Preliminary Study On The Pathogenicity Of Duck Adenovirus Type 2 And The Characteristics Of Induced Cells To Produce Immune Factors

Aviandenovirus?Aviadenovirus?is an important pathogen of poultry,and the pathogenicity varies greatly between the strains.Duck Aviadenovirus type 2?Duck Aviadenovirus 2 type,DAd V-2?is a newly discovered duck adenovirus in recent years.In this study,a DAd V-2 strain was isolated from the clinic and studied from the following three aspects.First,the entire genome of the virus is amplified and its pathogenicity is identified.In this study,liver duck livers were collected from the Annong Poultry Disease Diagnostic Center,and SPF chicken embryos were inoculated through the allantoic cavity after grinding.The allantoic fluid and chicken embryo kidneys of dead chicken embryos after 7days were collected.The virus DNA was extracted and the virus specificity was amplified by PCR.The gene fragment was finally determined as DAd V-2 by sequencing and comparison.According to the reported DAd V-2 sequence,13 pairs of specific large fragment primers were designed to amplify the genome,splice the target sequence and analyze the viral gene sequence.The results showed that the viral genome contains43024bp,designated as the HF-AH-2020,the BLAST results show Guangdong strain have been reported and a GR homology to strains,respectively 98.6%and 89.4%.Isolates can kill chicken embryos in about 7 days.The main organs of sick and dead chicken embryos were extracted,and the absolute viral quantitative PCR was used to calculate the viral load.The results showed that the liver had the highest viral load,followed by the duodenum,spleen,kidney,glandular stomach,muscular stomach,and heart.The brain is the lowest.Determine the virus titer according to 10^3.24TCID₅₀/0.1m The amount of L per animal was inoculated with 15-day-old SPF chickens and muscovy ducks.The results showed that the isolate could not cause animal death,but the liver,heart and kidney of the inoculated animal could detect the presence of the virus,which shows The virus can replicate in animal organs but is less pathogenic.Second,the mechanism by which the isolates induce cells to produce interferon.After the purified isolates infected primary kidney cells for 5-7 days,the total RNA of the cells was extracted and reverse transcribed.The relative fluorescence quantitative PCR was used to detect the type I interferon IFN-?and type II interferon IFN-?its related pathway genes.It was found that after infection with the virus,the expression of the genes related to the STING pathway MDA5,STING,MAVS and IRF7 could be induced,resulting in a significant increase in the expression of the IFN-?gene.At the same time,it can induce the expression of IFN-?gene mediated by NF-k B.This shows that the infection of the virus can cause a great increase in IFN,thereby helping the animal body to resist the infection of the virus.Finally,the intracellular localization analysis of the main structural proteins of the virus.Recombinant plasmids pm Cherry-C1-Hexon and pm Cherry-C1-Fiber2 containing viral Hexon and Fiber2 gene fragments were constructed and transfected into eukaryotic cells 293T,and the protein localization was confirmed by laser confocal technology.The results show that the proteins expressed by Hexon and Fiber2 are located in the nucleus and cytoplasm,respectively.