Browsing by Author "Bowen, Richard, committee member"
Now showing 1 - 15 of 15
Results Per Page
Sort Options
Item Open Access Bats as reservoir hosts: exploring novel viruses in New World bats(Colorado State University. Libraries, 2018) Malmlov, Ashley, author; Schountz, Tony, advisor; Bowen, Richard, committee member; Dinsmore, Page, committee member; Pabilonia, Kristy, committee memberTo view the abstract, please see the full text of the document.Item Open Access Development and efficacy testing of broad alphavirus vaccines and antivirals and characterization of alphavirus neuroinvasion(Colorado State University. Libraries, 2016) Rico, Amber, author; Olson, Ken, advisor; Ebel, Greg, committee member; Powers, Ann, committee member; Bowen, Richard, committee member; Chen, Chaoping, committee memberAlphaviruses are mosquito-borne pathogens that cause worldwide disease and death in humans and animals. Several alphaviruses are select agents and are a legitimate biosafety and bioweapon concern. Additionally, several alphaviruses are emerging infectious diseases. Climate change and urbanization have expanded mosquito populations and increased human-mosquito interactions within this decade and will continue into future decades. As mosquito populations expand, naïve human populations are exposed to arthropod-borne viruses, including alphaviruses, and vector-borne diseases have surged. The increasing prevalence of arthropod-borne disease has highlighted the global need to develop measures that prevent or treat arthropod-borne disease infection. Currently, vaccines to prevent alphavirus infection are limited to investigational new drug status and no therapeutics are available to treat alphavirus disease. This dissertation will describe projects aimed at preventing or treating alphavirus infection and characterizing the process of alphavirus neuroinvasion. To address the concern of potential outbreaks of an intentional or natural nature, alphavirus vaccines based on the ectodomain of alphavirus E1 were designed and tested. Cationic liposomes complexed with nucleic acid adjuvants and alphavirus E1 protein (lipid-antigen-nucleic acid complexes; LANACs) provided the best platform for alphavirus E1 vaccination. Interestingly, western equine encephalitis virus (WEEV) E1 (LANAC WEEV E1) protected against both mouse WEEV and eastern equine encephalitis virus (EEEV) challenge but not Venezuelan equine encephalitis virus (VEEV); whereas, VEEV E1 (LANAC VEEV E1) protected against both VEEV and EEEV challenge but not WEEV. LANAC VEEV E1 + WEEV E1 vaccination protected mice against EEEV, VEEV, and WEEV challenge. Mice immunized with LANACs (LANAC WEEV E1, LANAC VEEV E1 or LANAC VEEV E1 + WEEV E1) mounted strong humoral immune responses, but were lacking neutralizing antibody. Hamsters immunized with LANAC WEEV E1 failed to mount humoral immune responses and were not protected from challenge. Antibody derived from E1 vaccination binds infected cells and purified E1, but not intact virions. E1 antibody is non-neutralizing yet protective against CHIIKV, EEEV, SINV, VEEV, and WEEV in vitro. In vivo we have demonstrated that antibody is protective against all three new world alphaviruses (NWAs0. Antibody affects late stages of the viral life cycle and likely inhibits virus release or cell death. Following a screen, conducted by our collaborators, of FDA approved and ex-US approved compounds for effectiveness against viral encephalitic and hemorrhagic fever viruses, we tested compounds for anti-alphavirus activity to develop therapeutics to treat alphavirus infection. Yield reduction assays identified four compounds that inhibited virus replication by two to four logs. These compounds were further tested in vitro for mechanism of action. Compounds P-75802, P-75803, P-75805, and P-75811 affected early stages of replication. Compound P-75802 was tested in vivo and was found to possess limited antiviral activity. The process of alphavirus neuroinvasion, particularly following peripheral inoculation, is poorly defined. In the studies described here, encephalitic alphavirus neuroinvasion following EEEV, VEEV, and WEEV intranasal and subcutaneous inoculation were described relying on bioluminescent in vivo and ex vivo imaging, CLARITY imaging, and histopathology. We found that neuroinvasion occurs through two routes dependent on inoculation method. The cranial nerves, particularly the olfactory tract nerve, were involved in neuroinvasion following intranasal inoculation. Neuroinvasion from the periphery, footpad inoculation, occurred via a route independent of the olfactory tract. Neuroinvasion occurred in areas where the blood brain barrier is naturally absent including the hypothalamus, anterioventral third ventricle region (AV3V), area postrema, and the pineal body.Item Open Access Epidemiology, ecology, and evolution of canine influenza virus H3N8 in United States dogs(Colorado State University. Libraries, 2012) Pecoraro, Heidi Lee, author; Landolt, Gabriele, advisor; Blair, Carol, committee member; Bowen, Richard, committee member; Huyvaert, Kathryn, committee memberTo view the abstract, please see the full text of the document.Item Open Access Evaluating the efficacy of a Mycobacterium bovis vaccine in feral swine(Colorado State University. Libraries, 2017) Topliff-Yee, Elizabeth Kathrine, author; Holt, Timothy, advisor; Nol, Pauline, committee member; Bowen, Richard, committee memberBovine tuberculosis (bTB) is a globally significant zoonotic disease caused primarily by Mycobacterium bovis (M. bovis) transmission between wildlife, domestic livestock, and humans. Unfortunately in wildlife reservoirs of bTB, disease rates are increasing worldwide due to ecological dynamics and challenges in wildlife management. Despite effective, long-standing M. bovis eradication programs in the US, expanding wildlife reservoir habitat and importation of people, animals, and products from the Mexican dairy industry have become sources of zoonotic bTB infection. Currently, no tuberculosis vaccine is labeled for use in animals, although a vaccine could provide a new tool in preventing bTB in wildlife and domestic livestock. Bacille Calmette-Guerin (BCG), a live, attenuated M. bovis strain vaccine used for tuberculosis prevention in humans has been variably effective in reducing bTB development in studies on various species. We hypothesize that Texas-origin feral swine vaccinated orally with either modified-live BCG or inactivated M. bovis vaccine will have fewer, less severe lesions than non-vaccinated feral swine after virulent M. bovis challenge. In this study we test this hypothesis along with the immunologic response to vaccination and infection by measuring antibody levels in vaccinated and unvaccinated swine. Our results demonstrate that vaccination with BCG or inactivated strains of M. bovis do not confer protection against infectious challenge with a virulent Michigan strain of M. bovis.Item Open Access Explorations in West Nile virus ecology and evolution(Colorado State University. Libraries, 2021) Byas, Alexandria D., author; Ebel, Gregory D., advisor; Bowen, Richard, committee member; Kading, Rebekah, committee member; Sloan, Daniel, committee memberWest Nile virus (WNV) continues to be a major cause of human arboviral neuroinvasive disease. Susceptible non-human vertebrates are particularly diverse, ranging from commonly affected birds and horses to less commonly affected species such as alligators. The literature review in Chapter 1 summarizes the pathology caused by West Nile virus during natural infections of humans and non-human animals. While the most well-known findings in human infection involve the central nervous system, WNV can also cause significant lesions in the heart, kidneys and eyes. Time has also revealed chronic neurologic sequelae related to prior human WNV infection. Similarly, neurologic disease is a prominent manifestation of WNV infection in most non-human non-host animals. However, in some avian species, which serve as the vertebrate host for WNV maintenance in nature, severe systemic disease can occur, with neurologic, cardiac, intestinal and renal injury leading to death. The pathology seen in experimental animal models of WNV infection and knowledge gains on viral pathogenesis derived from these animal models are also briefly discussed. A gap in the current literature exists regarding the relationship between the neurotropic nature of WNV in vertebrates, virus propagation and transmission in nature. This and other knowledge gaps, and future directions for research into WNV pathology, are addressed. In Chapter 2, experimental evolution work is described. For arboviruses, the vertebrate and invertebrate hosts in which they circulate shape viral evolution and can lead to the emergence of new genotypes. Previous work in mosquitoes and birds has identified species-specific effects on viral populations when species were assessed in isolation. We united mosquito and bird species to perform experimental evolution studies which paired Culex (Cx.) pipiens with American crows, Cx. quinquefasciatus with American crows and Cx. quinquefasciatus with American robins. Crow and Cx. pipiens transmission cycles were the most successful and robin and Cx. quinquefasciatus transmission cycles were the least successful at reaching three complete rounds of bird-to-mosquito transmission. These findings suggest that crows may be more important to WNV maintenance in nature over robins. The greater success of crow cycles when paired with Cx. pipiens in comparison to crows paired with Cx. quinquefasciatus may also suggest fitness losses associated with Cx. quinquefasciatus. In multiple rounds of transmission, infection rates (WNV-positive mosquito midgut) and transmission-capability (WNV-positive mosquito saliva) decreased with each subsequent round of transmission, suggesting that pairings in isolation experience fitness losses. Competitive fitness assays of transmission cycles exhibited cyclical increases and decreases in fitness as virus moved through crows and mosquitoes, respectively. That the stronger competitive fitness tended to occur with samples from the avian host while virus from mosquitoes tended to have decreased fitness may be consistent with genetic restriction and strong purifying selection in birds and genetic expansion and weak purifying selection in mosquitoes. Sequencing is needed to assess whether differences in transmission cycle success and competitive fitness can be attributed to genetic changes. In Chapter 3, the avian single cell viral environment is assessed. Error-prone replication of RNA viruses generates the viral diversity required for adaptation to rapidly changing environments. This is crucial for arboviruses whose viral populations exist as mutant swarms maintained between both mosquito and vertebrate hosts. By infecting cells and birds with barcoded WNV stock and sequencing single cells, we demonstrated that the richness and frequency of rare variants in crows far exceeded that found in robins. Moreover, those rare occurring variants were maintained by crows more than they were by robins. We further demonstrated that bird viremia functions as a determinant of multiplicity of infection in peripheral blood mononuclear cells (PBMCs), a significant site of viral replication. We found that increased viremia leads to increased polyinfections of individual PBMCs with maintenance of defective genomes and less prevalent variants, specifically in crows, presumably through complementation. When two pairings of variably-fit viruses were used to co-infect American robins and American crows, we observed increases in replication for one of the less fit viruses when viremia was higher. The ability of the low fitness virus to better replicate at higher viremia is likely a result of polyinfections and complementation at the cellular level. Our findings suggest that weak purifying selection in highly susceptible crows is attributable to higher viremia, polyinfections and complementation while viral divergence and fewer variants rising to fixation in robins is a result of overall lower levels of viremia and fewer polyinfections. In Chapter 4, the potential contributions of American alligators to natural WNV ecology are examined. West Nile virus (WNV) overwintering is poorly understood and likely multifactorial. Interest in alligators as a potential amplifying host arose when it was shown that they develop viremias theoretically sufficient to infect mosquitoes. We examined potential ways in which alligators may contribute to the natural ecology of WNV. We experimentally demonstrated that alligators are capable of WNV amplification with subsequent mosquito infection and transmission capability, that WNV-infected mosquitoes readily infect alligators and that water can serve as a source of infection for alligators but does not easily serve as in intermediate means for transmission between birds and alligators. These findings indicate potential mechanisms for maintenance of WNV outside of the primary bird-mosquito transmission cycle. We performed a diverse array of experiments which utilize novel techniques and technologies to characterize the mechanisms of WNV evolution. We also identified a potential non-avian WNV amplifier host in alligators. This work represents a significant contribution to the West Nile virus literature by working with the unique species which contribute to virus propagation and assessing their effects on viral evolution and ecology.Item Open Access Ivermectin-treated bird feed to control West Nile virus transmission(Colorado State University. Libraries, 2018) Nguyen, Chilinh, author; Foy, Brian, advisor; Ebel, Greg, committee member; Bowen, Richard, committee member; Huyvaert, Kathryn P., committee memberWest Nile virus is the leading cause of arboviral fever and encephalitis in the United States. The highest WNV disease incidence occurs along the Great Plains region of the United States, as the ecology and land use provide a supportive habitat for the main WNV enzootic and bridge vector of the region, Culex tarsalis. However, due to the lack of dense human population, this area often does not benefit current WNV control measures as applied by conventional mosquito control districts. Based on the ecology of WNV transmission in the Great Plains region, a strategy that targets Cx. tarsalis through its ornithophilic blood feeding behavior could disrupt WNV transmission. Given that the majority of Cx. tarsalis blood meals on the northern Colorado plains may come from doves and passerine species during the WNV transmission season, effective targeting of these or other local preferred hosts with endectocide-treated bird feed could result control of WNV transmission. This study develops and characterizes the effects of IVM-treated bird feed in birds and biting Cx. tarsalis mosquitoes in both a laboratory and field setting. In Chapter 2, the effects of IVM on Cx. tarsalis survival were examined using both in vitro membrane blood meals and direct blood feeding on IVM-treated birds. Chickens and wild Eurasian Collared Doves fed solely on IVM-treated bird feed concentrations up to 200 mg IVM/kg feed exhibited no signs of toxicity, and most Cx. tarsalis that blood fed on these birds died compared to controls. Mosquito survivorship following blood feeding correlated with IVM serum concentrations at the time of blood feeding, which dropped rapidly after the withdrawal of treated feed. These results suggested IVM-treated bird feed should be further explored as a hyper-localized control strategy for WNV transmission. Chapter 3 presents the development of a method to detect and quantify IVM in individual blood meals of Anopheles gambiae and Cx. tarsalis, which will be important in measuring the coverage of this intervention in the field, and accurately assessing IVM's mosquitocidal effects in field situations. This ability to detect IVM in mosquito blood meals was similar between blood fed Cx. tarsalis and An. gambiae, and between sampling times of 0 or 12 hours post blood feed. The quantity of IVM ingested in individual mosquitoes was also compared to the venous serum concentrations of live animals. Chapter 4 presents promising results from two separate pilot field trials of IVM-treated bird feed that were conducted during the summers of 2016 and 2017. Results from 2016 showed that wild birds frequently visit the IVM-treated feeders. In addition, there was an observable trend where "far" traps that are expected to be beyond the zone of control had more WNV-positive pools compared to "near" traps at both ELC and ARDEC South sites. Results from the 2017 study continued to be promising, where birds were again visiting IVM-treated feeders and IVM could be detected in the sera of birds sampled by IVM feeders. There was also a trend of higher VI for the control sites compared to IVM sites for the 2017 season. The efficacy of IVM-treated bird feed was evaluated in two pilot trials where natural WNV transmission cycles occurred in wild birds and Cx. tarsalis, but should be followed up with field seasons with many control and IVM sites to allow for a robust analysis of IVM effects. This study introduces the novel concept of using systemic endectocides for controlling WNV transmission, and this concept could be explored for other arboviruses.Item Open Access Laboratory mouse models for bartonella bacterial infection: bacteremia, host specificity, and pathology(Colorado State University. Libraries, 2011) Colton, Leah, author; Foy, Brian, advisor; Michael, Kosoy, advisor; Gage, Kenneth L., committee member; Bowen, Richard, committee memberBartonella bacterial species are globally distributed in a diverse variety of mammalian reservoir hosts. Natural host infections are generally characterized by persistent bacteremias of long duration, seemingly without adverse host effect, whereas non-natural host infections can produce mild, self-limiting illnesses or more severe disease such as endocarditis. Incidental host infections seem to most closely resemble natural host infections when the taxonomic distance between the two hosts is small. The greater the taxonomic distance between the host of origin and the incidental host, the more likely it seems that the incidental host will either clear the bacteria or develop pathology following exposure. This level of bacterial host specificity has been demonstrated consistently and presents an enormous obstacle to the development of animal models, particularly murine models that reproduce characteristics of natural host infection or pathology consistent with human incidental infections. In this dissertation laboratory mouse models for bartonella infection are described following the introduction and literature review (Chapter 1). Chapter 2 reports infection of mice with bartonella strains from wild Mus species, simulating a cross-species host switch for the bacteria. Infected mice exhibited characteristics consistent with reports of natural rodent host infection. Chapter 3 reports on a mouse infection study using four rat bartonella strains, simulating a cross-genus host switch for the bacteria. Only one of the strains infected mice and alterations in bacteremia duration and magnitude were observed relative to those reported for natural host infections. Mice also displayed organ pathology following bacteremia resolution. Chapter 4 presents a mouse infection study using an Asian house shrew Bartonella elizabethae strain inoculated into three different laboratory mouse stocks. Mice of all three stocks developed bacteremia following bacterial exposures, a demonstration of cross-order host switching by the bacteria. No obvious differences in infection response were observed among the mice despite differences in their genetic backgrounds. Chapter 5 describes inoculation of aged mice with either a mouse bartonella strain or human Bartonella tamiae strains. Mice infected with the mouse strain developed bacteremia, whereas mice infected with B. tamiae did not, consistent with the idea that taxonomic distance between host of origin and incidental host can be a predictor of infection outcome. Chapter 6 details results of a study where aged mice were exposed to three different B. tamiae strains. The mice developed disease consistent with reports of human illness symptomatology. In summary (Chapter 7), these laboratory mouse models are presented as defined, scientific resources for research on Bartonella species host ecology, bacteria: host interactions, and transmission dynamics.Item Open Access Minipigs as a neonatal animal model for TB vaccine efficacy(Colorado State University. Libraries, 2016) Ramos Arriaza, Laylaa, author; Gonzalez-Juarrero, Mercedes, advisor; Bowen, Richard, committee member; Izzo, Angelo, committee member; Guth, Amanda, committee member; Dow, Steven, committee memberCurrently, the only vaccine available to prevent tuberculosis (TB) is Bacillus Calmette-Guerin (BCG). The vaccine lacks efficacy against pulmonary disease or reactivation of latent TB but prevents disseminated TB in children and is thus widely used in countries with endemic TB as part of the neonatal vaccine regimen. There are several new vaccines that have shown efficacy against TB in adult animal models yet fail to protect infants from TB disease in clinical trials. Failure in the development of new pediatric vaccines may be due to incomplete knowledge in the elicited immune response to BCG vaccination and testing of vaccine efficacy in adult rather than neonatal animal models. In this novel approach, we used the mini-pig as a neonatal animal model for evaluation of immune responses to BCG vaccine. We demonstrate young mini-pigs are susceptible hosts to the highly virulent Mycobacterium tuberculosis (Mtb) strain, HN878 and that the pathological course of infection resembles that seen in human TB. In this study we longitudinally monitored the immune response of neonatal mini-piglets vaccinated with BCG until adulthood, with the same monitoring applied to a group of unvaccinated mini-piglets. Further, we challenged both vaccinated and non-vaccinated animals via the aerosol route with HN878 and we characterized important changes between the two groups in the course of immune responses following challenge. Based on comparison of immune responses to BCG in mini-pigs and infants, our findings suggest that mini-pigs have the potential to serve as an effective neonatal animal model for TB vaccine development.Item Open Access Molecular analysis of the genetic determinants that contribute to virulence in lineage 2 West Nile virus(Colorado State University. Libraries, 2017) Romo, Hannah Elizabeth, author; Ebel, Greg, advisor; Brault, Aaron, advisor; Blair, Carol, committee member; Bowen, Richard, committee member; Huyvaert, Kate, committee memberThe ability of arboviruses to impart significant global disease burdens is related to the corresponding capacity of arboviruses to emerge in naïve environments or re-emerge in endemic environments. The introduction of West Nile virus (WNV) into North America was marked by rapid spread across the continent, high rates of neuroinvasive disease in humans and horses, and subsequent displacement by newer evolved genotypes. In the last 12 years, an underrepresented lineage of WNV, lineage 2 (L2) has similarly emerged from sub-Saharan Africa into areas of Europe and Russia, causing widespread neurological disease and recurrent enzootic transmission. Given the potential for further geographic spread of L2 WNV and to understand mechanisms that drive emergence events for WNV, I sought to characterize L2 WNV in a comprehensive and comparative manner by investigating potential molecular mechanisms of pathogenesis in mosquitoes, birds, and mice (as models for human disease). A more thorough understanding of the mechanisms that dictate rapid dispersal and endemic maintenance of arboviruses will improve our ability to predict emergence events, increase the effectiveness of surveillance mechanisms, and develop effective intervention strategies. Within lineage 1 (L1) WNV, the role of the NS3-249P amino acid in modulating severe virogenesis in American Crows (AMCRs) has been well established and is predicted to be involved in facilitating the emergent capacity of L1 WNV. The evolution of a proline at the same NS3-249 locus in L2 WNV was initially observed during the first L2 WNV associated outbreak in Europe. However, no bird mortality was observed during the NS3-249P associated L2 WNV outbreak, and the extent of L2 WNV pathogenesis in birds is unclear. In this aim, I examined the viremia titers and mortality profiles of North American AMCRs and house sparrows following infection with African and European L2 WNV strains with and without amino acid mutations at the NS3-249 locus. Our results demonstrate that L2 WNV strains can elicit severe virogenic and fatal outcomes in AMCRs and HOSPs. Additionally, I found that the NS3-249 locus is modulating AMCR viremia titer outcomes, similar to what has been previously observed for the NS3-249 locus in L1 WNV strains. I also demonstrated the 3' UTR of NS10 reduces viremia titers of AMCRs at later time points. The vast majority of our understanding regarding the vector competence of Culex mosquitoes for WNV originates from studies performed with L1 WNV strains, and as such, little information is available regarding the competency of Culex mosquitoes for L2 WNV. To remediate this, I assessed the vector competence phenotypes of two different North American Culex mosquito species for multiple L2 WNV strains. Our results demonstrate that Culex pipiens and Culex quinquefasciatus mosquitoes can effectively transmit L2 WNV. I also identified a L2 strain harboring an NS3-249P mutation (NS10) that limited infection to the midgut of Culex pipiens mosquitoes. The competence of North American Culex mosquitoes to transmit L2 WNV taken together with the ability of AMCRs and HOSPs to serve as reservoir hosts for L2 WNV demonstrates the capacity for L2 WNV transmission in the Western Hemisphere. Previous studies generated in this dissertation demonstrated that high viral titers in AMCRs were modulated by the NS3-249P mutation in the NS10 L2 WNV strain and that this same strain also generated lower infection rates in Culex pipiens compared to other L2 WNV strains, suggesting that the NS3-249 locus might be involved in concurrently modulating vector competence in Culex pipiens and viral titers in AMCRs. To conclusively determine the role the NS3-249P mutation in facilitating emergence of L2 WNV, I examined the phenotype of NS3-249P and NS3-249H L2 WNV mutations in a transmission cycle inclusive manner. Specifically, I found that the NS3-249P mutation was directly involved in decreasing fitness in Culex pipiens. Furthermore, I found that following infection in mice, the NS3-249 residue did not modulate neuroinvasive disease phenotypes and suggests that the emergence of L2 WNV in Greece was potentially facilitated by increases in force of transmission related to the occurrence of the NS3-249P mutation, rather than the emergence of a more neuroinvasive L2 genotype.Item Open Access Mucosal immunization of mice with a recombinant Salmonella choleraesuis that expresses a multimeric gonadotropin releasing hormone fusion protein(Colorado State University. Libraries, 2011) Kemp, Jeffrey M., author; Graham, James, advisor; Huyvaert, Kathryn P., committee member; Bowen, Richard, committee member; Miller, Lowell, committee member; Rhyan, Jack, committee memberTo view the abstract, please see the full text of the document.Item Open Access Of bats and bugs: characterizing arboviral transmission at the human-wildlife interface(Colorado State University. Libraries, 2021) Fagre, Anna C., author; Kading, Rebekah C., advisor; Bowen, Richard, committee member; Schountz, Tony, committee member; Stenglein, Mark, committee member; Stoner, Kathryn, committee memberTo view the abstract, please see the full text of the document.Item Open Access T cell independent mechanisms for protection against Mycobacterium tuberculosis infection(Colorado State University. Libraries, 2019) Bickett, Thomas, author; Izzo, Angelo, advisor; Dow, Steven, committee member; McLean, Jennifer, committee member; Bowen, Richard, committee member; Argueso, Lucas, committee memberThe live attenuated Mycobacterium bovis strain Bacille Calmette Guérin (BCG) is a potent innate immune stimulator. Innate Immunity provides the host with the ability to immediately respond to invasion by pathogens and can be utilized through the use of molecular adjuvants to trigger specific innate mechanisms leading to adaptive immunity. In the C57BL/6 mouse model of tuberculosis, BCG stimulated immunity causes a significant reduction of M. tuberculosis burden after pulmonary infection. Our studies indicate that BCG induced protection against pulmonary M. tuberculosis through early monocyte recruitment is present as early as 7 days after vaccination. This protection showed longevity, as it did not wane when mice were infected 30 days post vaccination. As BCG induced mycobacterial killing after 7 days, we sought to identify the contribution of different innate immune components to better understand mechanisms required for mycobacterial killing. When BCG was administered through subcutaneous inoculation, we found that there was significant monocyte recruitment in the lungs within 7 days after vaccination. Further studies revealed that killing of mycobacterium is dependent on BCG being viable and is monocyte derived, independent of trained innate immunity, highlighting a novel mechanism for killing M. tuberculosis. With the rise of drug resistant strains of Mycobacterium tuberculosis, new vaccine development is paramount. A better understanding of the BCG vaccine will hopefully lead to the development of a more effective alternative.Item Open Access The role of freshwater snails in the transmission of influenza A viruses(Colorado State University. Libraries, 2011) Oesterle, Paul Thomas, author; Huyvaert, Kathryn P., advisor; Root, J. Jeffrey, advisor; Bowen, Richard, committee member; Myrick, Christopher, committee memberWaterfowl are the natural reservoirs for avian influenza (AI) viruses. Avian influenza virus infections in these birds are generally subclinical, but they shed infectious virus through feces for several days, typically into water. Further, AI viruses can remain infectious in water for weeks. This characteristic enhances transmission of AI viruses among waterfowl because transmission is not constrained by direct contact. The prevalence of AI virus infection in waterfowl populations follows a cyclical pattern; prevalence is highest in the population after the breeding season. Shedding of AI viruses is nearly undetectable in these waterfowl populations by winter, yet the prevalence cycle repeats itself annually. Somehow, AI viruses are reintroduced to host populations. The mechanisms that drive the prevalence patterns observed in waterfowl are likely numerous and complex, but AI viral persistence in water is probably critical. Persistence of AI viruses in water also potentially exposes other organisms to the virus. Aquatic invertebrates, such as snails, are likely exposed to AI viruses while feeding on detritus in aquatic habitats, and gastropods are a common food source for many species of waterfowl. This trophic interaction may potentially serve as an additional route of AI virus transmission and maintenance. In this study, two species of freshwater snails (Physa acuta and P. gyrina) were experimentally exposed to avian influenza virus (H3N8) to determine: 1) whether the snails have cellular receptors capable of binding to AI viruses, 2) whether snails can bioaccumulate AI viruses, 3) how long bioaccumulated AI viruses are maintained and remain infectious in snail tissues, and 4) whether Physa spp. can serve as mechanical vectors of AI viruses. My results indicated that, while Physa spp. snails sequestered infectious AI virus, the duration was short-lived and no transmission occurred. These data suggest that the snail species examined do not directly impact AI virus transmission among waterfowl; however, in the process of feeding on snails, waterfowl may be exposed to AI viruses both via water and ingestion such that other avenues of investigation are warranted.Item Open Access The use of human monoclonal antibodies to study the structure and function of the West Nile virus prM protein(Colorado State University. Libraries, 2012) Calvert, Amanda Elaine, author; Blair, Carol, advisor; Roehrig, John, advisor; Bowen, Richard, committee member; Chisholm, Stephen, committee memberSeveral medically important flaviviruses cause severe disease in humans including West Nile virus (WNV) and dengue viruses (DENVs). No licensed vaccines exist for these viruses, and live-attenuated vaccines may be unsafe in certain populations such as the elderly and immunocompromised. Alternatives to traditional vaccines such as human monoclonal antibodies (hMAbs) would complement prevention and treatment of these diseases. While hMAbs could be used in the prevention and treatment of flavivirus infections, they are also useful tools in expanding our knowledge of the anti-flavivirus human antibody response and the complex antigenic structures of these viruses. In this dissertation production of hybridomas producing hMAbs were attempted, and hMAbs reactive to WNV were used to determine epitopes on the prM protein important in human infection. These epitopes were also studied for their unique involvement in particle secretion and prM presentation. In order to produce hMAbs to flaviviruses a competent human fusion partner cell line, MFP-2, was fused with several different sources of B cells including peripheral blood lymphocytes (PBLs) from people with previous vaccinations or infections with flaviviruses, and splenocytes from humanized mice vaccinated or infected with DENV. hMAbs secreted from hybridomas were able to secrete IgG and IgM antibodies; however, none was specific for anti-flavivirus antibody. Vaccinated and infected humanized mice produced very low and variable levels of virus specific antibody which did not class switch from IgM to IgG even after repeated booster immunizations or secondary infections, a feature consistent with a T-cell independent response. Hybridoma cell lines (2E8, 8G8 and 5G12) producing fully human monoclonal antibodies (hMAbs) specific for the prM protein of WNV were developed using MFP-2 cells and PBLs from a blood donor diagnosed with WNV fever in 2004. Using site-directed mutagenesis of a WNV-like particle (VLP), 4 amino acid residues in the prM protein unique to WNV were identified as important in the binding of these hMAbs to the VLP. Residues V19 and L33 were important amino acids for the binding of all three hMAbs. Mutations at residues T20 and T24 affected the binding of hMAbs 8G8 and 5G12 only. These hMAbs did not significantly protect AG129 interferon-deficient mice or Swiss Webster outbred mice from WNV infection, which was consistent with their inability to neutralize virus infectivity in vitro. While producing mutant WN VLPs to map epitope specificity of these hMAbs, 4 mutations (T20D, K31A, K31V, or K31T) resulted in undetectable VLP secretion from transformed COS-1 cells. K31 mutants formed intracellular prM-E heterodimers; however, these proteins remained in the endoplasmic reticulum, ER-Golgi intermediary compartments and Golgi of transfected cells. The T20D mutation affected glycosylation, heterodimer formation, and WN VLP secretion. When infectious viruses bearing the same mutations were used to infect COS-1 cells, K31 mutant viruses exhibited delayed growth and reduced infectivity compared to WT virus; however, the effect of these mutations on infectious virus was not as dramatic as what was shown in WN VLP. Epitope maps of WN VLP and WNV prM were also different. These results suggest that while mutations in the prM protein can reduce or eliminate secretion of WN VLPs, they have less effect on virus. This difference may be due to the quantity of prM in WN VLPs compared to WNV or to differences in maturation, structure, and symmetry of these particles.Item 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 memberA 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.