Identification Of Pathogenicity Of Duck Adenovirus 3(DAdV-3) And Establishment Of Indirect ELISA Detection Methods

Aviadenovirus is a common pathogen in poultry,and the virulence varies greatly between strains.In 2014,Duck Aviadenovirus 3（DAd V-3）was first detected in China,and in recent years,the virus has been detected in many places in Anhui.In this study,the pathogenic characteristics of DAd V-3 on ducks were studied,the optimal amplification pathway of screening inactivated vaccine strains and the main domain differences between different virulent strains were compared,and a method for detecting DAd V-3 indirect enzyme-linked immunosorbent assay（ELISA）was established.First,the DAd V-3 strain was isolated and identified,and the pathogenicity,pathological changes and virulence of each organ were determined.The DNA of the liver tissue of the hepatitis lesion was extracted and identified as DAd V-3 by PCR and named HF-AH-2022（Gen Bank registration number:OP432083）.Virus is inoculated with 10-day-old SPF duck embryos to obtain viral fluid.The viral half-cell infection volume(TCID₅₀)was determined to be 10^-5.32/0.1 m L.Animal experiments using duck embryo virus fluid found that the lethality rate of isolates in Muscovy ducks under 8 days of age was 42.9%,and pathological phenomena such as pericardial effusion,liver hemorrhage,piebald kidney and enteritis were found on autopsy.The results of pathological sections showed that typical basophilic inclusion bodies,deformation and necrosis of solid organs such as kidneys and digestive organs such as cecum showed deformation and necrosis of typical basophilic inclusion bodies in the dead Muscovy duck,and intestinal mucosal detachment of digestive organs such as cecum.SYBR Green was further applied to detect the viral load of various organ tissues,and the test results found that DAd V-3 could be detected in the collected organs,indicating that the isolated DAd V-3 was a strong strain.Second,the main domain differences of different virulent DAd V-3 strains were compared and analyzed.Extracting the viral genome and sending it to the company for whole-genome sequencing of the virus.The results showed that the whole genome of the virus contained43841 bp,and after BLAST comparison,it was found that the isolate had a homology of99.93%with the weak strain of HF-AH-2020 DAd V-3 previously found in our laboratory,and a homology of 93.3%with DAd V-2（GR）,which was determined to be DAd V-3.The Fiber-2 gene genetic tree found that HF-AH-2022 and HF-AH-2020 weak strains were in the same branch.Compared with the weak strain of HF-AH-2020,HF-AH-2022 isolates had a deletion of 2 threonine（Thr）in the ORF19B genome,and a point mutation in ORF66 led to the early termination of the gene.Third,in order to better select the optimal vaccine strain amplification route,the isolates infected with SPF chicken embryos and duck embryos were infected to obtain two viruses allantoic fluid.LMH cells were infected with two different viral fluids,and the cell virus fluid was received blindly passed on for 3 generations.The q PCR method was used to detect the viral load in cellular viral fluid,chicken embryo virus fluid and duck embryo virus fluid,and the results showed that the viral load of chicken embryo virus liquid was much higher than that of duck embryo,indicating that chicken embryo amplification virus could be used as the preferred route for vaccine strains.Fourth,an indirect ELISA method was established to detect DAd V-3.20 m L of chicken embryo virus liquid was purified by ultracentrifugation to obtain DAd V-3 whole virus at a concentration of 8.0 mg/m L,and rabbit anti-DAd V-3 polyantiserum was prepared and an ELISA method was established.Chicken embryovirus was used as antigen and rabbit serum as positive antibody to optimize the reaction conditions of ELISA method and perform repeatability and specificity detection.The results showed that the intra-batch repeatability coefficient of variation of the established ELISA method was less than 10%.At the same time,the detection rate of antibodies in Muscovy duck was 100%,which indicated that the method had good specificity,reproducibility and sensitivity.In summary,a highly pathogenic DAd V-3 strain was first isolated.The virus can infect multiple organs in ducks and cause pathological changes such as pericardial effusion,liver bleeding,piebald kidney and enteritis.Secondly,this study successfully proliferated the virus using the method of chicken embryo culture.Finally,a DAd V-3 indirect ELISA detection method is preliminarily established.These studies provide a theoretical basis for further pathogenesis research,vaccine preparation and clinical detection of DAd V-3.