Tripure

Nasal washes, oral swabs, plasma, tissues, PBMCs, BALF, and BALP were collected as outlined in Figure A1. Tissue was homogenized in 5 mL PBS (10% w/v homogenates) using a lysing kit and homogenizer from Precellys^® according to the manufacturer’s protocol (BERTIN Corp., Rockville, MD, USA). Total RNA was extracted from 100 µL of liquid samples in 900 µL TriPure (Roche) and from the whole PBMC and BALP pellets lysed in 1 mL TriPure according to the manufacturer’s protocol. The amount of EBOV in each sample was determined by a semi-quantitative real-time polymerase chain reaction (qRT-PCR) assay targeting the L-gene [31 (link)]. The detection limit for this test was 3.6 log₁₀ RNA copies/mL. Samples that were PCR-positive were subjected to virus isolation using a microtiter immunostained plaque assay as previously described [31 (link)].

Total RNA was isolated from the OSCC cell lines using TriPure (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer's instructions. 1 μg of total RNA for one 20 μL reaction volume was reverse transcribed to cDNA using the transcriptor first-strand cDNA synthesis kit (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer's instructions. The first-strand cDNA synthesis was performed by the use of Applied Biosystems® Veriti PCR (Veriti, Thermo Fisher, USA). qPCR was performed using a LightCycler® 480 SYBR Green I Master (Roche Diagnostics, Germany), following thermal cycling parameters: preincubation at 95°C for 5 min, followed by 40 cycles of amplification (95°C for 10 s, 60°C for 20 s, and 72°C for 30 s), melting curve (95°C for 5 s, 65°C for 60 s, and 97°C-), and cooling (40°C for 10 s). Expression levels were analyzed using the ΔΔCq method [25 (link)], and gene expression was normalized to the internal control, glyceraldehyde 3-phosphate dehydrogenase. The primers are shown in Table 1. All samples were analyzed in triplicate.