The Role Of EVs In EV71 Infection Of Central Nervous System

Objective: To investigate the functions of EVs in the EV71 infection,the transport mechanism of EVs at blood-brain barrier and the role of EVs in the CNS infection.Methods:(1)EVs were separated from EV71 infected RD cells culture supernatant by density gradient centrifugation,and then identified by TEM,NTA and Western-blot.(2)Blood-brain barrier model in vitro was established by bEnd3 cells and astrocytes,the permeability of BBB was detected by HRP and FLU.EVs and EV71 were incubating with blood-brain barrier model respectively to compare the efficiency of crossing the blood-brain barrier by using qRT-PCR to detect the level of EV71 RNA.(3)The colocalization of EVs with caveolin-mediated transcytosis maker Caveolin-1,macropinocytosis maker FITC-Dextran and clathrin-mediated transcytosis marker FITC-Transferrin was detected by IF respectively.FCM was used to detect the change in transcytosis of bEnd3 cells respectively after inhibition of clathrin-mediated transcytosis by Chlorpromazine,inhibition of caveolin-mediated transcytosis by Nystatin and inhibition of macropinocytosis by Amiloride.(4)The expression level of PI3 K was detected by ELISA respectively after EVs and EV71 crossed the blood-brain barrier model.After LY294002 inhibited PI3K-AKT signal pathway in the BBB model,the change in transcytosis of bEnd3 cells was detected by FCM and the tight junction protein claudin5,occludin and ZO-1 was detected by IF.(5)EVs and EV71 were inoculated respectively in one-day-old ICR mice by intraperitoneal injection.The mice model was assessed by the level of viral RNA replication in brain and gut which detected by qRT-PCR,the degree of brain lesions analyzed by pathological examination,and the survival curves.The expression level of PI3 K in brain was detected by ELISA after EVs infected mice model,and Z0-1 and GFAP in brain were detected by Western-blot and IF.(6)qRT-PCR was used to detect viral RNA in the EVs of patients’ plasma and stools.Results:(1)EVs secreted from EV71-infected RD cells contained viral RNA and could infect other cells.The TEM images showed a larger number of EVs without viral particles and a small number of EVs with viral particles.(2)The results of function tests showed the BBB model in vitro was established.At the same level of EV71 RNA,EVs crossed the blood-brain barrier model more efficiently than EV71(P<0.001).(3)The colocalization of EVs with Caveolin-1,FITC-Dextran and FITC-Transferrin indicated that EVs could cross the BBB by caveolin-mediated transcytosis,macropinocytosis and clathrin-mediated transcytosis.Chlorpromazine,Amiloride and Nystatin could inhibited EVs crossing the blood-brain barrier model,but Amiloride was most effective.The results showed that EVs could cross the blood-brain barrier model by macropinocytosis mainly.(4)The expression level of PI3 K in the BBB model was down-regulated(P<0.001)after EVs had crossed the BBB model and then infected astrocytes.There was no change in transcytosis of bEnd3 cells,but the tight junction of bEnd3 cells was broken down after LY294002 had inhibited PI3 K signal pathway in the BBB model.(5)In mice model,the weight of mice in EVs infected group was lighter and the level of EV71 RNA replication in brain and gut was higher than EV71 infected group.In EVs infected group,the brain lesions were more serious,the morbidity and mortality were higher.The expression level of PI3K(P<0.001)and GFAP in brain was downregulated,and the tight junction was broken down after EVs had infected mice model.(6)Viral RNA could be detected in the EVs of severe patients’ plasma.Conclusion:(1)Viral RNA could be detected in the EVs of EV71-infected cells and the EVs were infectious,a some number of EVs may clock intact EV71 virions.(2)EVs could cross the BBB model by transcytosis and mancropinocytosis was the major way.Astrocytes were infected and then PI3 K signal pathway was inhibited resulting in the breakdown of tight junction after EVs had crossed the blood-brain barrier.(3)The dectection of EVs infected mice model and clinical samples showed the characteristics of EVs crossing the BBB model may be related to CNS infection.