Interferon (IFN) is a protein which is classically designated as inducing an antiviral state in cells. In a variety of systems, other cytokines have occasionally been observed to exhibit apparent antiviral activity. We have examined 25 cytokines for protection of A549 human lung carcinoma cells from the cytopathic effect (CPE) of encephalomyocarditis virus. Four cytokines, IL-1beta, TNF-alpha, IL-4 and IL-13 appear to induce an antiviral state in these cells in the 100 to 800 pg/ml range independent of exogenous IFNs. None of the other cytokines enhance CPE alone or enhance or inhibit the CPE activities of IFN-alpha, -beta or -gamma. Antibodies specific for the 4 cytokines neutralize the effect as does an interferon receptor chain 2 specific neutralizing antibody, suggesting that type I IFN plays a role in the effect. A sheep polyclonal antibody which neutralizes all the human IFN alpha subtypes has no effect on the CPE activity of these 4 cytokines, while a sheep polyclonal which neutralizes human IFN-beta effectively inhibits the antiviral activity. Incubation of the cells with the cytokines alone leads to no measurable production of IFN, while addition of virus to the cells after cytokine incubation leads to significant IFN-beta production, but little or no IFN-alpha production when measured by ELISA. Cytokines which exhibit no apparent CPE did not induce interferon beta either before or after viral challenge in these assays. This suggests that 4 cytokines may be priming the cells to produce IFN when pattern recognition receptors are stimulated by the virus. The finding that IL-4 and IL-13 appear to prime IFN production is surprisinig since these cytokines often act antagonistically with IFN. Additional cell types were examined and endothelial cells were found to upregulate ISGs in response to TNF-alpha and IL-1beta while a proprietary mesothelioma cell line was found to be relatively insensitive to addition of any of the 25 cytokines. These results suggest caution is necessary when interpreting cell-based assay results in situations where other cytokines may be present.