Browsing by Author "Blair, Carol, committee member"

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Open Access
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.
Open Access
DENVax live attenuated chimeric dengue vaccine
(Colorado State University. Libraries, 2010) Mulhern, Kaitlyn, author; Nyborg, Jennifer, advisor; Livengood, Jill, advisor; Stinchcomb, Dan, committee member; Blair, Carol, committee member
Dengue viruses are endemic in regions inhabited by roughly one half of the world’s population and can cause symptoms ranging from a headache and fever to even death. There is no vaccine to prevent infection by dengue viruses and the only current protection and prevention is mosquito control. The dengue viruses and the Aedes aegypti mosquito that carries them are spreading to more areas of the world and are therefore posing an even greater threat to public health. There is an urgent need for an effective vaccine that confers protection against infection by all four dengue serotypes. Several companies are currently developing dengue vaccines using different technologies including a live attenuated virus vaccine, DNA vaccine, and a chimeric vaccine in the yellow fever virus vaccine backbone. Inviragen, Inc., a biotech company, in collaboration with the Center for Disease Control and Prevention (CDC), is developing a chimeric live attenuated virus vaccine that is currently in clinical testing. The Inviragen/CDC dengue vaccine is based on an attenuated dengue-2 virus, called PDK-53, which was developed by passaging the virus 53 times in primary dog kidney cells (PDK). The attenuating mutations in the dengue-2 PDK-53, which are in the nonstructural genes of the virus, are well defined and characterized. This is essential in a live attenuated vaccine in order to monitor the stability of the mutations throughout vaccine manufacture and development. The four separate serotype-specific vaccine viruses are constructed by inserting the appropriate structural E and prM gene sequences into the nonstructural genes of the attenuated dengue-2 PDK-53 backbone. The phenotypic properties including plaque size, temperature sensitivity, peak titers, efficiency of replication, and neurovirulence, showed that the ehimeric viruses for each serotype were attenuated in comparison to the wild type parental viruses. Tetravalent formulations with the chimeric D2/1, D2/3 and D2/4 viruses as well as the D2-PDK53 virus were tested for antibody responses in AG 129 mice. Two separate projects involving Inviragen’s dengue vaccine, DENVax, are discussed in this thesis. In the first project, assays were developed to purify and quantify the residual host cell DNA in the vaccine using real time qPCR. Initially, the purification of host cell DNA present in research grade vaccine lots was optimized and internal controls were generated to monitor the efficiency of DNA extraction from the samples. The purified DNA was then amplified by PCR and quantified using two fluorescence-based methodologies, SYBR Green and TaqMan. The SYBR Green method was used to test the virus samples and reproducible results were obtained. The TaqMan assay allows for multiplexing, which allows simultaneous detection of two PCR products; Vero cell DNA present in virus samples and an internal control. The multiplexing TaqMan reactions need to be further optimized. In the second project, three separate “second generation” DENVax4 viruses were designed and generated. Of the three, two viable infectious viruses were generated and tested for phenotypic growth in vitro, and sequenced. These viruses grew to similar titers and phenotypes to the existing DENYax4, and immunogenicity testing in AG 129 mice is planned.
Open Access
Epidemiology, ecology, and evolution of canine influenza virus H3N8 in United States dogs
(Colorado State University. Libraries, 2012) Pecoraro, Heidi Lee, author; Landolt, Gabriele, advisor; Blair, Carol, committee member; Bowen, Richard, committee member; Huyvaert, Kathryn, committee member
To view the abstract, please see the full text of the document.
Open Access
Molecular analysis of the genetic determinants that contribute to virulence in lineage 2 West Nile virus
(Colorado State University. Libraries, 2017) Romo, Hannah Elizabeth, author; Ebel, Greg, advisor; Brault, Aaron, advisor; Blair, Carol, committee member; Bowen, Richard, committee member; Huyvaert, Kate, committee member
The ability of arboviruses to impart significant global disease burdens is related to the corresponding capacity of arboviruses to emerge in naïve environments or re-emerge in endemic environments. The introduction of West Nile virus (WNV) into North America was marked by rapid spread across the continent, high rates of neuroinvasive disease in humans and horses, and subsequent displacement by newer evolved genotypes. In the last 12 years, an underrepresented lineage of WNV, lineage 2 (L2) has similarly emerged from sub-Saharan Africa into areas of Europe and Russia, causing widespread neurological disease and recurrent enzootic transmission. Given the potential for further geographic spread of L2 WNV and to understand mechanisms that drive emergence events for WNV, I sought to characterize L2 WNV in a comprehensive and comparative manner by investigating potential molecular mechanisms of pathogenesis in mosquitoes, birds, and mice (as models for human disease). A more thorough understanding of the mechanisms that dictate rapid dispersal and endemic maintenance of arboviruses will improve our ability to predict emergence events, increase the effectiveness of surveillance mechanisms, and develop effective intervention strategies. Within lineage 1 (L1) WNV, the role of the NS3-249P amino acid in modulating severe virogenesis in American Crows (AMCRs) has been well established and is predicted to be involved in facilitating the emergent capacity of L1 WNV. The evolution of a proline at the same NS3-249 locus in L2 WNV was initially observed during the first L2 WNV associated outbreak in Europe. However, no bird mortality was observed during the NS3-249P associated L2 WNV outbreak, and the extent of L2 WNV pathogenesis in birds is unclear. In this aim, I examined the viremia titers and mortality profiles of North American AMCRs and house sparrows following infection with African and European L2 WNV strains with and without amino acid mutations at the NS3-249 locus. Our results demonstrate that L2 WNV strains can elicit severe virogenic and fatal outcomes in AMCRs and HOSPs. Additionally, I found that the NS3-249 locus is modulating AMCR viremia titer outcomes, similar to what has been previously observed for the NS3-249 locus in L1 WNV strains. I also demonstrated the 3' UTR of NS10 reduces viremia titers of AMCRs at later time points. The vast majority of our understanding regarding the vector competence of Culex mosquitoes for WNV originates from studies performed with L1 WNV strains, and as such, little information is available regarding the competency of Culex mosquitoes for L2 WNV. To remediate this, I assessed the vector competence phenotypes of two different North American Culex mosquito species for multiple L2 WNV strains. Our results demonstrate that Culex pipiens and Culex quinquefasciatus mosquitoes can effectively transmit L2 WNV. I also identified a L2 strain harboring an NS3-249P mutation (NS10) that limited infection to the midgut of Culex pipiens mosquitoes. The competence of North American Culex mosquitoes to transmit L2 WNV taken together with the ability of AMCRs and HOSPs to serve as reservoir hosts for L2 WNV demonstrates the capacity for L2 WNV transmission in the Western Hemisphere. Previous studies generated in this dissertation demonstrated that high viral titers in AMCRs were modulated by the NS3-249P mutation in the NS10 L2 WNV strain and that this same strain also generated lower infection rates in Culex pipiens compared to other L2 WNV strains, suggesting that the NS3-249 locus might be involved in concurrently modulating vector competence in Culex pipiens and viral titers in AMCRs. To conclusively determine the role the NS3-249P mutation in facilitating emergence of L2 WNV, I examined the phenotype of NS3-249P and NS3-249H L2 WNV mutations in a transmission cycle inclusive manner. Specifically, I found that the NS3-249P mutation was directly involved in decreasing fitness in Culex pipiens. Furthermore, I found that following infection in mice, the NS3-249 residue did not modulate neuroinvasive disease phenotypes and suggests that the emergence of L2 WNV in Greece was potentially facilitated by increases in force of transmission related to the occurrence of the NS3-249P mutation, rather than the emergence of a more neuroinvasive L2 genotype.
Open Access
Optimization and analysis of live attenuated DENVax-4 constructs
(Colorado State University. Libraries, 2013) Benjamin, Sarah, author; Nyborg, Jennifer, advisor; Livengood, Jill, advisor; Blair, Carol, committee member
Dengue virus is a flavivirus that infects millions of people every year, causing high fever and rash and resulting in death in some cases. There are four serotypes (DENV-1, DENV-2, DENV-3, and DENV-4) of dengue virus that are transmitted by the Aedes aegypti mosquito, endemic to tropical and subtropical regions of the world. Currently no vaccine for dengue fever is available. The rising number of confirmed cases and the increased habitat of Aedes aegypti increase the urgent need for a vaccine. Together with the Centers for Disease Control, Inviragen Inc. has developed a tetravalent live attenuated chimeric vaccine (DENVax) that is currently in phase II clinical trials. DENVax is based on an attenuated DENV-2 backbone. DENV-2 strain 16681 was passaged 53 times in primary dog kidney (PDK) cells. This strain is the current DENVax-2 strain used in Inviragen's vaccine. There are nine attenuating mutations, three of which are silent. Attenuating phenotypes of DENVax-2 include temperature sensitivity, decreased plaque size, and decreased replication efficiency in mosquito cells. To generate DENVax-1, DENVax-3, and DENVax-4, prM and E genes from wild type DENV-1, DENV-3, and DENV-4 strains were cloned into the infectious cDNA clone of the attenuated DENVax-2 backbone, resulting in chimeras. Tetravalent DENVax has shown significant immunogenic responses in AG129 mice and non-human primates, and is currently in phase II clinical testing. Data from preclinical tests showed that DENVax-4 is less immunogenic in AG129 mice and non-human primates compared to the other DENVax strains. Two projects in this thesis were completed to reengineer the current DENVax-4 strain to increase immunogenicity. The first project uses blind serial passaging of DENVax-4 first generation and reengineered DENVax-4b second generation as a method to select for strains better fit to grow in vivo. These passaged strains were tested for increased growth kinetics and immunogenicity in both AG129 mice and non-human primates. The second project uses sequencing data from the serial passaging to identify several adaptive mutations in each DENVax-4 construct. These mutations were cloned into DENVax-4 sequence to potentially optimize the strains for Vero cell growth. Three new DENVax-4 constructs were introduced, each containing a different mutation. In addition three new DENVax-4 constructs with wild type reversions of non-critical attenuating mutations were generated. Growth kinetics for all six new DENVax-4 clones were characterized, and testing was done in AG129 mice to determine neutralizing antibody titers.
Open Access
Population genetics and vector competence of Aedes aegypti in West Africa
(Colorado State University. Libraries, 2014) Dickson, Laura B., author; Black, William C., IV, advisor; Miller, Barry, committee member; Foy, Brian, committee member; Blair, Carol, committee member; Huyvaert, Kate, committee member
The mosquito, Ae. aegypti is the primary vector for all four serotypes of dengue (DENV 1-4) and yellow fever viruses worldwide. Dengue remains an important public health problem with an estimated 390 million cases per year and yellow fever outbreaks are still continuously reported even though a safe and effective vaccine exists. Aedes aegypti (L) is found globally in tropical and sub-tropical climates and exists as two subspecies: Ae. aegypti aegypti (Aaa) and Ae. aegypti formosus (Aaf) which have previously been defined by the presence or absence of scales on the first abdominal tergite. This classification system was developed in East Africa and is contradictory in West Africa where this mosquito is genetically diverse and exhibits great variation in susceptibility to DENV and YFV, which is a quantitative genetic trait. Understanding the population genetics, the vector competence, and the way in which genetic diversity contribute to vector competence of Ae. aegypti can improve our general understanding of mosquito/virus interactions and lead to potential ways to control these mosquitoes in nature. In this dissertation, the distribution of FST values calculated from deep sequencing data between mosquitoes from two diverse locations in Senegal, one location in Mexico, and one location in Thailand across the entire genome were compared to identify the degree of genomic divergence as well as identify genes involved in speciation between the various populations. The distribution of FST values were also compared in different gene regions and mutation types to identify which parts of the genome provide the greatest resolution of subspecies population structure. Genetic crossing experiments and deep sequencing of the sex determining locus of Ae. aegypti from Senegal and Thailand was used to demonstrate discrete genetic differences between the subspecies. The vector competence for a local sylvatic isolate of DENV-2 and two genetically diverse YFV isolates was compared between various collections of Ae. aegypti throughout Senegal to demonstrate that vector competence in these mosquitoes is dependent on the viral isolate. The genetic diversity of an important immune sensing gene, Dcr2 of the exo-siRNA pathway, was determined from mosquitoes with various vector competence phenotypes to test how the genetic diversity of this gene in individuals and populations of mosquitoes contribute to vector competence. Overall, data in this dissertation suggest 1) allopatric speciation between Ae. aegypti from Senegal and Mexico or Thailand, and sympatric speciation within Senegal based on the distributions of FST values and variations in the sex determining locus between populations, 2) vector competence of Ae. aegypti from Senegal is dependent on the flavivirus species and viral genotype, and 3) increased genetic diversity of Dcr2 in individual Ae. aegypti, but not from populations of Ae. aegypti, from Senegal is correlated with the ability of Ae. aegypti to control DENV-2 infection. These results aid in our general understanding of the role of genomic divergence in speciation, as well as our understanding of genetics, vector competence, and the genetics of vector competence of West African Ae. aegypti, which could provide insight into the way we identify subspecies and make predictions about vector competence in this region.
Open Access
Sindbis virus usurps the cellular HUR protein to stabilize its transcripts and promote infections of mammalian and mosquito cells
(Colorado State University. Libraries, 2010) Sokoloski, Kevin J., author; Wilusz, Jeffrey, advisor; Wilusz, Carol, advisor; Blair, Carol, committee member; Peersen, Olve, committee member; Quackenbush, Sandra, committee member
Members of the genus Alphavirus are recognized as significant human pathogens. Infection of vertebrate hosts often results in febrile illness and occasionally severe encephalitis. The archetypical alphavirus is Sindbis virus, which we have utilized in these studies. The genomic and subgenomic RNAs of Sindbis virus strongly resemble cellular mRNAs as they are capped at their 5’ ends and polyadenylated at their 3’ termini. These features allow the viral RNAs to act like cellular mRNAs and make them prime substrates for the cellular mRNA decay machinery. Sindbis virus RNAs are indeed subject to degradation by the cellular mRNA decay machinery in cell culture models of infection. Nevertheless, they decay by a mechanism that is different from the majority of cellular mRNAs as the decay of Sindbis virus transcripts is predominantly deadenylation-independent. As cellular mRNAs are often regulated by elements present in their 3’ untranslated regions (UTR), we hypothesized that these viral 3’UTR elements were functioning similarly to cellular mRNA stability elements resulting in the enhancement of viral infection. The primary goal of the research described in this dissertation was to characterize in mechanistic detail how the Sindbis virus 3’UTR represses deadenylation. To this end we used both cell free extracts and tissue culture systems to assay the effects of the viral 3’UTR on transcript stability. Interestingly, multiple elements were found to be independently repressing deadenylation in mosquito cytoplasmic extracts. Further examination revealed that a major stability determinant was the U-rich element (URE) observed in the 3’UTR of many alphaviruses. The ability to repress deadenylation in our cell free extract system was similarly observed with the UREs of Venezuelan equine, eastern equine, western equine and Semliki Forest viruses. Taken together, these data strongly assert that the repression of deadenylation via the URE is evolutionarily conserved. Prior to this study, the URE had no ascribed function. The repression of deadenylation imparted by the URE correlated with the binding of a cellular 38kDa factor. This 38kDa factor was determined to be the cellular HuR protein. Both the human and mosquito HuR proteins were found to bind with high affinity to the Sindbis virus 3’UTR. Reduction of cellular HuR protein levels using RNAi resulted in an increase in the rate of viral RNA decay. Furthermore, a significant decrease in the titer of progeny virus was observed. A similar effect on viral titer was observed when the predominant HuR binding site, the URE, was deleted from the viral 3’UTR. Taken together these observations identify a novel Alphavirus/ host interface that significantly impacts viral biology. Furthermore these studies have confirmed our hypothesis that the members of genus Alphavirus have indeed evolved RNA stability elements that resemble cellular mRNA stability elements for the purpose of enhancing viral infection. Furthermore these studies identify a potential therapeutic anti-viral target - the cellular HuR protein.
Open Access
Studies of Aedes a egypti immune pathways in response to dengue virus infection: evaluation of genetically modified mosquito fitness, immune pathway expression and natural genetic variation
(Colorado State University. Libraries, 2016) Raban, Robyn, author; Olson, Ken, advisor; Black, William, committee member; Blair, Carol, committee member; Kondratieff, Boris, committee member
Mosquito-borne diseases, such as dengue, are global health priorities, since they affect hundreds of millions of people per year. Vector control is one of the most successful tools for preventing mosquito-borne disease transmission and characterization of anti-viral mechanisms has led to the development of novel vector control strategies. One of the main mechanisms of mosquito antiviral defense is the small interfering RNA (siRNA) pathway, which has been shown to influence Aedes aegypti dengue viral infection. The RNA interference (RNAi) response of this pathway has been utilized to create transgenic Ae. aegypti lines, which are refractory to dengue virus type-2 (DENV2) infection. Additionally, genetic studies can also provide insights into function and natural variation of anti-viral pathways, potentially leading to the development of new approaches to vector control. The recent advancements in transgenic technologies are increasing the potential of genetically-modified vectors for disease management. In this project an RNAi based genetically-modified mosquito, Carb109, was evaluated for fitness after a backcrossing to a genetically diverse laboratory strain (GDLS). This method improved the mosquito fitness and transgene stability over previous non-backcrossed strains, making it more feasible for use in genetic vector control programs. However, positional effects of the transgene made the homozygote less fit and stable regardless of introgression into a GDLS. The next two aims explored the genetics of siRNA genes in a gene expression and a population genetic study. Gene expression of multiple potential anti-viral immunity genes in Ae. aegypti strains artificially selected for differences in midgut infection rates showed some evidence for the involvement of dicer2 (Dcr2), a siRNA gene in DENV2 midgut escape in these strains. Gene expression also varied naturally diurnally and over the lifetime of the mosquito, which demonstrated the importance of keeping consistent sample collection schedules for gene expression studies. Lastly, four non-synonymous substitutions in the Dcr2 gene were evaluated for association with susceptibility to DENV2 Jamaica 1409 infection, but no association was found.