Identification Of Virulence Site Of Emerging Fowl Adenovirus 4 And Construction And Evaluation Of Recombinant Virus Vaccine Candidates

Fowl adenovirus 4(FAdV-4),belonging to the family Adenoviridae and Aviadenovirus genus,is a double-stranded DNA virus with a genome length of about 43～45 kb.Since 2015,a novel genotype of FAdV-4 has emerged in poultry farms in China,and the clinical symptoms of infected chickens were characterized by hepatitis-hydropericardium syndrome(HHS)and high mortality,causing substantial economic losses to the poultry industry.FAdV-4 isolated from healthy chicken flocks in foreign countries was not pathogenic to chickens,but the emerging FAdV-4 can cause up to 100% mortality in chicken flocks.Therefore,there is an urgent need to elucidate the molecularmechanisms responsible forthe enhanced virulence of emergent FAdV-4,which is crucial forunderstanding the pathogenic mechanism of FAdV-4 and developing novel vaccines against the emerging FAdV-4.At present,commercialized vaccines against HHS are mainly prepared by inactivating FAdV-4 highly pathogenic isolates into multiple inactivated vaccines,which require multiple virus strains to be cultured during the preparation process.The FAdV-4 virulent strain can be developed into novel live orinactivated vaccines by attenuating its virulence,which can provide new vaccine candidates forthe prevention and control of HHS.Employing the attenuated FAdV-4 strain as a vecorto express protective antigens of otherpathogens is expected to develop novle FAdV-4 multiplex vaccines,which can not only reduce the cultivation of virus strains,but also simultaneously prevent HHS and otherdiseases.Firstly,the FAdV-4 reverse genetic system was established based on infectious clone in this study.Using this system,the FAdV-4 genome could be site-specific deleted,inserted and replaced in vitro,and the FAdV-4 recombinant viruses could be generated rapidly and efficiently.This study provides an important tool forthe study of FAdV-4 pathogenic mechanism and recombinant virus vaccines.Next,to explore the molecularmechanism of the enhanced virulence of emerging FAdV-4,the Hexon and Fiber-2 genes of the highly pathogenic FAdV-4 HLJFAd15 strain were respectively replaced with the corresponding genes of non-pathogenic ON1 strain using the FAdV-4 reverse genetics system,and the recombinant chimeric viruses rHN20(replacement of Hexon)and rFB2(replacement of Fiber-2)were constructed.The results of animal experiments proved that Hexon gene was the virulence gene responsible forthe enhanced pathogenicity of emerging FAdV-4.To furtheridentify the key site forthe enhanced virulence of emerging FAdV-4,the recombinant viruses with single amino acid point mutation were constructed,and the results of animal experiments showed that the mutation of the 188 th amino acid(Hexon aa-188)of Hexon protein from arginine(R)to isoleucine(I)could significantly reduce the pathogenicity of HLJFAd15 strain;while the mutation of Hexon aa-188 from I to R enhanced the pathogenicity of the rHN20 strain.Furthermore,comparing the sequences of emerging FAdV-4 Hexon gene with otherFAdV-4 strains Hexon gene isolated in the past,and the results revealed that the Hexon aa-188 of highly pathogenic FAdV-4 was R,while the Hexon aa-188 of non-pathogenic FAdV-4 was I,which was consistent with the findings of this study.In addition,the replication ability of the virulent and attenuated recombinant strains constructed in this study showed no difference in vitro,but the viral loads of the attenuated strains were significantly lowerthan those of the virulent strains in SPF chickens,indicating that the high pathogenicity of FAdV-4 virulent strain may be caused by it being able to infect target organs more efficiently in vivo.Then,to develop novel recombinant FAdV-4 vaccines,the immunoprotective effect of the rHN20 strain was furtherevaluated,and the results indicated that whetherrHN20 was immunized as a live attenuated vaccine oran inactivated vaccine candidate strain,it could both induce neutralizing antibody responses in SPF chickens and provide 100% protection forchickens against FAdV-4.Lastly,the VP2 gene of the very virulent infectious bursal disease virus(vvIBDV)was inserted into the rHN20 strain to generate the FAdV-4 recombinant strain rHN20-vvIBDV-VP2,and the results demonstrated that the recombinant strain rHN20-vvIBDV-VP2 was used whetheras a live vectorvaccine oran inactivated vectorvaccine candidate,it could both induce chickens to produce FAdV-4 and vvIBDV-specific neutralizing antibodies and provide 100% protection forchickens against FAdV-4 and vvIBDV.In summary,this study identified that the Hexon gene was the virulence gene responsible forthe enhanced pathogenicity of emerging FAdV-4,and revealed that the aa-188 of Hexon was the key virulence site,which provides an important target forthe development of vaccines and drugs against FAdV-4.Moreover,the recombinant FAdV-4 vaccine candidates and the bivalent FAdV-4/vvIBDV vectorvaccine candidates constructed in this study had good immune protection,which can provide new strategies forthe prevention and control of hepatitis-pericardial effusion syndrome as well as the development of multiple vaccines forpoultry diseases.