Study On Biological Characterization And Pathogenicity Of Novel Fowl Adenovirus Group I

Fowl adenovirus group I?FAdV-I?has been clustered into five species?A-E?with 12serotypes.The five species of FAdV-I include species A?FAdV-1?,species B?FAdV-5?,species C?FAdV-4 and-10?,species D?FAdV-2,-3,-9,and-11?and species E?FAdV-6,-7,-8a,and-8b?.Although these species had spread globally prior to 2015,FAdV infections mostly caused subclinical symptoms or were avirulent.However,from the summer of 2015,the outbreaks of high pathogenic serotype 4 fowl adenovirus?FAdV-4?had emerged in many provinces of China.These outbreaks typically occurred in broiler and layer flocks,were associated with inclusion body hepatitis?IBH?,hydropericardium syndrome?HPS?,gizzard erosion and ulceration.In contrast to a mild disease caused by previous FAdV outbreaks,severe FAdV infections in the past two years have caused a huge economic loss in the poultry industry.In this study,six FAdV strains were isolated from clinical chickens from 2015 to 2016.After purification,the whole genomes of these viruses were sequenced.The strains were characterized and determined by the pathogenesis in vitro and in vivo.These preliminary studies are necessary and would provide useful information for diagnosis and developing efficient vaccines to control the disease.1.Isolation and biological identification of FAdV-I variantsFrom 2015 to 2016,outbreak of acute inclusion body hepatitis-pericarditis syndrome in chickens,we collected the liver samples in the chicken farms in Anhui and Jiangsu Provinces.The viruses were isolated and identified.First,the liver samples were grinded and inoculated in 6-7 days-old specific-pathogen-free?SPF?chicken embryonated eggs by yolk sac.The results showed that the dead embryos were thin,dysplasia,surface flushing,and bleeding;the livers were bleed with yellow Brown and the spotted necrosis.Then,the total DNAs were extracted and amplified by polymerase chain reaction?PCR?with the primers,which were designed according to the reference sequences of hexon and 52k genes.All the 6 isolates were characterized as FAdV-I,designated as HN,AQ,JS7,AH712,AH720,AH726,respectively.HN,AQ,JS7,AH712and AH726 were classified into group I Fowl adenovirus serotype 4?FAdV-4?;AH720 was classified into group I Fowl adenovirus serotype 8?FAdV-8?.The viruses were passaged in 7 days-old SPF chicken embryonated eggs for three times.By the identification of the physicochemical properties,morphological characteristics and Electron microscope of the viruses,the results were consistent with the virus characteristics of Fowl adenovirus group I.2.Sequencing and analysis of the whole genomes of the isolatesIn order to identify the molecular pathogenicity of the isolates,the whole genome sequences of 5 isolates of FAdV-4.The results showed that the full length of strain HN was43,724 bp,that of strain AH712 was 43,725 bp,and that of the rest three strains were 43,723 bp,and the G+C content was 54.87%.Compared with strain JSJ13 isolated at 2013,there were 33 nt deletions in the ORF29 gene in the five strains.Compared with the classical strain ON1,there were different GAGA repeats in the isolates at 19531-19571 nt.Strains HN,JS7,AH726,AH712,and AQ were similar to the highly virulent strain HLJ/15118 reported recently.They were located in the genetype II of Group I FAdV serotype 4.While strain AH720 was located at another subgroup of FAdV-E.3.Establishment of Taqman probe fluorescence quantitative PCR detection method to detect FAdV-4In order to rapidly and accurately detect and quantify FAdV-4,in this study,a highly sensitive Taqman probe fluorescence quantitative polymerase chain reaction?qPCR?method was established.According to ON1 strain?FAdV-C,GenBank Accession No.GU188428?1293¹387 nt of hexon gene were selected to design specific primers and Taqman specific probe,the PCR product was subcloned into the pGEM-Teasy vector to establish pGEM-Teasy-hexon plasmid which was used as template to generate the FAdV-4DNA standard curve.Taqman qPCR method was established,and evaluated the sensitivity,stability and specificity.The results showed it was successfully established with a good linear standard curve?y=-3.5237X+35.98,R²=0.998?.The detection limit was as low as22.8 copies/?L.The sensitivity was two tenfold orders of magnitude higher than the ordinary PCR method.The method laid the foundation for further detection of FAdV-4isolates in vitro and in vivo infection,as well as a tool for rapid clinical diagnosis of the disease.4.Pathogenesis of the strains on SPF chickensIn order to verify the pathogenicity of the isolates,animal challenge experiments were performed in this study.Nine chickens of each 3-week-old SPF chicken group were inoculated with strain HN,AQ,AH712,AH720,AH726 via 10⁵ TCID₅₀/100?L intramuscularly,respectively.The chickens in the negative control group were inoculated with PBS solution.The results showed that all the strains showed classical symptoms of acute avian adenovirus infection within 14 days after inoculation.The results showed that there are some significant lesions with yellow and hemorrhagic livers,pericardial effusion and bursa of Fabricius atrophy.Tissue samples were collected;all the tissues were separated,one part was stored at-80°C,and the other part was placed in 10%neutral formalin solution.The results showed that the mortality of HN,AQ and AH712 were100%,the mortality of AH726,JS7 and AH720 were 83.3%,80%,and 30%,respectively.Next,the total DNAs of the tissue samples from the FAdV-4infectious groups were extracted and detected by Taqman qPCR,the fixed samples were identified by H&E staining.Taken strain HN for example,after 2 days post infection?dpi?,the oral and cloacal could be found with high titers of amplified viruses.The virus amount of the liver was most,followed by the virus amount in the duodenum,jejunum and caecum samples.Even in the brain samples,the virus was positive with a low amount.H&E staining results showed that the livers revealed typical basophilic inclusions,pathology changes were also found in the other organs in the dead chickens.H&E staining showed that the liver showed a large number of typical basophilic inclusion bodies,a large number of inflammatory cell infiltration in the heart,severe disorder of the bursa of Fabricius,severe structural and significant reduction in thymus lymphocytes.Obviously,the isolates were the main cause of acute inclusion body hepatitis-pericarditis syndrome in clinic.