Study On The Responses Of Toll-like Receptors And Interferon Regulatory Factors To IBDV Infection

Infectious bursal disease(IBD)is an immunosuppressive disease caused by infectious bursal disease virus(IBDV).Since its discovery,it has been one of the main causes of economic losses in poultry industry.Innate immune response is the first line of defense against virus infection,which mainly recognizes viral pathogen-related molecular patterns(PAMP)through pattern recognition receptors(PRR),inducing a series of signal cascade reactions,resulting in a variety of cytokines such as interferon(IFN).Interleukin(IL)and tumor necrosis factor(TNF)mediate the antiviral effect of the body.Toll-like receptor(TLR)is one of the pattern recognition receptors.TLR activation induces interferon production after pathogen infection.Interferon regulatory factor(IRF)is a kind of transcription factor that regulates interferon expression,and its primary function is to induce interferon production.At present,there are many studies on TLRs and IRFs in mammals,but the expression changes of chicken TLRs and IRFs on pathogen infection have not been systematically reported.Therefore,this thesis takes chicken as the research object to study TLRs and its downstream signal pathway IRFs,and based on the establishment of chicken-infectious bursal disease virus infection model,to study the changes of host TLRs and IRFs expression caused by infectious bursal disease virus from a molecular point of view,so as to further deepen our understanding of the immune regulatory network of chicken TLRs and IRFs.Then we explored the subcellular localization of different cut chicken IRF4 genes in DF-1 cells and analyzed their preliminarily functions.Chapter 1: Effects of IBDV infection on expression of ch TLRs in chicken bursaIn the present study,layer chickens were infected with IBDV and the expression of chicken TLRs(ch TLRs)was assayed by quantitative real-time PCR(q RT-PCR).The results showed that the expression of ch TLR1 a,1b,2a,3,4 and 15 was upregulated in the bursa of chickens infected with IBDV compared with noninfected chickens,while ch TLR2 b,5,7 and 21 expression was downregulated.Correlation analysis showed that ch TLR3 expressions was directly associated with IBDV VP2 m RNA expression in bursa.These results suggested that different TLRs have different responses to the same viral infection.Some TLRs were activated early on,some later,and some were suppressed.This is the first study to report on the response of all ch TLRs to one virus.This provids a valuable overview of the expression pattern of ch TLRs when chickens are challenged by pathogens.Chapter 2: Tissue distribution and developmental changes of interferon regulatory factors in chickens and effects of infectious bursal disease virus infectionIn the present study,q RT-PCR was used to study the tissue distribution of IRFs in chickens at D15(the 15 th day of raising)and developmental changes of all chicken interferon regulatory factors(ch IRFs)in BF from E15(the 15 th day of incubation)to D15.The effects of IBDV infection with chickens on the transcriptional level of ch IRFs were also investigated.The results showed:(1)Ch IRF1 m RNA was expressed much more abundantly in intestinal tract,ch IRF2,ch IRF6,ch IRF7,ch IRF8 and ch IRF10 distributed mainly in liver or/and kidney.The expression of ch IRF5 was mainly in spleen and ch IRF4 distributed uniquely abundantly in BF.(2)The m RNA expression levels of ch IRF5,ch IRF7,ch IRF8 and ch IRF10 was low before hatching of chicken and at D1 and increased significantly from D5 till to the experiment end and the fold change of ch IRF5 at D10 and ch IRF7 at D5 reached 41.0-fold and 15.7-fold compared to that of E15,respectively(P < 0.05).Ch IRF4 m RNA level was always high during the whole experiment except for E15 and it was 11.9-fold at the highest time point than that of E15(the lowest time point).(3)When chicken was infected with IBDV,the expression levels of ch IRF2,ch IRF7 and ch IRF10 m RNA had the tendency of increasing first and then decreasing but they peaked at 1 dpi,2 dpi,and 3 dpi,respectively.The expression of ch IRF5 m RNA was suppressed obviously during the whole experiment stage in IBDV-infected chicken.And ch IRF4 expression was up-regulated transitorily at 1 dpi and then was suppressed on a very low level till to the experiment end.Conclusion: The ch IRFs were constitutively expressed in different tissues examined and has tissue-specific expression.Of them,ch IRF2,ch IRF4,ch IRF5,ch IRF7,ch IRF8 and ch IRF10 were related closely with the development or immune response of BF,and when chicken was infected with IBDV,some of them were activated,earlier or later on,some of them were suppressed.These findings would help to sieve out a few antiviral ch IRF candidate gene to improve the host’s innate immune.Chapter 3: Cloning and subcellular localization of the full coding region of IRF4 gene in different chicken breedsThe complete coding region of IRF4 gene of AA broiler,817 broiler,white leghorn laying hen and Hyline brown laying hen was cloned by RT-PCR technique.By comparing with the original amino acid sequence of Gen Bank,we found three different splicing changes,which were consistent with the original amino acid sequence,missing one amino acid and missing 36 amino acids respectively.In order to further explore the function of different splicing IRF4 genes,we constructed the corresponding eukaryotic expression vector and observed the subcellular localization by transfection into DF-1 cells.The results showed that p EGFP-N1-IRF4 and p EGFP-N1-IRF4(△36)eukaryotic expression vectors were located in the nucleus,and p EGFP-N1-IRF4(△1)was distributed in both cytoplasm and nucleus.The subcellular localization of the three splices did not change after poly(I:C)stimulation.These results lay a foundation for further exploration of the functions of IRF4.