Browsing by Subject "mosquito"
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Item Field and laboratory studies of venezuelan equine encephalitis virus ecology in Chiapas, Mexico.(2009-03-05) Eleanor Rose Deardorff; Scott WeaverThe emergence of Venezuelan equine encephalitis virus in Chiapas Mexico was examined from a field approach and from a laboratory approach. This virus was not previously associated with equine disease in Mexico. The evolution of the equine virulent phenotype was thought to have resulted in a mosquito vector switch from Culex (Melanoconion) taeniopus to Aedes (Ochlerotatus) taeniorhynchus as a result of land-use changes. Wild rodents and mosquitoes were captured over the course of one year and little evidence of virus circulation was found. Wild rodents from five species were then imported into the lab for experimental evaluation as virus amplifying hosts. It was found that a VEEV strain from the study area may use a variety of rodents as amplifying hosts in the laboratory. Lastly a breeding colony of Culex (Mel.) taeniopus mosquitoes was established and experimentally evaluated for the ability of these mosquitoes to transmit equine virulent VEEV. It was found that equine virulent virus infects and is transmitted by this mosquito with high efficiency and is likely maintained in transmission foci by Culex (Mel) taeniopus during inter-epizootic periods.Item Identification and characterization of germline-specific promoters for remobilization of transgenes in the mosquitoes, Aedes aegypti and Anopheles gambiae(2009-05-15) Hagen, Darren ErichThe development of genetic transformation systems in insects has revolutionized the field of entomology. Transgenic insects provide tools to identify, isolate and analyze insect genes and to genetically modify insects for the purposes of insect control or disease vector modification. When transformation frequencies are high, multiple transgenic lines can be generated with relative ease. However, in most mosquito species, the results of transformation experiments have been suboptimal. Increased mosquito transformation efficiency is a research priority. Additionally, incorporation of refractory transgenes will not be sufficient to modify natural populations. A gene drive system will be required to allow transgenes to proliferate throughout populations and potentially reach fixation. This study proposes the use of germline-specific regulatory elements to promote confined, regulated transposase expression within the germ tissue. Creation of helper constructs utilizing endogenous promoters will potentially increase genetic transformation frequencies. The generation of lab strains of mosquitoes expressing an endogenous source of transposase within the germline will also serve as a powerful research tool. Endogenous sources of transposase will allow for comparative analysis of integration rates using different donor plasmids. Finally, the generation of autonomous transposable elements will provide a gene drive mechanism to move a tightly-linked refractory gene into a population. Four genes have been identified, cloned, and characterized, revealing expression patterns expected of germline-specific genes. Transcription profiles and in situ hybridization data support these conclusions. Putative cis-acting regulatory elements have been cloned and incorporated into DNA plasmid constructs. These elements are cloned in a manner such that they will regulate fluorescent gene expression. Additionally, similar elements have been cloned upstream of the Mos1 open reading frame, within the inverted terminal repeats of the mariner transposable element, thus creating autonomous elements and a potential gene drive mechanism.Item The role of mosquito saliva on host immune response and pathogenesis of West Nile virus\r\n(2006-11-03) Bradley S Schneider; Stephen Higgs; Lynn Soong; Judith Aronson; Eric Smith; Donald ChampagneWest Nile virus is a positive sense single-stranded RNA virus in the family Flaviviridae that emerged globally following the appearance of a more neurotropic subtype. Recently outbreaks of WNV disease have occurred in the Middle East, Europe, Africa, South America, and North America. Vertebrates typically become infected when an infectious mosquito pierces the host epidermis to take a blood meal, depositing virus principally in the extravascular tissue. Accumulating evidence has demonstrated that the mosquito saliva, which carries WNV into the vertebrate, is not simply a transport medium, but can have a profound effect on vertebrate immunity, pathogen transmission efficiency, pathogenesis, and disease course. In the past most small animal models of arbovirus disease have used needle-inoculation, but recent evidence suggests that because of the potential effects of mosquito saliva on the immune system, it is important to re-evaluate the pathogenesis of these infections in the presence of mosquito saliva. A central question of this dissertation project was to determine if mosquito feeding or mosquito saliva could impact WNV disease. To evaluate the potential for mosquito saliva to alter WNV infection, the mouse model of disease employed in this dissertation revealed a potentiation of WNV disease when mosquitoes were allowed to feed at the virus inoculation site immediately before injection of WNV. Further, with higher titer inoculations of WNV subsequent to the feeding of mosquitoes more progressive infection, higher viremia, and accelerated neuroinvasion developed than the mice inoculated with an equivalent titer of WNV alone. To determine the mechanism of this potentiation, the in vivo expression of key TH1, TH2, inflammatory, and antiviral cytokines was quantified during peripheral arbovirus infection in the presence or absence of mosquito saliva. Data clearly showed that during early arbovirus infection mosquito salivary proteins down-modulates specific antiviral cytokines while enhancing production of TH2 and immunosuppressive cytokines. To understand the source of these shifts in immune signalling, the in vitro response of DCs and macrophages was investigated. Following exposure to Ae. aegypti SGE, APCs recently exposed to arbovirus displayed reductions in IFN-? and iNOS expression and transient amplification of IL-10 mRNA levels. Macrophages appeared to be more susceptible to the modulating effects of mosquito saliva than DCs. The influence of mosquito saliva on immune cell migration patterns both into the dermal site of WNV inoculation and the draining lymph node was also evaluated. The principal observation from this study was that the inclusion of mosquito saliva/feeding at the inoculation site of WNV leads to a suppression of lymphocytes, particularly CD4+ T cells, and a corresponding increase in DCs. Finally, although mosquito exposure and sensitization to it is widespread, the effect of prior exposure to mosquitoes on subsequent arbovirus infection had up until this point been unexplored. Accordingly, the potential for an immune response directed against mosquito salivary proteins to have a protective or confounding effect on naturally transmitted WNV infection was investigated by comparing early WNV pathogenesis in mosquito naïve and sensitized populations of mice. Previous exposure to Ae. aegypti feeding results in significantly higher mortality rates associated with elevation of inflammation, APC recruitment, and IL-4 expression concurrent with a decrease in lymphocytes mainly the CD4+ subtype. Mosquito sensitization-mediated amplification of WNV disease is facilitated by the humoral response to mosquito salivary proteins. This dissertation confirms that by ignoring the possible effects of the vector we may misinterpret the early immune response to arboviruses and also possibly aspects of the overall pathogenesis of arboviral infections. The information from the present study provides insight into early host responses to arbovirus infection, and suggests further determinants of WNV virulence.