Isolation And Identification Of Chicken Infectious Anemia Virus In Dandong Area And The Effect Of VP3 Protein On Apoptosis

Chicken infectious anemia(Chicken infectious anemia CIA)commonly known as blue wing disease,is caused by chicken anemia virus(Chicken anemia virus,CAV)caused by chicken anemia and immunosuppression as the main feature of infectious diseases.The disease was first discovered in Japan in 1979,and now has all over the world of the country has chicken,is an important infectious disease against the poultry industry.Chicken is the only host of CAV.The typical pathological changes of chicks after infection with CAV are:anemia,bone marrow yellowing,thymus atrophy and other histological lesions.In this study,CAV was isolated and identified by CAV infection in four commercial broiler farms in Dandong area of Liaoning province by means of clinical observation,pathological autopsy,PCR amplification of gene fragments,chicken embryo inoculation and animal regression test.The Results:One CAV was successfully isolated and named DD-2016.The full length of DD-2016 gene was 2297bp,encoding 764 amino acids.The DD-2016 and F507715.1 South kores showed the highest homology with the F57715.1South kores,which was 99.17%,compared with AF313470.1USA had the lowest homology of 95.59%.The isolate could cause typical pathologic damage such as anemia,subcutaneous hemorrhage,thymus atrophy and bone marrow.Compared with the reference strain CUX-1,the isolates DD-2016 showed that the base sequence of the VP3 gene of DD-2016 had two base mutations,and resulted in the corresponding 116th and 118th positions The amino acid of the point changes.In order to elucidate whether the mutations of these two sites could affect their pro-apoptotic effect,the 5-day-old SPF chicken embryos were infected with DD-2016,and the genomic DNA of chicken embryos was infected at 19 days,And the VP3 gene was amplified and cloned into the prokaryotic vector pET-32a.Then,the MDCC-MSB1 cells were transfected into Marek's tumor cells and detected by flow cytometry.The apoptotic effect of VP3 protein was studied.Results:The prokaryotic expression vector pET-32a-VP3 of DD-2016 VP3 gene was successfully constructed.The expression of CAV VP3 protein could significantly promote the apoptosis of MDCC-MSB1 cells:84.5%of the cells in normal growth state 9.1%,1.1%of apoptotic cells and 5.3%in apoptotic cells.The cells in the experimental group accounted for 24%of the cells,1%of the dead cells,5.2%of the early cells and 69.7%of the late apoptotic cells.