Venezuelan equine encephalitis virus variants lacking transcription inhibitory functions demonstrate highly attenuated phenotype.

UNLABELLED: Alphaviruses represent a significant public health threat worldwide. They are transmitted by mosquitoes and cause a variety of human diseases ranging from severe meningoencephalitis to polyarthritis. To date, no efficient and safe vaccines have been developed against any alphavirus infection. However, in recent years, significant progress has been made in understanding the mechanism of alphavirus replication and virus-host interactions. These data have provided the possibility for the development of new rationally designed alphavirus vaccine candidates that combine efficient immunogenicity, high safety, and inability to revert to pathogenic phenotype. New attenuated variants of Venezuelan equine encephalitis virus (VEEV) designed in this study combine a variety of characteristics that independently contribute to a reduction in virulence. These constructs encode a noncytopathic VEEV capsid protein that is incapable of interfering with the innate immune response. The capsid-specific mutations strongly affect neurovirulence of the virus. In other constructs, they were combined with changes in control of capsid translation and an extensively mutated packaging signal. These modifications also affected the residual neurovirulence of the virus, but it remained immunogenic, and a single immunization protected mice against subsequent infection with epizootic VEEV. Similar approaches of attenuation can be applied to other encephalitogenic New World alphaviruses. IMPORTANCE: Venezuelan equine encephalitis virus (VEEV) is an important human and animal pathogen, which causes periodic outbreaks of highly debilitating disease. Despite a continuous public health threat, no safe and efficient vaccine candidates have been developed to date. In this study, we applied accumulated knowledge about the mechanism of VEEV replication, RNA packaging, and interaction with the host to design new VEEV vaccine candidates that demonstrate exceptionally high levels of safety due to a combination of extensive modifications in the viral genome. The introduced mutations did not affect RNA replication or structural protein synthesis but had deleterious effects on VEEV neuroinvasion and virulence. In spite of dramatically reduced virulence, the designed mutants remained highly immunogenic and protected mice against subsequent infection with epizootic VEEV. Similar methodologies can be applied for attenuation of other encephalitogenic New World alphaviruses.