Browsing by Author "Olson, Kenneth Edward, committee member"
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Item Open Access Analysis of virus-derived small RNAs reveals that the RNA silencing response to flavivirus infection differs dramatically between C6/36 and Aag2 mosquito cell lines(Colorado State University. Libraries, 2010) Scott, Jaclyn Christine, author; Blair, Carol D., advisor; Olson, Kenneth Edward, committee member; Wilusz, Carol J., committee member; Peersen, Olve, committee memberThe exogenous small RNA pathway has been shown to be an important antiviral defense in mosquitoes against arboviruses such as dengue virus (DENV), but little is known about how the pathway and the virus interact in the cell. The studies described in this dissertation examine the how small RNA pathways interact with DENV and a mosquito-only flavivirus, cell-fusing agent virus (CFAV), in mosquito cell cultures. Deep sequencing of virus-specific small RNAs in Aedes aegypti Aag2 cells indicates that DENV2 is targeted by the exogenous RNA interference (RNAi) pathway in this cell line, which is consistent with the DENV2-specific small RNAs seen in DENV2-infected A. aegypti mosquitoes. When the DENV2-specific small RNAs from the Aedes albopictus C6/36 cell line were analyzed, the size and polarity of the small RNAs was not consistent with the exogenous small interfering RNA (siRNA) pathway. Further molecular analysis of the C6/36 cell line indicated that it appears to lack functional Dicer2 processing of long double-stranded RNA (dsRNA). CFAV small RNAs were also discovered in the Aag2 cell line during the deep sequencing analysis. It appears that this cell line is persistently infected with this mosquito-only flavivirus, and the virus is also targeted by the exogenous siRNA pathway in the cells. Sequence comparisons between CFAV and DENV2 RNA did not show long regions of sequence identity between the two viruses, indicating that a sequence-specific mechanism for virus-derived small RNAs from one virus to interfere with replication of the other virus during dual infections seems unlikely. The C6/36 cell line was inadvertently infected with CFAV, but the CFAV-specific small RNAs in C6/36 cells did not appear to be generated from the exogenous siRNA pathway, consistent with the DENV2-specific small RNAs in this cell line. The larger sized, mostly positive sense virus-specific small RNAs found in the C6/36 cells suggest that virus infections may be targeted by another small RNA pathway (such as the piwi-interacting pathway) in this cell line. These studies provide a better understanding of the interactions of DENV2 with the mosquito antiviral RNAi pathway in infected mosquito cells and have revealed a dysfunctional RNAi pathway in the C6/36 cell line. This work also provides a basis for further studies examining the interactions between mosquito-only flaviviruses, arboviruses and the antiviral RNAi pathway.Item Open Access Flavivirus surveillance in mosquitoes from northern Colorado, with the detection and description of two insect-specific flaviviruses(Colorado State University. Libraries, 2010) Bolling, Bethany Gayle, author; Blair, Carol D., advisor; Moore, Chester G.(Chester Gunn), advisor; Olson, Kenneth Edward, committee member; Olea-Popelka, Francisco J., committee memberVector-borne diseases remain a major public health concern worldwide. The studies described here underline the importance of combining field surveillance activities with laboratory experiments to provide a comprehensive understanding of the dynamics of vector-borne disease systems. Entomological measures of West Nile virus (WNV) risk were found to be strongly associated with human WNV disease cases in northeastern Colorado. Specifically, Culex tarsalis abundance and the Vector Index for WNV-infected Cx. Tarsalis females (weekly mean per trap night x weekly proportion of WNV-infected females) were associated with weekly numbers of WNV human disease cases with lag times of 4-7 weeks and 1-2 weeks, respectively. This provides information that can be utilized for decision-making processes concerning when to initiate mosquito control activities and how to best utilize limited resources. These studies also describe the first detection of insect-specific flaviviruses in mosquitoes collected in Colorado. Culex flavivirus (CXFV), first described in Japan in 2007, and a new insect-specific flavivirus, designated Calbertado virus, were detected in Culex spp. mosquitoes. Experiments were conducted to explore the transmission dynamics of CXFV in a naturally infected Culex pipiens laboratory colony and also the potential effects of CXFV infection on vector competence for WNV. Results indicated that vertical transmission is the primary mechanism for viral persistence in the colony, with venereal transmission perhaps playing a supplemental role. Vector competence experiments suggested possible suppression of WNV replication by persistent CXFV infection. These findings are important as insect-specific flavivirus transmission dynamics have not been described yet and studies investigating putative interactions between insect-specific flaviviruses with arboviruses, like WNV, are lacking. This work provides a better understanding of local vector-borne disease systems, providing a basis for additional studies to further characterize the dynamics of co-circulating flaviviruses in vector mosquitoes.Item Open Access Identification of Culex tarsalis D7 salivary protein and role of salivary protein vaccine on subsequent West Nile virus infection(Colorado State University. Libraries, 2010) Reagan, Krystle Lynn, author; Blair, Carol D., advisor; Olson, Kenneth Edward, committee member; Foy, Brian D., committee member; Chen, Chaoping, committee member; Wang, Tian, committee memberMosquito salivary proteins (MSPs) modulate the host immune response, leading to enhancement of arboviral infections. Identification of protein factors in saliva responsible for immunomodulation should lead to new strategies to prevent and protect against arboviral infection. D7 salivary proteins are among the most abundant in mosquito saliva, and they function as both vasodilators and suppressors of local inflammation. Here we identify D7 salivary proteins in Culex tarsalis, an important disease vector in the western United States. Recombinant D7 proteins were used to analyze the systemic and local immunomodulatory properties of the host. In this project, we immunized mice with recombinant D7 and tested for protection against subsequent challenge with West Nile virus (WNV) (NY99) delivered by mosquito bite. The vaccine was able to elicit a specific immune response. However, it enhanced WNV infection in the mouse model. We suggest that WNV enhancement is due to three factors. First, vaccinated mice had significant cellular infiltrates at the mosquito bite site, which included WNV permissive monocytes and dendritic cells. Increases in these cell populations at the mosquito bite site leads to an increase in initial viral infection and dissemination. Others have shown that higher peripheral viral levels lead to a worse disease outcome from infection. Secondly, mortality curves in infected mice receiving passive transfer of serum containing antibodies from vaccinated mice mimicked those from vaccinated animals. Antibody neutralization of mosquito salivary proteins that are critical in completing a successful blood meal may lead to increased probing time by the mosquito. Increased probing by the mosquito results in an increase in amount of saliva being deposited, therefore an increase in initial viral dose. Lastly, the cytokine profile observed in vaccinated mice showed an increase in the Th2 cytokine IL-4 and regulatory cytokine IL-10 and a decrease in Th1 cytokines such as IL-12p70 and IFNγ. A protective immune response to WNV includes high levels of Th1 cytokines. Production of IL-4 by mice that received the rD7 vaccine directly inhibits the Th1 cytokines necessary for protection. This work has increased our understanding of the complex nature of immunity to MSPs. Vector saliva vaccines have been successful in protecting against other blood feeding arthropods transmitted diseases. Nevertheless, differences in vector and pathogen physiology may preclude this approach from being successful for mosquito virus systems.Item Open Access Isolation and characterization of dengue virus membrane-associated replication complexes from Aedes aegypti(Colorado State University. Libraries, 2010) Poole-Smith, Betty Katherine, author; Blair, Carol D., advisor; Olson, Kenneth Edward, committee member; Foy, Brian D., committee member; Ross, Eric D., committee memberUltrastructural 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.