Preliminary Study On Pathogenicity Of Isolate FAdV-4 And The Characteristics Of Inducing Cells To Produce Immunons

Group I fowl adenovirus group I?FAdV-I?is an important pathogen of birds,and is divided into five subgroups of A,B,C,D and E.Among them,adenovirus type 4?FAdV-4?It is the serotype with the highest morbidity and mortality in the clinic.In this study,FAdV-4 was isolated and identified from the clinic and its pathogenicity,cellular immune mechanism and major structural proteins were analyzed.First,isolate the FAdV-4 strain and identify the pathogenicity of the virus.Collect the diseased liver of suspected adenovirus chickens in clinic,inoculate SPF chicken embryos,collect allantoic fluid and kidneys of diseased chicken embryos and extract the DNA of the virus,perform PCR amplification with avian adenovirus-specific primers,and the products are further sequenced and Comparison analysis.The results showed that the isolate was group I subgroup C subtype 4 avian adenovirus,named AH-FAdV-4.The TCID50 of this strain was determined to be 1?10^6.52 TCID₅₀/m L.15-day-old SPF chickens and 15-day-old muscovy ducks were inoculated,and it was found that the inoculated SPF chickens died within 3 days and the typical pericardial effusion lesions were observed on necropsy,but the ducks did not die and the typical lesions were not on necropsy.The results indicate that the AH-FAdV-4 isolated this time is a virulent strain,which has a very high lethal rate to chicks but no obvious pathogenicity to ducks.Second,research on the production of interferon by AH-FAdV-4.Isolate and culture 16-day-old SPF chicken embryo primary kidney cells,infect the primary kidney cells with 1MOI?multiplicity of infection?,5 days later,extract the RNA of the cells and perform reverse transcription,apply real-time fluorescence PCR?RT-PCR?method to detect signaling pathway regulatory molecules?STING and NF-?B?,cytokines?IFN-?,IFN-?,IL-1??,related membrane receptors?MDA5,NLRP3?,partial antigen presentation Molecular?Ii,MHC Ia,MHC II??and signal protein?MAVS?gene expression,it was found that AH-FAdV-4 can activate the STING,NF-?B pathway,can increase the transcription of IFN-?,IFN-?genes level,which indicates that the infected cells promote the expression of interferon by regulating the SRING and NF-?B signaling pathways to resist viral invasion;meanwhile,the inhibitor dithiocarbamate peptide pyrrolidine?PDTC?is used to inhibit NF-?B After activation,it was found that after the virus infected the cells,inhibition of NF-?B expression could reduce the expression of antigen presenting molecules Ii,MHC Ia,MHC II?,and NLRP3.This shows that the virus regulates the expression of antigen-presenting molecules by regulating NF-?B and indirectly regulates the cellular immune response mediated by T cells.Finally,the main structural protein Fiber2 was analyzed for cell localization and protein expression.Third,the analysis of cell localization and protein expression of the main structural protein Fiber2 of the virus.Construction of eukaryotic recombinant plasmids containing AH-FAdV-4-Fiber2 gene pm Cherry-C1-Fiber2,pm Cherry-N1-Fiber2,transfected 293T cells using laser confocal microscope to observe its localization in the cell,the results show that Fiber2is mainly located in the nucleus.The prokaryotic recombinant expression plasmid p ET-32a-Fiber2 of the Fiber2 gene was constructed and the protein was expressed and purified.The results of SDS-PAGE and Western Blot identification showed that a more pure Fiber2 protein was obtained.This is the AH-FAdV-4 subunit vaccine.Preparation laid the foundation.