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.

Plaque assays quantitating infectious HRV were performed as previously described, with modifications (Lee et al., 2015b) . In brief, HRV-B14 virus stock or cell supernatants were serially diluted in DPBS containing calcium, magnesium, and 0.1% BSA fraction V (Sigma-Aldrich). 200 µl diluted samples were added in duplicate to confluent monolayers of H1-HeLa cells in 6-well plates. After virus adsorption for 1 h at room temperature, the cells were overlaid with 0.8% Noble agar (Sigma-Aldrich) in 1x P6 medium (1x sMEM [Gibco], 26.2 mM sodium bicarbonate, 40.6 mM magnesium chloride hexahydrate, and 0.1% BSA fraction V [Sigma-Aldrich]). Nutritive medium was then overlaid to obtain final concentrations of 1x P6 medium, 2 mM l-glutamine (Gibco), 1.2 mM pyruvic acid (Sigma-Aldrich), 2 mM oxaloacetic acid (Sigma-Aldrich), and 0.1% glucose (Corning). After incubation at 35°C for 2 to 3 d, monolayers were fixed with 10% buffered formalin (Sigma-Aldrich) for 15 min at room temperature, overlaid agar removed, and plaques visualized by staining with 0.1% crystal violet (Sigma-Aldrich) in 20% ethanol for 1 h. Plaques were counted and calculated as PFU/ml of original virus stock or cell supernatant.