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Introduction/Aim: Reactive oxygen species (ROS) are crucial for the elimination of pathogenic bacteria, but the impact of ROS on viral pathogenicity is yet to be clearly defined. Our aim is to determine (1) the site of subcellular ROS generation, (2) the identity of the enzymes that generate ROS and (3) the impact of ROS on the pathogenesis of influenza A virus infection in vivo. Method(s): Confocal fluorescence microscopy was used to assess the subcellular distribution of viruses, Toll-like receptors (TLRs) and NOX enzymes, and to assess endosomal superoxide production in human and mousemacrophages that were infected with various ssRNA viruses (influenza A virus, HIV, Dengue, rhinovirus, respiratory synctitial virus, human parainfluenza virus and human metapneumovirus), dsRNA viruses (rotavirus) and dsDNA viruses (herpes simplex 2 and vaccinia virus). Mice (C57Bl/6) were infected intranasally with influenza A virus (Hong Kong X-31 strain, 105 PFUs/mouse) for assessments of airways inflammation, viral titers, cytokine expression and serum antibody levels. Site-directed mutagenesis was used to mutate cysteine residues to alanine on TLR7 for assessments of oxidatively modified receptor. Result(s): NOX2 oxidase co-located with virus and TLR7 in early endosomes, providing a spatially targeted platform for ROS production that operated within minutes after the internalization of the single-stranded RNA and DNA viruses into endocytic compartments. NOX2 oxidase activation was critically dependent on endosomal acidification and the engagement of TLR7 for ssRNA viruses or TLR9 for DNA viruses. NOX2-dependent endosomal H2O2 caused modification of crucial cysteine residues on the ectodomain of TLR7, resulting in suppression of innate anti-viral cytokine expression. Inhibition of NOX2 by genetic deletion or by novel endosome targeted pharmacological inhibitors of NOX2 resulted in a significant suppression in airways inflammation and viral titers following influenza A virus infec