Department of Microbiology, Immunology, and Pathology

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https://hdl.handle.net/10217/100473

These digital collections include theses, dissertations, faculty publications, and datasets from the Department of Microbiology, Immunology, and Pathology. Due to departmental name changes, materials from the following historical department are also included here: Microbiology.

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Open Access
Growth factors, oncogenes, and antioncogenes in the pathogenesis of osteopetrosis and osteosarcoma
(Colorado State University. Libraries, 1991) Rebatchi, Abdelaziz, author; Norrdin, Robert W., advisor; Smith, Ralph, advisor; Pearson, Leonard D., committee member; Collins, James K., committee member; Gasper, Peter, committee member
MAV-2(0) induced avian osteopetrosis is characterized by periosteal bone proliferation in long bones. Comprehensive cellular investigations to detect and understand factors involved in the proliferation of osteopetrosis cells have not been reported. In addition, osteopetrosis has never been shown to evolve to malignancy or neoplasia and this characteristic of the disease has not been studied. To investigate the evolution to neoplasia through the study of gene expression, canine osteosarcoma samples were also included in these investigations. cDNA probes specific for tumor suppressor genes, growth factors and oncogenes, were used to determine the expression of these genes in osteopetrotic, non-inoculated controls, 10 day-old inoculated chickens, and canine osteosarcoma samples. Prior to these investigations, a protocol for bone RNA extraction was developed. In these studies, mRNA specific for Wilms' tumor suppressor gene was detected in osteopetrotic samples. This gene was not expressed in non-infected chicken controls, 10 day-old-inoculated chickens or in canine osteosarcoma samples. The expression of a potent mitogenic factor c-erb B, confirmed the proliferative nature of osteopetrosis. Since osteopetrotic cells display some level of differentiation as opposed to canine osteosarcoma cells which are not differentiated, it is concluded that Wilms' tumor gene acts as a differentiating factor preventing osteoblastic cells from entering the cycle of neoplasia, since the action of Wilms' tumor gene is not antioncogenic. These results indicate that avian osteopetrosis appears to be the result of a concomitant expression of both an oncogene (c-erb B) feeding the proliferative phenotype, and a tumor suppressor gene (Wilms' tumor suppressor gene) that keeps these cells in check. Other supporting results were obtained; platelet-derived endothelial cell growth factor was significantly expressed in both osteopetrotic and osteosarcoma samples, suggesting a common pathogenic aspect of both clinical entities. Bone morphogenic protein-1 (BMP-1) was highly expressed in one sample only. Platelet-derived growth factor was weakly expressed in one osteopetrotic sample. These results suggest a sequel of a previous involvement in the pathogenesis of osteopetrosis and confirm that osteopetrotic cells are at higher stage of differentiation. BMP-2 and BMP-3 were not expressed in this system suggesting that they might be brought to the bone matrix by the circulation, or act at an earlier stage of differentiation. Finally, the observation that 10 day-old inoculated chickens did not show any expression of any mitogenic factor appears to confirm that these chickens do not develop osteopetrosis because their bone cells are differentiated and therefore have a different set of gene regulatory proteins which makes them non-permissive to the proliferative action of the virus.
Open Access
Effects of chemotherapy on innate and adaptive immune responses
(Colorado State University. Libraries, 2007) Biller, Barbara J., author; Dow, Steve, advisor
Chemotherapy has historically been viewed as immunosuppressive; however studies suggest that some chemotherapeutic agents possess immunostimulatory effects. Despite the promise offered by new treatment modalities such as immunotherapy, novel therapies appear most effective when used in combination with traditional treatments such as chemotherapy. Therefore, there is considerable interest in the identification of chemotherapeutics that can be synergistically combined with cancer immunotherapy. Little is known, however, concerning the immunomodulatory effects of chemotherapy on many aspects of immunity.
Open Access
Analyzing the role of the Aedes triseriatus inhibitor of apoptosis 1 gene in transovarial transmission of La Crosse virus
(Colorado State University. Libraries, 2007) Beck, Eric Thomas, author; Beaty, Barry J., advisor
Aedes triseriatus is the primary vector of La Crosse virus (LACV) in North America. The following studies were performed using field collections to elucidate the role of the Ae. triseriatus inhibitor of apoptosis 1 gene (AtIAP1) in conditioning TOT and to compare LACV ovarian titers in field collected mosquitoes with several laboratory Ae. triseriatus strains.
Open Access
The role of midgut serine proteases in Aedes aegypti vector competence
(Colorado State University. Libraries, 2007) Brackney, Douglas Eugene, author; Olson, Kenneth E., advisor
Numerous gut-associated viruses utilize host proteolytic enzymes to facilitate enhancement of infection. Similarly, arboviruses infecting the invertebrate host (vector) through the alimentary tract may exploit serine proteases in the midgut to enhance vector infection. Recent genetic and biochemical experiments have demonstrated that dengue virus type 2 (DENV-2) may require proteolytic processing by midgut trypsins to efficiently infect Aedes aegypti. These results suggest midgut serine proteases may influence A. aegypti vector competence. The requirement of serine proteases in DENV-2 infection of the vector provides unique targets for development of novel control strategies through approaches such as transmission blocking vaccines.
Open Access
Characterization of the enzymes involved in the methylerythritol phosphate pathway with a view to development of broad-spectrum antibiotics including anti-tuberculosis drugs
(Colorado State University. Libraries, 2007) Eoh, Hyung-Jin, author; Brennan, Patrick, advisor; Crick, Dean, advisor
Isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) are precursors of all isoprenoids, many of which play an essential role for the survival of organisms. To date, two separate pathways have been revealed for their biosynthesis. The mevalonate (MVA) pathway is utilized by eukaryotes, algae, archaca-bacteria and some Gram-positive bacteria. Besides the MVA pathway, an alternative route (the rnethylerythritol phosphate pathway; MEP pathway) has been discovered relatively recently. The MEP pathway is utilized exclusively by Gram-negative bacteria, plants and some Gram-positive bacteria. The enzymes in the MEP pathway are considered as potential targets for novel broad-spectrum antibacterial drugs, since they are absent in humans and the disruption of any genes encoding the enzymes in this pathway in E. coli showed lethal phenotypes. The severity of bioterrorist threats has been increased by the emergence of antibiotic-resistant bacilli. An ideal state of preparedness for pending bioterrorist attacks would be achieved by continuous development of novel antibiotics. The Centers for Disease Control and Prevention (CDC) and the National Institute of Allergy and Infectious Diseases (NIAID) have categorized lists of biological diseases/agents based on their potential lethality. Most of the organisms utilize the MEP pathway. Thus, the enzymes in the MEP pathway can provide potential drug targets to overcome drug resistant bacilli. In order to improve the quality of bioterrorism preparedness and tuberculosis control, we have identified and characterized the enzymes in the MEP pathway of the human pathogens; Salmonella typhi, Vibrio cholerae, Burkholderia mallei, and M. tuberculosis. In addition, we developed in vitro high throughput screening (HTS) assays to find specific inhibitors. In the present dissertation, 4-(cytidinc 5'-diphosphate)-2-C-methyl-D-erythritol synthase and 4-(cytidine 5'-diphosphate)-2-C-methyl-D-erythritol kinase were cloned, overexpressed, and purified for the purpose of characterizing the enzymes and developing in vitro HTS assays. In addition, in vitro enzyme assay of M. tuberculosis 1-deoxy-D-xylulose 5-phosphate synthase was optimized and applied to screen specific inhibitors. In vitro HTS assays used in this study are facile, direct, and relatively inexpensive compared to NMR spectroscopy or the HPLC based assays, previously employed for characterizing the orthologs of other organisms. We expect inhibitors screened through the in vitro HTS assays to show broad-spectrum activity. We anticipate the enzymes the MEP pathway studied in this dissertation are potential targets for developing novel broad-spectrum antibiotics and it would open up an entirely new class of antibiotics.
Open Access
Pathogenesis and immunity of rabies virus infection in bats
(Colorado State University. Libraries, 2007) Davis, April, author; Bowen, R. A., advisor; Blair, Carol D., advisor
Rabies is one of the oldest known viral diseases and, with a >99% fatality rate, it is also one of the most deadly. Although a major public health concern, human deaths due to rabies in the developed world are rare. Worldwide, statistics are very different as rabies kills in excess of 50,000 humans per year, most often the result of canine rabies variants. During the 1950's, canine rabies cases began decreasing in the United States as a result of vaccination efforts, followed by a decrease in human rabies cases. Rabies in insectivorous bats was first reported in the US in the 1950's and has now been reported in most North American bat species. Over the last 20 years, 92% of the human rabies cases have been the result of a bat variant. This purpose of this work was to expand our knowledge of rabies variants associated with silver hair bats, Mexican free-tailed bats, and big brown bats. The goal of the first study was to characterize disease progression between two closely related big brown bat rabies variants. The data from this experiment indicated that changes in the rabies virus genome may have profound effects on infectivity and virulence. The second study was designed to determine if rabies virus could be transmitted to bats through the aerosol route. Outbred mice and two species of bats were exposed to three variants of rabies virus by aerosol exposure. Although all bats survived the aerosol exposure, 44% of the mice died of rabies. Following exposure, anti-rabies virus neutralizing antibodies were demonstrated in all bats. Following an intramuscular inoculation of rabies virus six months after the aerosol exposure, the number of seropositive bats that developed rabies was equal to the control bats. The third study examined the dynamics of rabies virus infection in bats from colonies in Texas and Colorado. The ability of healthy wild bats incubating rabies to transmit rabies virus was also examined. Rabies virus antigen was found in 50% of the salivary glands from rabid bats yet infectious rabies virus was isolated from less than 35% of rabid bats. Evaluation of of bats from both the field and in captive colonies demonstrated that approximately 0.5-2% of clinically healthy bats were in the incubation phase of rabies virus infection.
Open Access
Characterization of mosquito densonucleosis virus non-structural protein NS2
(Colorado State University. Libraries, 2008) Azarkh, Yevgeniy "Eugene," author; Carlson, Jonathan, advisor
Mosquito densonucleosis viruses (or mosquito densoviruses) are insect parvoviruses that present practical interest as potential bio-insecticides and theoretical interest as a distinct parvoviral group with unique properties. Non-structural protein NS2 of the mosquito densonucleosis viruses is poorly characterized and bears no sequence similarity to NS2 proteins of other parvoviruses. It was hypothesized that mosquito densovirus NS2 is required for efficient viral propagation in cell cultures and during mosquito host infections, and that direct interactions between NS2 and other viral constituents are likely to play a role in NS2-mediated stages of viral life cycle.
Open Access
Investigations of the evolutionary, epidemic, and maintenance potential of La Crosse virus
(Colorado State University. Libraries, 2008) Reese, Sara M., author; Beaty, Barry J., advisor
Arthropod-borne viruses are resurging and emerging worldwide, and La Crosse virus (LACV) is a prototypical emergent virus in the United States. In this dissertation, the evolutionary, epidemic, and maintenance potential of LACV is investigated. In laboratory and field studies, LACV has shown significant evolutionary and epidemic potential through point mutations and segment reassortment. Through sensitive molecular epidemiological techniques, significant genetic variation was observed in LACV RNA amplified from field-infected Aedes triseriatus mosquitoes, suggesting the potential for frequent segment reassortment of LACV in nature. Maximum parsimony phylogenetic analysis and linkage disequilibrium analysis revealed that 25-38.6% of the mosquito samples contained reassortant viruses. The geographical, environmental and temporal factors that condition the genetic structure of LACV were also investigated. The analysis revealed that there are no physical barriers to viral flow in the study site, indicating that the more virulent LACV strains could traffic and be transmitted throughout the entire 15,360 km2 study range (southeastern Wisconsin, southwestern Minnesota and northeastern Iowa). Although there were no barriers to viral gene flow and no isolation by distance, a significant temporal association with viral genotype was revealed. The maintenance of LACV in nature is not well understood. Mathematical models have revealed that field infection rates are well below those required to maintain the virus in nature. However, the mathematical models have not considered the possibilities of stably-infected Ae. triseriatus mosquitoes or a LACV induced mating advantage for infected females. Super-infected Ae. triseriatus mosquitoes in nature were identified in southeastern Wisconsin and southwestern Minnesota in these studies (0.011% prevalence rate) suggesting that LACV could be maintained in nature through a stabilized infection in a small number of females. LACV maintenance in nature may also be assisted by a mating advantage for Ae. triseriatus females infected with LACV. In this study, LACV transovarially-infected female mosquitoes become inseminated faster than uninfected mosquitoes, and this could increase the chance for transovarial transmission as well as venereal transmission of the virus. The evolutionary and maintenance potential of LACV was investigated in this dissertation and the results provide insight into the determinants of arbovirus emergence and epidemic potential in nature.
Open Access
Diversity and dynamics of rodent-borne Bartonella bacteria are regulated by rodent community and host population parameters
(Colorado State University. Libraries, 2008) Bai, Ying, author; Calisher, Charles H., advisor; Kosoy, Michael Y., advisor
Bartonella infections are widely distributed in rodents of many species with high prevalences and high genetic diversity. Some rodent-associated bartonellae are also associated with human illnesses. After reviewing the current knowledge of bartonellae (Chapter 1), this dissertation focuses on analyses of associations of bartonellae with their rodent hosts in order to understand how dynamics and diversity of bartonellae are regulated by parameters of rodent communities and populations (Chapter 2).
Open Access
Modification of the innate immune response during feline immunodeficiency virus infection
(Colorado State University. Libraries, 2008) Lehman, Tracy L., author; Avery, Paul, advisor; Hoover, Edward, advisor
Lentiviruses such as the human immunodeficiency virus (HIV) and the feline immunodeficiency virus (FIV) have successfully evolved to both use and subvert the host innate and adaptive immune responses to establish long-term infections. Investigation into the mechanisms lentiviruses use to overcome host immune response allows the development of potential therapies and elucidates the intricacies of the immune response. Dendritic cells are professional antigen presenting cells that are intricately involved in innate immune responses and in coordinating the adaptive immune response. However, these same cells have been implicated in initial lentiviral infection, transfer of infection to other cells of the immune system, and alteration of the immune response to allow chronic and progressive infection of the host. To better understand the effects of lentiviral infection on myeloid dendritic cells (mDC), we used the FIV model and bone marrow-derived mDC to evaluate differences in growth, phenotype, and function. We found that chronic FIV infection did not affect mDC growth in culture, phenotype, or maturation as assessed by CD11c, MHC class II, CD80, and CD1a and ability to uptake dextran particles. However, mDC from FIV-infected cats were found to have significantly decreased ability to stimulate proliferation of allogeneic CD4+ T cells in the mixed leukocyte reaction. To begin a mechanistic examination of FIV-induced alteration of mDC function, we examined cytokine responses to Toll-like receptor (TLR) ligands and CD40L. We documented changes in the ratio of the immunoregulatory cytokines IL12 and IL10 in response to select TLR ligands and CD40L, which could result in impaired immune responses, impaired T cell interactions, and enhanced viral survival. Having identified alterations in DC function with FIV infection, we attempted to augment the antiviral effects of mDC by supplementing IL-12 levels in vivo using an adenoviral vector. Consistent with the known complexity of the immune response, increased IL12 levels proved toxic and thereby failed to be a viable means of enhancing the innate immune response to lentiviral infection. Our research documents functional changes induced in bone marrow-derived mDC by chronic FIV infection and provides a means of further investigation into the development, mechanisms, and therapies for those changes.
Open Access
Avian immunity to West Nile virus
(Colorado State University. Libraries, 2008) Nemeth, Nicole M., author; Bowen, R. A., advisor; Spraker, Terry R., advisor
As West Nile virus (WNV) becomes endemic throughout much of North America, it continues to have detrimental effects on countless birds of various taxonomic groups. However, many birds survive infection, mounting an effective immune response. This dissertation focuses on the avian immune response to WNV, including naturally and experimentally-induced antibody duration and passive transfer of immunity. In addition, persistent WNV infection is a potential factor in altering pathogenesis if immunity were to wane. The duration and protection provided by anti-WNV antibodies was documented in house sparrows (Passer domesticus) and raptors for 3-4 years. Antibody levels were relatively stable over time, and protected against viremia in the former and recurrence of clinical disease in the latter. Passive transfer of WNV immunity from hen to eggs and chicks was characterized in domestic chickens (Gallus gallus domesticus). Eggs from both seropositive and seronegative hens were either sacrificed to test for WNV antibody in yolks or chicks artificially inoculated to examine viremic and serologic responses. Concurrently, age-associated differences in response to WNV infection were documented. The passive transfer experiment was repeated in house sparrows to explore this phenomenon in a passerine species; passive transfer was less prevalent in sparrow versus chicken chicks, was of shorter duration, and was less protective. Persistent WNV shedding, viremia, and tissue infection was examined in house sparrows, with juveniles sampled more intensively on a shorter time scale (30-65 days) and adults sampled at 1, 6, 12, 18, and 24 months post-infection. Infectious WNV was isolated from an oral swab, spleen, and kidney of several individuals at 30 DPI, but not from sera after 6 DPI or swabs after 15 DPI. However, WNV was detected in an oral swab by RT-PCR at 44 DPI and was in multiple tissues from most sparrows at 30 DPI, and from kidney and spleen of two individuals at 65 DPI. These findings suggest that WNV infection in tissues may persist beyond the acute stage of infection, while implications for natural transmission and avian health remain unknown.
Open Access
Of mice, genes, and radiation: the genetics of non-hereditary breast cancer explored using the common laboratory BALB/c mouse
(Colorado State University. Libraries, 2008) Ramaiah, Lila, author; Ullrich, Robert L., advisor; Benjamin, Stephen A., advisor
In this dissertation we describe the generation and characterization of two novel strains of mice carrying alternate genetic variants of the DNA repair gene Prkdc (DNA-PKcs). Strains congenic for the common (PrkdcB6) and variant (Prkdc BALB) alleles of Prkdc are developed, genotypically validated, and used to examine the functional consequences of Prkdc BALB and its linkage with radiation susceptibility. DNA-PKcs protein expression, post-irradiation double strand break repair, post-irradiation cell survival, breeding depression, and constitutive and radiation-induced gene expression are examined. By western blot we demonstrate that PrkdcBALB is required and sufficient to decrease DNA-PKcs protein expression. Using three different DSB repair quantification methods we show that PrkdcBALB is required for reduced radiation-induced DSB rejoining in BALB/c. We also show that Prkdc BALB is both sufficient and required for decreased cell survival after exposure to ionizing radiation. Thus we demonstrate that Prkdc BALB modulates and even diminishes the ability of cells to maintain genomic homeostasis. Using our newly developed congenic mice, we present the first evidence that PrkdcBALB has a significant effect on gene expression in unirradiated as well as irradiated mice. Microarray analysis of gene expression reveals that PrkdcBALB may have a greater impact on overall gene expression than does radiation, and that Prkdc may play a role in constitutive and DNA damage-induced apoptotic and transcriptional responses. The results presented within this dissertation support the hypothesis that the main role of PrkdcBALB in radiation-induced breast cancer is the initiation of mammary epithelial cells. Our data show that PrkdcBALB is strongly associated with diminished DNA-PKcs expression and function, diminished survival, and altered transcriptional regulation. The congenic strains developed and characterized in this proposal will be instrumental in ongoing studies aiming to clarify the role of Prkdc and genomic instability in radiation-induced mammary carcinogenesis in the BALB/c mouse. Future studies should endeavor to quantify DNA-PKcs specific kinase activity and protein metabolism and to evaluate cytogenetic instability, with particular emphasis on telomeres. The congenic strains developed and characterized in this work serve as compelling rodent models of sporadic and radiation-induced human breast cancer, and provide proof of principle for the role of genetic polymorphisms and genomic instability in breast cancer susceptibility.
Open Access
Characterization of walleye dermal sarcoma virus Orf B during tumor development
(Colorado State University. Libraries, 2008) Daniels, Candelaria Christina, author; Quackenbush, Sandra L., advisor
Walleye dermal sarcoma virus is a complex retrovirus associated with walleye dermal sarcomas (WDS). These sarcomas develop and regress on a seasonal basis, providing a unique model to study mechanisms of tumor development and regression in vertebrates. WDS is experimentally transmissible to walleye with cell-free, regressing tumor homogenates. During the fall, low levels of spliced accessory gene transcripts, A and B, are present in developing tumors suggesting that their encoded proteins, rv-cyclin and Orf B, may play a role in oncogenesis. Infectious virus and high levels of full-length viral RNA and spliced accessory and env transcripts are expressed during tumor regression, the following spring. The three accessory proteins Orf A (rv-cyclin), Orf B, and Orf C function in tumor development and regression. In explanted tumor and mammalian cells stably expressing the 35kDa Orf B protein, Orf B is localized at the cell periphery in structures similar to focal adhesions and along actin stress fibers. Results from these studies demonstrate Orf B interacts directly or in a complex with several cellular proteins important in signal transduction pathways: receptor for activated C kinase (RACK1), protein kinase C alpha (PKCα), Src, phosphatidylinositol-3-kinase (PI3K), and protein phosphatase 2A (PP2A). The cellular proteins BAD, 90kDa ribosomal S6 kinase (p90RSK), PKCα, and protein kinase B (AKT), which are important in controlling apoptosis and/or proliferation, are activated in Orf B-expressing cells. Orf B protects cells from staurosporine-induced apoptosis and induces cell proliferation of Orf B-expressing cells under serum-deprived conditions suggesting a mechanism of action for tumor development. Expression of Orf B induces transformation of NIH3T3 cells in vitro and a PI3K and mTOR inhibitor prevented transformation, providing the first evidence that Orf B induces a transformed phenotype. The regulation of cell signaling pathways is one way in which viruses induce oncogenesis. Orf B ensures the establishment of dermal sarcoma by activating signal transduction pathways that control cell survival and proliferation such as PKC and Akt.
Open Access
Characterization of epitope-blocking ELISA for differential diagnoses of secondary flavivirus infections
(Colorado State University. Libraries, 2008) Loroño Pino, Maria Alba, author; Beaty, Barry J., advisor
West Nile virus (WNV) and Dengue virus (DENY) infections cause significant public health and animal health problems in countries around the world. Accurate laboratory results and diagnoses are essential elements of effective treatment of patients. On a broader scale, accurate diagnoses are critical for public health officials to select appropriate control and prevention measures. However, accurate diagnoses of WNV and DENY infections are currently complicated in areas where multiple flaviviruses circulate. To address this complication, the dissertation project investigated the ability of WNV-specific monoclonal antibodies to compete actively in binding the epitopes on the NS1 peptides and to distinguish between antibodies induced by different flaviviruses. Developing a test distinguish between antibodies to different flaviviruses would significantly improve differential diagnostic capabilities, and reduce false positive WNV diagnoses for humans and horses potentially infected by other flaviviruses. For the diagnosis of WNV infections in humans, an epitope-blocking enzyme-linked immunosorbent assay (b-ELISA) using the WNV-specific monoclonal antibody (MAb) 3.1112G and the flavivirus-specific MAb 6B6C-1 was evaluated. Sera from patients previously diagnosed with WNV infections and with dengue infections were tested. The WNV-specific b-ELISA was efficacious in diagnosing WNV infections in humans with primary infections. The sensitivity and specificity of this test were 90.8% and 91.9%, respectively. However, in tropical regions where people have experienced multiple flavivirus infections, the use of the b-ELISA for WNV diagnosis is contraindicated, due to the 8o% false positive rate using this protocol. Arrays of synthetic peptides of the non-structural-1 (NS1) and the envelope (E) proteins of WNV were also evaluated as diagnostic reagents for peptide-based ELISA for WNV. All WNV peptides investigated accurately diagnosed WNV infections; however, the WNV NS1-1 peptide was found to be the best peptide to distinguish between recent dengue infections and sera classified as negative for flavivirus infections. Finally, b-ELISA was evaluated for its ability to detect antibodies to WNV in 14 horses sequentially infected with WNV and SLEV, SLEV and WNV, or DENY and WNV. The sensitivity and specificity of b-ELISA for detecting antibodies to WNV were 90.9% and 91.7%, respectively in these test results. B-ELISA was specific for detecting antibodies to WNV.
Open Access
The acquisition of dendritic cell tolerance during malaria infection results in differential T-cell activation
(Colorado State University. Libraries, 2008) Perry, James A., author; Avery, Anne, advisor
Malaria is caused by intracellular protozoan parasites belonging to the genus Plasmodia. These single cell eukaryotes have a complex life cycle requiring both mammalian (and in certain Plasmodium species, avian) and mosquito hosts. Clinical malaria in humans and other animals is the result of red blood cell (RBC) infection. Although infection direcdy destroys erythrocytes, causing anemia, a significant degree of anemia and morbidity is the result of the host immune response. Inflammatory cytokines have been implicated in the pathogenesis of severe malaria anemia (SMA) and cerebral malaria (CM), two diseases that are responsible for most malaria-related morbidity. Therefore, understanding the regulation of host immunity and inflammatory cytokine production during malaria infection will improve our understanding of malaria related illness.
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
Counter-selection markers for allele replacement in Burkholderia pseudomallei
(Colorado State University. Libraries, 2008) López Peláez, Carolina María, author; Schweizer, Herbert P., advisor
Burkholderia pseudomallei is a gram negative bacillus that lives in the soil of tropical regions around the planet and causes melioidosis in humans, a disease endemic in regions of Southeast Asia and Northern Australia. The United States government has classified B. pseudomallei, and its relative Burkholderia mallei, as potential bioterrorism agents. The increased interest in these complex pathogens initiated a quest to better understand the biology of these bacteria at the molecular level. Completed genome sequences of diverse strains have provided a wealth of information that opened new venues for further study. Many genetic tools have been successfully adapted for use in Burkholderia species, but others are yet to be discovered. The ability to introduce unmarked single nucleotide changes or other genetic modifications into the B. pseudomallei genome, by way of the host's natural homologous recombination pathways, has been hampered by the lack of a suitable counter-selection marker that works efficiently in different wild-type strains. Counter-selection markers allow for the positive selection of strains that have lost the marker and other unwanted sequences around them. This dissertation describes the search for a system that allows isolation of unmarked mutations and single nucleotide changes in the B. pseudomallei genome. Two different systems were proven effective and provide alternative options for isolation of allelic mutants of genes of interest. The first method uses a mutated allele of the B. pseudomallei pheS gene. This gene encodes for a subunit of phenylalanine tRNA synthase. A specific PheS mutant protein exhibits relaxed substrate specificity, allowing for incorporation of a toxic chlorinated phenylalanine analog into proteins resulting in death of cells expressing the mutant protein. Counter-selection based on the mutant pheS gene of B. pseudomallei allowed for the creation of amrRAB-oprA deletion mutants of different B. pseudomallei strains. The AmrAB-OprA efflux pump is responsible for intrinsic resistance to aminoglycosides and macrolides in B. pseudomallei. Consequently, efflux pump mutants became sensitive to selected aminoglycosides. Also, as a proof of concept experiment, a clean unmarked purM mutant was created. purM mutants are thiamine and adenine auxotrophs and have been shown to result in a strong attenuation of virulence in a mouse model of melioidosis. A second system based on the I-SceI homing endonuclease of Saccharomyces cerevisiae was also developed. Expression of the endonuclease in cells containing chromosomal I-SceI recognition sites integrated in their chromosomes in place of counter-selection markers via homologous recombination, leads to the selection of isolates that have lost the sites and thus unwanted sequences containing them. This is because I-SceI creates double-strand breaks and promotes recombination between nearby homologous sequences. As a proof of concept experiment this system was also used to create a B. pseudomallei purM mutant. Furthermore, by creating a temperature sensitive fabD mutant due to a point mutation in the fabD gene proved that I-SceI could be used to create point mutations. FabD is an essential enzyme of the bacterial fatty acid biosynthesis pathway. In summary, this report describes the first counter-selection markers that work in wild-type B. pseudomallei strains. Availability of the markers will allow the routine generation of mutants required for studies of the biology and pathogenesis of this understudied pathogen and the related B. mallei.
Open Access
Host cell antigen and T-lymphocyte subset contribution to simian immunodeficiency virus pathogenicity
(Colorado State University. Libraries, 2008) Stump, Debora Shawn, author; VandeWoude, Sue, advisor
The continuity of the host cell plasma membrane and the simian immunodeficiency virus (SIV) envelope at the time of budding results in the incorporation of host membrane antigens. Of these host antigens, major histocompatibility complex class II (MHCII), is abundantly represented on the virion surface. In Chapter 1, the investigation the potential of antibodies specific for MHCII to block viral infection by binding viral envelope MHCII in vitro is presented. Our results did not demonstrate viral neutralization associated with anti-MHCII antibodies but illustrate that viral infectivity is influenced by target cell membrane and immunological signaling characteristics. In Chapter 2 we investigated the utility of alloimmunization of genetically divergent rhesus macaques in eliciting immune responses specific for host cell antigens capable of limiting SIV infectivity in vivo. Our results suggest that alloimmune responses are not sufficient to protect animals from SIV challenge. We were also able to assess differences in response to pathogenic SIV infection in rhesus macaques of Chinese origin (ChRh) compared to Indian origin (InRh) in Chapter 3. ChRh in our study were better able to control viral replication and resist disease progression compared to InRh. Peripheral immunocyte kinetics were evaluated using four color flow cytometry in order to define parameters of the differential immune response. No consistent differences were evident, demonstrating that peripheral immune correlates of viral control and disease progression remain unknown. Natural SIV infection has been identified exclusively in primate species inhabiting continental Africa. Serological evidence of exogenous lentiviral infection has been noted in wild lemurs in Madagascar. In Chapter 4, we investigated evidence of a naturally occurring lentivirus, possibly related to African SIVs, in samples from a captive population of L. catta at the Indianapolis Zoo. We show confirmatory serological reactivity to diverse lentiviral antigens but failed to amplify lentiviral specific sequences using established degenerate primer sets. In total, this work represents investigations that interrogate important aspects of nonhuman primate lentiviral pathogenicity. While results were primarily negative in nature, these studies provide important new information and point to additional studies required that will continue investigations into the complex nature of lentiviral host: virus relationships.
Open Access
Vector and virus interactions: La Crosse encephalitis virus and the mosquito vector Aedes (stegomyia) albopictus
(Colorado State University. Libraries, 2008) Sutherland, Ian W., author; Beaty, Barry J., advisor
La Crosse encephalitis continues to be an important cause of pediatric arboviral encephalitis in the United States. Since 1985, the invasive mosquito vector, Aedes albopictus, has spread across the country and into La Crosse virus endemic regions. As an aggressive, daytime feeder, this vector has the potential to change the epidemiology of La Crosse encephalitis. This study investigated 4 components of the La Crosse virus- Ae. albopictus system: (1) time course of disseminated and filial infection rates (FIR) among recently colonized field strains, (2) anatomic basis of ovarian infection during the 1st gonotrophic cycle, (3) mitochondrial DNA (mtDNA) variation among geographically dispersed populations in the U.S., and (4) development of transovarially susceptible and refractory strains of Ae. albopictus. All geographic strains tested are susceptible to La Crosse virus oral infection and capable of transovarial transmission (TOT). No regional or geographic patterns emerged with respect to dissemination or TOT. 1st gonotrophic cycle vertical transmission was observed at low levels, with an FIR averaging 1%. 2nd gonotrophic cycle FIR averaged approximately 10% and was significantly lower than that of the natural vector, Ae. triseriatus. La Crosse virus antigen (Ag) was detected in ovaries by Day 2 after oral infection and prior to detection in head tissues. Ag was not detected in follicles through Day 7, suggesting vertical transmission. Examination of variation in the ND5 mtDNA marker revealed high levels of homogeneity among U.S. Ae. albopictus populations. Only 2 haplotypes were observed from 16 geographically dispersed states, including Hawaii. Such broad homogeneity could be due to multiple factors, including founder effects and cytoplasmic incompatibility. Ae. albopictus responded poorly to selection based on FIR for the development of susceptible and refractory strains. This study supports prior literature suggesting Ae. Albopictus may be an important secondary vector of La Crosse virus.
Open Access
Tumor-associated macrophage recruitment and regulation of angiogenesis
(Colorado State University. Libraries, 2008) U'Ren, Lance W., author; Dow, Steve W., advisor
Tumors are no longer thought of as purely a mass of transformed cells. A major component of the cellular composition of a tumor is infiltrating immune cells. Macrophages can constitute a large proportion of infiltrating immune cells. In many cases, increased numbers of Tumor-Associated Macrophages (TAMs) can be associated with a poorer prognosis. Utilizing mice which lack a functional type I interferon receptor (IFN-α/βR-/-), we found that endogenous levels of type I IFNs control tumor growth and angiogenesis. We also determined that tumors grown in IFN-α/βR-/- mice have an increase in macrophage infiltrate. In vitro assays suggest that suppression of macrophage responsiveness to CSF-1 by type I IFNs was responsible for the increased macrophage accumulation in tumors of mice unable to respond to type I IFNs. These results indicate that endogenous production of type I IFNs by tumor cells or inflammatory cells may be an important means of suppressing the accumulation of TAMs and their subsequent induction of angiogenesis. The ability of TAMs to produce VEGF is one of the major means by which TAMs are known to induce tumor angiogenesis. Since VEGF expression is in part induced by hypoxia, it has been speculated that the hypoxic tumor environment is responsible for driving TAM VEGF production. As an alternative possibility we suggest that the engulfment of apoptotic tumor cells can stimulate TAM production of VEGF. We determined that the use of Liposome DNA-complex (LDC) therapy can induce anti-tumor immunity through the combined activation of systemic innate and adaptive immune responses. We show that LDC can traffic into macrophages and induce expression of activation markers. In vitro results show that LDC therapy can inhibit the production of VEGF by macrophages after their consumption of apoptotic cells, suggesting that LDC may be an effective way to circumvent the pro-tumor function of TAMs. Additionally, we determined that LDC combined with chemotherapy can be used as a safe and effective immunotherapy for the treatment of canine hemangiosarcoma. Taken together, these findings could uncover new avenues in which TAMs can be targeted and identified a novel immunotherapy as a potential candidate.