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Western equine encephalitis virus: development and application of a new world alphavirus transducing system


A recombinant western equine encephalitis virus (WEEV) was generated that expressed firefly luciferase (FLUC) as a marker of infection. In vivo imaging technology was used to visualize bioluminescence in the context of WEEV infection of outbred (CD-1) and inbred (C57/BL6) strains of mice as well as Culex tarsalis mosquitoes. Bioluminescent imaging permitted us to follow a neurovirulent strain of WEEV in the living tissue of a single animal over time. The recombinant virus also permitted detection by bioluminescence of WEEV in the mosquito vector, Culex tarsalis. In vivo imaging was used to test the hypothesis that an alphavirus transducing system could be used to predict efficacy of a cationic lipid RNA complex (CLRC) immunomodulator in the suppression of WEEV infection. Bioluminescent imaging in screening potential antivirals for activity against WEEV in vivo was confirmed to be consistent, clear, and in agreement with traditional survival curve analysis. WEEV is maintained in an enzootic cycle through transmission by Culex tarsalis to passerine bird species. Tangential transmission to equine or human hosts has been associated with severe outbreaks of disease in the past. These hosts are considered to be dead-end hosts as they may become infected during epizootics but do not generate sufficient viremia titers to infect a bloodfeeding mosquito. Understanding the determinants of transmission to the vector from the host, dissemination within the vector, and secretion in saliva of WEEV are crucial to understanding the overall cycle. The recent development of a WEEV transducing system facilitated the study of WEEV interaction with the midgut, ovary, and salivary gland tissue of C. tarsalis. The expression by a recombinant alphavirus of monomeric cherry fluorescent protein allowed an overall picture of infection, dissemination, and transmission with both enzootic (IMP181) and epidemic (McMillan) strains of WEEV. Salivary gland infection rate was hypothesized to be greater for IMP181 than McMillan. IMP181 was hypothesized to be transmitted at a higher rate compared to McMillan and present in higher viral titers in saliva. The barriers to McMillan infection of salivary glands or transmission were hypothesized to be dose dependent. Increased viral titer of injected McMillan was expected to result in a higher salivary gland infection rate, transmission rate, and amount of virus detected in the saliva. A midgut barrier to infection was circumvented by injection of each virus strain into mosquitoes. There was no significant difference in McMillan and IMP181's ability to infect salivary glands or transmit at 7 and 14 days post infection. IMP181 infection resulted in higher viral titers found in expectorated saliva. The use of chimeric recombinant WEEV also revealed WEEV sequence determinants in the structural coding regions and 3'UTR of IMP181 that enhanced virus titers in expectorated C. tarsalis saliva. The transmission rate and not the salivary gland infection rate were found to be dose dependent after intrathoracic injection with both strains of WEEV.


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Culex tarsalis
in vivo imaging
western equine encephalitis virus


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