Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 84-100).

Thesis (Ph.D.) - University of Queensland, 2004. / Includes bibliography.

Thesis (Ph. D.)--Ohio State University, 2005. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2008 Nov 30

Techn. Hochsch., Diss., 2005--Aachen.

Thesis (Ph.D.)--Baylor University, 2009. / Includes bibliographical references (p. 107-129).

Numerous toxins and pathogens gain entry into mammalian cells using cell surface glycans. The Iyer group at Georgia State University is working on the development of glycoconjugates for the accurate detection of infectious agents. In this thesis, I have focused on the development of glycans to detect influenza virus and norovirus. In the first section, I have focused on influenza viruses. A panel of synthetic glycans was synthesized as receptor mimics for the specific capture of influenza viruses. The synthetic glycans were printed onto commercial glass slides using a free amine at the end of a spacer to generate a small focused microarray. This glycan printed microarray was evaluated for its ability to capture three strains of influenza viruses. The analytical limit of detection is ~10 pfu/ml, (plaque forming units/milliliter) which is clinical relevant as 102 viral particles are typically required to cause infection. We also tested the drug susceptibility of current antivirals, Zanamivir and Ostelamivir using the microarray and determined the feasibility of this system to determine antiviral resistance for different strains. In addition to optical detection, I developed an electrochemical assay to rapidly detect influenza viruses. Here, we utilized an unique property of influenza viral surface enzyme, Neuraminidase (NA), which cleaves terminal N-Acetyl Neuraminic acid (sialic acid) from cell surfaces and proteins. We designed an electrochemical assay that uses glucose bearing sialic acid substrates. Glucose is released when exposed to viral NA or intact viruses. The released glucose can be detected using repurposed glucose meters. Thus, personal glucose meters that were designed to assist diabetics and prediabetics monitor blood glucose can potentially be used to detect pathogens. Using this approach, we have detected 19 unique strains of influenza viruses. We also demonstrated drug susceptibility using this assay. The limit of detection of this assay is 102 pfu/sample, which is clinically relevant. The results were validated plaque assays and polymerase chain reaction (PCR). In the second part of this thesis, I focused on norovirus detection. I developed a focused glycan microarray that comprised of a library of histo blood group antigens (HBGAs). The HBGAs were attached to a carrier protein and printed onto activated glass slides. A panel of norovirus virus like particles (VLPs) and strains that included different genogroups was exposed to the microarray. We found that different VLPs and strains give rise to unique binding patterns. When the binding pattern of VLPs for a particular strain were compared to the corresponding intact virus, the binding patterns didn't match well, presumably because the virus does not recognize the same antibody as the VLPs. Unfortunately, antibodies for the virus cannot be generated because the virus cannot be grown in a laboratory setting. Indeed, all norovirus samples are obtained from human challenge studies. I also used surface plasmon resonance (SPR) studies in an effort to determine the binding affinities. Divalent biotinylated H type glycans were synthesized and their binding affinities with different VLPs and viral strains were determined. Initial studies suggest that the binding affinities are strain specific. These results demonstrate that glycans can be used to capture and isolate norovirus, although more research is required to develop glycan based norovirus detection kits.

Made available in DSpace on 2014-06-11T19:32:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-08-10Bitstream added on 2014-06-13T19:22:41Z : No. of bitstreams: 1 morais_ats_dr_sjrp.pdf: 1276143 bytes, checksum: 62a89d8b555ac92b5faf4baa19e4db2f (MD5) / O vírus da Febre Amarela (YFV) pertence ao gênero Flavivirus e causa uma importante doença. Nos últimos anos, uma alarmante ressurgência da circulação viral e expansão do vírus em áreas endêmicas têm sido detectadas na África e América do Sul. NS5 é uma proteína viral não estrutural com duas atividades essenciais para a replicação viral, uma de metiltransferase e outra de RNA Polimerase dependente de RNA (RdRp). Para o melhor entendimento dos mecanismos de replicação viral, interações entre NS5 e proteínas celulares têm sido amplamente estudadas. Assim, os objetivos desse estudo foram caracterizar a interação da proteína NS5 e eIF3L, avaliar a função de eIF3L na replicação do vírus da febre amarela, e caracterizar estruturalmente a proteína eIF3L. Métodos. Para identificar a interação de NS5 YFV com eIF3L, foi realizado ensaios em sistema duplo-híbrido usando RdRp NS5 YFV contra eIF3L. Para o mapeamento da interação, foram construídos mutantes deletantes de RNApol e analisados em sistema duplo-híbrido. A região de interação de RNApol foi segmentada em três fragmentos e analisada na presença de eIF3L. Para mapear os resíduos de NS5 críticos para a interação, foi realizada mutagênese sítio-dirigida no segmento 3 de ID. A interação foi analisada em ensaios in vitro e em cultura de células de mamíferos. A significância de eIF3L para a replicação do YFV foi investigada usando superexpressão de eIF3L em células BHK21-RepYF17D LucNeoIres. A proteína eIF3L foi purificada usando uma combinação de cromatografia de afinidade e de exclusão molecular para subsequente caracterização estrutural. Resultados. Nesse estudo, foi caracterizada a interação de NS5 com o fator eucariótico de início de tradução... / Yellow fever virus (YFV) belongs to the Flavivirus genus and causes an important disease. An alarming resurgence of viral circulation and expansion of the YFV endemic zones have been detected in Africa and South America in recent years. NS5 is a viral protein that contains the methyltransferase and RNA-dependent RNA polymerase domains, which are essential during viral replication. Interactions among NS5 and cellular proteins have been studied for the understanding of viral replication. The aim of this study was to characterize the interaction of NS5 protein with EIF3L and evaluate the role of EIF3L in yellow fever replication. Methods. To identify the interaction of YFV NS5 with cellular proteins, we performed a two-hybrid screen using YFV NS5 RdRp domain as bait and a human cDNA library. For mapping the interaction, RNApol deletions mutants were performed and analyses in two-hybrid system. The RNApol region of interaction was segmented in three fragments and analyses into yeast containing eIF3L. To map residues of NS5 that are critical for its interaction, we performed a site-direct mutagenesis in segment 3 of ID. The interaction was confirmed in vitro assays and by in vivo coimmunoprecipitations. The significance of eIF3L for replication of YFV was investigated using overexpression of eIF3L in BHK21-RepYF17D LucNeoIres cells. eIF3L was purified using a combination of affinity and subsequent size exclusion chromatography for subsequent structural characterization. Results. In this work we describe and characterize the interaction of NS5 with the translation factor eIF3L. The interaction between NS5 and eIF3L was confirmed by in vitro binding and in vivo coimmunoprecipitation assays. This interaction occurs in a region (Interaction Domain of RNApol domain) that is conserved in several... (Complete abstract click electronic access below)

Les virus zoonotiques sont une menace pour les humains à cause de leur capacité à passer des réservoirs animaux à l'homme. La rage est provoquée par un virus zoonotique (Virus de la Rage) qui a de multiples réservoirs animaux. La rage est contractée lors de la morsure par un animal infecté et est incurable et létale après l'apparition des premiers symptômes. Un défi visant à éradiquer la rage chez les chiens à l'horizon 2030 a été proposé. Il imposera de stopper la transmission du virus aux populations de chiens à partir des autres réservoirs animaux. Pour cela, il est essentiel de comprendre les mécanismes de transmission entre hôtes potentiels du virus. Dans notre thèse, nous faisons l'hypothèse que le passage d'un hôte à un autre est lié à la diversité des populations virales chez un hôte donné, appelée "hétérogénéité virale".Pour étudier cette hétérogénéité virale, des méthodes de séquençage des populations virales ont été développées. La transmission du virus de la rage entre chiens a été analysée et un évènement de transmission entre chien et renard a été étudié. Une plus importante hétérogénéité virale a été observée chez le renard après sa contamination par le chien en comparaison avec d'autres renards infectés par des congénères de la même espèce. Ceci suggère que l'hétérogénéité virale est importante dans le phénomène de transmission inter-espèce. Ces résultats sont importants pour améliorer notre compréhension de l'évolution du virus de la rage chez un nouvel hôte et pourront aider les efforts d'éradication de la maladie. / Zoonotic viruses are a threat to humans, jumping from animal reservoirs into humans. Rabies is caused by rabies virus (RABV), a zoonotic virus, with many animal reservoirs. Rabies is contracted from a bite of infected animal and once symptoms appear, death is inevitable. A challenging target date of 2030 to eliminate rabies in dogs has been set. One challenge will be stopping RABV re-entering the dog population from other animal reservoirs. Understanding how RABV switches hosts is important to prevent it happening. In this thesis, I hypothesise that successful host switching is due to the diverse population of viruses within the host termed ‘viral heterogeneity’. To investigate viral heterogeneity, methods to sequence the virus populations within clinical samples were developed. Transmission of RABV within dogs was analysed and a host shift event from dogs to foxes was investigated. High viral heterogeneity was seen in foxes after the host shift than in other foxes, suggesting it is important for a successful host shift. These data will be important to improve our understanding of how viruses evolve in new hosts, helping governments to eradicate disease.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Programa de Pós-Graduação em Biotecnologia, Florianópolis, 2011 / Made available in DSpace on 2012-10-25T19:49:50Z (GMT). No. of bitstreams: 1 289322.pdf: 1352290 bytes, checksum: 60582a492810471a9e9e523e508c5f7a (MD5) / A preocupação com a contaminação do ambiente aquático e, consequentemente, dos recursos hídricos, têm crescido devido a uma possível escassez da água potável no planeta. A contaminação das águas costeiras por vírus entéricos é causada pelo despejo de resíduos de origem fecal, afetando as águas utilizadas em atividades de recreação, pesca e no cultivo de moluscos bivalves, sendo responsável pela maioria dos surtos de gastroenterites que ocorrem no mundo. Indicadores bacterianos de contaminação fecal como os coliformes fecais, têm sido utilizados para o controle da qualidade das águas, porém já é estabelecido em todo o mundo que esta contaminação não está correlacionada com a presença de vírus entéricos. O desenvolvimento de novos métodos de concentração e detecção de vírus entéricos é uma alternativa para o controle da contaminação destas águas a fim de assegurar a qualidade de uso das mesmas. Os objetivos deste estudo foram: 1) Padronizar a metodologia de floculação utilizando leite desnatado acidificado na concentração de vírus em amostras de água do mar natural e artificial semeadas com quantidades conhecidas de adenovírus do sorotipo 2 (HAdV2). Comparar os resultados obtidos na recuperação viral com a metodologia já padronizada de filtração em membrana eletronegativa e reconcentração em Centriprep; 2) Aplicar metodologia padronizada na concentração e detecção de adenovírus humanos, poliomavírus cepa JC, vírus da hepatite A (HAV) e norovírus humano (HuNoV genogrupos GI e GII) em amostras de água do mar de 11 praias ao redor da Ilha de Santa Catarina, Florianópolis, Brasil durante um ano (Agosto de 2009 a Julho de 2010). 3) Analisar a presença de coliformes fecais (E. coli) e avaliar parâmetros físico-químicos, temperatura, pH, salinidade e oxigênio dissolvido. Para as detecções virais, o material genético das amostras foi extraído e diluído 1:10 para a redução de inibidores das reações enzimáticas. Nested-PCR qualitativo foi usado na detecção de HAdV e para a realização do sequenciamento. A detecção e quantificação de todos os vírus pesquisados foi realizada por PCR em tempo real. Os resultados da recuperação viral quando a metodologia de floculação foi empregada foram de 14% e 13,6% e no método de filtração, foram de 2,8% e 21% para as amostras de água do mar natural e artificial respectivamente. Das 132 amostras de campo analisadas, 16% foram positivas nas reações de nested-PCR para HAdV. Nas análises de PCR em tempo real, o vírus mais prevalente foi o HAdV, com 55% das amostras positivas, com intervalos de detecção de 8,40 x 103 a 1,9 x 107 CG/ L, seguido pelo HAV, com 51,5% de positividade, variando de 1,60 x 101 a 2,30 x 103 CG/ L. Para o JCPyV, 3% das amostras foram positivas, com intervalos de 1,90 x 104 a 1,70 x 105 CG/ L e para o NoV 7,5% e 4,5% das amostras foram positivas, com intervalos entre 2,41 x 103 a 5,14 x 106 e 3,73 x 104 a 2,10 x 106 CG/ L respectivamente para o GI e GII. As médias obtidas para os valores de coliformes fecais detectados foram de 8,0 a 1,325 UFC/100 mL em todos os pontos analisados. Somente dois dos pontos analisados não ultrapassaram o tolerável (Joaquina e Mole). Os demais pontos tiveram valores acima do permitido em algum mês do período analisado, destacando-se a Beira-Mar Norte com os valores mais elevados. As análises físico-químicas demonstraram valores concordantes com os previstos na legislação. O sequenciamento das amostras positivas para o HAdV demonstraram prevalência do adenovírus humano respiratório sorotipo 2. Os resultados obtidos evidenciam o destaque da contaminação viral nas amostras de água do mar e confirmam a não correlação da presença destes em relação à contaminação bacteriana. Este é o primeiro trabalho que avalia as condições das praias a redor da Ilha de Santa Catarina de forma mais abrangente, destacando diferentes tipos de contaminação. / The concern about aquatic environment contamination and consequently water resources is increasing mainly due to the warning about potable water scarcity in our planet. The contamination of coastal waters by enteric viruses is caused by discharge of residues of fecal origin, affecting waters used for recreational activities, fishing and shellfish growing, being responsible for most outbreaks of gastroenteritis occurring in the world. Bacterial indicators such as fecal coliforms have been used as parameters of water quality, however it has been clearly established worldwide that bacterial contamination show no relationship with the presence of human enteric viruses. The development of new methods of water concentration for enteric viruses detection is an alternative to monitor the contamination of these waters in order to ensure its quality.The objectives of this study were: 1) Standardize the methodology of flocculation using acidified skimmed milk for viral concentration in natural and artificial seawater samples previously spiked with known amounts of human adenoviruses serotype 2 (HAdV2). Results obtained with viral recovery were compared with the standardized methodology of water filtration in electronegative membrane and reconcentration by Centriprep#; 2) Apply standardized methodology in the concentration and detection of HAdV, poliomavirus (JCPyV), hepatitis A vírus (HAV) and noroviruses (HuNoV GI and GII) in seawater samples harvested in 11 different beaches in Santa Catarina Island, Florianópolis, Brazil in a period of one year (August 2009 to July 2010). Were also measured the presence of fecal coliform (E. coli) and physical-chemical parameters such as temperature, pH, salinity and dissolved oxygen. To reduce the presence of inhibitors of PCR reactions, a tenfold dilution of DNA/RNA of each sample was used. Nested-PCR was used for qualitative detection of HAdV and for conducting sequencing analysis. The detection and quantification of all the viruses evaluated was performed by real time PCR. The results of viral recovery by the standardized method of flocculation were 14% and 13.6% and for the filtration method, were 2.8% and 21% for samples of natural and artificial seawater respectively. From 132 field samples analyzed, 16% of these were positive for HAdV in the nested-PCR assay. In the analysis of real-time PCR, the most prevalent virus was HAdV, with 55% of positive samples with intervals of detection of 8.40 x 103 to 1.90 x 107 GC/ L, followed by HAV, with 51,5% of positivity, ranging from 2.30 x 101 to 1.60 x 103 GC/ L. For JCPyV, 3% of samples were positive, with ranges between 1.90 x 104 to 1.70 x 105 GC/ L and for NoV 7.5% and 4.5% of samples were positive, with intervals between 2.41 x 103 to 5.14 x 106 and 3.73 x 104 to 2.10 x 106 GC/ L respectively for GI and GII. The mean values of fecal coliforms detected were from 8.0 to 1.325 CFU/100 mL at all sites analyzed. Only two sites analyzed did not exceed the allowed by current legislation (Joaquina and Mole). The other sites showed, sometimes, values above the allowed during the period analyzed, with emphasis for Beira-Mar Norte site presenting the highest values. The physical-chemical analysis demonstrated values consistent with those recommended by legislation. The sequencing analysis of the samples positive for HAdV in qualitative PCR showed prevalence of human adenovirus respiratory serotype 2. The results show the highlight of viral contamination in the seawater samples and confirm the lack of correlation of their presence in relation to the bacterial contamination. This is the first study evaluating the conditions of the beaches around the Santa Catarina Island, highlighting different types of contamination.

New affinity molecules such as nucleic acid aptamers are in demand in the science and medical fields. Current aptamer selection technologies can generate unique aptamers with desired properties to targets of interest. My thesis describes a series of investigations on the protection of an oncolytic virus, the isolation of target cells from biological fluids, and aptamer-facilitated biomarker discovery. We tested individual aptamers and constructed a tetramer aptamer structure (quadramer) to increase virus infectivity. The quadramer protects vesicular stomatitis virus (VSV) during freeze–thaw cycles, shields the virus from neutralizing antibodies and increases viral active units. In addition to aptamers, we screened carbohydrate-based ice recrystallization inhibitors for the possible elimination of the cold chain of Vaccinia virus, VSV, and Herpes virus-1. N-octyl-gluconamide provides the longest shelf life for Vaccinia virus and Herpes virus-1 as tested according to the World Health Organization’s requirements for viral vaccines efficiency during transportation and distribution. We generated switchable aptamers capable of isolating cells expressing LIFR, NRP1, DLL4, uPAR, or PTCH1. These aptamers bind to the receptor positive cells in the presence of Mg2+ and Ca2+, and release the pure cells upon addition of EDTA. The aptamers were applied for a sequential positive immunomagnetic isolation of cells from mice bone marrow. We also utilized fluorescence-activated cell sorting (FACS) in our aptamer selections to develop switchable aptamers to positive isolation of monocytes from human blood. Moreover, we have selected non-switchable aptamers as an affinity probe to the cells expressing Axl receptor for immunofluorescent analysis and cell sorting. We determined aptamers to CD107a and applied them for biomarker discovery with mass spectrometry and found that CD107a was co-expressing with PD-1. Furthermore, we identified CD91 as binding partners to our aptamers in human monocytes using FACS and orbitrap mass spectrometry.