Evaluating Endothelial Barrier Function in Yellow Fever

To evaluate the putative effects of serum from acutely YFV-infected patients on endothelial barrier function, we used the TEER assay as described previously (26 (link), 27 (link)). In brief, human umbilical vein endothelial cells, kindly donated by Dr. Miriam Fonseca-Alaniz (Instituto do Coração, InCor, University of São Paulo, Brazil) were seeded (6×10⁴ cells/well) in Transwell polycarbonate membrane inserts (0.4 μm pore, 6.5 mm diameter; Corning Inc.) in endothelial cell growth basal medium 2 supplemented with an Endothelial Cell Growth Medium-2 (EGM-2TM) supplemental bullet kit (Lonza). After 72h of incubation at 37°C and 5% CO₂, cells were treated with human sera (10% final vol/vol concentration) obtained from YFV-positive severe and non-severe patients or YFV-negative blood donors (healthy controls). TEER values, expressed in Ohms (Ω), were collected at sequential 2-h time-points 2–10h following treatments using an Epithelial Volt Ohm Meter (EVOM) with a “chopstick” electrode (World Precision Instruments). Resistance of inserts with no cells (blank) and inserts with cells (untreated) containing medium alone, were used to calculate relative TEER as a ratio of the corrected resistance values as (Ω experimental condition - Ω blank)/(Ω untreated - Ω blank). Recombinant YFV NS1 (Native Antigen Co.) at 10 μg/mL was used as positive control.