Recurrent rhinovirus infections in a child with inherited MDA5 deficiency

Lamborn, Ian T.; Jing, Huie; Zhang, Yu; Drutman, Scott B.; Abbott, Jordan K.; Munir, Shirin; Bade, Sangeeta; Murdock, Heardley M.; Santos, Celia P.; Brock, Linda G.; Masutani, Evan; Fordjour, Emmanuel Y.; McElwee, Joshua J.; Hughes, Jason D.; Nichols, Dave P.; Belkadi, Aziz; Oler, Andrew J.; Happel, Corinne S.; Matthews, Helen F.; Abel, Laurent; Collins, Peter L.; Subbarao, Kanta; Gelfand, Erwin W.; Ciancanelli, Michael J.; Casanova, Jean-Laurent; Su, Helen C.

Nasal airway epithelial cells were collected from the lower surface of the inferior nasal turbinate using a sterile cytology brush (Cytosoft; Medical Packaging Corporation) as previously described (Poole et al., 2014; Reynolds et al., 2016) . Cells were placed in Ham's F-12 medium (Gibco) supplemented with 10% DMSO (Sigma-Aldrich), 30% heat inactivated FBS (Gibco), and 0.1% Y-27632 Rho kinase inhibitor (ApexBio). The collection medium was also supplemented with 1.25 µg/ml amphotericin B (Sigma-Aldrich), 2 µg/ml fluconazole (Gallipot), and 50 µg/ml gentamicin (Gibco). Cells were stored cryopreserved until needed for culture, using adapted methods that combined use of an irradiated fibroblast feeder layer and Rho kinase inhibition (Suprynowicz et al., 2012) . The major modification was the use of irradiated 3T3 fibroblasts (ATCC) as the feeder cell layer. Nasal airway epithelial cells were cultured in T75 flasks to ∼80% confluence and harvested using the previously described multistep trypsinization procedure (Suprynowicz et al., 2012) . Harvested cells were cryopreserved using the aforementioned medium but without antibiotic supplementation. Cells of the same passage number and no greater than passage three were used for experiments.