Theses and Dissertations

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Open Access
Spatial analyses of vector-borne disease risk for allocation of disease prevention and control resources
(Colorado State University. Libraries, 2009) Winters, Anna M., author; Eisen, Lars, advisor; Moore, Chester G., advisor
Vector-borne diseases are major threats to human health and well-being, inflicting enormous health and economic burden around the world. Meeting the challenge of emerging and resurging vector-borne diseases is a difficult undertaking as vaccines are limited, and future vaccine prospects are slow in coming. This underscores the importance of appropriately applied vector control and disease prevention efforts to decrease morbidity and mortality caused by vector-borne diseases. The objective of this dissertation was, therefore, to investigate spatially and temporally-explicit methods to enhance targeting of vector control and disease prevention efforts. Three diseases were investigated: West Nile virus disease in Colorado, USA; human plague in the West Nile region of Uganda; and dengue in Merida, Mexico. Various methods were explored to present spatial information representing risk of vector-borne diseases. Spatial models were developed indicating the risk of human exposure to WNV in the state of Colorado; and Yersinia pestis, the causative agent of plague, in Uganda. The resulting models provided robust validations indicating that the spatial risk maps contained useful information for disease prevention and control efforts. The final chapter expanded from spatially-explicit methods to also account for the temporal aspect of disease outbreaks or epidemics. This chapter describes the evaluation of the feasibility of an early detection system to identify outbreaks in a timely fashion in Merida, Mexico. The early detection system has the potential to provide Merida public health authorities with a resource to clearly recognize when the current dengue burden is exceeding historical norms and may be applicable and useful in other dengue endemic areas. The chapters composing this dissertation describe the application of cross-cutting methods used to determine spatial risk in different vector-borne disease systems. Furthermore, the methods used are applicable to the current public health situation where new vector-borne diseases are emerging and "old" diseases are resurging, further underscoring the importance of targeting limited public health resources.
Open Access
Drug efflux systems and antibiotic resistance in Burkholderia pseudomallei
(Colorado State University. Libraries, 2009) Trunck, Lily, author; Schweizer, Herbert P., advisor
Burkholderia pseudomallei, the etiologic agent of melioidosis, is intrinsically resistant to most antibiotics. A predicted 10 RND efflux transporters are encoded by the B. pseudomallei genome; 3 have been characterized (AmrAB-OprA, BpeAB-OprB, and BpeEF-OprC) as major contributors to the intrinsic aminoglycoside, macrolide, chloramphenicol, and trimethoprim resistance of this organism. AmrAB-OprA is constitutively expressed in most strains and confers resistance to aminoglycosides. Gentamicin susceptible clinical isolates have been identified and work in this dissertation demonstrates that such susceptibility occurs as a result of either insufficient expression or deletion of amrAB-oprA. The mechanisms regulating expression in these strains are unclear, but are not related to mutations in this operon's putative repressor (AmrR) or mutations in the regulatory regions of amrAB-oprA. Expression analysis of seven B. pseudomallei RND efflux pumps (amrB, bpeB, bpeF, bpeH, BPSL0309, BPSL1267, and BPSL1567) in 60 clinical and environmental B. pseudomallei isolates from Thailand demonstrated (i) efflux pump expression is prevalent in both clinical and environmental strains (ii) bpeH is expressed at a higher level in clinical isolates as compared to environmental isolates and (iii) efflux pump expression correlates with resistance/susceptibility to several antibiotics. These data suggest possible substrates for uncharacterized efflux pumps or, alternatively, co-regulation of resistance determinants. Since a deficit in efflux resulted in susceptibility to otherwise clinically useful drugs and that expression of efflux pumps was prevalent in B. pseudomallei, we suggest that efflux pump inhibitors would broaden the spectrum of antibiotics useful for treatment of melioidosis. To facilitate discovery and characterization of such compounds, we have developed a panel of efflux deficient B. thailandensis strains that express the B. pseudomallei efflux pumps, amrAB-oprA and bpeAB-oprB, which can be handled under BSL2 conditions. When expressed in the surrogate background, these pumps have the same substrate profile and can be inhibited by clinically insignificant efflux pump inhibitors to a degree similar to that observed in the native background. In summary, we have assessed the contribution of efflux to antibiotic resistance in B. pseudomallei and described a tool for discovery and characterization of efflux pump inhibitors for pumps expressed in this organism.
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.
Open Access
Investigating molecular determinants of FIV pathogenesis
(Colorado State University. Libraries, 2009) Thompson, Jesse Alan, author; VandeWoude, Susan, advisor
Five FIV clades have been identified and are distinguished by envelope sequence. Two isolates, FIV-PPR and FIV-CPG (molecular clone FIV-C36), belonging to clades A and C, respectively, are variable with regard to disease potential. Chimeric viruses constructed between phenotypically distinct strains of FIV are potentially useful tools to identify molecular determinants of virulence. Several chimeric constructs were therefore developed by exchanging elements between FIV-C36 and FIV-PPR. FIV-PCenv and FIV-PC3'LTR were two resulting chimeras that were capable of competent in vitro replication. Studies in Chapter One aimed to characterize these viruses in the domestic cat model and to test the hypothesis that elements surrounding the env region contribute to in vivo pathology. FIV-PC3'LTR, containing FIV-C36 rev2 and 3' LTR was infectious, although attenuated compared to parental constructs. FIV-PCenv, containing FIV-C36 vif, otfA, env, and the first exon of rev, displayed a phenotype intermediate to parental viruses with regard to replication kinetics, and CD4+ T-cell and neutrophil declines, but peak viral load and development of clinical disease was delayed by three weeks compared to FIV-PPR, FIV-PC3'LTR, and FIV-C36. Studies in Chapter Two evaluate potential mechanisms for the delayed phenotype of FIV-PCenv. To test the hypothesis that delayed kinetics observed during FIV-PCenv infections was due to adaptation of the construct during first round infections, pooled plasma from the in vivo study described in Chapter One was used to inoculate a second cohort of cats. Passaged FIV-PCenv again displayed intermediate phenotype in terms of viral replication and immunopathology, but onset of acute viremia was no longer delayed. To further pinpoint particular genes that contribute to FIV pathogenesis, three additional chimeras were generated using PCR-driven overlap extension as described in Chapter Three. Overlapping-PCR was utilized to produce chimeras with specifically substituted ORF genes encoding the FIV-C36 regulatory proteins Vif and OrfA for those from FIV-PPR; chimeras FIV-PCvif, FIV-PCvif/orfA, and FIV-PCorfA were successfully constructed. Upon infection of the feline MYA-1 T-cell line, all three chimeras produced measurable RT activity. Further, in vitro analysis of FIV-PCvif/orfA demonstrated that this construct had replication properties equal to those of FIV-C36 as measured by viral capsid and RNA genome production.
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
Characterization of the mannosyltransferase RV3779, which forms polyprenyl phosphomannose for the biosynthesis of phosphatidylinositol mannoside and lipoarabinomannan, and elucidation of the polymerization stages of galactan biosynthesis in mycobacteria
(Colorado State University. Libraries, 2008) Scherman, Hataichanok, author; Brennan, Patrick J., advisor
The cell wall complex of mycobacteria, dominated by highly unique structural polysaccharides and a rather impermeable layer of lipids, can give these bacteria the ability to resist the immune response and cause a prolonged and deadly illness. Various lipoglycans and glycolipids in the cell wall complex of mycobacteria, such as lipoarabinomannan (LAM), as well as the synthetic precursors of LAM; lipomannan, and the phosphatidyl myo-inositol mannosides, are essential for the normal growth and viability of mycobacteria, although the specifics of their synthesis are still not fully defined. Their synthesis are governed by a myriad of enzymes, including a class of enzymes called glycosyltransferases, which are one of the most diverse and important groups of enzymes in nature. A particular glycosyltransferase, Rv3779, is the primary focus of this study, and from sequence and bioinformatic analysis, we identified it as a putative mannosyltransferase belonging to the GT-C superfamily found to be involved in various aspects of synthesis of the higher forms of the phosphatidyl myo-inositol mannosides and the subsequent lipoglycans. Rv3779 is present in a prominent gene cluster involved in cell wall biosynthesis. From the experimental evidence gathered from construction and analysis of a Rv3779 knockout mutant of M. tuberculosis, the in vitro assay of the membrane fractions of an Rv3779-overexpressed strain of M. smegmatis, and analysis of the reaction products by thin layer chromatography and mass spectrometry, we have concluded that Rv3779 possesses polyprenyl phosphomannose synthase activity in a homologous manner to Ppm1. Polyprenyl phosphomannose is the key mannosyl donor that is utilized heavily by later enzymes in the extracytoplasmic hypermannosylation of the higher forms of these lipoglycans. The galactan chain serves as the covalent attachment point for mycolated arabinan and is a central structural polymer in the cell wall complex that is the second focus of this study. The exact stages of the buildup galactan are not fully elucidated, and involve unique polymerization steps. We have utilized various organic extraction and analytical techniques from a cell-free assay utilizing UDP-D-galactofuranose as a donor and the results of these studies suggest that the galactan chain is polymerized one galactofuranosyl residue at a time.
Open Access
Molecular epidemiology of leprosy in Cebu, Philippines and essentiality and characterization of an arabinosyltransferase involved in the cell wall synthesis of mycobacteria
(Colorado State University. Libraries, 2009) Sakamuri, Rama Murthy, author; Vissa, Varalakshmi D., advisor
Leprosy is a chronic granulomatous infectious disease caused by Mycobacterium leprae, which affects the skin, peripheral nerves and mucous membranes. Approximately a quarter million new cases are detected annually throughout the world. Since M. leprae is an obligate intracellular bacterium, not cultivable under in vitro conditions, and with a long incubation time, the causes for continued prevalence in endemic countries are still unknown. In this regard, molecular tools for differentiation of isolates of M. leprae are needed to track and control transmission of leprosy. Genome sequencing of M. leprae facilitated the identification of genetic markers such as variable number of tandem repeat (VNTR) and single nucleotide polymorphic (SNP) loci. We standardized methods for DNA extraction from the clinical samples and also developed rapid, high throughput and inexpensive methods like multiplex-PCR and fragment length analysis (FLA) for VNTR analysis; polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for SNPs to facilitate molecular strain typing of clinical samples. We have applied these methods on the DNA extracts of stored and new samples obtained from leprosy patients from Leonard Wood memorial (LWM), Cebu, Philippines. We compiled a database containing epidemiological information from over 200 leprosy patients. VNTR alleles were found to be highly concordant when obtained from biopsies and slit skin smears (SSS) from the same patients indicating that either can be used for strain typing. Based on parsimony cluster analysis of 207 clinical isolates, five major groups were observed. VNTR markers alone were able to differentiate the SNP type 3 isolates from the majority of the SNP type 1 isolates. VNTR profiles of the M. leprae isolates within the multicase families (MCFs) were similar indicating common source of transmission. In a continuation study, VNTR data was obtained for another seventy samples from newly diagnosed leprosy patients from LWM, Cebu, Philippines. Multiplex PCR and FLA methods were found to be more efficient and accurate compared to short tandem repeat (STR) amplification and DNA sequencing. Intra-patient VNTR variability using different SSS was found to be minimal. Population structure of M. leprae isolates from Cebu remained similar with stable clusters in the phylogentic tree, even with the inclusion of 70 additional samples. Drug resistance is a cause for concern in the treatment and control of any infectious disease. Drug resistance in leprosy was known to be caused during the dapsone monotherapy control program and patients' irregular treatment. WHO MDT which includes anti-leprosy drugs such as dapsone, rifampicin and clofazimine was introduced in 1982. However drug resistance was reported even in the areas where the MDT was implemented successfully. So to evaluate the primary drug resistance in newly diagnosed leprosy patients who are being treated with the standardized MDT regimen, we developed a multiplex PCR for amplifying the drug resistance determining regions (DRDR) in genes rpoB, folP1, gyrA and gyrB, which are targets of the drugs rifampicin, dapsone and oflaxacin, respectively. No mutations were detected in rpoB and folP1 DRDRs for 77 samples and in the gyrA DRDR for 15 samples tested thus far. A separate chapter describes the studies on the cell wall biosynthesis. The cell envelope of mycobacteria are comprised of a core macromolecule known as mycolyl arabinogalactan (mAG) which is covalently attached to the peptidoglycan layer forming the mAGP complex and other macromolecules such as lipomannan and lipoarabinomannan. The mAGP complex is essential for survival of the bacteria and is a permeability barrier. The synthesis and assembly of this mAGP complex is still not fully understood. In this regard, through comparative genome approaches we identified Rv3805c as a putative glycosyltransferase located in the cell wall synthesis gene cluster. Rv3805c predicted to have ten transmembrane domains, contains a conserved glycosyltransferase and proline rich motif also present in Emb proteins which are involved in cell wall biosynthesis. In this study we identified that, Rv3805c encodes an arabinosyltransferase through an in vitro arabinosyltransferase assay. Furthermore, a knock out mutant of an ortholog gene NCgl2780 was generated in C. glutamicum. The structural analysis of AG of NCgl2780 mutant in C. glutamicum revealed the loss of β(1→2) Araf residues at the non-reducing ends. We also indentified that the ortholog MSMEG_6400 is essential for M. smegmatis, as we were unable to obtain a genetic knock out mutant in the absence of an additional functional copy of the gene.
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
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
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
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
Proteomic and biological diversity of closely related clinical isolates of Mycobacterium tuberculosis
(Colorado State University. Libraries, 2009) Mehaffy, Martha Carolina, author; Dobos, Karen, advisor
Recent studies have indicated that some Mycobacterium tuberculosis {Mtb) strains may be more easily transmitted and successful in causing disease. While this phenotypic diversity exits and is well documented, protein variation between Mtb strains appears to be small. Historically, the analysis of virulence determinants in Mtb has focused on the comparison of virulent to avirulent Mtb strains as well as on the comparison of single gene knock-out mutants to Mtb reference strains. Biological and biochemical comparison of Mtb clinical isolates with different degrees of virulence, although more complex than those described above, might provide additional information regarding the natural phenotypic variability that exists in Mtb. The focus of this study was to determine if a group of closely related clinical isolates of Mtb (BE, C28 and H6) as well as the more distantly related Mtb CDC 1551 present differences in both, virulence in the guinea pig model of TB infection and in the protein expression profiles of secreted and cytosolic fractions. The three closely related Mtb isolates comprise the S75 group, which has been reported to be very successful in causing disease. In contrast, Mtb CDC 1551, although reported to be highly transmissible, is associated with only a few number of active TB cases. To test the hypothesis that closely related Mtb strains would have similar virulence between each other, but higher virulence when compared to Mtb CDC 1551, guinea pigs were infected with each Mtb strain and time-to-death analysis was performed. Proteomes of secreted and cytosolic fractions of these strains were also analyzed and compared by 2D-GE and isobaric tag labeling (iTRAQ) followed by tandem mass spectrometry in order to test whether protein levels of these strains would present statistically significant differences when compared to each other and when compared to Mtb CDC1551.
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
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
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
In vitro amplification and enhanced trans-species transmission of chronic wasting disease prions
(Colorado State University. Libraries, 2009) Kurt, Timothy Daniel, author; Hoover, Edward A., advisor; Wilusz, Jeffrey, advisor
Chronic wasting disease (CWD) is a prion disease of deer, elk and moose that is spreading rapidly in North America. Like all prion diseases, CWD is associated with conversion of a normal protein, PrPC, to a protease-resistant conformer, PrPRES (or PrPCWD). Little is known about the mechanisms of prion conversion or how it could lead to the rapid spread of CWD among cervids in nature. In this dissertation, I demonstrate the in vitro conversion of PrPC to PrPCWD via two protocols: non-denaturing amplification and serial protein misfolding cyclic amplification (sPMCA). Serial PMCA using brain substrate from transgenic mice that express cervid PrPC [Tg(CerPrP)1536 mice] produced PrPCWD amplification of >6.5 x 109-fold after six rounds. Efficient in vitro amplification of PrPCWD is a significant step toward potential ante-mortem detection of PrPCWD in CWD-infected animals. Whether CWD presents a threat to non-cervid species is not known. To predict non-cervid susceptibility to CWD, I used sPMCA to amplify PrP CWD in normal brain substrates from several non-cervid species. I show that brain homogenates from several CWD-susceptible species, such as ferrets and hamsters, support amplification of PrPCWD by sPMCA, whereas brain homogenates from CWD-resistant species, such as laboratory mice and transgenic mice expressing human PrPC, do not. Three common rodent species (including prairie voles and field mice) that share the environment with infected cervids supported PrPCWD amplification, whereas several other species (including prairie dogs, cats and coyotes) did not. Analysis of PrP sequences suggests that ability to support amplification of PrPCWD in trans-species sPMCA correlates with the presence of asparagine at position 170 of the substrate species PrP. I then inoculated CWD from deer into prairie voles (Microtus ochrogaster) and found they are somewhat susceptible to CWD. Inoculation of prairie voles with trans-species sPMCA products resulted in shorter and more consistent incubation periods. Furthermore, immunohistochemical analysis revealed an altered pattern of CWD prion deposition in infected voles compared to infected Tg(CerPrP)1536 mice, suggesting a different CWD strain. These results indicate that sPMCA can be used to increase PrPCWD detection sensitivity, predict susceptibility to CWD, accelerate adaptation in non-cervid species, and create new strains of CWD.
Open Access
Analysis of T cell subsets induced in response to Mycobacterium tuberculosis infection
(Colorado State University. Libraries, 2009) Henao-Tamayo, Marcela I., author; Orme, Ian, advisor
Tuberculosis, an ancient disease, still kills more people each year than does any other bacterial infection. The global epidemic of tuberculosis (TB) results in eight million new tuberculosis cases per year and two million deaths; 98% of these occur in developing countries. At present the only available vaccine against tuberculosis, M. bovis Bacillus Calmette-Guerin (BCG), has proven unreliable and only minimally protects against pulmonary tuberculosis in adults. The reasons why the BCG vaccine is not fully protective are still very unclear, and even though novel vaccines are being developed, there is not a clear understanding of what kind of immune response they should elicit in order to provide maximum protection. The overall aim of these studies, therefore, was to investigate the T cell subsets generated by M. tuberculosis [including clinical strains] as well as further analysis of those generated by BCG, in order to explore their role in protection against M. tuberculosis infection.
Open Access
The role of cellular RNA decay pathways in Sindbis virus infection
(Colorado State University. Libraries, 2009) Garneau, Nicole L., author; Wilusz, Jeffrey, advisor; Wilusz, Carol, advisor
Sindbis virus is the prototypic species of the Alphavirus genus. Members of this genus can cause febrile illness, arthritic pain and potentially fatal encephalitis. The alphaviral lifecycle generates single-stranded, positive-sense genomic and subgenomic RNAs which are capped on the 5' terminus, contain 5' and 3' untranslated regions (UTRs), and are polyadenylated at the 3' terminus. These characteristics make alphaviral RNAs similar in structure to cellular mRNAs. Such features allow alphaviruses, such as Sindbis, to benefit from the host cell translation process; however, they also could make the viral transcript vulnerable to the cellular mRNA decay enzymes. mRNA decay is a form of post-transcriptional regulation of gene expression found in both mammalian and mosquito hosts of Sindbis virus. The interaction between Sindbis viral RNAs and mRNA decay pathways was investigated in this dissertation. Using a novel in vivo viral RNA decay assay to accurately assess the rate of alphavirus RNA decay during infection, we found a correlation between Sindbis viral RNA stability and viral replication efficiency, demonstrating mRNA decay potentially represents a novel host cell restriction factor. We established that the RNAi pathway likely plays a dominant role in the decay of the viral RNAs during infection in mammalian cells. These data represent a novel demonstration that the RNAi pathway is potentially an effective antiviral response in the mammalian host as it is in the mosquito host. With the development of a highly sensitive method to assess poly(A) tail length, we were able to demonstrate the importance of the viral 3'UTR as a repressor of deadenylation of viral RNAs in vivo. Lastly, we found that Repeat Sequence Element 3 (RSE 3), the third and final in a series of three RSEs within the viral 3'UTR, hinders the processivity of the cellular deadenylases on viral RNAs in vitro, providing the first evidence for a function of this conserved alphaviral genome element. Taken together, these results shed light on the much understudied area of viral RNA decay. Our data support the notion that the interaction between viral RNAs and the cellular RNA decay machinery is very important to the biology of the virus.
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.