Insufficiency in airway interferon activation defines clinical severity to infant RSV infection

Chin-Yi Chu; Xing Qiu; Matthew N. McCall; Lu Wang; Anthony Corbett; Jeanne Holden-Wiltse; Christopher Slaunwhite; Qian Wang; Christopher Anderson; Alex Grier; Steven R. Gill; Gloria S. Pryhuber; Ann R. Falsey; David J. Topham; Mary T. Caserta; Edward E. Walsh; Thomas J Mariani

RSV GFP-A2 strain (gift of Dr. Mark Peeples) was generated in Hep2 cells as previously described 46 . ALI-differentiated PHLE cultures were washed twice with PBS and infected apically with RSV at a multiplicity of infection of 1. After one hour at 37°C, the inoculum was removed and the apical surface was rinsed with PBS twice to remove unbound viral particles. Infected PHLE were maintained at ALI for up to 48 hours, and monitored for infection using a fluorescence microscope. After 48 hours, cells were harvested and RNA isolated, and subjected to qPCR as described above. Infection was quantified by measuring GFP fluorescence with ImageJ software (https://imagej.nih.gov/ij/) and RSV-M gene quantification using real-time polymerase chain reaction (qPCR). Where indicated, interferon ligands (30 ng/ml IL28 or 2.5 ng/ml IFN-β; R&D Systems) were provided to PHLE cells for an hour prior RSV infection and maintained throughout the All rights reserved. No reuse allowed without permission.