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.

In other experiments, nasal epithelial cells were digested from feeder cells and seeded in 12-well plates at 100,000 cells per well in 1 ml epithelial culture medium (Promocell; C21060) with 10 µM Y-27632 (ApexBio) and incubated at 37°C 5% CO 2 . The cells were seeded into plates that had been previously coated with 300 µl rat tail collagen (BD; 354236) at 30 µg/ml in PBS for 45 min at room temperature, washed twice with PBS, and air dried for 20 min. The next d, the cells were washed once with DPBS and then infected with HRV-B14 at a MOI of 1 in DPBS with 0.25% BSA (Sigma-Aldrich). Virus was adsorbed for 40 min at room temperature and then 90 min at 35°C. After washing three times with DPBS, the cells were cultured in epithelial culture medium with 10 µM Y-27632, for 48 or 72 h at 35°C. Total RNA isolated from cells was used to measure virus transcripts by qRT-PCR, as described below in the next section.