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.

Virus stocks were prepared from HRV-B14 and HRV-A16 seed stocks (a gift from W.-M. Lee) as previously described (Lee et al., 2015a) . In brief, H1-HeLa cells were maintained in suspension at 37°C using an incubator shaker set at 230 rpm. Cells were cultured in MEM suspension medium with Earle's salts and no calcium (sMEM; GIB CO BRL #11380-037), supplemented with 10% FBS (Hyclone), 1x MEM nonessential amino acids, 2 mM l-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.1% Pluronic F-68 (Gibco). Cells were incubated with high-titer HRV-B14 at a multiplicity of infection (MOI) of 15, in Dulbecco's PBS (DPBS) containing calcium and magnesium (Lonza) for 1 h at room temperature. After adsorption, infected H1-HeLa cells were cultured in complete medium for an additional 8 h at 35°C, with shaking at 120 rpm. Cell pellets were subjected to three freeze-thaw cycles in 10 mM Hepes, pH 7.2 (Quality Biological), and 0.5% Nonidet P-40 (Calbiochem) added before lysates were clarified by high-speed centrifugation. After incubating clarified lysates with 400 µg RNase A (Invitrogen) for 30 min at 35°C, 0.9% N-laurylsarcosine (Sigma-Aldrich) and 28.5 µM 2-ME (Sigma-Aldrich) were added. Virions were concentrated and partially purified by ultracentrifuging for 2 h at 40,000 rpm, 16°C, over a 30% (wt/vol) sucrose cushion (Sigma-Aldrich) containing 16.7 mM Tris acetate pH 7.5 (Sigma-Aldrich) and 0.833 M sodium chloride (Quality Biological). The virus pellet was resuspended in DPBS containing calcium and magnesium, with 0.01% BSA fraction V (Sigma-Aldrich). Virus aliquots were stored at -80°C until use.