Preliminary Identification Of Virulence-associated Genes For Highly Pathogenic Fowl Adenovirus Serotype 4

Since 201 5,outbreaks of Hepatitis-hydropericardium syndrome(HHS)caused by a novel genotype of fowl adenovirus 4(FAd V-4)have been reported in major provinces of China with a mortality reaching 80%,causing significant economic losses to the poultry industry.Research has shown that comparing to the non-pathogenic FAdV-4 isolates,the highly pathogenic FAdV-4 Chinese isolates have a 1966bp natural deletion and amino acid mutations in structural proteins fiberl,fiber2,penton and hexon.This study aimed to investigate the association between the naturally absent 1966bp,fiberl,fiber2,hexon and penton genes and the virulence of highly pathogenic FAdV-4.To do so,five recombinant viruses,namely rHNJZ-ON1/1966,rHNJZ-ON1/fiber2,rHNJZ-ON1/hexon,rON1-HNJZ/fiber2 and rON1-HNJZ/hexon,were constructed by using the reverse genetic technique,and the biological characteristics of the five recombinant viruses were compared with the parental viruses.It was preliminarily determined that fiber2 and hexon genes are closely associated with the virulence of the emerging and highly pathogenic fowl adenovirus 4,whereas the natural deletion of 1966bp has no effect on the virulence.Furthermore,the virulence of the highly pathogenic FAdV-4 is independent of fiber-1 and penton,proving by constructing recombinant viruses rHNJZ-ON1/fiber 1 and rHNJZ-ON1/penton and studying their biological characteristics.The thesis is composed of the following three aspects.1.Construction and identification of the fiber2,hexon and 1966bp replaced FAdV-4 recombinant virusesIn this study,fiber2 or hexon gene of the highly pathogenic strain HNJZ and the non-pathogenic strain ON1 was exchanged with each other,and the 1966bp in the ON1 genome was inserted into HNJZ by using the Reda(3 recombineering and ccdB counter-selection techniques.The recombinant infectious clones were identified by restriction enzyme digestion analysis.The stability of the recombinant region was identified through virus rescue and multiple passages.The gel electrophoresis results of recombinant infectious clones after BamH I digestion were consistent with expectation.The correct recombinant infectious clones were linearized by Pme I digestion and transfected into LMH cells for recombinant virus rescue.Sequencing results proved that recombinant viruses rHNJZ-ON 1/1966,rHNJZ-ON1/fiber2,rHNJZ-ON1/hexon,rONl-HNJZ/fiber2 and rON1-HNJZ/hexon are genetical stable after serial passages.2.Study on the biological characteristics of the fiber2,hex on and 1966bp replaced recombinant virusesTo study the association between naturally absent 1966bp,fiber1,fiber2,hexon and penton genes and the virulence of highly pathogenic FAdV-4,the biological characteristics of recombinant viruses rHNJZ-ON1/1 966,rHNJZ-ON1/fiber2,rHNJZ-ON1/hexon,rON1-HNJZ/fiber2 and rON1-HNJZ/hexon were compared with the parental strains HNJZ and ON1.The replication dynamics of the recombinant viruses and parental viruses on LMH cells were studied by virus titer assay.The results showed that all viruses replicated in a similar manner.Three-week-old SPF chickens were then orally infected with the recombinant viruses and the parental viruses with the same dose.The mortality rate of chickens infected with rHNJZ,rHNJZ-ON1/1966 and rON1-HNJZ/fiber2 was 100%.The mortality rate of chickens infected with rON1-HNJZ/hexon was 50%.No death was observed in the control group and groups infected with rHNJZ-ON1/fiber2,rHNIZ-ON1/hexon,or rON1.Various degrees of pathological leisions were observed in multiple organs of chickens infected with rHNJZ,rHNJZ-ON1/1966,rON1-HNJZ/hexon and rON1-HNJZ/fiber2,whereas no gross leisions were present in chickens of the control group and groups infected with rHNJZ-ON 1/fiber2,rHNJZ-ON1/hexon,and rON 1.The viral loads in different organs of chickens in rHNJZ,rHNJZ-ON1/1966,rON1-HNJZ/fiber2,and rON1-HNJZ/hexon infected groups were significantly higher than that of chikens in rON1,rHNJZ-ON1/fiber2,and rHNJZ-ON1/hexon infected groups.Chickens in all virus-infected groups excreted viruses,but there were significant differences in the shedding viral titers.In conclusion,the natural 1966bp-deletion region in the genome of the highly virulent FAdV-4 novel genotype is dispensable for viral replication and virulence,while the fiber2 and hexon genes are closely associated with the virulence of the emerging and highly pathogenic fowl adenovirus 4.3.Construction and biological characteristics of the the fiber1 and penton replaced recombinant virusesTo further investigate the association between fiber1 and penton genes and the virulence of highly pathogenic FAdV-4,recombinant viruses rHNJZ-ON1/fiber1 and rHNJZ-ON1/penton were constructed by replacing the fiber1 and penton genes in the HNJZ strain with the corresponding genes of the ON1 strain.Results of continuous passages and sequencing indicated that the recombinant viruses replicated in a similar manner with the parental viruses and are genetically stable.Three-week-old SPF chickens were orally infected with the same dosage of the recombinant viruses and the parental viruses.The results of pathogenicity test showed that the mortality rate of chickens infected with rHNJZ,rHNJZ-ON 1/fiber1 and rHNJZ-ON1/penton was 100%.No death was observed in the control group and groups infected with rON1.Histopathologic analysis indicated that chickens infected with rHNJZ,rHNJZ-ON1/fiber1 and rHNJZ-ON1/penton had various degrees of lesions in the tissues,while chickens in the control group and groups infected with rON1 did not present any gross lesions.The viral loads in infected chicken organ tissues was detected by real-time quantitative PCR,and the results indicated that chickens infected with rHNJZ,rHNJZ-ON1/fiber1 and rHNJZ-ON1/penton had significantly higher viral loads than the chickens infected with rON1.Chickens in all virus-infected groups had viral shedding,but chickens infected with rHNJZ-ON1/fiber1 and rHNJZ-ON1/penton had significantly higher virus shedding than chickens infected with rON1.In conclusion,the increased virulence of hypervirulcnt fowl adenovirus 4 is independent of fiber1 and penton.