The Isolation, Pathogenicity And Preliminary Establishment Of Indirect Immunofluorescence Of Serum Type 4 Avian Adenovirus

Fowl Adenovirus(FAdV)belongs to the family Adenovirus,Aviadenovirus.The fowl adenovirus has been grouped into 5 species(FAdV-A to FAdV-E)with 12 serotypes(FAdV-I to 8a and 8b to 11)based on the restriction enzyme digestion pattern and serum cross-neutralization test.FAdV is susceptible to all day-old chickens with a worldwide distribution.Chickens infected with FAdV are generally characterized as subclinical symptoms,whereas the acute infections are responsible for inclusion body hepatitis(IBH),hepatitis-hydropericardium syndrome(HPS)and gizzard erosion.Since 2013,the clinical cases of HPS caused by serotype 4 fowl adenovirus(FAdV-4)have been increasing in most domestic chickens in China.resulting in considerable economic losses for the poultry industry.To investigate the molecular characterization,pathogenesis and develope serological diagnostic test for endemic FAdV-4 in China,in this study,several domestic FAdV-4 were first isolated and sequenced,and the fiber 1 gene of FAdV-4 was cloned and expressed.And an indirect immunofluorescence assay for the detection of antibody against FAdV-4 was established based on the Fiberl expression product.1.Isolation,identification and sequence analysis of serotype 4 fowl adenovirusSamples suspected with the infection of fowl adenovirus in 2015 were used for viral isolation and identification.Results showed that a total of 9 isolates of FAdV were isolated and identified from five provinces in China.Among the nine isolates,one isolate was identified as FAdV-8 and the other eight isolates were belonged to FAdV-4.In comparison with the FAdV-4(JSH13 and JH13)isolated in 2013 in China,the deletions with different sizes in ORF29 were found in these FAdV-4 isolated in 2015.A 66-nucleotide deletion was present in ORF29 from one isolate,whereas a 33-nt deletion was found in others.Moreover,different from the FAdV-4 from other countries,a deletion of 1961-nt in FAdV-4 recently isolated in China was revealed.In addition,the fiber 1 and fiber 2 protein associated with the antigenicity for these Chinese FAdV-4 showed 94.4%-94.7%and 93.3%-94.5%homology respectively to those from other countries.These data demonstrated that FAdV-4 with novel genotype is epidemiology in China,indicating the deletion or mutation in the genome of these FAdV-4 found might contribute to the outbreak of FAdV-4 in China.2.Pathogenicity of serotype 4 fowl adenovirusIn order to investigate the pathogenicity of FAdV-4,three FAdV-4 isolates(SD15,JSCZ15 isolated in 2015,JH13 isolated in 2013)were tested in vivo and in vitro.Viral growth kinetics in chicken liver cell showed that the three isolates of FAdV-4 had similar replication pattern.The viral titer of SD15 could reach to 1 × 108TCID50/mL.Through intramuscular inoculation with the dose of 103TCID50,the mortality of the SPF chickens infected with SD15,JSCZ15 and JH13 were 80%,90%and 60%respectively.Viral shedding showed that the cloaco continued to shed viruses until 10 days after infection.A typical case of hepatitis-hydropericardium syndrome could be found in the dead chickens.HE staining showed that the basophilic intranuclear inclusions and fatty degeneration could be found in hepatocytes.These data demonstrated that the three FAdV-4 isolates tested were highly pathogenic serotype 4 fowl adenovirus.However,the molecular mechanism of the pathogenicity of these FAdV-4 isolates needs further investigation.3.Preliminary establishment of indirect immunofluorescence assay for detection of antibody against FAdV-4The fiber 1 gene of FAdV-4 was first amplified by PCR,and inserted into the eukaryotic expression vector pcDNA3.1 through ligation by recombinant enzyme ExnaseTM II,and the recombinant plasmid was named as pcDNA3.1-F1.The expression of pcDNA3.1 and its antigenicity was confirmed through indirect immunofluorescence and Western blot respectively by using chicken serum against FAdV-4.An indirect immunofluorescence assay(IFA)for the detection of antibodies against FAdV-4 was established using 293T cells transfected with pcDNA3.1-F1.Specificity analysis showed that the IFA only reacted with the serum anti FAdV-4 and not reacted with serum against Marek’ s virus,avian leukosis virus,avian influenza virus,Newcastle disease virus,infectious bursal disease virus and infectious bronchitis virus tested.These data demonstrated that the IFA based on fiberl developed here showed potential application for detection of antibodies against FAdV-4.