Preliminary Study On The Role Of PI3k/Akt Signaling Pathway In MDV Infection

Marek ?s disease(MD)is a chicken neoplastic disease caused by the chicken’s Marek ?s disease virus(MDV),which can cause a chicken’s semi-paralysis or complete paralysis.With the immunization pressure of MDV vaccine,MDV has an increasing trend,and it has been reported that super virus strains can break through the immune protection of existing vaccine CVI988.Therefore,it is urgent to study the pathogenic mechanism of MDV to more effectively control MD.The cell contains a variety of signal transduction pathways,which play an important role in the regulation of biological activity of the organism.The PI3K/Akt signaling pathway is an important member of the signal transduction pathway and has a regulatory role in the infection of many viruses,but its research in MDV infection has not been reported.Therefore,to explore whether the PI3K/Akt signaling pathway can be activated during MDV infection and its role in the MDV infection process is of positive significance for further revealing the pathogenic mechanism of MDV.The virus used in this study was a Malicious strain Md5,which infects CEF cells in vitro,and to analyze the changes of PI3 K expression in different periods of infection from the gene expression level by using the fluorescence quantitative PCR method.The results showed that the expression of PI3 K in CEF cells increased after infection with MDV.Next,use the method of Western blot to detect Akt phosphorylation levels from protein expression levels.We found that phosphorylation of Akt was significantly elevated at 4 hpi(hours post-infection)until a high level of phosphorylated Akt was detected at 60 hpi.LY294002,a specific inhibitor of PI3 K,treats cells with it and then infects MDV,Western blot analysis revealed that Akt phosphorylation was significantly inhibited.The above results indicate that the PI3K/Akt signaling pathway is activated during MDV infection of CEF cells in vitro.In order to clarify the effect of PI3K/Akt signaling pathway on the proliferation of MDV,the CEF cells infected with MDV after LY294002 treatment were used as the post-inhibition virus group,and the virus titer was measured separately from the normal virus group without the use of inhibitors.The results showed that the virus titer of the post-infection group was significantly lower than that of the normal group at different time points after infection.In order to further verify this result,a real-time PCR detection method using Meq as a target gene was established in this study.It was found through detection that the level of Meq gene transcription was significantly lower in the post-infection group than in the normal group,in summary,the PI3K/Akt signaling pathway has a significant role in promoting the replication and proliferation of MDV.The function of the PI3K/Akt signaling pathway itself mainly depends on the regulation of the activities of many downstream factors.Therefore,this study examined the downstream related factors of the PI3K/Akt signaling pathway.First Western blot analysis of whether PI3K/Akt/GSK-3β pathway is activated by MDV infection,the results showed that phosphorylated GSK-3β was detected at different time points in MDV infection,and when PI3K/Akt signaling pathway was inhibited,the phosphorylation of GSK-3β protein expression was significantly downregulated,and it can be seen that MDV infection can activate PI3K/Akt/GSK-3β signaling pathway.In addition,we used fluorescence quantitative PCR technology to observe the changes of GSK-3β,mTOR,p53,and Bad expression levels of PI3K/Akt downstream key factors.The results showed that there was no significant difference in the expression of p53 and mTOR between the cells of the virus group after the inhibition and that of the normal virus-injected group.The expression levels of GSK-3β and Bad genes were significantly different,and showed significant up-regulation compared with the normal recipient group.Therefore,we hypothesize that GSK-3β and Bad genes may participate in the process of viral replication of MDV-infected CEF cells.The results of this study are conducive to revealing the interaction mechanism between virus infection and host cells,which is conducive to a deep understanding of MDV infection and pathogenic mechanisms,and provide new directions and ideas for the prevention and control of MD.