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.

Recombinant wild-type RSV strain A2 in which enhanced GFP was inserted between the P and M genes was propagated, sucrose-purified, and titered by plaque assay on Vero cells, as previously described (Munir et al., 2008) . The virus stock was sequenced and had no adventitious mutations, as confirmed by Sanger dideoxy sequencing. A549 cells were seeded and transfected with siRNA to MDA5, RIG-I, MAVS, or a nonsilencing negative control as described for HRV infections. Alternatively, primary nasal epithelial cells were digested from feeder cells and seeded in 12-well plates at 150,000 cells per well in 1 ml epithelial culture medium (Promocell) with 10 µM Y-27632 (ApexBio) and incubated at 37°C in 5% CO2. The cells were seeded on plates previously coated with 300 µl rat tail collagen (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 once with PBS. Transfected A549 cells or primary nasal epithelial cells were infected with RSV-GFP at an MOI of 1 or 0.2, respectively (diluted in appropriate complete medium, 300 µl per 12-well), for 1 h at room temperature, and washed twice with PBS to remove unadsorbed virus, and then medium was replaced (complete F-12K for A549; epithelial culture media without Y-27632 for nasal epithelial cells) and cultures were returned to 37°C in 5% humidified CO 2.