| Infectious bursal disease（IBD）is an acute immunosuppressive disease caused by infectious bursal disease virus（IBDV）.The incidence rate of this disease is high and its potential spread is wide.It is easy to cause great economic losses to poultry breeding industry.When the virus infects the host,it can realize immune escape by using the membrane cycle between plasma membrane and endosome to disguise itself as a part of the host itself.Membrane circulation usually involves vesicle transport and membrane fusion.ADP ribosylation factor 6（ARF6）is a GTPase that can regulate endocytosis and vesicle transport,and has been proved to play different roles in different virus infections.The study of IBDV found that the multifunctional VP3 protein has the ability to bind to the endosome and can replicate using the host membrane to escape the monitoring of host cells,but the effect of ARF6 on IBDV infection has not been reported;Interferon induced transmembrane protein 3（IFITM3）is an effector protein of the natural immune system.It has strong internal cellular resistance to the infection of a variety of envelope viruses.It prevents virus proliferation by mainly inhibiting the fusion of viral membrane and endosomal membrane by mediating the changes of physical properties of host endosome.IFITM3 has been proved to exist in avian cells and can resist influenza virus,but its role and mechanism against envelope free viruses such as IBDV are not clear.Therefore,this paper takes chicks as the research object to explore the effect of IBDV infection on ARF6 and the transcriptional expression of IFITM3,and tries to explore the regulatory mechanism of ARF6 and IFITM3 on IBDV infection and replication from the perspective of cell membrane by using DF-1 cells,which provides new ideas for the study of the pathogenic mechanism of IBDV.Therefore,this topic intends to study from the following aspects.1.Effect of IBDV infection on the expression of ARF6 in chicken bursa and DF-1 cells in this studyIn this study,IBDV infected chicks and chicken fibroblasts（DF-1）cells for in vivo and in vitro experiments,and samples at different times after infection were collected.The pathological changes of chicken bursa,the expression of ARF6 and the expression levels of Vp2,ARF6 and VP3 in DF-1 cells were detected by immunohistochemistry,quantitative real-time PCR（q RT-PCR）and Western blot（WB）.The results showed that:（1）Compared with the control group,IBDV-VP3 protein was expressed in the bursa of Fabricius at 3,5 and 7 days after infection,and the expression was the highest on the 3 days post infection（dpi）,and then decreased;（2）The expression of ARF6 was significantly up-regulated after IBDV infected bursa Fabricius（P<0.05）;（3）Compared with the control group,after IBDV infected DF-1 cells,the expression of ARF6 increased with the increase of Vp2,and there was a significant difference at 12 hours post infection（hpi）（P<0.05）and increased sharply at 24 hpi（P<0.001）.（4）The change of protein level of ARF6 is consistent with the trend of transcription level.2.Effect of ARF6 on IBDV replicationIn order to explore the effect of ARF6 on IBDV replication,ARF6 eukaryotic expression vector pEGFP-ARF6 and point mutants affecting ARF6 activity,pEGFP-ARF6-T27N and pEGFP-ARF6-Q67L,as well as the synthesis of specific small interfering RNA（si RNA）for ARF6 were constructed.The effects of changes in ARF6 expression level on IBDV-Vp2 gene and VP3 protein expression and IBDV titer were studied by q RT-PCR,WB,TCID50 and laser confocal technique,The results showed that:（1）overexpression of ARF6 could significantly promote the replication of IBDV:the expression of IBDV-Vp2gene and VP3 protein were significantly up-regulated with the increase of ARF6 expression（P<0.01）,and there was a dose-dependent relationship between ARF6 and IBDV;（2）Knockdown of ARF6 could significantly reduce the replication of IBDV:compared with the control group,after reducing the expression of endogenous ARF6,the expression of IBDV-Vp2 gene and VP3 protein decreased significantly（P<0.05）,and the titer of IBDV decreased significantly（P<0.01）;（3）Laser confocal microscopy showed that the activity enhanced ARF6-Q67L and wild-type ARF6 showed aggregation dot distribution in DF-1 cells,while the activity decreased ARF6-T27N showed diffuse distribution;（4）Compared with wild-type ARF6,ARF6-T27N could significantly reduce the expression of Vp2 and VP3（P<0.01）,and also significantly reduce the titer of IBDV（P<0.05）.3.The preliminary study on the mechanism of ARF6 promoting IBDV replicationAccording to the above results,ARF6 can promote the replication of IBDV,so we try to further explore the mechanism.Combined with the function of ARF6,the mechanism was explored by using laser confocal technology,q RT-PCR,WB and CCK-8 kit to detect the effect of compound pitstop 2 on cell viability.The results showed that:（1）ARF6 has co-localization with membrane associated organelle mitochondria（Mito）,endoplasmic reticulum（ER）and Golgi apparatus;（2）In the early infection stage of IBDV,compared with the control group（only infected with IBDV）,the expression level of Vp2 was significantly up-regulated after overexpression of ARF6（P<0.05）;After knockdown of ARF6,the expression of Vp2 decreased significantly（P<0.05）;（3）Using targeted clathrin inhibitor pitstop 2,the expression levels of Vp2 gene and VP3 protein were significantly down regulated（P<0.01）;（4）When ARF6 was overexpressed and treated with pitstop 2,the expression of IBDV-Vp2 gene and VP3 protein did not increase compared with the control group.4.Effect of IFITM3 on replication of IBDVIn this study,chickens and DF-1 cells were infected with IBDV,and overexpression and knockdown experiments were used.Samples at different times after infection were collected and the expression levels of Vp2 and Ifitm3 were detected by q RT-PCR.The results showed that:（1）after IBDV infected bursa Fabricius,the expression of Ifitm3 was significantly up-regulated at 24 h（P<0.05）and peaked at 72 h.Although it decreased later,it was still significantly higher than the normal level;（2）Compared with the control group,the expression of Ifitm3 in DF-1 cells infected with IBDV increased sharply at 24 h and was significantly higher than that in the control group（P<0.05）;（3）Overexpression of IFITM3 could significantly down-regulate the expression of Vp2;（4）Knockdown of IFITM3 significantly increased the expression level of Vp2（P<0.05）.The above results showed that IBDV infection can up-regulate the expression of ARF6 in vivo and in vitro;ARF6 can promote the replication of IBDV,and T27N site may play an important role in this process;In addition,ARF6 may promote the replication of IBDV by affecting the early entry process of IBDV into cells through clathrin.Adding clathrin inhibitor can effectively block this process.On the one hand,this study confirmed the effect of ARF6 on IBDV infection and preliminarily discussed its mechanism.On the other hand,the results of this study preliminarily show that IFITM3 can reduce the replication of IBDV. |
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