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.

SV40-transformed fibroblasts were seeded at 50,000 cells/well in 48-well tissue cultures plates in complete DMEM. 16-24 h later, cells were infected with A/Netherlands/602/2009 (H1N1) or A/Puerto Rico/8/1934 (H1N1) at the indicated MOI for 60 min at 37°C in HBSS supplemented with 0.3% BSA (Sigma-Aldrich). Cells were washed twice with PBS and cultured at 37°C in DMEM supplemented with 0.1% FBS (Hyclone) and 0.3% BSA, 2 mM glutamine (Gibco), and 55 µM 2-ME (Sigma-Aldrich) in the presence of 1 µg/ml TPCK-trypsin (Sigma-Aldrich). Cell supernatants were collected at the indicated time points after infection and stored at -80°C. Once all samples were collected, supernatants were thawed and influenza titers were determined by infectious plaque assay on MDCK cells, as previously described (Balish et al., 2013) . In brief, MDCK cells were plated in 12-well plates and grown to 100% confluency. Cells were washed twice with PBS, and serial dilutions of influenza infection supernatants diluted in PBS were absorbed onto MDCK cells for 1 h at room temperature. Cells were then overlaid with agar medium of MEM, 28 mM sodium bicarbonate, 2 mM l-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (all from Gibco), 0.4% BSA, 1 µg/ ml TPCK-trypsin (both from Sigma-Aldrich), and 1% Oxoid Agar (Thermo Fisher Scientific). After 36-60 h, plaques were counted by direct visualization or by fixation and crystal violet counterstain as described in HRV plaque assay, and then calculated as PFU/ml of influenza infection supernatant. qrt-Pcr for influenza-induced proinflammatory cytokines A549 cells in which MDA5, RIG-I, MAVS, or negative control were silenced by transfecting in siRNA were infected with influenza strain A/Victoria/361/2011 (H3N2) as described above. Total RNA were isolated from influenza-infected A549 cells using TRIzol extraction (Invitrogen). 2 µg total RNA per sample was reverse transcribed using High-Capacity cDNA Reverse Transcription kit with RNase Inhibitor (ABI). All quantitative RT-PCR were performed by the SYBR green method on a 7500 Real Time PCR System (ABI). Primer sequences are as follows: human TNF forward primer, 5′-CTG CTG CAC TTT GGA GTG AT-3′; human TNF reverse primer, 5′-AGA TGA TCT GAC TGC CTG GG-3′; human IL-1α forward primer, 5′-ACT GCC CAA GAT GAA GAC CA-3′; human IL-1α reverse primer, 5′-CCG TGA GTT TCC CAG AAG AA-3′; human IL-6 forward primer, 5′-AGT GAG GAA CAA GCC AGA GC-3′; human IL-6 reverse primer, 5′-GTC AGG GGT GGT TAT TGC AT-3′; human IL-8 forward primer, 5′-TCC TGA TTT CTG CAG CTC TGT-3′; human IL-8 reverse primer, 5′-AAA TTT GGG GTG GAA AGG TT-3′; human β-actin forward primer, 5′-GCA CAG AGC CTC GCC TT-3′; human β-actin reverse primer, 5′-GTT GTC GAC GAC GAG CG-3′.The expression of mRNA for proinflammatory cytokine genes of interest was normalized to the expression of β-actin, and then normalized to the control groups at 8 h after infection. For quantitation of IFN-β, supernatants were diluted twofold and were assayed for Human IFN-β using the VeriKine Human Interferon Beta ELI SA kit (PBL Assay Science). For quantitation of influenza-induced cytotoxicity, supernatants were diluted 2.5-fold and were assayed for LDH release using the Cytotoxicity Detection kit Plus (Roche) to measure enzymatic activity. Colorimetric absorbance was measured according to kit manufacturers' recommendations, using a VIC TOR X4 Multi-label Plate Reader (PerkinElmer).