Study On The Role Of Interferon Regulatory Factor 7(IRF7) Gene In Anti-Chicken ALV-J

Subgroup J Avian Leukosis(ALV-J),as a neoplastic infectious disease,has caused huge economic losses to the poultry industry.The research on genes related to ALV-J resistance is the basis and key to research on disease resistance at the genetic level.This study was based on the differential expression data of chicken AVL-J before and after infection using transcriptome sequencing technology.Through the joint analysis and host-virus interaction analysis,six possible disease resistance genes were obtained,which interferon the expression difference of regulatory factor 7(IRF7)was the largest.Therefore,this study used IRF7 as a target gene to explore its role in chicken anti-ALV-J process from the following aspects.First,we tested the expression changes of IRF7 and its related genes in the body resist ALV-J through ALV-J challenge test,and assisted tissue section HE staining to observe the morphological and pathological changes of immune tissues before and after ALV-J infection.Then,using chicken embryo fibroblasts as a cell model,the expression of IRF7 in cell ALV-J infection and polyI:C transfection was detected,and combined with interference and overexpression technology,to explore the molecular mechanism of IRF7 in resisting ALV-J immune process at the cellular level.Through the above series of experiments,we have the following results:(1)After SPF chickens were treated with ALV-J,HE staining was performed on three immunological tissues of thymus,liver and bursa of 15,24 and 40 days old.The results showed that there were significant differences in morphology between the three immune tissues before and after infection with ALV-J.Among them,there were obvious pathological differences in the thymus and bursa of 15 d with slight fatty infiltration accompanied by a small amount of congestion.There was no significant difference in liver tissue;there were obvious pathological changes in the thymus,liver and bursa of 24 d and 40d: the thymus contained a lot of adipose tissue,and it was obviously loose and swollen,and the medulla area also showed an expanded state.The arrangement of hepatocytes is disordered,the fat infusion is relatively more,and some of the cells in the hepatocytes are pyknosis and apoptosis.The bursa of Fabricius is infiltrated by a large amount of fat,forming a vacuole-like shape,the structure of the capsule is disordered,and the nodular tissue is loose and swollen.The above results indicate that the individual challenge test was successful.(2)Real-time PCR was used to detect the expression of IRF7 gene in the spleen,thymus,liver and bursa of the 19 th,24th and 40 th days.The results showed that the expression of IRF7 gene in the ALV-J group was significantly higher than that in the control group(P<0.05),and the related genes IFN-α,IFN-β,IRF1,STAT1,MyD88,TLR3 and TLR7 in different immune tissues.There is an increasing trend in both,indicating that IRF7 and its related genes are involved in the body’s anti-ALV-J response.(3)The chicken embryo fibroblasts(CEFs)were used as the cell model for ALV-J infection and polyI:C transfection experiments.It was found that the expression of IRF7 gene showed a trend of increasing first and then increasing after ALV-J challenge.The expression of IRF7 gene was higher than that of the control group(0h).After the challenge,the related genes showed a trend of increasing first and then decreasing,and the expression levels were higher than 0h.After transfection of polyI:C,IRF7 and its related genes all showed a trend of increasing first and then decreasing.Western-blot results showed that the abundance of IRF7 protein increased first and then decreased after ALV-J and polyI:C treatment at 0h,24 h,48h and 72 h,and reached the highest level at 48 h.From the above experiments,IRF7 and its related genes are involved in the anti-ALV-J reaction after ALV-J infection on fibroblasts.(4)IRF7 interference assay was performed on chicken embryo fibroblasts.Compared with the control group,the expression of IRF7 gene was significantly decreased at 24 h and 48 h after interference(P<0.05),IRF1,IFN-β,IFN-α,The expression level of the STAT1 gene was down-regulated relative to the control group,while MyD88,TLR3,and TLR7 were up-regulated compared to the control group.Subsequently,the overexpression test of IRF7 was carried out.Compared with the control group,the expression of IRF7 gene was significantly increased at 24 h and 48 h after overexpression(P<0.01),IRF1,IFN-β,IFN-α,STAT1.MyD88 and TLR3 are up-regulated and TLR7 is down-regμLated.The results of this experiment revealed that IRF7 has a positive regulatory effect on IRF1,IFN-β,IFN-α,and STAT1.(5)Plasmid DNA of IRF7 was over-expressed in chicken embryo fibroblasts.After 24 h,partial samples were collected for Real-time PCR to ensure successful overexpression,followed by ALV-J and polyI:C treatment for 24 h.In the ALV-J challenge group and the non-ALV-J challenge group,the expression level of IRF7 gene in the overexpression group was significantly higher than that in the negative control group(NC)(P<0.01);In the polyI:C transfection group and the non-polyI:C transfected cells,the expression of IRF7 gene was significantly increased in the overexpressing group compared with NC(P<0.05);ALV-J after IRF7 overexpression Treatment,related genes were up-regulated;IRF7 over-expression was treated with polyI:C,and IFN-α,IFN-β,IRF1,STAT1 and TLR3 were up-regulated.The above results indicate that IRF7 can positively promote the anti-ALV-J pathway.In summary,IRF7 and its related genes are involved in the anti-ALV-J response,and IRF7 can participate in anti-ALV-J by positively promoting Toll-like receptor signaling pathway.In addition,IRF7 has a positive regulatory effect on IRF1,IFN-β,IFN-α,and STAT1.