Complete inactivation of Venezuelan equine encephalitis virus by 1,5-iodonaphthylazide Anuj Sharma a,b , Yossef Raviv c,d , Anu Puri c , Mathias Viard c,d , Robert Blumenthal c , Radha K. Maheshwari a, * a Centre for Combat Casualty and Life Sustainment Research, Department of Pathology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814, USA b Birla Institute of Technology and Science, Pilani, India c CCR Nanobiology Program, Centre for Cancer Research, National Cancer Research Institute, Frederick, MD, USA d Basic Research Program SAIC Frederick Received 10 April 2007 Available online 26 April 2007 Abstract Hydrophobic alkylating compounds like 1,5-iodonaphthylazide (INA) partitions into biological membranes and accumulates selec- tively into the hydrophobic domain of the lipid bilayer. Upon irradiation with far UV light, INA binds selectively to transmembrane proteins in the viral envelope and renders them inactive. Such inactivation does not alter the ectodomains of the membrane proteins thus preserving the structural and conformational integrity of immunogens on the surface of the virus. In this study, we have used INA to inactivate Venezuelan equine encephalitis virus (VEEV). Treatment of VEEV with INA followed by irradiation with UV light resulted in complete inactivation of the virus. Immuno-fluorescence for VEEV and virus titration showed no virus replication in-vitro. Complete loss of infectivity was also achieved in mice infected with INA treated plus irradiated preparations of VEEV. No change in the structural integrity of VEEV particles were observed after treatment with INA plus irradiation as assessed by electron microscopy. This data sug- gest that such inactivation strategies can be used for developing vaccine candidates for VEEV and other enveloped viruses. Published by Elsevier Inc. Keywords: 1,5-Iodonaphthyl-azide; Venezuelan equine encephalitis Virus; Photoactive; Inactivation Venezuelan equine encephalitis virus (VEEV) is a mem- ber of arbovirus group, family togaviridae in genus alpha- virus and is transmitted by mosquito in nature by subcutaneous inoculation [1–3] and causes biphasic infec- tion [4–6]. Clinical signs of disease in humans include fever, headache, malaise and myalgia [7,4]. Recent outbreaks of VEEV have resulted in its identification as an emerging pathogen [8]. It is highly infectious in aerosol [9] and has been developed as a bioweapon [10]. Current live attenuated TC-83 vaccine for VEEV is under new-investigational drug status and is given to labo- ratory personal at risk. Formaldehyde inactivated TC-83, known as C84, is used as a booster following immunization with live attenuated TC-83 vaccine [11,12]. These vaccines have limitations such as, adverse reaction, short-lived immunity and several non-responders [13–15,11]. There- fore, it is important to develop new safe vaccine candidates for VEEV. INA selectively penetrates into the hydrocarbon core of biological membranes. Upon irradiation, with UV light these compounds directly bind to proteins and lipids embedded in the bilayer [16–21]. The resulting inactivation of multiple components in the membrane is efficient and selective so that ectodomain of proteins or lipids outside the bilayer are not affected [22,16]. Recently this approach was used to produce inactivated HIV and SIV virions for vaccine application [23]. INA has also been used for inac- tivation of Ebola and Influenza virus (Raviv et al., personal communication). In this study, we demonstrate a complete 0006-291X/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.bbrc.2007.04.115 * Corresponding author. Fax: +1 301 295 1640. E-mail address: rmaheshwari@usuhs.mil (R.K. Maheshwari). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 358 (2007) 392–398