiber-2 protein,and to develop the live attenuated vaccines against FAdV-4 through editing the Fiber-2 protein using CRISPR/Cas9 genome editing technology.1.Fiber-2 of FAdV-4 interacted with host protein KPNA3/4To explore the pathogenesis and molecular mechanism of Fiber-2,co-immunoprecipitation,silver stain and mass spectrum analysis were carried out to identify and study the interaction proteins binding with Fiber-2 using the monoclonal antibodies against Fiber-2.After screening and identification,host protein KPNA3/4 were found to interact with the Fiber-2 of FAdV-4.Further investigation revealed that the overexpression of KPNA3/4 could facilitate viral replication,while knock-out of KPNA3/4 could inhibit viral replication at early stage.Epitope mapping revealed that N terminus of Fiber-2(N-1-40aa)was identified to be responsible for the interaction with KPNA3/4.Further sequence alignment analysis uncovered that several mutations,deletions or substitutions were found within N-1-40aa of the low pathogenic FAdV-4 strains compared with the high pathogenic strains.Identification of the interaction between Fiber-2 and KPNA3/4,the epitope mapping of Fiber-2,and the effects of KPNA3/4 to FAdV-4 replication would provide targets for further study the effect of Fiber-2 on the pathogenesis and molecular mechanism of FAdV-4.2.N terminus of Fiber-2 interacted with KPNA3/4 affected the pathogenicity of FAdV-4To investigate whether the interaction of Fiber-2 with KPNA3/4 affects the pathogenicity of FAdV-4,the interaction domain of FAdV-4 Fiber-2 responsible for the interaction with KPNA3/4 was deleted using the CRISPR/Cas9 genome editing technology.In our strategy,the recombinant virus FA4-EGFP expressing the fusion protein EGFP-Fiber-2 was firstly generated and purified,then the EGFP was deleted together with interaction domain on the Fiber-2,and finally the recombinant virus FAV4-Del with the interaction domain deleted on Fiber-2 was obtained through the limit dilution assay.The following animal experiments demonstrated that both recombinant viruses were highly attenuated,and showed non-pathogenic to SPF chickens compared with wild-type FAdV-4.Notably,both recombinant virus could induce high level of neutralizing antibodies eficiently in vivo,thus providing efficient protection against the lethal challenge of FAdV-4.Taken together,the highly attenuated FAV4-Del demonstrate that the interaction domain on Fiber-2 responsible for the interaction with KPNA3/4,laying within 7～40 amino acids on Fiber-2 N terminus,determines the virulence of FAdV-4.Moreover,the recombinant virus generated in the study showed great potential to be vaccine candidates for the prevention and control of FAdV-4 as live attenuated vaccines.In addition,the construction of highly attenuated FA4-EGFP would provide inserting site for foreign genes to generate FAdV-4-based genetic engineering vaccines,which paves the way for generating FAdV-4-based multiple vaccines.3.Fiber-2 protein was not necessary for both virus assemble and challengeAs one of the most important structural proteins of FAdV-4,Fiber-2 was reported to provide better protection against the lethal challenge of FAdV-4 than other structural proteins,including Fiber-1,penton and hexon protein.However,compared with inactivated FAdV-4 vaccines,SPF chickens vaccinated with the recombinant Fiber-2 proein could not produce detectable neutralizing antibodies,indicating that Fiber-2 may not contribute for the production of neutralizing antibody against FAdV-4.Therefore,we hypothesize that Fiber-2 may not necessary for FAdV-4.To test our hypothesis,the Fiber-2 gene was replaced with EGFP using the CRISPR/Cas9 genome editing technology.As a result,the recombinant virus FAV4-EGFP-rF2 was successfully generated and purified,indicating that Fiber-2 was not necessary for FAdV-4 assemble.Further animal experiments showed that the recombinant virus FAV4-EGFP-rF2 with the deletion of Fiber-2 was highly attenuated,but could provide efficient protection against the lethal challenge of FAdV-4.All these data demonstrated that Fiber-2 was not necessary for viral assemble,as well as providing protection against challenge.Moreover,the rescue of highly attenuated FAV4-EGFP-rF2 indicated that Fiber-2 might be an efficient inserting site for foreign genes to generate FAdV-4-based genetic engineering vaccines to prevent both FAdV-4 and other pathogens.