Study On The Mechanism Of EV71 Invading Blood-Spinal Cord Barrier In Tree Shrews

Enterovirus 71(EV71)belongs to the genus Enterovirus of the picornavirus family and is one of the main pathogens causing hand,foot and mouth disease in infants.In addition to causing symptoms of hand,foot and mouth disease,it also has neurophagy,a few severe cases of central nervous system complications such as aseptic meningitis,brainstem encephalitis,neurogenic pulmonary edema,and acute flaccid paralysis.However,the pathogenesis of EV71 is not completely clear.The path of how EV71 invades the nervous system and causes nerve damage is still unclear.Some studies suggest that EV71 virus may spread from blood to the central nervous system(Nervous System,CNS)through the blood-brain barrier(BBB).but there is no conclusive evidence.Based on the previous research,this study isolated the spinal cord microvascular endothelial cells by in vitro culture,explored the molecular characteristics of EV71-infected receptors and the permeability of the blood-spinal barrier structure,and combined the infection experiments of tree mites to explore the EV71 invasion nerve.A systematic mechanism that contributes to clinical critical care treatment and vaccine development.Firstly,the spinal cord microvascular endothelial cells(SCMECs)and spinal astrocytes(SCASTs)of tree shrews were isolated and purified in vitro,and their purity was identified by cellular immunofluorescence.Subsequently,the two cells were infected with EV71,and the corresponding antigen molecules of EV71 were detected by cell immunofluorescence.The viral load in the cells and culture supernatant was detected by real-time PCR and the virus proliferation curve was drawn.The main research was on EV71 infection of spinal cord microvascular endothelial cells.The characteristics of EV71-infected SCMECs-associated receptors and barrier-associated proteins were detected by real-time PCR by selecting multiple infection time points.Secondly,an in vivo infection experiment of tree shrews was carried out.A total of 16 tree shrews of 3 months old were selected,and 12 of the experimental group were challenged by intragastric and nasal injection to simulate the natural infection route.The viral load in the serum was measured every other day,and the body temperature and body weight were measured.In the control group,4 cells were administered with the same maintenance route.The viral load in the tissues of the tree shrews on the 7th and 20th day of the challenge was detected.The distribution of EV71 in the brain and spinal cord tissues was detected by immunohistochemistry,and the pathological examination was performed on each organ tissue.Meanwhile,the Evans blue dye was injected.After the brain and spinal cord were taken,frozen sections were made,and the change of barrier permeability was analyzed according to the fluorescence range.The results showed that the established separation and purification method could obtain two cells with higher purity and used in subsequent experiments.After EV71 infection of SCMECs and SCASTs,the cells showed obvious cytopathic effects.Cellular immunofluorescence could detect EV71 antigen molecules in both cells.The viral proliferation dynamics curve indicated that EV71 proliferated rapidly in both cells,and the load reached a peak(1×109 copies/mL)around 24 hours after infection,indicating that SCMECs and SCASTs can be infected by EV71 virus.qPCR was used to detect EV71-infected receptors and barrier-associated proteins in SCMECs.The results showed that SCMECs had the main receptors for EV71 infection:SCARB2,Anx2,and increased expression after infection;blood-spinal cord barrier-associated molecules ZO-1 and JAM-1 The expression level decreased significantly.Occludin,Claudin-1 and Claudin-5 showed a downward trend and then increased.MMP-9,MMP-2 and MMP-12 showed up-regulation after infection.MFSD2a showed up-regulation 12 h before infection.,there is a rapid downward adjustment after 12 hours.In vivo infection experiments in tree shrews showed symptoms of deteriorating heat and slowing(thinning)after challenge,and EV71 load was continuously detected in serum,peaking on day 7(1.31×105copies/mL);in heart,liver and spleen EV71 viral load was detected in lung,kidney,brain and spinal cord.The viral load in brain and spinal cord reached 4.98 × 105 copies/mL and 8.27× 105 copies/mL,respectively.It can still be detected in brain and spinal cord until infection for 20 days.Virus,immunohistochemistry also detected EV71 in the brain and spinal cord;pathological results showed that a certain degree of pathological abnormalities appeared in the organs of various tissues,especially subarachnoid vascular congestion,cerebral cortex hyperemia,neuronal vacuolar degeneration And spinal cord tissue vascular congestion;frozen section results also showed that the Evans blue in the brain and spinal cord of the challenge group diffused into the extravascular infiltration tissue,and the vascular permeability was significantly enhanced.These results indicated that the tree shrews produced obvious viremia after challenge,and EV71 invaded various tissues to cause tissue lesions,including the brain and spinal cord.The presence of EV71 infection-related receptors in tree shrews may mediate the invasion process of viruses;EV71 The invasion is accompanied by changes in the permeability of the blood-spinal barrier,which is likely to be closely related to its invasion of the nerve and the formation of nerve damage.In summary,the isolated SCMECs and SCASTs in this study can be used in subsequent related experimental studies;EV71 can infect SCMECs and SCASTs,SCMECs have EV71 infection-associated receptors,and have significant up-regulation after infection,blood-spinal barrier correlation The molecular protein expression changes significantly;it is concluded that EV71 can invade the nervous system by infecting SCMECs or other barrier-associated cells,and is introduced into the cell by the relevant infectious receptor,and the invasion of EV71 can destroy the integrity of the blood-spinal barrier or This leads to an increase in barrier permeability,which in turn causes nerve damage to the spinal cord tissue.Tips Tree can be used as a good animal model for EV71 research.