The Mechanism Of High Expression Of VEGFA Of SFTSV-infected FRCs Target Cells To Promote Vascular Endothelial Hyperpermeability

Objective:Severe Fever with thrombocytopenia syndrome（SFTS）,brought about by SFTSV,is a rising tick-borne viral hemorrhagic fever first mentioned in China in 2009,which can lead to symptoms such as hemorrhagic manifestations,disseminated intravascular coagulation,persistent thrombocytopenia,and involvement of multiple organs with lesions.The mortality rate is as high as 30%,and 80%of patients who died have hemorrhagic manifestations.However,the pathogenesis of SFTSV and the motives of the deadly result are no longer thoroughly understood.The aim of this study is to elucidate the molecular mechanism of changes in vascular endothelial cell permeability caused by SFTSV infection with FRCs in secondary lymphoid organs,to look into the pathogenesis of SFTSV and comparable hemorrhagic fever viruses,and to make a contribution to the improvement of centered tablets to supply new ambitions for broad-spectrum antiviral tablets and ailment prevention and control.Methods:The expression levels of VEGFA in the serum of SFTS patients and healthy individuals and in the serum of type I interferon receptor knockout mice(IFNAR^-/-mice)and healthy controls were measured by ELISA assay.The changes of VEGFA expression levels in the spleen of IFNAR^-/-mice with the prolongation of virus infection were detected by immunohistochemical staining.The source of VEGFA protein expression in the spleen of IFNAR^-/-mice was detected by laser confocal assay.At the cellular level,the viruses were infected with FRCs and HMEC-1,and each set up a control group.Also,the two cells vitality was previously determined using the CCK-8 assay,and q RT-PCR was performed to identify changes in viral load and VEGFA expression in the two cells at various stages of viral replication.Moreover,an ELISA assay was used to find VEGFA expression in the culture supernatant of FRCs that were infected with SFTSV.By using a dextran extravasation assay,the variations in endothelial cell permeability were discovered.And the morphological changes were observed by microscopy.In order to understand the molecular basis of the changes in vascular endothelial cell permeability brought on by SFTSV infection of secondary lymphoid organs FRCs,immunofluorescence assay and Western blot assay were used.Results:（1）Immunohistochemical staining and laser confocal results showed that after SFTSV infection of IFNAR^-/-mice（lethal SFTSV infection mice model）,immunohistochemical staining of individual organs revealed that the spleen was one of the major target organs and one of the major organs with significantly elevated VEGFA protein expression levels,and the expression levels gradually increased with prolonged viral infection.Co-localization of FRCs with SFTSV was observed under laser confocal microscopy,and the virus did not directly infect vascular endothelial cells in vivo.ER-TR7⁺FRCs co-localized with VEGFA,a small number of F4/80⁺macrophages expressed VEGFA protein,and CD3ε⁺T cells and PAX5⁺B cells did not co-localize with VEGFA.（2）QRT-PCR and ELISA results showed that in vitro,SFTSV could directly infect FRCs cells and HMEC-1 cells and actively replicate in the cells,causing cytopathic effects.The level of VEGFA m RNA expression in FRCs cells was increased significantly with the increase of virus infection titer,and the level of VEGFA expression in cell culture supernatant was increased significantly,the level of VEGFA m RNA in HMEC-1 cells did not increase significantly compared with the control group.（3）The results of dextran extravasation assay showed that direct infection of HMEC-1by SFTSV for 48 hours did not cause a significant increase in HMEC-1 cells permeability.However,incubation in total cultures of SFTSV-infected FRCs or high concentrations of recombinant VEGFA protein for 48 hours could promote a significant increase in HMEC-1 cells permeability.Blocking the VEGFA signaling pathway could rescue the trend of elevated HMEC-1 permeability.（4）Immunofluorescence experiments and protein immunoblotting results showed that high concentrations of recombinant VEGFA protein or bioactive mediators released from FRCs infected with SFTSV caused a significant decrease in cell-linked protein expression in monolayer HMEC-1 cells,which was much greater than direct viral infection.Blocking the VEGFA signaling pathway effectively rescued the disruptive effect of bioactive mediators from SFTSV-induced FRCs on intercellular junctions in the vascular endothelium.