Ebola Virus Detection Workflow

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)].