Molecular characterization and pathogenesis of Eastern equine encephalitis viruses

Julie M. Strizki

doi:10.17918/00008069

Eastern equine encephalitis (EEE) virus is a highly neurotropic mosquito-borne virus which causes a severe and often fatal encephalitis in humans and horses. Two serotypes of the virus are known to exist: the North American (NA) and the South American (SA) serotypes. Despite the medical importance of this virus and its occurrence in the Americas, many central issues regarding the epidemiology and pathogenesis of EEE virus are not understood. In addition, very little was known at the onset of this thesis regarding the molecular relationships among different temporal and geographic isolates of the virus. In order to address certain epidemiological questions, we initially sought to establish the molecular relationships between and among field isolates of both NA and SA serotype viruses. Utilizing reverse transcriptase-PCR (RT-PCR), we demonstrated that it was possible to amplify entire structural gene regions from most virus isolates using a single "universal" primer set specific for each gene. RT-PCR amplification of EEE viral genes should permit rapid assessment of clinical and field specimens for the presence of EEE infection. Subsequent restriction enzyme digestion of PCR-amplified products permitted comparisons of specific genes and proved to be a rapid epidemiological tool for examining gene relationships between and among members of the two virus serotypes. This methodology should also be valuable for identification and grouping of new virus isolates. Our findings demonstrated that the structural genes of NA serotype viruses were highly conserved, while those of the SA serotype viruses were more heterogeneous. Analysis of EEE virion proteins both confirmed the findings of the above genetic studies and provided strong evidence that, among the SA type viruses, relationships appeared to correlate with geographic foci of infection. The molecular diversity evident between the two virus serotypes raised concerns regarding the immunity of EEE-vaccinated personnel to infection with diverse strains of virus. In a study to address this issue, it was found that human immune sera (elicited with a NA serotype vaccine) were strongly reactive with, and neutralized, NA serotype viruses. However, these sera reacted variably with, and did not neutralize, viruses of the SA serotype. This finding raised obvious concerns regarding the efficacy of the EEE vaccine to protect against infection with the SA type viruses. To study this important question, a "good" animal model of EEE viral pathogenesis (similar to clinical infection) was needed to permit evaluation of in-vivo protection. Since no such model existed, it was necessary to develop one. In this regard, infection of adult CD-1 mice inoculated by intravenous injection (to mimic natural inoculation) was found to be a relevant model of EEE pathogenesis. Analysis of virus dissemination and multiplication in organs of infected mice, the associated neuropathological changes, and the humoral antibody response were found to be similar to those observed in clinical infections, suggesting that this model is indeed relevant to natural infection. Development of this model allowed us to test the protective efficacy of human EEE virus vaccine against SA type viruses. Although the results of our pilot vaccination and challenge study were inconclusive, this model should prove immensely useful in future studies to test the protective efficacy of EEE vaccines and to perform more detailed mechanistic studies of EEE viral pathogenesis.

Molecular characterization and pathogenesis of Eastern equine encephalitis viruses

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Molecular characterization and pathogenesis of Eastern equine encephalitis viruses

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