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 some experiments, human primary dermal fibroblasts were transfected with negative siRNA control, Stealth siRNA to MDA5 (HSS127414), or siRNA to MAVS (Thermo Fisher Scientific; HSS148537), each at 2 µM, using the P3 Primary Cell 96-well Nucleofector kit and Nucleofector Program 96-DT-130 (Lonza). 3 d after transfection, cells were infected with HRV-A16 in DPBS buffer with 0.25% BSA fraction V (Sigma-Aldrich) at MOI of 10. Virus was adsorbed for 1 h at room temperature, and then another h at 35°C. Cells were washed with DPBS and then cultured in DMEM medium supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 55 µM 2-ME. Cells were infected for the indicated times. Uninfected cells were used for qRT-PCR normalization purposes, as described below in the next section.