Isolation And Identification Of Serotype ? Fowl Adenovirus Group ? And Sequence Analysis Of Cytotoxicity In Different Generations

Fowl Adenovirus?FAdV?belongs to the adenoviral family and avian adenovirus.Based on restriction mapping and serological cross-neutralization reactivity,FAdV can currently be divided into 5 species?A-E?and 12 serotypes?serotypes 1-7,8a,8b,9-11?.Epidemiological studies have found that FAdV is widely distributed worldwide and susceptible to poultry of different ages.Among them,Fowl Adenovirus serotype 4?FAdV-4?mainly causes chicken's"Hydropericardium-hepatitis syndrome"?HHS?,which was first reported in the United States in 1963.After being discovered,it then spread to all countries in the world.Since 2013,several provinces in China have also erupted HHS caused by FAdV-4 infection,causing serious economic losses to domestic chicken breeding.To understand the prevalence and evolution of FAdV-4 in Shandong Province,this study investigated the FAdV-4 strains isolated from multiple chicken farms in Shandong.The main contents are as follows:1.Isolation,Identification and Whole-Genome Sequencing of FAdV-4In this study,Hexon gene was amplified by polymerase chain reaction?PCR?from 19chickens suspected to be infected with FAdV-4 disease in Shandong province,and the PCR products were sequenced.The results of sequencing showed that FAdV-4 could be detected in19 samples.A strain of Avian adenovirus type 4 of serotype I was isolated and named as SDTA1512 strain by inoculating chicken embryo with yolk sac after grinding and filtering.Different gene regions were amplified and cloned and sequenced by different primer designs,and the whole genome was obtained by splicing the obtained gene fragments.The sequence was compared with that of different reference strains.The results showed that this isolate had the highest homology with HB1510 and JSJ13,but low homology with other isolates.Finally,the isolated SDTA1512 strain was inoculated intraperitoneally into 1-day-old SPF chicks,and the pathogenicity of the virus was studied.The results showed that after inoculating the virus,the chickens in the attack group had clinical symptoms such as depression,drooping wings and disheveled feathers.Death began on the second day after drug attack and the death rate reached 100 in 10 days.Dissecting the diseased chicken,we can see the accumulation of a large amount of yellow fluid in the pericardium;the yellow stain of the liver,some of which have obvious bleeding spots;the renal swelling and bleeding,the deposition of uric acid salt;the enlargement of the stomach of individual chickens.In the test group,the dissection of chickens was normal and no pathological changes were found.Animal experiments showed that the SDTA1512 strain isolated in this study had strong pathogenicity.2.Study on the Proliferation of FAdV-4 SDTA1512 Strain in Chicken Primary Embryonic Kidney Cells?CEK?In order to study the proliferation of FAdV-4 SDTA1512 strain in chicken embryo primary kidney cells?CEK?,a SYBR Green I fluorescent quantitative PCR method for detecting FAdV-4 Hexon gene was first established in this experiment.The results showed that the fluorescence quantitative PCR method established in this study showed a single peak in the dissolution curve,no primer dimer and other non-specific amplification products,a linear relationship between the standard curve and Ct value,linear regression curve correlation coefficient?R²?was 0.999,high specificity,high sensitivity,good repeatability and stability,and lays the foundation for studying the dynamics of virus proliferation.Subsequently,10⁴TCID₅₀0 SDTA1512 strains were inoculated into CEK cells,and the cell morphology was observed at 6,12,24,36,48,72,and 96 hours after the virus was inoculated.At the same time,cell samples were collected to extract DNA,and the fluorescence quantitative PCR was established.Methods To study the viral proliferation dynamics.The results show that the SDTA1512 strain can proliferate well in CEK cells and reach a higher titer.When the initial infection amount was 10⁴TCID₅₀,the 48-hour cells showed obvious lesions,and the cells became larger and rounder,gradually became compact,and plaques appeared,and eventually they died.The results of fluorescence quantitative PCR showed that the virus proliferated after 12 h of virus inoculation and reached the peak at 48 h,after which the total amount of virus remained unchanged.This study provided scientific data and support for the in vitro passage of FAdV-4.3.In vitro passage of FAdV-4 and analysis of sequence variation of different generations of viral genesThe FAdV-4 SDTA1512 strain isolated in our laboratory was serially passaged on CEK cells and passed to the 25th generation,and each generation retained virus and infected CEK cell samples.The 5th,10th,15th,20th and 25th generations of viruses were selected to extract their viral DNA and their Hexon,Penton,Fiber 1 and Fiber 2 genes were amplified by PCR.The amplified sequences were sequenced and analyzed.The results showed that the Penton gene and Fiber 2 gene were stable during the passage,while the Hexon gene and Fiber 1 gene were mutated.The relationship between these mutations and the weakening of the virus remains to be further verified.