Preliminary Study On The Role Of Three Kinds Of Zinc Finger Protein In NDV And AIV Infection

Innate antiviral factors play an important role in the immune response of antiviral infection,which is a hot issue in antiviral immunity.In recent years,it has been shown that zinc finger proteins are innate antiviral factors and play an important role in host immune system.Zinc finger proteins are involved in the regulation of cytokines expression(such as interleukin and interferon),and directly regulate the growth and differentiation of lymphocytes.Zinc finger proteins are very important in host innate immune responses as well.Nevertheless,little is known about avian zinc finger proteins and their functions.The study on chicken zinc finger proteins,especially antiviral zinc finger proteins,would be helpful to the development of new drugs and estalbishment of novel poultry disease prevention and control methods.As the experiments were carried out as follows:1)The expression of chicken Zinc Finger Proteins during NDV infectionIn previous study,the expression profile of zinc finger proteins in NDF-infected CEF cells was obtained by high-throughput sequencing.On this basis,this study screened a variety of post-viral infection in the expression of significant changes in the zinc finger protein for follow-up study.The RT-qPCR detection primers of zinc finger protein were first designed by using the zinc finger protein gene sequence predicted by GenBank.The changes of zinc finger protein expression in DF-1 cells were detected.The results showed that chZC3HAV1,chZC3H6 and chZEB1 were down-regulated after virus infection and were significantly up-regulated at the later stage,indicating that these three zinc finger proteins may be involved in the virus infection process.Subsequently,we amplified the complete gene sequences of the three zinc finger proteins from the cDNA samples of CEF cells and performed bioinformatics analysis.Chicken chZC3HAV1,chZC3H6 and other poultry homologous gene homologous close,followed by reptiles,and mammals have the lowest affinity,while the chicken chZEB1 protein in evolution with other poultry mammals,reptiles Source is very high,which shows that ZEB1 in evolution is very conservative.The results showed that the ZC3HAV1,ZC3H6 and ZEB1 proteins had similar functional domains to human proteins,and their zinc finger structures were relatively conserved in evolution,suggesting that chicken and human zinc finger proteins had similar functions.2)Preliminary identification of antiviral function of chicken zinc finger protein chZC3HAV1,chZC3H6 and chZEB1.In order to confirm whether chZC3HAV1,chZC3H6 and chZEBl were involved in the anti-infective immune response process of host cells after virus infection,the antiviral function of the above three zinc finger proteins was identified.At first,we optimized the primers and systems of qPCR,and finally obtained RT-qPCR detection method with high sensitivity and good specificity.Quantitative detection of zinc finger proteins in various organs,Spleen and bursa of Fabricius are relatively high.The results of viral infection showed that the expression of chZC3HAV1 and chZEB1 increased significantly in the late stage of NDV infection.AIV infection can potently stimulate the up-regulation of chZC3HAV1.Overexpression of chZEB1 showed that chZEB1 could not inhibit the proliferation of NDV,indicating that chZEB1 may not be directly involved in the immune response,it may be involved in the specific immune response process.Overexpression of chZC3HAV1 found that chZC3HAV1 on NDV proliferation inhibition is not strong,but it can limit the proliferation of AIV.3)chZC3HAV1 inhibits the mechanism of virus proliferation.So we did a further study.First,by overexpressing chZC3HAV1,we found that chZC3HAV1 has a strong anti-AIV effect and is't resistant to NDV.By comparing the expression of chZC3HAV1 at different time points after AIV and NDV infection,we found that NDV could not induce chZC3HAV1 gene uptake at the early stage of infection,and the expression of chZC3HAV1 was up-regulated during the whole AIV infection.Follow-up experiments confirmed this phenomenon,early NDV infection,chicken tissue organs chZC3HAV1 expression did not appear significantly increased.Since chZC3HAV1 is an early antiviral factor,We speculated that the hysteresis of chZC3HAV1 expression after NDV infection was one of the factors that could not effectively exert anti-NDV effect.This study further studied the antiviral mechanism of chZC3HAV1.By detecting the activation of NF-?B,IRF-3/7,IFN-P and other signaling pathways in chZC3HAV1 overexpression,we found that chZC3HAV1 did not act as an enhancement of IFN signaling,Indirect proof that ZC3HAV1 does not via interacting with RIG-I to enhance the antiviral effect of interferon signaling.In chZC3HAV1 overexpressing intrancellular and supernatant,the expression of viral RNA was much lower than that of the control group,suggesting that chZC3HAV1 may be a direct recognition of viral RNA and recruiting immune factors to degrade viral RNA to play antiviral effects.This inference needs to be demonstrated by subsequent experiments.