Study On The Autophagy Induced By Enterovirus 71 In Mouse Hippocampal Cells

Objective:Hand,foot,and mouth disease(HFMD)is a common acute infectious disease in children,mainly occurring in children under 5 years of age,and its pathogenesis is associated with enterovirus infection.Most of the common HFMD cases are self-limited,while severe cases are dangerous with rapid progress and high mortality rate.Enterovirus 71(EV71)is the main pathogen causing severe HFMD.It can lead to severe neurological complications such as viral meningitis,spinal cord gray matter syndrome and neurogenic pulmonary edema.The exact pathogenic mechanism remains unclear.Autophagy is an evolutionarily conserved and lysosome dependent catabolic pathway that has both physiological and pathological functions.Under normal circumstances,it can be used as a survival mechanism of cells to help clear microorganisms such as bacteria and viruses inside the cells,and play an antiviral natural immune effect.However,in some studies,it has been found that intracellular autophagy can be utilized by certain RNA viruses(such as poliovirus)to help them produce immune escape,thus leading to severe disease.As an RNA virus,whether EV71 can use autophagy pathway to produce immune escape during the occurrence of severe HFMD remain unclear.At present,there are studies on autophagy in neuroblastoma cells worldwide,but there are few reports on the occurrence of autophagy after the infection of normal nerve cells.In order to explore the key pathogenic mechanism of severe HFMD induced by EV71 infection,we took mouse hippocampal cells as normal cells to study the occurrence of autophagy after EV71 infection for the first time.Methods:In this study,we mainly infected mouse hippocampal cells with EV71 in vitro.At different time post infection,we detected the occurrence of autophagy and virus replication in the cells,and conducted a preliminary experimental study on the interaction between virus and autophagy.Specifically:1.Autophagy detection,firstly the formation of vacuoles in hippocampal cells of EV71 infected mice was observed by optical microscopy,and then the existence of autophagosomes was confirmed by transmission electron microscopy.Then,the expression of LC3-II protein,a marker of autophagy,was detected by flow cytometry and Western Blot,which confirmed the occurrence of autophagy.2.Virus replication detection,the expression of EV71 protein in infected cells was detected by Western Blot.The titer of EV71 in infected cells was confirmed by plaque assay,and virus replication was confirmed.3.Interaction between virus and autophagy,the interaction between EV71-RNA and autophagosomes in mouse hippocampal cells was detected by immunoelectron microscopy and living cell dynamic imaging techniques,respectively,to preliminarily verify whether the virus uses autophagy to produce immune escape.Results:24 hours after EV71 infection,the formation of intracellular vacuoles can be observed under optical microscope,a few autophagosomes were observed under transmission electron microscope.With the infection time extended to 48h,the vacuoles and autophagy cells increased gradually,the expression of LC3-II protein was detected by flow cytometry and Western blot,and the expression of LC3-II protein increased gradually with the prolongation of infection time.The expression of LC3-II protein was significantly decreased after the addition of autophagy inhibitor 3-methyladenine(3-MA).The results suggested that EV71 infection induced autophagy in mouse hippocampal cells.At the same time,we used Western Blot to detect the expression of EV71 protein in infected cells,and it was found that the expression of EV71 protein gradually increased with the extension of infection time.The results of virus plaque test showed that the expression of EV71 protein increased with the increase of infection time,and the expression of EV71 protein increased with the increase of infection time.The results of Virus plaque test also showed that the number of plaques increased gradually with time,that is,the titer of EV71 virus increased,suggesting that EV71 replication occurred in mouse hippocampal cells.After confirming autophagy and virus replication,a large number of EV71 protein wrapped by immunogold gold particles were found in the autophagy of EV71 infected cells by immunoelectron microscopy.The living cell dynamic imaging technique observed the co-localization of CTAB quantum dot labeled EV71-RNA and EGFP-LC3-labeled autophagosomes in mouse hippocampal cells,which preliminarily proved that there is an interaction between EV71-RNA and autophagosomes,and the virus may be largely locolized around the autophagosomes and not degraded.Conclusion:Based on the above experimental results,we believe that EV71 can not only replicate in the mouse hippocampus cells after infection,but also induce autophagy.In addition,it is speculated that EV71 may induce immune escape through autophagy pathway,leading to the occurrence of severe clinical phenotype.This study will provide a new research idea for exploring the key pathogenic mechanism of EV71 infection caused severe HFMD.