Browsing by Author "Olson, Kenneth, advisor"

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Open Access
The use of double-subgenomic Sindbis virus transducing systems as tools for understanding virus-vector interactions
(Colorado State University. Libraries, 2008) Cirimotich, Christopher Michael, author; Olson, Kenneth, advisor
Recombinant Sindbis viruses (SINV) have been developed that express foreign sequence from an engineered viral promoter. These SINVs are valuable tools for studying virus interactions with mosquito vectors. Currently, transducing systems constructed from the genomes of SINV strains TE12 and MRE16 allow researchers to study molecular determinants of virus infection in mosquitoes and manipulate endogenous pathways in multiple invertebrate species. However, many aspects of arbovirus transmission cycles including interaction of the infecting virus with the mosquito immune response and virus transmission to the vertebrate host remain incompletely characterized. Novel double-subgenomic systems were constructed to further study SINV interactions with the Aedes aegypti mosquitoes and develop new tools to analyze aspects of the Sindbis virus transmission cycle. A TE 12-based transducing system has been engineered to express Flock House virus protein B2, a known inhibitor of RNA interference (RNAi). Here I show that RNAi modulates SINV infection of Ae. aegypti mosquitoes and that the virus can become pathogenic when the vector's RNAi response is suppressed. RNAi may be necessary for persistent arbovirus infection of the mosquito vector. The mechanism of virus-induced gene silencing was examined in cell culture using SINVs expressing sequence of an endogenous mosquito gene (lysozyme) in sense or antisense orientation. TE12-based transducing systems can efficiently mediate silencing of lysozyme expression but MRE 16 systems cannot. The MRE 16 system does elicit production of siRNAs, the hallmark of RNAi-mediated silencing, but to a much lesser degree than the equivalent TE12 system. To improve on existing transducing systems, a panel of MRE16 viruses was constructed to express fluorescent and bioluminescent proteins to be used as markers of virus infection. Also, a TE12 virus with potential targeted infection capabilities was engineered. Although the functionality of the targeted virus could not be shown, the range of experiments that can be performed using SINV systems has been expanded. Together, this work provides insight into the interactions between SINV and the mosquito RNAi response, characterizes the use of SINV for RNAi-mediated silencing studies, and has produced viruses that can be used to better understand the molecular mechanisms of SINV infection of and transmission by the mosquito vector.
Open Access
Western equine encephalitis virus: development and application of a new world alphavirus transducing system
(Colorado State University. Libraries, 2012) Stauft, Charles Brandon, author; Olson, Kenneth, advisor; Brennan, Carol, committee member; Foy, Brian, committee member; Bowen, Richard, committee member
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.
Open Access
Western equine encephalitis virus: neuroinvasion, pathogenesis, and immunomodulatory treatment strategies
(Colorado State University. Libraries, 2013) Phillips, Aaron Timothy, author; Olson, Kenneth, advisor; Brennan, Carol Blair, committee member; Aboellail, Tawfik, committee member; Tjalkens, Ronald, committee member
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.