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 in vitro infections, A549 cells, seeded at 100,000 per well in 12-well tissue culture plates 1 d prior, were transfected when ∼50-70% confluent with Stealth siRNA to MDA5 (HSS127414), RIG-I (HSS119008), and nonsilencing negative control (Thermo Fisher Scientific) at 40 nM in triplicate wells using siLentFect transfection reagent (Bio-Rad Laboratories). For silencing of MAVS in parallel with controls, cells underwent a second round of Stealth siRNA (HSS183886) transfection 3 d after the first round. After transfection, cells were cultured for 48-72 more h at 37°C before infection. HRV-B14 virus stock, diluted in DPBS containing calcium and magnesium (Lonza), and 0.1% BSA fraction V (Sigma-Aldrich), was added at a MOI of 1. Virus was adsorbed for 1 h at room temperature followed by 1 h at 35°C. After washing five times with DPBS containing calcium and magnesium to remove unbound virus, infected cells were cultured in Ham's F-12K (Kaighn's) medium (Gibco), supplemented with 5% FBS (Hyclone), for 24-72 h at 35°C. Cell supernatants were collected to measure virion release by plaque assay, and total RNA was isolated from cells to measure virus transcripts by qRT-PCR, as described in the Pan-HRV and type I/III IFN qRT-PCR section. In some experiments, A549 cells were transfected with a silencer siRNA to MDA5 (S34498) and