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Browsing by Author "Chotiwan, Nunya, author"

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    Characterization of changes in metabolic pathways during dengue virus serotype 2 infection of the Aedes aegypti mosquito vector to identify control points for interrupting virus transmission
    (Colorado State University. Libraries, 2018) Chotiwan, Nunya, author; Perera, Rushika, advisor; Blair, Carol, committee member; Foy, Brian, committee member; Huang, Claire, committee member; Di Pietro, Santiago, committee member
    Dengue viruses (DENV) are mosquito-borne viruses that cause a wide range of acute symptoms from mild fever to lethal dengue shock syndrome in humans. DENV are transmitted primarily by Aedes aegypti (Ae. aegypti). These mosquitoes are widely distributed throughout tropical and subtropical areas around the world. Increasing globalization, urbanization and global warming are factors that enhance the spread of these vectors placing over 2.5 billion people at risk of contracting these viruses. Transmission of these viruses depends on their ability to infect, replicate and disseminate into several tissues in the mosquito vector. During DENV infection of its human and mosquito hosts, a visible rearrangement of lipid membrane architecture and alterations of the metabolic repertoire is induced. These events occur to facilitate efficient viral replication and virus assembly within the cell and to circumvent antiviral responses from the host. Interference with these virus-induced processes can be detrimental to virus replication and can prevent viral transmission. In this dissertation, we present the first insight into the metabolic environment induced during DENV serotype 2 (DENV2) replication in Ae. aegypti. Using untargeted high-resolution liquid chromatography-mass spectrometry, we explored the temporal metabolic perturbations that occur following dengue virus infection of the midgut, the primary site of the virus infection in the mosquito vector. Temporal changes of metabolites across early-, mid- and late-infection time points were identified. A marked increase in the /content of glycerophospholipids, sphingolipids and fatty acyls was coincident with the kinetics of viral replication. Elevation of glycerolipid levels and the accumulation of medium-chain acyl-carnitines suggested a diversion of resources during infection from energy storage to synthetic pathways and energy production. From the observations above, two active pathways, sphingolipid and de novo fatty acid synthesis pathways, were further validated to identify metabolic control hubs. Using inhibitor screening of the sphingolipid pathway, we determined that sphingolipid Δ-4 desaturase (DEGS), the enzyme that converts dihydroceramide to ceramide was important for DENV2 infection in cultured Ae. aegypti cells (Aag2). Long, double-stranded RNA-mediated knockdown of DEGS expression led to the imbalance of ceramide to dihydroceramide ratios and affected DENV2 infection in cell culture. However, the inhibitory effect to DENV2 replication was not observed during DEGS-knockdown in mosquito vectors. De novo fatty acid biosynthesis is the pathway that synthesizes the first lipid molecules, fatty acids, required in synthesizing complex lipid molecules, such as glycerophospholipids, glycerolipids and sphingolipids. As a result, this pathway serves as a bottle neck for the control of lipid metabolism. In this study, we annotated and characterized the expression of seven Ae. aegypti fatty acid synthase (AaFAS) genes in the different stages of mosquito development and upon exposure to different diets. We found that AaFAS1 shares the highest amino acid similarity to human fatty acid synthase (FAS) and is the dominant AaFAS that expressed in female mosquitoes. Knockdown expression of AaFAS1 expression showed a reduction in DENV2 replication in the Aag2 cells and in the midgut of Ae. aegypti mosquitoes during early infection. However, the correlation between viral infection and levels of AaFAS1 expression was difficult to elucidate. The work in this dissertation has highlighted metabolic pathways that are induced by DENV2 infection and the metabolic control points within these pathways that are critical for DENV2 infection in Ae. aegypti. Successful perturbation of metabolic homeostasis can potentially limit virus replication in the vector, presenting a novel avenue to block the transmission of DENV2 from the mosquitoes to humans.
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    Molecular determinants of dengue virus type-2 critical for early events in antibody-dependent enhancement of infection
    (Colorado State University. Libraries, 2013) Chotiwan, Nunya, author; Blair, Carol, advisor; Huang, Claire, advisor; Chen, Chaoping, committee member
    Antibody-dependent enhancement (ADE) of infection might be one of the major factors in the development of more severe forms of dengue disease in patients undergoing a secondary infection of dengue virus (DENV). The ADE is caused by cross-reactivity of subneutralizing or non-neutralizing antibodies (Abs), which form virus-Ab complexes and enhance virus infection by binding to the Fcγ receptors (FCγR) on FcγR-bearing cells. The early events in non-ADE infection have been previously studied, but the virus entry pathway and the DENV molecular determinants involved in ADE are still largely unclear. There are two hypotheses for the early entry pathway of ADE infection: (1) Ab opsonized DENV binds to FcγR and directly enters cells through phagocytic pathway; (2) FcγR plays an auxiliary role in concentrating the opsonized virus to the cell surface, but other cellular receptors are still required for virus entry into the endocytosis pathway. Herein, we investigated contributions of the DENV2 E proteins to ADE infection, as well as the role of FcγRIIA in the enhancement of infection and possible entry route of the Ab-opsonized virion. ADE of wild type (WT) DENV2 infection can be promoted in FcγRIIA-bearing K562 cells by using subneutralizing cross-reactive flaviviral monoclonal antibody (MAb), subcomplex DENV MAb or serotype-specific DENV2 MAb against the E protein, as well as non-neutralizing anti-DENV-prM MAb. The enhancement of infection was analyzed by comparing DENV2 infection under ADE conditions with DENV-Ab complex and non-ADE conditions with DENV alone. Numbers of DENV infected cells were determined by flow cytometry of infected cells stained with labeled MAb 2H2-AlexaFluor-488, while increases in viral output were quantified by qRT-PCR of viral genomes. We investigated multiple DENV mutants generated by reverse genetic technology to identify molecular determinants in the envelope protein (E) of DENV-2 that are critical for DENV attachment and viral-endosomal membrane fusion in both non-ADE and ADE infection of the virus in K562 cells. We determined that binding of virus-Ab complex with FcγRIIA alone is not sufficient for virus entry during ADE infection. Furthermore, the molecular determinants of E protein critical for virus entry and virus-mediated endosomal membrane fusion involved in non-ADE infection were also required for ADE infection. ADE was also tested in FcγRIIA-transfected CV-1 cells (CV-1- FcγRIIA). Surprisingly, no enhancement occurred with any of the tested MAb in this cell type. Numbers of cells expressing FcγRIIA and density of FcγRIIA molecules expressed on CV-1- FcγRIIA cell surface were similar to those of K562 cells. These results supported our findings that FcγRIIA alone was not sufficient to enhance viral infection. The results also suggested that cellular components that are present in K562 cells but absent in CV-1- FcγRIIA cells play a major role in ADE of infection.