Isolation,Identification And Pathogenicity Of Fowl Adenovirus Serotype 8b Isolates In Shandong Province

Fowl adenoviruses(FAd Vs)are non-enveloped double-stranded DNA virus.It's structure is typically icosahedral symmetry.It is divided into three groups according to the difference in antigenicity and gene sequence(FAd V-??III),group I is divided into 5 species(A?E)and 12serotypes(FAd V-1 to 11,FAd V-8 is divided into types a and b).FAd V-I can cause chicken hydropericardium syndrome(HPS),inclusion body hepatitis(IBH)and Gizzard erosions(GE).Since the outbreak of HPS in large area in China in 2015,a variety of FAd V serotypes have been widely spread in all kinds of poultry farms in China.With the change of the epidemic trend of the virus,FAd V-8 has become one of the main serotypes causing IBH,which has caused huge economic losses and severe epidemic prevention challenges to the poultry industry in China.In order to understand the infection status and pathogenicity of FAd V in Shandong province,samples of chickens suspected of IBH were collected from commercial broiler farms in different areas of Shandong Province,and virus isolation and epidemiological investigation were carried out.In the end,17 of 45 samples were positive for antigen,the positive rate was 37.8%.There were seven FAd V-8b antigen positive samples,Fadv-8b accounted for 41.2%of the total detection rate.In order to investigate the molecular characteristics of FAd V-8b Shandong strain and its pathogenicity to chickens,in this study,a strain SD2009 of FAd V-8b was isolated and identified by inoculation of SPF chicken embryos,the whole genome sequence and the coding sequence of three main structural proteins were analyzed.The complete genome sequencing of SD2009 isolate showed that the genome is44439 bp in length with a G+C content of 58.18%and A+T content of 41.82%.The base sequences of the major structural proteins,Fiber,Hexon and Penton,were 1566 bp,2856 bp and 1659 bp,respectively.ORF prediction was carried out by Gene Mark S,SD2009 strain complete genome contained 35 ORF coding regions.Genome-wide nucleotide homology comparisons revealed that the homology between SD2009 strain and FADV-8b reference strains UPM04217(KU517714.1)from Malaysia was 97.70%.which further confirmed that SD2009 isolated belonged to FAd V-8b.Subsequently,in order to explore the pathogenicity of SD2009 strain,1-day-old SPF chickens were artificially infected by oral and intramuscular injection,at the same time set up negative control group.Combined with the clinical symptoms,histopathological changes,viral load and antibody levels of the two groups after infection,the results showed that the virus widely distributed in chickens and could damage multiple organs.In this experiment,all the muscle group died within 6 days,and the fatality rate was 100%.The oral group still survived after the 14 dpi observation period,and the final survival rate was 30.7%-42.8%.The liver of the dead chicken was yellowish and greasy,enlargement and congestion,the quality was brittle and fragile.Kidney enlargement and lung congestion.Histopathologic examination revealed between groups of chicken liver,kidneys,lungs and other organs in different degree of pathological damage,the spleen,bursa of fabricius,thymus lymphocyte degeneration necrosis.Among them,the histopathological changes of the liver and kidney in the muscle group and the oral group were the most obvious.The liver shows hepatic cell plate arrangement disorder,necrosis foci,intercellular vacuolar steatosis,central venous congestion,abundant inflammatory cell infiltration.Renal hyperemia,central venous hyperemia,glomerular swelling and bleeding,renal tubule edema and granular degeneration.The result of viral load shows that the virus content in liver is the highest,the average liver viral load in the muscle group was more than 10~5copies/mg.According to the above results,it can be inferred that the isolate has high pathogenicity,and it has a high tissue tropism for liver.The virus also has damage to immune organs,resulting in reduced immunity of chickens,and easy to cause mixed infection with other diseases in the natural environment,resulting in increased mortality.The main constitutive proteins of FAd V include Penton,Hexon and Fiber,in which Fiber protein binds the virus to the host cell through the recognition of specific receptors on the host cell,facilitating the invasion and internalization of the virus.Therefore,the Fibre protein plays an important role in the infection,pathogenesis and diagnosis of fowl adenovirus.In this study,the Fiber gene of SD2009 was cloned based on the whole genome sequencing of SD2009,and constructed prokaryotic expression plasmid,designated p ET32-8b-Fiber,The p ET32-8b-Fiber was transformed into E.coli BL21 and induced by IPTG to obtain the recombinant fused protein.The antiserum was prepared by immunizing BALB/c mice with purified Fiber protein.In this study,an indirect immunofluorescence assay for FAd V-8b was established using the prepared antiserum.It is proved that the antiserum has good reactivity and specificity.In conclusion,this study investigated the molecular epidemiology of FAd V in commercial broiler farms in some areas of Shandong Province.Isolation and identification of FAd V-8b isolates,and the whole genome sequence was analyzed,and carried out an artificial infection test of 1-day-old SPF chickens.Enriched the gene sequence data of FAd V-8b in Shandong Province.To provide scientific basis for understanding the pathogenic characteristics of f Ad V-8b epidemic strain in Shandong Province,It also provides technical support for laboratory diagnosis of FAd V-8b.