BACKGROUND A photoactive hydrophobic agent 1,5-iodonaphthyl-azide (INA), has been previously shown to completely inactivate the enveloped viruses. INA sequesters into the lipid bilayer of the virus envelope and upon UV-irradiation bind to the hydrophobic domains of the envelope glycoproteins. In our earlier study, we have shown that the Venezuelan equine encephalitis virus (VEEV) genomic RNA was also inactivated during the inactivation of the virus with INA. RESULTS In the present study, we evaluated if the RNA inactivation property of INA can be used to inactivate non-enveloped RNA viruses. Encephalomyocarditis virus (EMCV) was used as a model non-enveloped virus. Treatment with INA followed by UV-irradiation resulted in complete inactivation of EMCV. RNA isolated from INA-inactivated EMCV was non-infectious and INA was found to be associated with the viral RNA genome. INA-inactivated EMCV induced robust total antibody response. However binding capacity of INA-inactivated EMCV to neutralizing antibody was inhibited. CONCLUSIONS This is the first study to show that INA can completely inactivate non-enveloped virus. Our results suggest that the amino acid composition of the neutralizing epitope may interfere with the protective antibody response generated by the INA-inactivated non-enveloped virus.