Effects Of Different Pathotype Ibv Infections On Tlr3 And Tlr7 Signaling Pathways In Chickens And Influence Of Isg On Ibv Replication

Avian infectious bronchitis virus(IBV)is a Gammacoronavirus in the family Coronaviridae,and causes infectious bronchitis(IB),which is an acute and highly contagious respiratory disease in chickens.The genome of IBV is easily mutated,resulting in diversification of virus serotype and pathogenicity,Furthermore,the cross protection between different serotypes is limited,which brings great economic losses to the poultry industry.Therefore,it is extremely important to prevent and control effectively the disease.Pathogenesis and immune mechanism of IBV infection is one of the main research directions of IB prevention and control.The innate immune response is the first line of defense against viral infections and plays an important role in host defense against IBV.Studies have shown that the regulation of coronavirus on innate immune responses was diverse and complex,leading to the differences in pathogenicity and immunity of coronaviruses.The pathogenicity of different IBV strains to different tissues may be related to the innate immune response caused by the virus.Therefore,it is of great significance to study the innate immune response of different target tissues caused by different pathogenic IBV infection.There are a variety of clinical pathogenic types caused by IBV strains,of which respiratory and nephropathic types are the two major pathogenic types.In this study,two different serotypes of respiratory and renal IBV strains were used to infect SPF chickens and the research on TLR3 and TLR7 signaling pathways in trachea and kidney of infected chicken were conducted.The effects of these two IBV infections on the expression of nuclear transcription factors IRF7 and NF-?B p65 were also studied,and on this basis,the effects of Viperin and IFIT5 on IBV replication were studied by overexpression or knockdown.1.Effects of IBV infection on TLR3 and TLR7 signaling pathway in the trachea and kidney of chickenIn this study,two different serotypes of IBV,the respiratory pathogenic M41 strain and nephropathogenic GX-YL5 strain were used to infect SPF chickens respectively.The pathological changes of tracheal mucosa and kidney tissues were observed after infection.The viral RNA and mRNA of key molecules in TLR3 and TLR7 signaling pathways,inflammatory cytokines,type IIFN and interferon stimulated genes(ISG),as well as the changes of TLR3 and cytokine protein were detected.The results showed that tissue lesions appeared in M41 and GX-YL5 infected tracheal mucosa at 1-11 days post infection(dpi),and the viral load increased rapidly.Mild tissue lesions and low viral loads appeared in M41 infected kidney,while GX-YL5 infected kidney showed severe nephritis and had high viral loads.The mRNA of key molecules in TLR3 and TLR7 signaling pathways were significantly up-regulated in tracheal mucosa at 1-21 dpi,while most of these molecules were not significantly changed in kidney.TLR3 protein was expressed and distributed in these two tissues.The mRNA and protein expression of IL-1? and IL-6 were significantly up-regulated in M41 infected tracheal mucosa and GX-YL5 infected tracheal mucosa and kidneys.The mRNA and protein expression of IFN-a was up-regulated in tracheal mucosa and down-regulated in kidney after infection of these two IBV strains.IFN-? expression was mainly up-regulated in the M41 infected tracheal mucosa and GX-YL5 infected kidney.ISG were significantly up-regulated in both tissues infected with two IBV strains.These results suggested that the different pathological changes of the tracheal mucosa and kidney caused by M41 and GX-YL5 strain infection were consistent with viral loads.There were tissue differences in TLR3 and TLR7 signaling pathway responses,and expressions of IL-1p and IL-6 were associated with inflammatory damage in tissues.There were tissue differences in IFN-a expression,and there were tissue and strain differences in IFN-? expression.ISG were up-regulated to exert an antiviral effect after IBV infection.The effects of different IBV strains infection on TLR3 and TLR7 signaling pathways in target tissues were analyzed in this study,providing a scientific basis for the innate immune response of different pathotype IBV strains2.Effects of IBV infection on the expression of nuclear transcription factors IRF7 and NF-?B p65Nuclear transcription factors are key signaling molecules in TLR and RLR signaling pathways of host innate immune and responsible for initiating transcription of type I IFN,pro-inflammatory cytokines and chemokines.In order to investigate the effects of IBV infection on nuclear transcription factors IRF7 and NF-?B p65,and provide a theoretical basis for the pathogenesis and immune mechanism of IBV.In this study,real time quantitative PCR and Western Blot were used to detect the expression of IRF7 and NF-?B p65 mRNA and protein in tracheal and kidney of SPF chickens infected with IBV M41 and GX-YL5 strains.The distribution of IRF7 and NF-?B p65 proteins in tracheal and kidney as well as N protein of IBV were observed by immunohistochemistry,The results showed that the mRNA and protein expressions of IRF7 in tracheal mucosa and kidney were significantly up-regulated and increased at 3-8 dpi after M41 strain infection and at 1-21 dpi after GX-YL5 strain infection.The mRNA and protein expressions of NF-?B p65 were significantly up-regulated and increased in tracheal mucosa at early stage of M41 strain infection and at 1-3 dpi after GX-YL5 strain infection.However,the mRNA of NF-?B p65 in kidney was significantly down-regulated or no change after two IBV strains infection,and NF-?B p65 protein was undetected by Western Blot.The results of immunohistochemistry showed that the expressions of IRF7 and NF-?B p65 proteins in tracheal mucosa and kidney tissues were increased significantly after IBV infection,which were consistent with the changes of viral loads and N protein.At the peak of viral replication,IRF7 and NF-?B p65 were transferred to the nucleus.Therefore,these results suggested that both M41 and GX-YL5 strain infection activated the IRF7 and NF-?B p65 signaling,and IRF7 and NF-?B p65 played important roles in anti-IBV innate immune responses3.Effects of viperin and IFIT5 on replication of IBV in vitroIn chapter 2,it was found that IBV infection activated the innate immune TLR3 and TLR7 signaling pathway in the tracheal mucosa and kidney of SPF chickens,and significantly up-regulated the expression of type I IFN and induced many ISG including viperin and IFIT5.On this basis,this section we carried out the effects of viperin and IFIT5 on the replication of IBV in vitro Firstly,the dynamic expression of viperin and IFIT5 mRNA in CEK and DF1 cells infected with IBV was analyzed.Secondly,IBV M41 and GX-YL5 strains were used to infect the DF1 cells of overexpression or knockdown of chicken viperin and IFIT5,which were get by transient expression of proteins or shRNA lentiviral infection to interfere with mRNA expression respectively.Finally,the effects of viperin and IFIT5 protein on IBV replication in DF1 cell was assessed based on the changes of viral RNA load and N protein expression after IBV infection.The results showed that the mRNA of viperin and IFIT5 in CEK and DF1 cells were significantly up-regulated at 6-72 h after infection with M41 and GX-YL5 strains.The peak of mRNA expression was consistent with the peak of viral RNA.Overexpression of viperin and IFIT5 protein in DF1 cells could significantly reduce the RNA and N protein of M41 strain;overexpression of IFIT5 protein in DF1 cells only could significantly reduce N protein of GX-YL5 strain,but failed to reduce RNA load of GX-YL5 strain;overexpression of Viperin protein in DF1 cells had no significant effect on the RNA load and N protein of GX-YL5 strain.Knockdown of Viperin expression promoted both replication of IBV RNA and expression of N protein,knockdown of chicken IFIT5 expression only could significantly promote N protein expression,but did not affect replication of viral RNA.The results suggested that chicken viperin and IFIT5 were involved in the innate immune response induced by IBV infection and had the function of resisting IBV replication in vitroIn summary,the present study demonstrated that both the infection of two IBV strains,the respiratory pathogenic strain M41 and nephropathogenic strain GX-YL5 could activate TLR3 and TLR7 signaling pathways in trachea and kidney,and activate nuclear transcription factors IRF7 and NF-?B p65 to induce the expressions of proinflammatory and anti-inflammatory cytokines,type IIFN and ISG,indicating that the innate immune response play an important antiviral role in the early stage of IBV infection.However,there were differences in innate immune response between different strains and different tissues.Chicken viperin could inhibit the RNA replication and protein expression of M41 strain at the same time,and chicken IFIT5 could inhibit the expression of IBV protein to play a role in anti-IBV replication.This study provides a theoretical basis for the study of pathogenesis and the mechanism of immunoresponse of IBV,and unique insights into the response and regulation of innate immunity after infection with different pathotype IBV strains,and that might provide some useful reference for the development of anti-IBV drugs.