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Isolation and characterization of dengue virus membrane-associated replication complexes from Aedes aegypti

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dc.contributor.authorPoole-Smith, Betty Katherine, author
dc.contributor.authorBlair, Carol D., advisor
dc.contributor.authorOlson, Kenneth Edward, committee member
dc.contributor.authorFoy, Brian D., committee member
dc.contributor.authorRoss, Eric D., committee member
dc.date.accessioned2007-01-03T04:41:48Z
dc.date.available2007-01-03T04:41:48Z
dc.date.issued2010
dc.description.abstractUltrastructural studies of flavivirus replication have long observed proliferation of host membranes. Membrane-bound replication compartments have recently been isolated and characterized from flavivirus-infected mammalian cells, providing insight into the morphology, organelle of origin, and protein components of the flavivirus membrane-associated replication complex. Our laboratory has proposed that a balance exists between dengue virus (DENV) replication in Aedes aegypti and the mosquito's RNA interference (RNAi) based antiviral response. Here, we have isolated and characterized membrane-bound replication compartments from mosquito cell culture and Ae. aegypti to evaluate the role that these membranes may play in shielding DENV double-stranded RNA (dsRNA) from RNAi. Membrane isolation techniques and immunofluorescent staining techniques for dsRNA identification were developed to isolate and characterize membrane-associated replication complexes in DENV-infected mosquito cell culture and Ae. aegypti. Here we show that double-membrane vesicles arise from the endoplasmic reticulum (ER) and are associated with DENV dsRNA in mosquitoes. These data suggest that DENV dsRNA replicative intermediates may be shielded from the RNAi response in the mosquito. DENV membrane-associated replication complexes were characterized in mosquito cell culture and Ae. aegypti using immunofluorescent staining for dsRNA, confocal microscopy, sucrose gradient cellular fractionation, and electron microscopy. In addition, we compared immunofluorescent staining for dsRNA between DENV and Sindbis virus (SINV). We also evaluated replication of DENV mutants in the DENV-resistant transgenic mosquito strain known as Carb77 and whether mutations in DENV genome sequence lead to evasion of the enhanced RNAi response of Carb77 mosquitoes. This is the first isolation of membrane-associated replication complexes and first characterization of dsRNA staining from DENV-infected mosquito cell culture and Ae. aegypti, providing knowledge which can be used to develop improved RNAi-based control strategies for DENV in mosquitoes.
dc.format.mediumborn digital
dc.format.mediumdoctoral dissertations
dc.identifierPooleSmith_colostate_0053A_10049.pdf
dc.identifierETDF2010100008MIPA
dc.identifier.urihttp://hdl.handle.net/10217/40476
dc.languageEnglish
dc.language.isoeng
dc.publisherColorado State University. Libraries
dc.relation.ispartof2000-2019
dc.rightsCopyright and other restrictions may apply. User is responsible for compliance with all applicable laws. For information about copyright law, please see https://libguides.colostate.edu/copyright.
dc.subjectDengue viruses -- Molecular aspects
dc.subjectAedes aegypti -- Molecular aspects
dc.subjectFlaviviruses -- Molecular aspects
dc.subjectRNA -- Synthesis
dc.titleIsolation and characterization of dengue virus membrane-associated replication complexes from Aedes aegypti
dc.typeText
dcterms.rights.dplaThis Item is protected by copyright and/or related rights (https://rightsstatements.org/vocab/InC/1.0/). You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s).
thesis.degree.disciplineMicrobiology, Immunology, and Pathology
thesis.degree.grantorColorado State University
thesis.degree.levelDoctoral
thesis.degree.nameDoctor of Philosophy (Ph.D.)

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