Browsing by Subject "arbovirus"
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Item An investigation and analysis of past, present, and future research on the dengue virus in Bangkok, Thailand emphasizing the research previously conducted at the Armed Forces Research Institute of Medical Sciences during the summer of 2011(2013-05-24) Hossenlopp, Cassady; Hossenlopp, Cassady; Wilke, R. Russell; Guardiola, Amaris R.; Jones, Crosby W., Jr.; Guardiola, Amaris R.; Jones, Crosby W., Jr.The dengue virus is the most common arbovirus in tropical and subtropical regions of the world. After analyzing the evolutionary history of the dengue virus, the health risks it presents, prevention methods, and the progress and setbacks that are currently being made in vaccine development, scientists understand the important role detection plays in combatting the virus. The research conducted in this thesis attempts to improve detection of the dengue virus using samples that were previously shown as negative by conventional RT-PCR but positive by ELISA. After designing new primers, conventional RT-PCR was able to detect 22% of the dengue mutants. The results from qRT-PCR, ELISA, and conventional RT-PCR were compared with the clinical data to recognize any possible trends between viral symptoms and the percentage of detection as well as account for possible differences in sensitivity.Item Characterization of the entry mechanisms utilized by the alphavirus venezuelan equine encephalitis virus to infect mosquito cells(2007-08-13) Tonya Michelle Colpitts; Robert Davey, PhD; Scott Weaver, PhD; Peter Mason, PhD; Lisa Elferink, PhD; Christopher Broder, PhDVenezuelan equine encephalitis eirus (VEEV) is a New World alphavirus that can cause fatal encephalitis in humans. VEEV is an enveloped, positive-strand RNA virus that is transmitted by a mosquito vector. Most research on alphavirus entry was done with the Old World alphavirus Semliki Forest virus (SFV) in mammalian cells. Not much is known about the entry of New World alphaviruses, especially in cells of the viral vector, the mosquito. Work with SFV has shown that Old World alphaviruses enter mammalian cells via receptor-mediated, clathrin-mediated endocytosis. This endocytic pathway utilizes several proteins in the mammalian cell, including the clathrin protein, the small GTPases known as Rab proteins and the large GTPase dynamin. These proteins have been shown to play a role in the entry of several viruses and are thought to be involved in alphavirus entry in mammalian cells. Here mosquito homologs of these proteins are identified, isolated and characterized in the mosquito cell. Rab5, Rab7 and dynamin are shown to be involved and necessary for VEEV entry and infection in mosquito cells. A novel entry assay is used to confirm that VEEV requires a low pH to enter mosquito cells. This work represents the first characterization of the involvement of mosquito endocytic pathways for infection of a New World alphavirus and sheds light on an important aspect of virus infection in an insect vector. The role of actin in VEEV internalization was also examined. Actin is known to be involved in the mammalian endocytic pathway and to act together with dynamin to coordinate endocytosis. Here mosquito actin is identified and shown to colocalize with mosquito dynamin. Both proteins also colocalize with internalized VEEV. Inhibiting actin polymerization prevents entry of the virus both by microscopic examination as well as utilizing the luciferase entry assay. This work shows that VEEV enters the mosquito cell via a pH-dependent endocytic pathway that requires functional endocytic proteins including Rab5, Rab7 and dynamin. It is also shown that F-actin must be present for VEEV to enter mosquito cells and that actin and dynamin act together during virus internalization.Item Ecology and transmission dynamics of Everglades virus(2005-10-20) Lark Lee Arwen Coffey; Douglas Watts; Daniel Brooks; Billy PhilipsEverglades virus (EVEV), an alphavirus in the Venezuelan equine encephalitis (VEE) serocomplex, circulates among rodents and vector mosquitoes in Florida and occasionally infects humans, causing a febrile disease sometimes accompanied by neurological manifestations. \r\nEVEV infections of cotton rats from Florida, and from Texas, a non-endemic area were performed to validate their role in enzootic transmission and to evaluate whether the viremia induced regulates EVEV distribution. Cotton rats from both localities developed viremia levels that exceeded the threshold for infection of the vector indicating that rat susceptibility does not limit EVEV distribution. \r\nSusceptibility experiments were performed with _Aedes taeniorhynchus_ and _Culex nigripalpus_, potential EVEV vectors, to evaluate their permissiveness to EVEV infection. In contrast to the high degree of susceptibility of the established vector _Culex (Melanoconion) cedecei_, these two species were relatively refractory to oral EVEV infection, indicating that they are probably not important vectors. \r\nPet dogs were used as sentinels of EVEV activity to detect recent circulation and to delineate EVEV distribution. Four percent of Florida dog sera contained EVEV antibody and many animals lived farther north than recorded EVEV activity, indicating that EVEV is widespread in the state and may be a cause of undiagnosed febrile illness in residents.\r\nDespite evidence that enzootic subtype ID Venezuelan equine encephalitis viruses (VEEVs), the closest relatives of EVEV, have emerged to cause significant human and equine disease, EVEV has not caused outbreaks in Florida. Venezuelan equine encephalitis virus emergence can be mediated by adaptation to epizootic mosquito vectors via mutations in the E2 glycoprotein gene. EVEV may accrue similar E2 mutations resulting in epizootic disease in Florida. The role of the EVEV E2 gene in epizootic vector infection was evaluated with a VEEV/EVEV E2 chimera. Insertion of the EVEV E2 into the VEEV backbone reduced infection in _Ae. taeniorhynchus_ by 40%, indicating that the epizootic VEEV E2 is necessary for infection of epizootic vectors. \r\nThe intrinsic plasticity of RNA viruses can facilitate changes in host range that may cause epidemics. However, the evolutionary processes that promote cross-species virus transfers are poorly defined, especially for generalist RNA arboviruses that replicate alternately in arthropods and vertebrates and often exhibit slower evolutionary rates than other RNA viruses with similar mutation frequencies that replicate only in vertebrates. The observed genetic stability of RNA arboviruses may result from constraints imposed by alternating between disparate hosts, where optimal replication in one host involves a fitness tradeoff for the alternate host. Accordingly, freeing RNA arboviruses from the alternate replication cycle, and thereby allowing them to specialize in a single host, will facilitate faster evolution and adaptation. To test this hypothesis in vivo, VEEV was passaged serially in mosquitoes or in vertebrates to eliminate host alteration, or alternately between mosquitoes and vertebrates. Virus lineages allowed to specialize in mosquitoes exhibited increased mosquito infectivity, and vertebrate-specialized strains produced higher viremias. Alternately passaged VEEV exhibited no detectable fitness gains in either host and serial passaged VEEV exhibited fitness declines in the bypassed host. These results support the hypothesis that arbovirus adaptation and evolution is limited by obligate host alternation. \r\n\r\nItem Experimental studies of the ecology and evolution of eastern equine encephalitis virus and implications for its emergence and classification(2010-06-25) Nicole Cherise Arrigo; Scott C. Weaver; Michael J. Turell; Ilya V. Frolov; Frederick A. Murphy; Douglas M. WattsEastern equine encephalitis virus strains from North (NA EEEV) and Central/South America (SA EEEV) have developed markedly different epidemiologic, pathogenic, antigenic, and genetic profiles, have distinct geographic distributions, and potentially occupy unique vector and vertebrate ecological niches. The goal of my research was to clarify the extent to which these viruses have diverged by further understanding their evolutionary history and adaptation to different ecological niches, and the impact that this divergence has had on their ability to emerge in reciprocal environments. My studies were designed to examine each of the three main aspects of the arboviral transmission cycle: the virus, the vertebrate host, and the mosquito vector. To investigate the evolutionary history and genetic divergence of NA and SA EEEV, I conducted a phylogenetic and Bayesian coalescent analysis of the structural polyprotein genomic region (26S) of all available SA EEEV, and additional NA EEEV, isolates spanning a broad geographic and temporal spectrum. In accordance with support provided by the evolutionary and phylogenetic analyses, I sought to apply a more direct and experimental approach to explore the adaptation of NA and SA EEEV to the use of different vertebrate host species. Wild cotton rats (Sigmodon hispidus) and house sparrows (Passer domesticus) were collected in Galveston and Houston, Texas, respectively, and evaluated for their potential to serve as amplification and/or reservoir hosts for NA and SA EEEV. Juvenile cotton rats experienced complete mortality with both NA and SA EEEV and provided me with a unique opportunity to compare the pathology resulting from NA and SA EEEV infection a wild vertebrate species. In order to better understand the directionality of NA EEEV divergence and adaptation and to further clarify the vector ecology of SA EEEV, I evaluated the relative susceptibilities of the NA enzootic vector, Culiseta melanura, and the presumed enzootic vector for SA EEEV, Culex taeniopus, and the probable epizootic EEEV mosquito vectors, Aedes (Ochlerotatus) taeniorhynchus and Ae. (Och.) sollicians, to sympatric and allopatric EEEV strains. Taken together, the results of my dissertation research emphasize the striking extent of evolutionary divergence between NA and SA EEEV and provide a greater understanding of the directionality of NA EEEV adaptation to North America subsequent to its divergence from an ancestral EEEV in Central/South America. My research has also clarified the vector and vertebrate usage of both NA and SA EEEV, providing support for the use of mammalian vertebrate host species by SA EEEV and highlighting its emergence potential in a novel North American environment.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 Pathogenesis and transmission of Venezuelan equine encephalitis virus(2006-04-03) Darci Renee Smith; Scott C. Weaver; Robert B. Tesh; Lynn Soong; Judith F. Aronson; Ilya V. Frolov; George V. LudwigVenezuelan equine encephalitis virus (VEEV) is an emerging arboviral pathogen that affects the Americas. Outbreaks can involve hundreds- of- thousands of equines and humans, spread over large geographic regions, and can last several years. The principal vector in most major coastal outbreaks is the mosquito Aedes taeniorhynchus. This species is more susceptible to most epidemic than to enzootic strains, and the adaptation of VEEV to this vector may be an important determinant of epidemic transmission. However, studies on the infection, dissemination, and transmission of VEEV regarding this important vector are lacking. \r\nThe major determinant of Ae. taeniorhynchus infection with VEEV is the E2 envelope glycoprotein, which interacts with cellular receptors. I therefore hypothesized that differential interactions of VEEV with receptors on midgut epithelial cells determine the ability of a representative epidemic versus a representative enzootic strain to infect this mosquito. In support of this hypothesis, I found that significantly more epidemic VEEV bound to and infected mosquito midguts compared to the enzootic strain. The dissemination from the midgut of an epidemic VEEV strain was compared to that of an enzootic strain. Following initial infection, the epidemic strain was pantropic in tissues of the mosquito, including the salivary glands, whereas the enzootic strain did not infect the midgut efficiently and replicated only in muscles and nervous tissue upon dissemination. \r\nFollowing the infection of the mosquito salivary glands with an epidemic strain, the amount of VEEV transmitted was estimated. I hypothesized that the method of mosquito infection and saliva collection significantly affects estimates of the amount of virus transmitted and that differing infection routes affect the viremia and mortality of mice. Both the mosquito species and infection route used affected the amount of virus detected in the saliva. The amount of VEEV transmitted in vivo by mosquitoes during blood feeding was significantly less than in vitro transmission estimates and mosquito transmission had little or no effect on murine viremia or mortality compared to needle inoculations. These results have important implications for evaluating the vector competence of Ae. taeniorhynchus and other VEEV vectors, for designing pathogenesis experiments, and for modeling transmission in nature.\r\nItem 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.