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Browsing by Author "Beaty, Barry J., advisor"

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    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.
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    Characterization of epitope-blocking ELISA for differential diagnoses of secondary flavivirus infections
    (Colorado State University. Libraries, 2008) LoroƱo 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.
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    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.
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    Metabolomics-based diagnosis and prognosis of dengue virus infections and NS1 antigen detection for diagnosis and surveillance in humans and mosquitoes
    (Colorado State University. Libraries, 2013) Voge, Natalia Victoria, author; Blair, Carol D., advisor; Beaty, Barry J., advisor; LoroƱo-Pino, Maria A., committee member; Eisen, Lars, committee member; Belisle, John T., committee member; Prenni, Jessica E., committee member
    Dengue (DEN) is a mosquito-borne viral disease of significant public health importance. There are currently no commercialized vaccines or accepted pharmacological treatments for DEN disease, making mosquito surveillance critical for the prevention of outbreaks of this disease. Aedes aegypti is the principal vector of DENV, although Aedes albopictus and other Aedes species have been reported to be able to transmit it. Improved surveillance methods for DENV in mosquito populations would be of great value for public health and vector control programs and would provide better risk assessment of potential DENV infections in humans. Improved mosquito-based surveillance would also improve vector control programs by targeting areas at higher risk for ongoing or potential epidemics for vector control. Non-structural protein 1 (NS1) detection by ELISA is a commercially available test with the ability to detect DENV NS1 protein in DENV infected samples. Studies were conducted to determine the ability of the NS1 antigen test to detect DENV in Aedes aegypti mosquitoes. The NS1 antigen detection test proved to be highly sensitive and specific for DENV-antigen detection in pools of mixed infected and non-infected mosquitoes under various field-simulated conditions and in different sizes of mosquito pools. This test could facilitate mosquito-based surveillance for early warning of DEN outbreaks. The capacity of this test to diagnose human DENV infections in non-invasive clinical specimens, i.e., urine and saliva, was also investigated. NS1 protein detection in acute phase, non-invasive clinical specimens was found to be less sensitive than NS1 detection in serum samples. Most dengue virus (DENV) infections are subclinical. The clinical manifestations of apparent infections range from DEN fever (DF), typically a self-limiting illness with fever and rash, to severe DEN hemorrhagic fever and shock syndrome (DHF/DSS). Upon presentation, there is no way to predict whether or not the patient will experience DF or will progress to the severe form of disease (DHF/DSS). A sensitive and specific test that utilizes acute phase clinical specimens and that provides diagnosis of DENV infection as well prognosis of progression to severe disease is sorely needed. Such a test would permit identifying those patients destined for severe disease for appropriate patient management and therapeutic intervention. An innovative metabolomics platform was used in these studies to determine a biosignature of small molecular biomarkers (SMBs) that can potentially differentiate patients with the most severe forms of disease from DF and non-DEN patients. SMBs that could potentially be diagnostic of DENV infections and prognostic of disease outcomes were identified in acute phase serum, saliva, and urine specimens obtained from DEN and non-DEN patients from Mexico and Nicaragua. Using acute phase serum specimens, a panel of six candidate SMB compounds was identified by tandem liquid chromatography-mass spectrometry (LC-MS/MS); five of these six biomarkers differentiated DF patients from those with DHF/DSS and have potential to diagnose and predict DEN disease or DEN severity (DHF/DSS) outcomes. Two candidate SMB compounds were identified in urine and one in saliva that could potentially be used for diagnosis and prognosis of DEN infections. Tandem mass spectra of candidate compounds and commercial standards were obtained and compared to identify the SMB metabolites. Some of the SMB identities were confirmed by using the NIST (National Institute of Standards and Technology) or METLIN spectrum libraries. The biochemical nature of the identified metabolites included phospholipids, fatty acids, amino acids, nucleosides, and vitamin D. These SMBs have potentially important roles in DEN pathogenesis and in endothelial cell metabolism (these cells are among the principal target cells affected during severe DEN disease). The immediate goal of this dissertation research was to identify a biosignature panel of LC-MS/MS-identified candidate SMB metabolites that differentiate the DEN disease diagnosis groups (DHF/DSS, DF, and ND) and that have potential for diagnosis and prognosis of DENV infections using acute phase serum and non-invasive clinical specimens. However, it is unlikely that LC-MS/MS technology will be applicable in the front-line clinics where DEN patients first present. Thus a long-term goal of the research project is to select a subset of these pathogenically and physiologically relevant SMBs and then determine the potential for the metabolite analyte or a surrogate (e.g., a protein involved in the metabolic pathway conditioning the metabolic change) to be incorporated into diagnostic formats amenable to point of care tests (POC), such as ELISA based formats. A diagnostic algorithm incorporating results from such a POC test and conventional laboratory and clinical biomarkers could provide dramatically improved capability for diagnosis and prognosis of DENV infections and would be of immense value to physicians in managing patients.
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    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.