Browsing by Author "Paulson, Sally L."

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    2021 Home Grounds and Animals PMG - Author Contact List
    Askew, Shawn D.; Wycoff, Stephanie B.; Bergh, J. Christopher; Bush, Elizabeth A.; Day, Eric R.; Del-Pozo, Alejandro; Derr, Jeffrey F.; Frank, Daniel L.; Hansen, Mary Ann; Hong, Chuan X.; Laub, Curtis A.; McCall, David S.; Miller, Dini M.; Nita, Mizuho; Parkhurst, James A.; Paulson, Sally L.; Pfeiffer, Douglas G.; Rideout, Steven L.; Wilson, James; Yoder, Keith S. (Virginia Cooperative Extension, 2021-02-12)
    This is a chapter of the 2021 Home Grounds and Animals PMG. This 2021 Virginia Pest Management Guide provides the latest recommendations for controlling diseases, insects, and weeds for home grounds and animals. This publication contains information about prevention and nonchemical control as alternatives to chemical control or as part of an integrated pest management approach. The chemical controls in this guide are based on the latest pesticide label information at the time of writing. Because pesticide labels change, read the label directions carefully before buying and using any pesticide. Regardless of the information provided here, always follow the latest product label instructions when using any pesticide. Commercial products are named in this publication for informational purposes only. Virginia Cooperative Extension does not endorse these products and does not intend discrimination against other products that also may be suitable.
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    2021 Home Grounds and Animals PMG - Nuisance Insects of the Home and Yard
    Miller, Dini M.; Paulson, Sally L. (Virginia Cooperative Extension, 2021-02-12)
    This is a chapter of the 2021 Home Grounds and Animals PMG. This 2021 Virginia Pest Management Guide provides the latest recommendations for controlling diseases, insects, and weeds for home grounds and animals. This publication contains information about prevention and nonchemical control as alternatives to chemical control or as part of an integrated pest management approach. The chemical controls in this guide are based on the latest pesticide label information at the time of writing. Because pesticide labels change, read the label directions carefully before buying and using any pesticide. Regardless of the information provided here, always follow the latest product label instructions when using any pesticide. Commercial products are named in this publication for informational purposes only. Virginia Cooperative Extension does not endorse these products and does not intend discrimination against other products that also may be suitable.
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    2021 Home Grounds PMG - Index
    Askew, Shawn D.; Wycoff, Stephanie B.; Bush, Elizabeth A.; Day, Eric R.; Del-Pozo, Alejandro; Derr, Jeffrey F.; Frank, Daniel L.; Hansen, Mary Ann; Laub, Curtis A.; McCall, David S.; Miller, Dini M.; Nita, Mizuho; Parkhurst, James A.; Paulson, Sally L.; Pfeiffer, Douglas G.; Rideout, Steven L.; Wilson, James; Yoder, Keith S.; Hong, Chuan X. (Virginia Cooperative Extension, 2021-02-12)
    This is a chapter of the 2021 Home Grounds and Animals PMG. This 2021 Virginia Pest Management Guide provides the latest recommendations for controlling diseases, insects, and weeds for home grounds and animals. This publication contains information about prevention and nonchemical control as alternatives to chemical control or as part of an integrated pest management approach. The chemical controls in this guide are based on the latest pesticide label information at the time of writing. Because pesticide labels change, read the label directions carefully before buying and using any pesticide. Regardless of the information provided here, always follow the latest product label instructions when using any pesticide. Commercial products are named in this publication for informational purposes only. Virginia Cooperative Extension does not endorse these products and does not intend discrimination against other products that also may be suitable.
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    Bed Bug Management in Low-Income, Multi-Unit Housing: An Evaluation of Resident Education and Cost-Effective, Minimally Toxic Suppression Methods
    Stedfast, Molly Logan (Virginia Tech, 2014-06-10)
    In the United States, we have been battling the bed bug (Cimex lectularius L.) resurgence for over ten years. Current treatment methods are labor intensive, time consuming, and very expensive. Many studies have evaluated the efficacy of treatment methods, but few have focused on bed bug suppression in multi-unit housing. Low income, multi-unit housing residents lack basic bed bug knowledge and are particulary vulnerable to bed bug infestations because they are unable to afford conventional treatment. In this study, diatomaceous earth (D.E.), an inexpensive desiccant dust labeled for bed bug control, was evaluated for its efficacy in killing bed bugs, and determined to be successful. A proactive bed bug suppression program that included D.E. was implemented in a low-income housing facility in Harrisonburg, VA. The program consisted of inexpensive, low toxicity, integrated bed bug management methods, including a novel strategy for applying a perimeter barrier of D.E. in apartment units (n = 121). Over the course of one year, both the number of initial infestations and the costs associated with bed bug treatments were reduced. Low-income, multi-unit housing residents (n = 479) from three cities (Harrisonburg and Richmond, VA; New Orleans, LA) were surveyed before and after an educational seminar to assess their bed bug. After attending the seminar, residents (n = 112) significantly improved (P < 0.0001) their bed bug knowledge, and were able to correctly answer more bed bug-related questions than they had before the educational seminar.
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    Biological rhythms in Aedes aegypti mosquitoes
    Eilerts, Diane Francine (Virginia Tech, 2021-06-03)
    Aedes aegypti mosquitoes are found globally and also act as the primary vector of Zika, dengue, and Chikungunya viruses, for which there are limited treatment options and no vaccines available. The use of insecticides as the main control strategy against diseases transmitted by this mosquito, is increasingly challenged by emerging resistance. Thus, there is a dire need for the development of novel approaches informed by an improved understanding of mosquito biology, to control mosquito populations and, ultimately, disease transmission. Rhythmic biological processes in mosquitoes help optimize resource exploitation by coordinating behaviors and physiology with fluctuating environmental conditions. Such synchronization enables organisms to adjust their physiology, metabolism, and behavior to predictable external cycles. In mosquitoes, circadian rhythmicity has been demonstrated in their biting and oviposition behavior, as well as their locomotor activity. However, little is known regarding how responses to long-range host cues are modulated by the circadian system. Here we show that both antennal sensitivity and olfactory behavior are time-of-day and odor-specific in Ae. aegypti females. Global transcriptomic analysis in whole heads of Ae. aegypti females reveal chemosensory genes differentially expressed throughout the day, providing insight into the molecular mechanisms behind daily variations in olfactory sensitivity and behaviors. We additionally show an odor-induced activation of mosquito behavior. Mosquito locomotion and behavior are also mediated by physiological state, and activity decreases after blood-feeding. Since the central clock components have been shown in other organisms to be redox-sensitive, we explored the role that diet heme plays in mediating behavioral changes following blood ingestion using artificial blood diets. We found that the transcription of the timekeeping gene period is reduced in the head immediately after feeding on a meal containing hemoglobin, but peripheral period transcription is reduced throughout the course of digestion following ingestion of a protein meal independent of hemoglobin inclusion. Overall, our results show that Ae. aegypti behavioral rhythms mediated by rhythmic gene expression are plastic and susceptible to external host cues and host blood digestion. This work can be leveraged for future studies investigating mosquito host-seeking and blood digestion to identify novel targets for vector control.
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    Bionomics of Ochlerotatus triseriatus Say (Diptera: Culicidae) and Aedes albopictus Skuse (Diptera: Culicidae) in emerging La Crosse virus foci in Virginia
    Barker, Christopher M. (Virginia Tech, 2001-07-10)
    Recently, the number of human cases of La Crosse encephalitis (LACE), an illness caused by mosquito-borne La Crosse (LAC) virus, has increased in southwestern Virginia, resulting in a need for better understanding of the virus cycle and the biology of its vectors in the region. This project examined the spatial and temporal distributions of the primary vector of LAC virus, Ochlerotatus triseriatus, and a potential secondary vector, Aedes albopictus. Ovitrapping surveys were conducted in 1998 and 1999 to determine distributions and oviposition habitat preferences of the two species in southwestern Virginia. For virus assay, adult mosquitoes were collected at a tire dump and a human case site during 1998 and 1999, and ovitrap samples were taken from a human case site in 2000. In a separate study, a landcover map of Wise County was created by supervised classification of Landsat Enhanced Thematic Mapper imagery, and maps indicating posterior probabilities of high mosquito abundance were created by combining ovitrap survey-derived, landcover-based prior and conditional probabilities for high and low mosquito abundance using remote sensing techniques and Bayesian decision-making rules. Both Oc. triseriatus and Ae. albopictus were collected from all ovitrap sites surveyed in Wise, Scott, and Lee Counties during 1998. Numbers of Oc. triseriatus remained high from late June through late August, while Ae. albopictus numbers increased gradually through June and July, reaching a peak in late August and declining thereafter. Overall, Oc. triseriatus accounted for 90.1% of eggs collected during this period, and Ae. albopictus made up the remaining 9.9%. Abundance of the two species differed among the sites, and in Wise County, relative Ae. albopictus abundance was highest in sites with traps placed in open residential areas. Lowest numbers of both species were found in densely forested areas. Ovitrapping at a human LACE case site during 1998 and 1999 revealed that Aedes albopictus was well-established and overwintering in the area. An oviposition comparison between yard and adjacent forest at the Duncan Gap human LACE case site in 1999 showed that Ae. albopictus preferentially oviposited in the yard surrounding the home over adjacent forested areas, but Oc. triseriatus showed no preference. LAC virus was isolated from 1 larval and 1 adult collection of Oc. triseriatus females from the Duncan Gap human case site, indicating the occurrence of transovarial transmission at this site. The supervised landcover classification for Wise County yielded a landcover map with an overall accuracy of 98% based on comparison of output classification with user-defined ground truth data. Posterior probability maps for Oc. triseriatus and Ae. albopictus abundance reflected seasonal and spatial fluctuations in mosquito abundance with an accuracy of 55-79% for Oc. triseriatus (Kappa=0.00-0.53) and 70-94% for Ae. albopictus (Kappa=0.00-0.49) when model output was compared with results of an ovitrapping survey. Other accuracy measures were also considered, and suggestions were offered for improvement of the model.
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    Cache Valley Virus in Aedes japonicus japonicus Mosquitoes, Appalachian Region, United States
    Yang, Fan; Chan, Kevin K.; Marek, Paul E.; Armstrong, Philip M.; Liu, Pengcheng; Bova, Jacob E.; Bernick, Joshua N.; McMillan, Benjamin E.; Weidlich, Benjamin G.; Paulson, Sally L. (2018-03)
    We detected Cache Valley virus in Aedes japonicus, a widely distributed invasive mosquito species, in an Appalachian forest in the United States. The forest contained abundant white-tailed deer, a major host of the mosquito and virus. Vector competence trials indicated that Ae. j. japonicus mosquitoes can transmit this virus in this region.
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    Chagas Disease in the United States: the Emerging Threat and the Role Climate and Awareness Play in Its Spread
    Lambert, Rebecca Click (Virginia Tech, 2007-03-27)
    This study evaluates the roles of temperature variability and disease awareness in the emergence of Chagas disease (American trypanosomiasis). Chagas disease is endemic in Latin America and primarily spreads to humans directly via the triatomine vector. Hosts for most triatomine species are mainly rodents and occasionally dogs. The disease itself is caused by a parasitic protozoan, Trypanosoma cruzi (T. cruzi) which is found in the triatomine's feces and is often spread while the triatomine is consuming a blood meal. T. cruzi from feces enters the body via an abrasion on the skin, the mucous membranes, conjunctivae, or through consumption. To determine the risk of Chagas disease transmission one must define qualities that make the triatomine an effective disease vector as well as investigate the level of disease awareness among physicians and the population within the vector's range. This thesis maps triatomine species within the U.S. that harbor T. cruzi naturally and that exhibit qualities of domesticity. These qualities are defined by whether the species bites humans and dogs as well as reports that the species has been found in the domestic setting. Ranges illustrating temperature thresholds for increased triatomine activity for 2000 and 2030 are also depicted. Additionally, outcomes of a physician survey are presented to gauge the status of Chagas disease awareness in areas at higher risk for disease transmission. Results reveal limited consideration of Chagas disease in physician diagnosis despite the higher risk range which extends through the southern U.S. and is predicted to expand significantly by 2030.
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    Chromosomal evolution in mosquitoes - vectors of diseases
    Naumenko, Anastasia Nikolayevna (Virginia Tech, 2017-06-23)
    The World Health Organization estimates that vector-borne diseases account for 17% of the global burden of all infectious diseases and has identified the mosquito as the most dangerous of all disease-transmitting insects, being responsible for several million deaths and hundreds of millions of cases each year. The study of mosquito genomics provides a deeper understanding of the molecular mechanisms involved in every aspect of vector biology, such as sex determination, host-parasite interaction, ecology, feeding behavior, immunity and evolutionary trends and can be used for the development of new strategies for vector control. We developed the first map of the mitotic chromosomes of the major vector for West Nile fever and lymphatic filariasis, Culex quinquefasciatus. The map was then successfully utilized for mapping of approximately 90% of available genetic markers to their precise positions on the chromosomes. Idiograms were integrated with 140 genetic supercontigs representing 26.5% of the genome. A linear regression analysis demonstrated good overall correlation between the positioning of markers on physical and genetic linkage maps. This will improve gene annotation and help in distinguishing potential haplotype scaffolds and regions of segmental duplications. It will also facilitate identification of epidemiologically important genes that can be used as targets for the vector control and provide a better framework for comparative genomics that will help understanding of the evolution of epidemiologically important traits. In another study, we confirmed the presence of the newly described species, Anopheles daciae, in regions of Russia using molecular data. Although sympatric with its sibling species, Anopheles messeae, five nucleotide substitutions in the internal transcribed spacer 2 of ribosomal DNA can be used to distinguish the morphologically similar species. Chromosome rearrangements have a significant impact on mosquito adaptation and speciation. Using sequencing data in combination with karyotyping, we demonstrated that significant differences in inversion frequencies distinguish An. messeae from An. daciae, suggesting that these inversions are actively involved in adaptation and speciation. It is essential to have reliable toolbox for correct identification of these species and to know their range for future possible malaria outbreaks prevention.
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    Chromosome and Genome Evolution in Culicinae Mosquitoes
    Masri, Reem Abed (Virginia Tech, 2021-07-14)
    The Culicinae is the most extensive subfamily among the Culicidae family of mosquitoes. Two genera, Culex and Aedes, from this subfamily have world-wide distribution and are responsible for transmitting of several deadly diseases including Zika, West Nile fevers, chikungunya, dengue, and Rift Valley fevers. Developing high-quality genome assembly for mosquitoes, studying their population structure, and evolution can help to facilitate the development of new strategies for vector control. Studies on Aedes albopitcus as well as on species from the Culex pipiens complex, which are widely spread in the United States, provide excellent models on these topics. Ae. albopictus is one of the most dangerous invasive mosquito species in the world that transmits more than 20 arboviruses. This species has highly repetitive genome that is the largest among mosquito genomes sequenced so far. Thus, sequencing and assembling of such genome is extremally challenging. As a result, the lack of high-quality Ae. albopictus genome assembly has delayed the progress in understanding its biology. To produce a high-quality genome assembly, it was important to anchor genomic scaffolds to the cytogenetic map creating a physical map of the genome assembly. We first developed a new gene-based approach for the physical mapping of repeat-rich mosquito genomes. The approach utilized PCR amplification of the DNA probes based on complementary DNA (cDNA) that does not include repetitive DNA sequences. This method was then used for the development of a physical map for Ae. albopictus based on the in situ hybridization of fifty cDNA fragments or gene exons from twenty-four scaffolds to the mitotic chromosomes from imaginal discs. This study resulted in the construction of a first physical map of the Ae. albopictus genome as well as mapping viral integration and polyphenol oxidase genes. Moreover, comparing our present Ae. albopictus physical map to the current Ae. aegypti assembly indicated the presence of multiple chromosomal inversions between them. To better understand population structure and chromosome evolution in Culicinae mosquitoes, especially in the Culex pipiens complex, we studied genomic and chromosomal differentiation between two subspecies Cx. pipiens pipiens and Cx. pipiens molestus. For the species responsible for the spread of human diseases, understanding the population dynamics and processes of taxa diversification is important for an effective mosquito control . Two vectors of West Nile virus, Cx. p. pipiens and Cx. p. molestus, exhibit epidemiologically important behavioral and physiological differences, but the whole-genome divergence between them was unexplored. The first goal of this study was to better understand the level of genomic differentiation and population structures of Cx. p. pipiens and Cx. p. molestus from different continents. We sequenced and compared whole genomes of 40 individual mosquitoes from two locations in Eurasia and two in North America. Principal Component, ADMIXTURE, and neighbor joining analyses of the nuclear genomes identified two major intercontinental, monophyletic clusters of Cx. p. pipiens and Cx. p. molestus. The level of genomic differentiation between the subspecies was uniform along chromosomes. The ADMIXTURE analysis determined signatures of admixture in Cx. p. pipens populations, but not in Cx. p. molestus populations. Thus, our study identified that Cx. p. molestus and Cx. p. pipiens represent different evolutionary units with monophyletic origin that have undergone incipient ecological speciation. The second goal was to study differences at the chromosome level between these two organisms. We first measured whole chromosome and chromosome arm length differences between Cx. p. molestus and Cx. p. pipiens as a basic cytogenetic approach. In addition, we used the novel Hi-C approach to detect chromosomal rearrangements between them since Hi-C was successful in detecting a known inversion in Cx. quinquefasciatus. Cx. p. molestus and Cx. p. pipiens embryos were used to perform the Hi-C technique. Analysis of the Hi-C data showed the presence of two different inversions in Cx. p. pipiens and Cx. p. molestus heatmap, which could explain their different physiology and adaptation in nature. Developing modern genomic and cytogenetic tools is important to enhance the quality of genome assemblies, improve gene annotation, and provide a better framework for comparative and population genomics of mosquitoes; also it is the foundation for the development of novel genome-based approaches for vector control.
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    Combined Roles of Glandular-haired Alfalfa and Natural Enemies in Alfalfa Pest Managment in Virginia
    Dellinger, Theresa Ann (Virginia Tech, 2003-09-19)
    Both alfalfa weevil, Hypera postica (Gyllenhal), (Coleoptera: Curculionidae), and potato leafhopper, Empoasca fabae (Harris), (Homoptera: Cicadellidae), remain key pests of alfalfa in Virginia. Commercial varieties of potato leafhopper-resistant (or glandular-haired) alfalfa were released in the mid-1990s, but the impact of alfalfa weevil on these varieties has not been well documented. In 1999, two large-scale field experiments were initiated to compare the performance of a glandular-haired alfalfa variety against a standard, non-glandular-haired variety under both alfalfa weevil and potato leafhopper pest pressures in the southwestern and Piedmont regions of Virginia over a 3 year period. Results indicated that alfalfa weevil must be managed in potato leafhopper-resistant alfalfa to limit crop loss. Surprisingly, similar densities of potato leafhoppers were found in both the glandular-haired and standard varieties. Both varieties frequently had similar yields and forage quality. In general, the glandular-haired variety did not outperform the standard variety. Results also indicated that insecticide application did not always provide the expected benefits of higher yields and forage quality, despite reducing pest densities for 2-3 weeks after application. These data suggest that the economic thresholds for one or both of these pests in Virginia may require adjustment. The potential impact of glandular-haired alfalfa on the natural enemies of alfalfa weevil was examined as well. Bathyplectes anurus (Thompson) (Hymenoptera: Ichneumonidae) was the dominant parasitoid attacking weevil larvae at both locations. Parasitization of weevil larvae by Bathyplectes spp. did not appear to be adversely affected by the presence of glandular trichomes on the potato leafhopper-resistant variety. Glandular trichomes had little impact on the infection of weevil larvae by the fungus Zoophthora phytonomi as well, but this was not unexpected. The genetic variation of B. anurus was surveyed at both study sites using RAPD-PCR to establish or eliminate the possibility that differences in parasitization levels between the Piedmont and southwestern regions could be attributed to the presence of different parasitoid strains. Most of the detected phenotypic variation was attributed to within population variation, with very little variation occurring between the two populations. However, the between population variation was statistically significant in 2000, but not in 2001.
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    Cross-reactivity among alphaviruses provides insight into viral emergence and novel defense strategies
    Webb, Emily Morgan (Virginia Tech, 2022-04-13)
    Alphaviruses are a group of medically relevant arthropod-borne viruses (arboviruses) belonging to the Togaviridae family that are maintained by mosquito vectors. These zoonotic viruses are clustered into two groups: New World and Old World, depending on their geographical origin/distribution and clinical manifestations. Both of these groups cause disease symptoms of an acute febrile illness; however, each group has a distinct, hallmark disease symptom; New World alphaviruses, such as Eastern, Western, and Venezuelan equine encephalitis viruses (EEEV, WEEV, and VEEV, respectively), present with severe encephalitis while Old World alphaviruses, such as Sindbis, chikungunya, and Mayaro viruses (SINV, CHIKV, and MAYV, respectively) present with an incapacitating polyarthralgia that can persist for years following initial infection. To date, the most effective means of controlling these arboviral infections is through mosquito control programs. However, these programs have crucial limitations in their effectiveness; therefore, novel approaches are necessary to control the spread of these crippling pathogens and lessen their disease burden. Given the close phylogenetic and antigenic relationship between MAYV and CHIKV, we hypothesized that prior CHIKV immunity may affect the outcome of MAYV disease and/or limit its emergence in humans. Our work has shown that anti-CHIKV neutralizing antibodies can provide cross-protective immunity against MAYV disease. Alongside these studies, we have characterized the potency of a camelid-derived single-domain antibody (sdAb) that neutralizes a breadth of alphaviruses, including CHIKV and MAYV. With these data, we have designed and generated transgenic Aedes aegypti mosquitoes that express two anti-CHIKV sdAbs to target infection, dissemination, and transmission of MAYV and CHIKV within this deadly vector. These findings are particularly significant because they highlight the ability to co-target two emerging alphaviruses that are crippling public health and obliterating quality of life around the globe within a single defense strategy.
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    Determination of Allosteric Solvent Effects Between Acetylcholinesterase and Mosquito Selective Carbamates: Implications for High Throughput Screening of Insecticides
    Swale, Daniel Robert (Virginia Tech, 2009-12-04)
    Malaria is vectored by the mosquito Anopheles gambiae (Ag) in Sub-Saharan Africa and infects approximately 500 million people annually. The increasing prevalence of pyrethroid-resistant mosquitoes has amplified the need for development of new, selective mosquitocides for use on insecticide-treated nets. We have developed several phenyl-substituted N-methylcarbamates producing a high degree of selectivity for Anopheles gambiae acetylcholinesterase (AgAChE) over human AChE. Molecular models suggest alternate conformations (flexibility) of W84 and W431 (Ag numbering) at the hydrophobic subpocket of the AgAChE active site and poor flexibility within human AChE, allowing for the high selectivity of our novel carbamates. Initial selectivity data was obtained through screening of these insecticides while using ethanol as a solvent. Re-screening of these carbamates in the presence of 0.1% DMSO (v/v) resulted in antagonism of inhibition for AgAChE, thus reducing the AgAChE-selectivity by at least 10-fold. However, the presence of 0.1% DMSO did not antagonize the inhibition of human, Drosophila melanogaster, or Musca domestica AChE. Non-selective carbamates also displayed no solvent-dependent antagonism of inhibition in any species studied, including AgAChE. Molecular models provide an explanation for antagonism of inhibition when DMSO is present. I, and collaborators, propose that W84 and W431 in AgAChE comprise an allosteric pocket that is stabilized by DMSO and is responsible for the solvent-dependent antagonism of inhibition observed with AgAChE.
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    Development, Characterization, and Use of Molecular Tools to Study Immune-Driven Zika Virus Evolution
    Marano, Jeffrey Matthew (Virginia Tech, 2023-02-16)
    Emerging viruses represent a significant threat to human health. Understanding the drivers of emergence, such as viral evolution, is a critical avenue to combat these pathogens. One specific group of emerging pathogens of interest is flaviviruses. Flaviviruses are arthropod-borne viruses (arbovirus) in the family Flaviviridae. The medically relevant flaviviruses can be divided into two groups – tick-borne and mosquito-borne. Included within the mosquito-borne flaviviruses group are dengue viruses 1-4 (DENV 1-4), which causes 400 million infections annually, and Zika virus (ZIKV), which caused over 128 million infections from 2013-2018. These viruses, which are cocirculating, share high sequence similarity in key antigenic regions. Because of these similarities, pre-existing immunity to DENV has been correlated with altered pathogenesis of subsequent ZIKV infections. Despite this, there has been little analysis of the effects of pre-existing DENV immunity on the evolution of subsequent flavivirus infection, despite being characterized for many other viruses. Given that mutation that could arise from cross-reactive immune selection could alter pathology or transmissibility, it is critical to assess the role of cross-reactive immune selection as an evolutionary driver. However, this line of research has historically been difficult due to the inherent toxicity of flavivirus infectious clones in bacteria. To mitigate the toxic nature of flavivirus clones, we developed several entirely in vitro workflows using a combination of rolling circle amplification (RCA) and replication cycle reaction (RCR). We demonstrated that RCA was a comparable substitute to traditional plasmid propagation using an alphavirus infection clone. We further demonstrated that RCR could be used to generate infectious clones by producing infectious clones of DENV2 and SARS-CoV-2, as well as demonstrating it could be used to introduce mutations into infectious clones by producing a D614G SARS-CoV-2 mutations. With this technology in place, we used in vitro directed evolution system, where we passaged ZIKV in convalescent patient serum to assess the role of cross-reactive immune selection as an evolutionary driver. After passaging, we performed next-generation sequencing to assess the impacts of cross-reactive immune selection on the viral populations and to identify mutations that arose post-passaging. We observed that ZIKV passaged in convalescent DENV serum had reduced diversity and divergence in the premembrane region. Within the convalescent DENV passaged population, we identified two mutations of interest with the dominant antibody binding region – E-V335I and NS1-T139A. These mutations were then introduced using our in vitro workflows. The resulting mutant viruses were then assessed for their replicative fitness in mammalian cell culture and mosquito models and their sensitivity to neutralization. We observed that while both E-V355I and NS1-T139A have increased fitness in mammalian cells, they had reduced fitness in mosquitoes. These results align with the trade-off hypothesis, which states that in a multi-host system, adaptation to one host reduces fitness in the other hosts. When we assessed the neutralization sensitivity of the mutants, we observed that while NS1-T193A was resistant to neutralization, E-V355I was more sensitive to neutralization. These results indicate that neutralization escape is not necessary for enhanced post-passaging in convalescent DENV serum. Our findings demonstrate that cross-reactive immune selection can generate several mutations with altered fitness in mammalian cells and mosquitos. This research is significant for both highlighting novel technologies to facilitate molecular virology and demonstrating that cross-reactive immune selection has the potential to alter the evolutionary trajectory of flaviviruses. This work provides critical information to understand how flaviviruses are evolving and emerging, and therefore critical information to address their threat to human health.
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    Discrete and continuous mathematical investigation of juvenile mosquito dynamics
    Walker, Melody Anne (Virginia Tech, 2021-06-15)
    There are thousands of species of mosquitoes, but only a handful of these species carry pathogens that cause human diseases. Here, we study two species, Aedes albopictus and Aedes aegypti, which transmit infections such as dengue, Zika, Mayaro virus, and La Crosse virus. Curtailing these diseases is a good reason to consider control of mosquito populations. However, mosquitoes are quite hardy and spraying of pesticides is typically a short-term solution. Thus, more long-term solutions require careful thought about mosquito populations, including early juvenile aquatic stages: egg, larva, and pupa. In this dissertation, we examine the factors that affect the dynamics of aquatic stages by creating mathematical models. The goal is to assess what key biological features most impact the total population. Both Aedes albopictus and Aedes aegypti lay eggs in small containers, producing limitations on space and food. We investigate how restricting resources changes development time, survival to adulthood, and body mass at emergence. The interactions between these changes are complicated, so to disentangle their effects we create three different mathematical models. The first model is discrete in time and focuses on the best way to incorporate the influence of larval density. We compare the impact of larval density by inputting seven different functional forms altering survival and development time. Larval density used in the model is determined from the average of the population size over the past one to thirty-six days. The second model is also discrete in time but focuses on the interaction between survival, development time, and mass. This model considers three levels of mass. Here, we use the density-dependent function determined from our first model and fit the maximum value for development time from experimental data. Survival values are fit using constants and a density-dependent function. Finally, growth is built in as a function of food. Food decreases at each time point as a function of the total larvae in the environment. We compare between model formulations with Akaike information criterion. The third model examines the ramifications of constricting resources on growth verses death. We employ a partial differential equation that has three independent variables: time, age, and mass. We find that density dependence is highly influential in the maturation of mosquitoes, and it is more essential to include its impacts on development time than on survival. These findings can be incorporated into a larger framework of disease dynamics, and give insight into better control of mosquitoes and disease spread.
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    Dynamics of La Crosse virus: Surveillance, Control and Effect on Vector Behavior
    Yang, Fan (Virginia Tech, 2017-01-31)
    La Crosse virus (LACV) encephalitis is the most common and important endemic mosquito-borne disease of children in the U.S. with an estimated 300,000 annual infections. The disease is maintained in a zoonotic cycle involving the eastern treehole mosquito, Aedes triseriatus and small woodland mammals such as chipmunks and squirrels. The objectives of this study were 1) to conduct surveillance of LACV and other mosquito-borne viruses; 2) to evaluate the effect of virus infection on mosquito host-seeking and neurotransmitter levels, and 3) to determine the effectiveness of barrier sprays to control infected mosquito vectors. Our surveillance study demonstrated the involvement of an invasive species, Aedes japonicus, in the transmission cycle of Cache Valley virus (CVV). CVV is a mosquito-borne virus that is closely related to LACV. Thus, surveillance is a critical step in public health, providing pathogen distribution and frequency data as well as identifying and incriminating new vectors. LACV infection did not affect the host-seeking behavior of Ae. triseriatus females. Using high performance liquid chromatography with electrochemical detection (HPLC-ED), the levels of serotonin and dopamine were measured in infected and uninfected mosquitoes. Serotonin is known to affect blood-feeding and dopamine affects host-seeking. Serotonin levels were significantly lower in LACV-infected mosquitoes but dopamine levels were unaffected by virus. A previous study found that LACV infection caused an alteration in mosquito blood-feeding in a way that could enhance virus transmission. This work showed that LACV infection can reduce the level of serotonin in the mosquito, promoting virus transmission through altered blood-feeding without impairing the vector's ability to locate a host. Standard CDC bottle assays were used to evaluate the efficacy of two pyrethroids and two essential oil sprays on LACV infected and uninfected mosquitoes. LACV-infected Ae. triseriatus females were more susceptible to both pyrethroids than uninfected ones. Infection status did not affect the susceptibility of Ae. albopictus to either pyrethroid. The essential oils were inconsistent in their effects. These results demonstrate that barrier sprays may be a viable part of a mosquito control program, not just to reduce the biting rate but to potentially reduce the virus-infected portion of the vector population.
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    Ecology and Climate Tolerance of Emerging Tick Disease Vectors in Central Appalachia/Southwestern Virginia
    Whitlow, Amanda Marie (Virginia Tech, 2021-06-29)
    Little is known about the tick populations found within the central Appalachian/southwestern region of Virginia. The main focus of this research was to better assess local tick communities in the central Appalachian-Southwestern region of Virginia, which was addressed by determining species diversity, habitat associations, seasonal phenology, pathogen prevalence, and ecological factors that influences tick presence and abundance. A field study was conducted from June 2019 - November 2020 across 8 counties and 3 habitat types. Forested habitats exhibited greater tick species diversity than pasture and urban habitats. Each tick species was observed to be associated with particular habitats. The presence of B. burgdorferi sensu stricto (causative agent of Lyme disease), the human variant of A. phagocytophilum (causative agent of human granulocytic anaplasmosis), and Powassan virus (the causative agent of Powassan encephalitis) were detected in collected field specimens, suggesting a significant threat to public health. The detection of Powassan virus RNA in local Ixodes scapularis ticks is the first evidence of this viral pathogen within the region. The overwintering abilities of ticks, whose populations are expanding or becoming more invasive, including Haemaphysalis longicornis, Amblyomma americanum, and Amblyomma maculatum, were examined through a combination of laboratory and field experiments. Amblyomma americanum and H. longicornis nymphal ticks had a lower supercooling temperature than adult ticks, suggesting their potential to overwinter better; A. maculatum nymphs had similar average supercooling temperatures as the other two species at nymphal stage. Via a field experiment, A. americanum, H. longicornis, and A. maculatum were subjected to natural elements of a Virginian winter in a two-factor design investigating elevation and potential insulation coverage. Elevation and insulation coverage were found to have no significant impact on the overwintering survival of H. longicornis and A. americanum. However, the life-stage of the tick was determined to be a significant factor that dictated the survival of ticks of these species. Overwintering survival of Amblyomma maculatum nymphs was influenced by insulation (proxied by leaf litter); which may be attributed to this tick's preference of drier climate. Low overwintering survival suggests that a tick may not be able to establish a permanent population within the area.
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    The Effects of European corn borer on whole-plant yieldand root knot nematode fitness in corn
    Tiwari, Siddharth (Virginia Tech, 2007-04-26)
    Field studies were conducted over two growing seasons to evaluate the effect of different levels of third instar European corn borer, Ostrinia nubilalis Hübner (Lepidoptera: Crambidae), on whole-plant dry matter in corn grown for silage. Mean (± SEM) whole-plant dry matter was significantly greater by 18.8% in uninfested control plants than in plants with an infestation level of 6 larvae/plant in 2004. Whole-plant dry matter in 2005 was significantly greater by 10.5% in control plants than in plants with an infestation level of 5 larvae/plant. Economic injury levels were calculated for each year using regression equations between whole-plant dry matter and European corn borer infestation level. Plant growth stage and infestation level had no effect on percent acid detergent fiber, neutral detergent fiber, and crude protein values for either year. Greenhouse studies were conducted to examine the relationship between aboveground herbivory by European corn borer and belowground herbivory by root knot nematode, Meloidogyne incognita Chitwood (Tylenchida: Heteroderidae), in corn. Two experiments were conducted to measure belowground herbivory by M. incognita in juvenile penetrations and eggs/root system. In the first experiment, the main effects interaction was not significant for either M. incognita juvenile penetrations or eggs/root system. Overall mean juvenile penetrations/root system across all three growth stages, at infestation levels of 1 and 3 larvae/plant were significantly less than in the non-infested control. In addition, overall mean eggs/root system at an infestation level of 3 larvae/plant were significantly less than in the control. In the second experiment, the main effects interaction was significant for both juvenile penetrations and eggs/root system. At the 8 and 10 leaf growth stages, juvenile penetrations/root system at infestation levels of 1 and 3 larvae/plant were significantly less than in the control. In addition, eggs/root system at an infestation level of 3 larvae/plant were significantly less than in the control, at all growth stages. In the reciprocal study, which examined the effect of different M. incognita inoculation levels on European corn borer stalk tunneling, no significant effect of inoculation level on European corn borer stalk tunneling was found.
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    Effects of La Crosse virus infection on the host-seeking behavior and levels of two neurotransmitters in Aedes triseriatus
    Yang, Fan; Chan, Kevin K.; Brewster, Carlyle C.; Paulson, Sally L. (2019-08-09)
    Background La Crosse virus (LACV) infection has been shown to manipulate the blood-feeding behaviors of its main vector, Aedes triseriatus. Here, we investigated the effects of virus infection on serotonin and dopamine and their potential roles in host-seeking. In mosquitoes, serotonin depletion has been shown to interfere with blood-feeding but not host-seeking. Dopamine depletion does not affect either blood-feeding or host-seeking; elevations of dopamine, however, has been shown to inhibit host-seeking. The purpose of this study was to determine the effects of LACV infection on the host-seeking behavior of and neurotransmitter levels in Ae. triseriatus. Methods Host-seeking behavior was evaluated using a uni-port olfactometer and a membrane feeder assay. Levels of serotonin and dopamine in infected and control mosquito heads were measured using HPLC-ED. Results Infection with LACV significantly inhibited the activation and attraction of Ae. triseriatus females to a host. A higher proportion of uninfected Ae. triseriatus females were activated by the presence of a host compared to infected mosquitoes and more uninfected mosquitoes were full responders (95.7%) compared to infected ones (91.1%). However, infection with LACV did not significantly affect the landing, probing, or blood-feeding rates of female mosquitoes. LACV-infected mosquitoes had lower serotonin levels than controls (104.5 vs 138.3 pg/head) while the dopamine levels were not affected by infection status (282.3 vs 237 pg/head). Conclusions Our work suggests that virus-induced reduction of serotonin is related to previously reported blood-feeding alterations in LACV-infected mosquitoes and could lead to enhanced transmission and increased vectorial capacity. In addition, some aspects of host-seeking were inhibited by virus infection.
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    Efficacy of Entomopathogenic Nematodes and Entomopathogenic Fungi against Masked Chafer White Grubs, Cyclocephala spp. (Coleoptera: Scarabaeidae)
    Wu, Shaohui (Virginia Tech, 2013-05-07)
    Entomopathogenic nematodes (EPN) (Heterorhabditis bacteriophora and H. megidis) and entomopathogenic fungi (EPF) (Metarhizium anisopliae and Beauveria bassiana) were evaluated for efficacy against masked chafer white grub, Cyclocephala spp., under laboratory and greenhouse conditions, as well as their efficacy against various grub stages in the field.  Under both laboratory and greenhouse conditions, additive interactions were found between EPN and EPF in their combined application against Cyclocephala spp., except a few observations that showed antagonism or synergism.  Significantly greater control occurred from the combination of a nematode and a fungus compared with a fungus alone, but not compared with a nematode alone.  The combined effect did not differ significantly for nematode and fungi applied simultaneously or at different times.  EPF had no significant impact on EPN infection and production of infective juveniles (IJs) in grub carcasses.  Nematodes alone or in combination with fungi were comparable to the insecticide Merit 75 WP (imidacloprid) against 3rd instar Cyclocephala spp in the greenhouse. Efficacy of EPF and EPN varied dramatically between field sites and conditions; EPN and EPF applied alone or in combination were less effective than Merit 75 WP in >50% field trials, but some EPN + EPF treatments were more effective than the insecticide in reducing grub numbers.  EPN and EPF showed better potential than insecticides for providing extended control of white grubs in the subsequent generation.  In addition, the sub-lethal effects of EPF on southern masked chafer, C. lurida, were investigated.  Neither M. anisopliae nor B. bassiana had a sub-lethal effect on grub weight gain, adult longevity, oviposition, pupation and eclosion.  Finally, interaction between H. bacteriophora and M. anisopliae was examined to determine the potential of the nematode in improving fungal distribution in soil.  H. bacteriophora enhanced fungal distribution in sandy loam soil without grass thatch, but not in sandy soil with thatch.  In both soil types, soil depths significantly affected nematode and fungal distribution. In water profile, M. anisopliae conidia germinated hyphae that attached to sheath of H. bacteriophora IJs, which molted to detach from the fungus.  IJs mortality and virulence were not affected by the presence of M. anisopliae.