bIn 2014, the United States experienced a large outbreak of severe respiratory illness associated with enterovirus D68 (EV-D68). We used a homogeneous, cell-based assay to assess the antiviral activity of compounds developed for EV/rhinovirus infection or other indications. Three of 15 compounds were highly active against all four strains tested (the prototype and three 2014 strains), with 50% effective concentrations of 0.0012 to 0.027 M. Additional studies are needed to assess their in vivo efficacy against EV-D68. E nterovirus (EV) D68 (EV-D68), a rare EV until recently, has been associated with severe respiratory illness in children, often resulting in hospitalization (1-3). During late summer and fall of 2014, a large outbreak of severe respiratory disease in young children occurred across the United States, with laboratory confirmation of EV-D68 infection in Ͼ1,100 cases (http://www.cdc.gov/non -polio-enterovirus/outbreaks/EV-D68-outbreaks.html). The outbreak was characterized by a severe disease, often requiring intensive care and noninvasive ventilatory support, that particularly affected children with a history of asthma or reactive airway disease (4, 30). Cases were also reported in Canada and Europe (5, 6). Exacerbation of pre-existing asthma or reactive airway disease, similar to that associated with rhinovirus (RV) infection (7), was noted in a high proportion of cases, though some patients with no history of asthma also had asthma-like symptoms (4; Midgley et al., submitted).Despite decades of development, a significant disease burden, and more than 100,000 hospitalizations per year (8, 9), there are currently no approved antiviral drugs for the treatment of diseases associated with EV or RV infections (10). To identify potential therapeutic compounds to treat EV-D68 disease, we tested compounds developed specifically for RV or EV indications, drugs that inhibit influenza virus (given that EV-D68 was recently shown to also bind sialic acids on the cell surface [11]), and several drugs that are FDA approved for other indications. The compounds tested included the picornavirus capsid inhibitors pleconaril (12), pocapavir (V-073; ViroDefense, Washington, DC) (13), and vapendavir (BTA-798; Biota Holdings, Alpharetta, GA) (12); the picornavirus protease inhibitors rupintrivir (AG-7088; Pfizer, Groton, CT) (14) and V-7404 (ViroDefense) (15); and the viral polymerase inhibitor favipiravir (T-705; Toyama Chemical Co., Toyama, Japan) (16). DAS181 is an inhibitor of influenza virus binding to ␣2,6-linked sialic acids (Ansun Biopharma, San Diego, CA) (17). In addition to these antiviral compounds, we also tested several compounds that were originally developed and approved for other indications but have been shown subsequently to have antiviral activity against one or more EVs or RVs. These include fluoxetine (selective serotonin reuptake inhibitor antidepressant) (18), formoterol (bronchodilator) (19), and itraconazole (antifungal) (20). Two additional drugs, mefloquine (anti-malarial) and nitazoxanide (anti...