Mechanism Of Vimentin-Mediated Infected With Dengue Virus Type 2

BACKGROUDDengue virus(DENY)is a mosquito-borne single-stranded positive-strand RNA virus of the Flavivirus family.It is divided into four serotypes of DENV 1-4 according to different epitopes.Types can cause dengue fever(DF)and severe dengue(SD).Studies have shown that about 10%of patients with dengue have neurological complications during or after infection,and the pathogenesis may be that the virus induces neurological dysfunction by directly invading the nerve tissue.These neurological complications include encephalitis,meningitis,Myelitis,acute disseminated encephalomyelitis(ADEM),Guillain-Barre syndrome and optic neuritis.The main component of the blood-brain barrier(BBB),human brain microvascular endothelial cells(HBMEC),can infect dengue virus,so HBMEC may be one of the ways in which dengue virus invades nerve tissue.Vimentin is one of the structural proteins of the intermediate filament protein ?.It is involved not only in a variety of cellular processes,but also in the invasion and infection of pathogens.It has been found that the invasion of dengue virus can cause the phosphorylation of vimentin and cause vimentin.Rearrangement,but the role of vimentn in the process of dengue virus-infected cells has not been reported.In this study,we investigated the dynamic changes of phosphorylation,solubility,and expression levels of vimentin in DEN-V-2 infected HBMEC cells in vitro,and used in vitro experiments and animal models to explore the process of vimentin infecting host cells with DENV-2 infection.METHODS1.Enrich the virus and verify the cytopathic effect of DENV-2 infection with HBMECThe preserved rat brain tissue was ground and filtered,and the virus solution was collected and serially passaged on C6/36 cells,and the live virus titer was detected by the TCID50 method and the plaque formation assay.DENV-2 was infected with HBMEC,HBMEC(con),HBMEC(vim-ko)cells(MOI=5),and cell CPE was observed for 5 consecutive days.2.Mechanism of action of vimentin in DENV?2 infected nerve cellsThe rearrangement of vinentin after 0,1,2,6,12,and 24 h of DENV-2 infected HBMEC cells(MOI=5)was observed by irmmunofluorescence.DENV-2 was infected with HBMEC cells(MOI=5)for 0,1,2,6,12,24,36,and 48 h,and the expression levels,phosphorylation levels,and solubility changes of vimentin were detected by western blot.After DENV-2 infection of HBMEC(con)and HBMEC(vim-ko)cells(MOI=5),real-time unlabeled cell function analyzer was used to monitor the state of the two groups in real time,and real-time fluorescence quantitative QPCR was used to detect intracellular viral load.Quantities and detection of cell supernatant and intracellular viral load 1-3 days after infection.3.The role of vimentin in virus-infected nerve cells was explored on a DENV-2 infected suckling mouse model.SV129 and SV129(vim-ko)one-day-old suckling rats were intracranially injected with DENV-2.The body weight,signs and symptoms of the two groups were observed 1-5 days after infection.The mice were detected by real-time fluorescence quantitative QPCR.Viral load in serum and brain tissue on days 4 and 5RESULTS1.The live virus titer detected after continuous amplification of the DENV-2 standard strain was 2.6×106 PFU/mL.2.Vimentin rearranges after DENV-2 infection,and its expression level,phosphorylation level and solubility produce significant dynamic changes with changes in infection time.Compared with the control group HBMEC(con),the cytobiological state of DENV-2 invaded HBMEC(vim-ko)was more obvious,and the intracellular viral load was more(P<0.05).Compared with SV129,the synptoms of SV129(vim-ko)one-day-old suckling mice were more severe after intracranial injection of DENV-2.The viral load in the intracranial and serum was significantly higher oin the 4th day after infection(P<0.05).).CONCLUSIONThis study demonstrates that HBMEC,HBMEC(con),and HBMEC(vim-ko)cells are all susceptible to DENV-2.After repeated infection of DENV-2 in HBMEC cells,the vimentin was rearranged,and the expression of vimentin,phosphorylation level and solubility of the infection process were dynamically changed.Vimentin inhibits the invasion of DENV-2 in host cells and inhibits viral infection,replication and release at the animal level.