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Western equine encephalitis virus: neuroinvasion, pathogenesis, and immunomodulatory treatment strategies




Phillips, Aaron Timothy, author
Olson, Kenneth, advisor
Brennan, Carol Blair, committee member
Aboellail, Tawfik, committee member
Tjalkens, Ronald, committee member

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Western equine encephalitis virus (WEEV; Alphavirus) is a mosquito-borne virus that can cause severe encephalitis in humans and equids. WEEV is closely-related to eastern equine encephalitis virus (EEEV) and may model similar pathogenesis in a mouse model. Previous studies have shown that intranasal infection of outbred CD-1 mice with the WEEV McMillan (McM) strain result in high mortality within 4 days of infection, thus providing a model of exposure to airborne encephalitic alphavirus. In addition, WEEV McM causes high mortality within 5-7 days following peripheral inoculation of mice. Therefore, WEEV McM may be used to model infection following exposure to infected mosquitos. The route of WEEV entry into the central nervous system (CNS) is not well-understood. In the studies presented here, bioluminescence (BLM) imaging and recombinant reporter viruses based on WEEV McM were applied to detect and track virus in mice following intranasal or subcutaneous inoculation, and used to determine correlation between BLM and viral titer. Additionally, histopathology analysis was guided by corresponding BLM images and used to identify specific CNS regions affected during infection. The major findings from these studies indicate that WEEV McM uses a different route for entry into the CNS for each of the two inoculation methods (intranasal or footpad). Intranasal challenge resulted in neuroinvasion occurring primarily through cranial nerves, mainly in the olfactory tract. Olfactory bulb neurons were initially infected followed by spread of the infection into different regions of the brain. WEEV distribution was confirmed by immunohistochemistry as having marked neuronal infection but very few infected non-neuronal glial cells. Axons displayed infection patterns consistent with viral dissemination along the neuronal axis. The trigeminal nerve served as an additional route of neuroinvasion showing significant FLUC expression within the brainstem. Neuroinvasion from footpad inoculation demonstrated a consistent pattern in the spatiotemporal distribution of virus among the imaged brains, none of which involved the olfactory bulb. These studies support the hypothesis that neuroinvasion of WEEV likely occurs in areas of the CNS where the blood-brain barrier is naturally absent. These areas include the median eminence of hypothalamus (hypothalamic output), posterior pituitary, pineal body, and the area postrema. There are no antiviral therapies against alphaviral disease and current vaccine strategies target only a single alphavirus species. In an effort to develop new tools for a broader response to outbreaks, a novel alphavirus vaccine comprised of cationic-lipid-nucleic acid complexes (CLNCs) and the ectodomain of WEEV E1 protein (E1ecto) was designed and tested. Interestingly, the CLNC component alone had therapeutic efficacy as it increased survival of CD-1 mice following lethal WEEV infection. Immunization with the CLNC-WEEV E1ecto mixture (lipid-antigen-nucleic acid complexes; LANACs) using a prime/boost regimen provided strong protection in mice challenged with WEEV subcutaneously, intranasally, or via mosquito. In addition, the LANAC immunization protocol significantly increased survival of mice following intranasal or subcutaneous challenge with EEEV, indicating potential as a 'pan-alphavirus' vaccine candidate. Mice immunized with LANAC mounted a strong humoral immune response, but did not produce neutralizing antibodies.


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