PROJECT SUMMARY There have been globally distributed, regional outbreaks of a novel strain of adenovirus (Ad) 14, Ad14p1, that induces acute lung injury similar to that observed during outbreaks of infections with other Ad serotypes (e.g., Ad 3, 4 and 7). It has not, however, previously been possible to do comparative virology and pathogenesis studies, contrasting a prototype, parental Ad strain, such as Ad14, with a single, globally distributed genetic variant, such as Ad14p1. The reasons for the increased severity of these Ad14p1 infections are poorly defined. We have shown that cells undergoing Ad-induced cytopathic effect (CPE) from infection with Ad14 elicit an immunorepressive activity of activated human alveolar macrophages by repressing IL-1b, IL-6, IL-8 and TNF-alpha. In contrast, Ad14p1 CPE corpses enhance production of these same cytokines, due to decreased expression of viral E1B 20K in Ad14p1 CPE corpses. Consistent with these in vitro observations, infection of Syrian hamsters with prototype Ad14 resulted in minimal inflammation and lung injury, whereas Ad14p1 infection induced a marked acute lung injury and ARDS-like pathology. It has been shown that apoptotic cells have altered expression of microRNA (miRNA) that can repress pro- inflammatory responses and that these miRNA from apoptotic cells can be delivered to macrophages. We postulated that a similar mechanism could be occurring with Ad14 infected cells but not cells infected with the Ad14p1 variant. To test this, we infected human cells with Ad14 or Ad14p1 and did small RNA-seq on the respective CPE corpses. The data showed that Ad14 CPE corpses are enriched in four miRNA (Ad14 miRNA), all of which have been shown to repress NF-kB dependent transcription of pro-inflammatory cytokines, compared to Ad14p1 CPE corpses. Our hypothesis is that increased expression of these specific miRNA in Ad14 CPE corpses and their transfer to responder macrophages are the mechanisms through which Ad14 CPE corpses repress pro-inflammatory responses. The studies we propose here will test those hypotheses. We will test the role of Ad14 miRNA in modulating inflammatory responses of macrophages using miRNA mimics and whether E1B 20K modulates Ad14 miRNA expression during infection. We will test the role of the most abundantly expressed Ad14 miRNA, miR-181a-5p, as the mediator of Ad14 CPE immunorepression by using 3’ UTR reporter constructs and inhibiting miR-181a- 5p with miRZips. Finally, we will do translational studies to determine the role miR-181a-5p plays in the immunopathogenesis of Ad14p1 infection in the Syrian hamster. Upon completion of this project, we expect to have an increased understanding of the role of virally induced cellular miRNAs in modulation of macrophage responses during acute lung injury in response to Ad14p1. The positive impact of our studies will be the determination of the potential of miRNAs as therapeutic interventions for ARDS.