Isolation And Identification Of Fowl Adenovirus During 2015-2017 And Generation Of Recombinant Newcastle Disease Viruses Expressing The Penton And Fiber2 Proteins From Fowl Adenovirus

Hydropericardium syndrome(HP S)is a highly contagious disease caused by fowl adenovirus(FAV).It was first reported in 1987 in the Angara region of Pakistan and therefore also known as"Angara disease".Fowl adenovirus genome is a non-enveloped,double stranded DNA and the virus capsid is composed of hexon,penton and fiber proteins.Penton is the important structural protein associated with virus attachment and entry into the cells.The antibodies against penton possess the high ability in adenovirus neutralization,and penton vaccine can provide high protection efficacy against FAV infection.Fiber combine with penton by its N-terminal part,and its head domain plays a key role in virus infection process by binding receptors on cell surface.As previous report,the fiber2 expressed from both eukaryotic and prokaryotic systems can provide strong immune protection for SPF chickens.In recent years,the infection of FAV in china has caused serious economic losses,but no commercial vaccine has been approved for practice until now.In order to develop live vaccines for prevention of this disease,we use Newcastle disease virus as a vector to express FAV penton and fiber2 proteins respectively,and the immune efficacy of these recombinant viruses were also tested in this study.1.Isolation,identification and phylogenetic analysis of FAVA method of culturing fowl adenovirus in LMH cell was established by optimizing the culture medium and conditions.The titer of FAV cultured by LMH cell can reach 108.5TCID50/ml,indicating that high titer of FAV can be harvested by LMH cell culture,and the titer is 100 times higher than the traditional method.From 2015 to 2017,a total of 27 samples suspected as FAV infection were received from the diseased chicken flocks.After identification by PCR,18 type I FAVs were isolated,among which 16 strains belonged to serotype 4 and 2 strains belonged to serotype 1,indicating that HPS in china mainly was caused by the serotype 4 virus.Phylogenetic analysis showed that the hexon,penton and fiber2 genes of serotype 4 viruses each exhibited 100%genetic identities with the domestic reference strains.However,phylogenetic distance was present between these isolates and foreign isolates,among which the Indian strains is relatively close.The amino acid sequence analysis showed that the 195 amino acid(aa)residues of hexon from the domestic isolates was Q,while the corresponding amino acid residues from foreign strains was E,respectively.The 426aa and 486aa residues of penton from the domestic strains were V and T,while the corresponding residues from foreign strains were I and S,respectively.Therefore,the Chinese fowl adenovirus has characteristic amino acid residues both in hexon and penton,which can be used to distinct the domestic and foreign FAVs effectively.Meanwhile,three serotype 4 FAVs isolated from different years were inoculated into SPF chickens and all the viruses can cause 100%morbidity and typical pathological change in chickens,indicating that the disease was successfully reproduced.2.Generation of recombinant Newcastle disease viruses expressing the Penton and Fiber2 proteins from Fowl AdenovirusThe genotype ? attenuated Newcastle virus AI4 was selected as a vector,which possesses high ability of replication and immunogenicity.The penton and fiber2 genes from serotype 4 FAV isolate JSJY2017 were inserted into P and M genes of AI4,respectively,resulting two recombinant NDV full-length plasmids pNDV/rAI4-penton and pNDV/rAI4-fiber2.The full-length plasmids and three helper plasmids were co-transfected into BSR cell and rescued recombinant viruses were named as rAI4-penton and rAI4-fiber2,respectively.The mean death time of recombinant viruses was over 120h(typical characteristic of lentogenic NDV),and possessed good replication ability in embryonated eggs.The HA titers ofallantoic fluids from rAI4-penton and rAI4-fiber2 were 9log2 and 7log2,and possessed 1.5×108EID50/ml and 3.2×107 EID50/ml respectively.The growth curve test showed that the recombinant viruses and vector virus had the similar growth kinetic characteristics.The two recombinant viruses were sequentially passaged in 9-day old embryonated eggs and the foreign genes can still express successfully in 5th passage,which confirmed the good stability for these recombinant NDVs.SPF chickens were immunized via oculonasal route with 106EID50 recombinant NDV per bird.Three weeks post-immunization,recombinant virus rAI4-penton can induce neutralization antibodies against FAV with the VN titer as 1:64.The immune-challenge test showed that the recombinant viruses rAI4-penton can provide effective immune protection.The work here provides an useful information for development of live recombinant vaccines against the FAV infection.