The Biotechnological Applications of an Insect-Specific Alphavirus

The coupling of viral and arthropod host diversity with evolving methods of virus discovery has resulted in the identification and classification of a growing number of novel insect-specific viruses (ISVs). These viruses appear to be evolutionarily related to many human pathogens but have either lost or have yet to gain the ability to replicate in vertebrates. The discovery of ISVs has raised many questions as to the origin and evolution of many human pathogenic viruses and points to the role that arthropods may play in this evolutionary process. Furthermore, the use of ISVs to control the transmission of arthropod-borne viruses has been proposed and demonstrated experimentally. Herein, we aim to further expand the application-scope of ISVs and propose their use in vaccine and diagnostic development. Previously, our lab reported on the discovery and characterization of Eilat virus (EILV), an insect-specific alphavirus that phylogenetically groups within the mosquito-borne clade of medically relevant alphaviruses including eastern (EEEV) and Venezuelan equine encephalitis (VEEV), as well as chikungunya (CHIKV) viruses. Despite its evolutionary relationship to these human pathogens, EILV is unable to replicate in vertebrate cells due to blocks at attachment/entry and RNA replication. We hypothesized that, using a chimeric-virus approach, EILV could be utilized as a platform for vaccine and diagnostic development, serving as a proof-of-concept for other ISVs. To test this hypothesis, we generated EILV/CHIKV, EILV/VEEV, and EILV/EEEV chimeras by replacing the structural protein open reading frame of EILV with those of CHIKV, VEEV, or EEEV. All three chimeras replicate efficiently in mosquito cells yet retain the host-restricted phenotype of EILV, unable to replicate in a variety of vertebrate cell lines as well as in a highly susceptible suckling mouse brain model. Structural analyses revealed that the chimeric viruses are indistinguishable from their pathogenic counterparts. Following immunogenicity and efficacy experiments, we demonstrated that EILV-based chimeras can induce single-dose neutralizing antibody responses as well as enhance the activation of T-cells and provide complete protection against challenge in multiple animal models. Furthermore, as diagnostic antigens, EILV chimeras can be produced easily and provide for the sensitive and specific detection of recent and past infection, making them ideal reagents for diagnostic assays. Due to the vast abundance of ISVs in many virus taxa, they may be an untapped resource for the development of vaccines and diagnostics for a variety of human pathogens and further work in this area is warranted.