Impact of Tick-Borne Orthoflaviviruses Infection on Compact Human Brain Endothelial Barrier
Abstract
1. Introduction
2. Results
2.1. Barrier Integrity Is Not Affected by Virus Inoculation
2.2. Virus Translocation Across Compact hCMEC/D3 Barrier
2.3. Influence of Modulators of Endocytosis on LGTV Translocation
2.4. Blocking of Laminin-Binding Protein and Vimentin Reduces Translocation of TBEV Across Endothelial Barrier
3. Discussion
4. Materials and Methods
4.1. Viruses
4.2. Human Cerebral Microvascular Endothelial Cells
4.3. In Vitro Transwell Barrier Model
4.4. Barrier Permeability Assay
4.5. Virus Translocation Across hCMEC/D3 Monolayer Cultured on Transwell Inserts
4.6. RNA Extraction
4.7. Virus Quantification RT-qPCR
4.8. Immunocytochemistry of hCMEC/D3 Inoculated with Virus
Primary Antibody | Secondary Antibody | ||||
---|---|---|---|---|---|
Antigen/Target | Origin | Clonality | Dilution | Provider | |
Tick-borne encephalitis virus E-protein | Mouse | Monoclonal (Clone 1786) | 1:100 | Matthias Niedrig [56] | Goat-anti-mouse IgG Alexa Fluor® 568 (abcam, Cambridge, UK) |
Human laminin-binding protein | Rabbit | Polyclonal | 1:500 | Merck, Darmstadt, Germany | Goat-anti-rabbit IgG Alexa Fluor® 488 (Thermo Scientific invitrogen, Carlsbad, CA, USA) |
Human Vimentin | Goat | Polyclonal (antiserum) | 1:50 | Merck, Darmstadt, Germany | Donkey-anti-goat IgG Alexa Fluor® 488 (Thermo Scientific invitrogen, Carlsbad, CA, USA) |
4.9. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
BBB | Blood–brain barrier |
BFA | Brefeldin A |
BHQ1 | Black hole quencher 1 |
BPL | β-propiolactone |
CCD | Cytochalasin D |
CNS | Central nervous system |
CSF | Cerebrospinal fluid |
DENV | Dengue virus |
DMSO | Dimethyl sulfoxide |
dpi | Days post inoculation |
EGM+ | EndoGRO®-MV complete culture medium supplemented with hFGF-2 |
E-protein | Envelope protein |
FAM | Carboxy fluorescein |
FITC | Fluorescein isothiocyanate |
hCMEC/D3 | Human Cerebral Microvascular Endothelial Cell Line (D3) |
hFGF-2 | Human fibroblast growth factor 2 |
hpi | Hours post inoculation |
ICC | Immunocytochemistry |
JEV | Japanese encephalitis virus |
LBP | Laminin-binding protein |
LGTV | Langat virus |
PBS | Phosphate-buffered saline |
PBS-T | Phosphate-buffered saline supplemented with 0.05% Tween20 reagent |
RNA | Ribonucleic acid |
RTC | Rat tail collagen |
RT-PCR | Reverse transcription polymerase chain reaction |
RT-qPCR | Reverse transcription real-time polymerase chain reaction |
TBE | Tick-borne encephalitis |
TBEV | Tick-borne encephalitis virus |
TCID50 | Tissue culture infectious dose |
TEER | Transendothelial electrical resistance |
WNV | West Nile virus |
ZIKV | Zika virus |
ZO-1 | Zonula occludens protein 1 |
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Schweitzer, F.; Letoha, T.; Osterhaus, A.; Prajeeth, C.K. Impact of Tick-Borne Orthoflaviviruses Infection on Compact Human Brain Endothelial Barrier. Int. J. Mol. Sci. 2025, 26, 2342. https://doi.org/10.3390/ijms26052342
Schweitzer F, Letoha T, Osterhaus A, Prajeeth CK. Impact of Tick-Borne Orthoflaviviruses Infection on Compact Human Brain Endothelial Barrier. International Journal of Molecular Sciences. 2025; 26(5):2342. https://doi.org/10.3390/ijms26052342
Chicago/Turabian StyleSchweitzer, Felix, Tamás Letoha, Albert Osterhaus, and Chittappen Kandiyil Prajeeth. 2025. "Impact of Tick-Borne Orthoflaviviruses Infection on Compact Human Brain Endothelial Barrier" International Journal of Molecular Sciences 26, no. 5: 2342. https://doi.org/10.3390/ijms26052342
APA StyleSchweitzer, F., Letoha, T., Osterhaus, A., & Prajeeth, C. K. (2025). Impact of Tick-Borne Orthoflaviviruses Infection on Compact Human Brain Endothelial Barrier. International Journal of Molecular Sciences, 26(5), 2342. https://doi.org/10.3390/ijms26052342