Life-threatening influenza pneumonitis in a child with inherited IRF9 deficiency

Hernandez, Nicholas; Melki, Isabelle; Jing, Huie; Habib, Tanwir; Huang, Susie S.Y.; Danielson, Jeffrey; Kula, Tomasz; Drutman, Scott; Belkaya, Serkan; Rattina, Vimel; Lorenzo-Diaz, Lazaro; Boulai, Anais; Rose, Yoann; Kitabayashi, Naoki; Rodero, Mathieu P.; Dumaine, Cecile; Blanche, Stéphane; Lebras, Marie-Noëlle; Leung, Man Chun; Mathew, Lisa Sara; Boisson, Bertrand; Zhang, Shen-Ying; Boisson-Dupuis, Stephanie; Giliani, Silvia; Chaussabel, Damien; Notarangelo, Luigi D.; Elledge, Stephen J.; Ciancanelli, Michael J.; Abel, Laurent; Zhang, Qian; Marr, Nico; Crow, Yanick J.; Su, Helen C.; Casanova, Jean-Laurent

HRV-A16 and recombinant RSV derived from the A2 strain and containing the enhanced GFP were used as previously described (Lamborn et al., 2017) . rgPIV3, a recombinant human PIV type 3 derived from the JS strain and containing the enhanced GFP, was a gift from Dr. P. Collins, National Institute of Allergy and Infectious Diseases (Zhang et al., 2005) . SV40-transformed fibroblasts were seeded at 100,000 cells per well in 12-well tissue culture plates in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (all from Invitrogen), and 55 µM 2-ME (Sigma-Aldrich). After overnight culture, cells were infected with RSV-GFP (MOI = 0.5) or PIV3-GFP (MOI = 0.1). At 24 and 48 h after infection, cells were harvested, and flow cytometric detection of GFP in RSV-infected viable cells was performed as previously described (Lamborn et al., 2017) . Flow cytometric detection of GFP in PIV3-infected viable cells was performed under similar conditions. For IRF9 siRNA knockdown experiments, human primary dermal fibroblasts were transfected with negative siRNA control, Stealth siRNA to IRF9 (HSS173591; Thermo Fisher Scientific), or siRNA to MAVS (HSS148537; Thermo Fisher Scientific), using the P3 Primary Cell 96-well Nucleofector kit (Lonza) as described (Lamborn et al., 2017) . 3 d after transfection, cells were infected with RSV (MOI = 0.5), PIV (MOI = 0.1), or HRV16 (MOI = 10). RSV, PIV, and HRV replication was quantitated as described above.