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.

For infection of A549 cells, cells were seeded at 100,000 per well in 12-well tissue culture plates 1 d before and transfected when ∼50-70% confluent with Stealth siRNA targeting MDA5 (HSS127414), RIG-I (HSS119008), MAVS (HSS127415), and nonsilencing negative control (12935300; all from Thermo Fisher Scientific) at 40 nM in triplicate wells using siLentFect transfection reagent (Bio-Rad Laboratories). After transfection, cells were cultured for an additional 72 h at 37°C before infection. Alternatively, primary nasal epithelial cells were digested from feeder cells and seeded in 24-well plates at 100,000 cells per well in 0.5 ml epithelial culture medium (C21060; Promocell) with 10 µM Y-27632 (ApexBio) and incubated at 37°C in 5% CO 2 . The cells were seeded on plates previously coated with 150 µl rat tail collagen (354236; BD) at 30 µg/ml in PBS for 45 min at room temperature, washed twice with PBS, and air dried for 20 min. 36 h later, the cells were washed twice with PBS be-fore infection. Transfected A549 cells were infected with A/ Victoria/361/2011 (H3N2) at MOI of 0.1 (diluted in PBS containing 0.3% BSA (Sigma-Aldrich) to a final volume of 300 µl/12-well) for 1 h at room temperature, washed twice with PBS to remove unadsorbed virus, and the medium was replaced with F-12K supplemented with 0.1% BSA, 0.1% FBS (Hyclone), 2 mM glutamine, 55 µM 2-ME, and 1 µg/ml TPCK-trypsin (Sigma-Aldrich). Primary nasal epithelial cells were infected with A/Victoria/361/2011 (H3N2) at MOI: 0.02 (diluted in complete epithelial culture medium without Y-27632 to a final volume of 150 µl/24-well) for 1 h at room temperature, washed twice with PBS to remove unadsorbed virus, and medium was replaced with complete epithelial culture medium plus 1 µg/ml TPCK-trypsin (Sigma-Aldrich) and without Y-27632. After infection, all cultures were returned to 37°C in 5% humidified CO 2 .