Graduation Year


Document Type




Degree Granting Department

Global Health

Major Professor

Thomas Unnasch, Ph.D.

Committee Member

Lillian Stark, Ph.D.

Committee Member

Azliyati Azizan, Ph.D.


DIVA, nonstructural protein, serological diagnosis, horses, cloning


Eastern Equine Encephalitis virus (EEEV) causes a fatal mosquito-borne virus that is vaccine preventable for horses. The conventional serological tests measure antibodies to the structural proteins of EEEV which are also found in the vaccine. This makes it difficult to differentiate infected and vaccinated animals (DIVA). Detection of antibodies to non-structural proteins (NSPs) is a theoretical strategy that would allow you to survey natural infections among vaccinated populations. This test would also allow for more accurate representations of the natural infection rate, vaccination rate, and help identify vaccine failures. The potential uses of the NSPs of Eastern Equine Encephalitis virus as diagnostic antigens were examined in this study. Each of the four NSP encoding genes of EEEV strain FL93-939 was separated into two parts, inserted into expression vector pDEST17, and expressed in Escherichia coli strain BL21-AI. Recombinant forms of the protein were used as an antigen for an indirect IgG ELISA to measure the serological response of horse sera to the NSPs. Serum samples collected from infected, vaccinated, and unvaccinated horses were tested for NSP antibodies. A decrease in the optical densities (ODs) for the vaccinated horse sera was seen when using the NSPs compared to whole EEEV antigen. However, the ODs for the vaccinated horses were lowered to the same level as those infected, leaving no quantitative difference between the two. The use of the IgGa secondary antibody decreased the ODs even more for the vaccinated samples, but it was still impossible to differentiate the infected and vaccinated sera due to the samples' ODs being below the cutoff point. The IgGa ELISA however, was the only ELISA where the infected samples were consistently above the vaccinated samples. Based on the results of the study, it was not possible to accurately differentiate between infected and vaccinated animals. Future research should be conducted in other ways to use the NSP recombinants for the DIVA strategy. This could include the use of an IgM ELISA or microsphere immunoassay (MIA), using different IgG subtypes for the assays, using epitope mapping to develop a new recombinant protein, or the development of a DIVA vaccine.