RESEARCH PROJECTS

Genetic Determinants of Virulence and Attenuation of EAV

Geographically and temporally distinct strains of EAV have been identified which differ in the severity of clinical disease they induce and in their abortigenic potential. While the majority of field strains of EAV are associated with subclinical or inapparent infection, some strains give rise to moderately severe signs of EVA.  Virulence is intimately intertwined with the pathogenesis of virus infection; however, the genetic determinants of EAV virulence have not been identified.

We are utilizing two full-length infectious cDNA clones of EAV and reverse genetic techniques to characterize the virulence determinants of the virus. Specifically, we obtained the infectious cDNA clone (EAV030) of a highly cell culture adapted strain of EAV that was developed by Eric Snijder’s group at Leiden University. Inoculation of stallions with the recombinant virus derived from this infectious clone caused only subclinical infection, and the cloned virus was genetically stable during in vivo replication. Because the virus derived from the original infectious cDNA clone was avirulent, we developed an infectious cDNA clone of the virulent horse adapted Bucyrus strain (velogenic Bucyrus strain [VBS] of EAV) to further investigate the virulence determinants of EAV.  We recently showed that horses inoculated with the recombinant virus derived from our new infectious cDNA clone developed marked clinical disease typical of severe equine viral arteritis. 

Furthermore, the sequencing of a series of viruses, ranging from the virulent horse-adapted VBS to the fully attenuated modified live virus (MLV) vaccine (ARVAC®) derived from it, has allowed us to identify the nucleotides/amino acids that likely are responsible for attenuation of the virulent virus. The in vivo virulence (attenuation) phenotype of each virus in this panel has been previously characterized through experimental infection of horses.  The significance of individual nucleotide and amino acid differences between virulent EAV VBS strain and avirulent MLV vaccine strain of EAV will be determined by site-directed mutagenesis of the virulent infectious cDNA clone. We will develop a panel of recombinant viruses with specific nucleotide/amino acid changes to confirm the putative attenuation mutations of MLV vaccine strain of EAV.  This study will clearly identify the attenuation mutation(s) present in the current MLV vaccine strain of EAV.

This information can be used to develop a recombinant plasmid with multiple engineered attenuating mutations, which then might serve as a genetically marked MLV vaccine. Such recombinant plasmids carrying complete copies of attenuated EAV might ultimately also be used as stable validated repositories of seed viruses for live virus vaccine production, which would ensure a genetically homogeneous virus stock. Furthermore, data from these studies eventually will facilitate the rapid identification of new strains of EAV with enhanced virulence, better delineate the role of the carrier stallion in generating them, and guide the logical development of improved vaccines for EAV.