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INTRODUCTION: In the Americas, dengue disease which is associated with the Dengue virus has the highest incidence of all mosquito-borne viruses with cyclic epidemics every 3 to 5 years. It is estimated that the number of dengue cases is underreported globally as most cases of infection are asymptomatic, affecting both clinical surveillance and timeliness (Angelo et al., 2020). Wastewater-based epidemiology has recently gained much attention and has the potential to provide a snapshot view of community health (Irene Xagoraraki, 2019). Research into the feasibility of wastewater monitoring for the detection of viral outbreaks has been emerging. Detection of non-waterborne viruses including arboviruses Zika, West Nile virus, Dengue virus, and yellow fever virus in urine samples which suggests the concept of wastewater-based epidemiology could be used in the future (Irene Xagoraraki, 2019). AIM: In the current study, we investigate the concentration and presence of dengue virus nucleic acid in various fluids of infected patients. The hope is to further contribute to the growing data on the feasibility of the use of WBE to monitor DENV infections as a tool for community-level public health surveillance. METHODS: De-identified secondary data was obtained from a community-based enhanced surveillance study in Pau da Lima community, Salvador, Brazil and included the results of analysis of serum, oral fluid, and urine. Literature review and evidence gap mapping was completed to inform on existing research and gaps in knowledge. RESULTS: A total of 96 participants were confirmed positive for dengue virus in laboratory testing. Of these, 53 (55%) were positive in acute-phase serum, 5 (5%) in acute-phase urine, 2 (3.5%) in convalescent-phase urine, and 1 (2%) in acute-phase oral fluid. There were no observed statistically significant associations between demographic and clinical manifestations and positive nucleic test detection of dengue virus RNA in acute-phase urine. DISCUSSION: Testing of acute- and convalescent-phase urine resulted in an overall 7% positivity across all confirmed positive patients via serum. Higher frequencies of detection in urine were seen during the acute-phase (days 0-7 days) than in the convalescent-phase (days 10-40 days). Future research into detection rates and viral shedding in feces or the use other highly sensitive assays could provide valuable information for in the feasibility of wastewater surveillance.

Dengue virus (DENV) is transmitted to human cells in the skin after the bite of a mosquito. As an abundant cell type in the skin, fibroblasts are hypothesized as one of the cell types in the skin susceptible to DENV infection and replication. Their innate immune response to DENV might be critical for regulating DENV early during infection, although this has yet to be fully characterized. Thus, the aim of this work was to characterize DENV infection of fibroblasts, and gain insight surrounding the innate immune response to infection. This was evaluated by comparing their characteristics in vitro against BHK-21 cells, a fibroblast cell line known to grow rapidly in culture and that is also highly susceptible to DENV. Results show that regardless of seeding density, fibroblasts demonstrate slower growth kinetics compared to BHK-21 cells in vitro. Infection with DENV-1 16007 and DENV-2 16681 at 0.01 and 0.1 MOI does not induce cytopathic effects in cultures of human fibroblasts. Infectious DENV titers in viral supernatants collected from fibroblasts are also lacking at low MOIs, especially for DENV-1. Conversely, BHK-21 cells proved to be permissible to both DENV-1 and DENV-2 infection at 0.01 and 0.1 MOI as early as 1 day post infection and infectious DENV titers were measured for both serotypes and MOIs. When the seeding density of human fibroblasts and the MOI of infection were increased, differences in the amount of floating cells were observed for infection at 0.1 and 1 MOI. At 5 and 10 MOI though, cytopathic effects were observed by days 10 and 6 for DENV-1 and DENV-2 infected fibroblasts respectively. Infection from DENV-2 also appeared to be produce stronger CPE, similarly to BHK-21 cells. Infectious DENV titers in viral supernatants collected from DENV-1 infected fibroblast did not appear to increase post infection for any MOI while titers for DENV-2 slightly increased after day 0. Lastly, an 18-plex analyte Luminex kit identified several cytokines and chemokines (IL-6, IL-8, CCL2, CXCL10, CCL5, CCL7, and CXCL1) that were strongly expressed by fibroblasts infected with DENV-2 at 10 MOI days 0-9 post infection. Overall, these results establish an infection model for investigating DENV-1 and DENV-2 infection in human fibroblast. Further insight surrounding the role human fibroblasts may play during the innate immune response to DENV-1 and DENV-2 is also uncovered.

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.

Dengue virus, consisting of four distinct serotypes (DENV1-4), is a mosquito-borne flavivirus that causes over a hundred million human infections annually. DENV infections can lead to a range of outcomes, from asymptomatic infection to classical dengue fever (DF) to dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). B cell immune responses are critical for clearing flavivirus infections through serum antibodies from long-lived plasma cells (LLPCs) and the activation of memory B cells (MBCs). Conversely, B cells and antibodies from a previous infection can be associated with severity in a subsequent secondary DENV infection. The recent explosive epidemic of Zika in the Americas and the co-circulation of Zika virus (ZIKV) with the phylogenetically related DENV underscored the relevance of defining the role of MBC and antibody responses in these antigenically related viruses that overlap or succeed one another in space and time. In this dissertation, the effect of pre-existing DENV immunity in the development of B cell immune responses after a ZIKV infection and the analysis of the polyclonal MBC response to the four DENV serotypes plus ZIKV during DENV infection are studied. As a means to study MBCs at a polyclonal level, we developed, optimized and implemented a Multi-Color FluoroSpot (MCF) to enable the analysis of type-specific versus cross-reactive B cell populations at a single-cell level for all four DENV serotypes and ZIKV. This novel assay permitted the analysis of unique human samples from long-term cohort and hospital studies of pediatric DENV and ZIKV infections in Nicaragua. We then analyzed long-term MBC alongside serum antibody responses after ZIKV infection in patients who had experienced prior DENV infections or were DENV-naïve. We showed that ZIKV infection generates strong type-specific MBC responses and that the majority of late convalescent anti-ZIKV serum neutralizing activity was attributable to ZIKV-specific antibodies, despite prior DENV immunity. Moreover, the initial high levels of ZIKV and DENV cross-reactive MBC responses declined over time in patients with prior DENV immunity. Overall, our results suggest that despite the phylogenetic and antigenic relatedness between DENV and ZIKV, immunity to DENV only modestly shapes the breadth of B cell responses to ZIKV.The development of polyclonal MBCs to the four DENV serotypes and ZIKV during DENV infection is not fully understood. Here, we analyzed polyclonal MBCs from primary and secondary DENV infections over time using the MCF assay. We showed that DENV elicits robust type-specific MBC responses after primary and secondary DENV infection, with cross-reactive responses always being significantly higher. Despite the comparable number and proportion of type-specific and cross-reactive MBCs between primary and secondary DENV infections, we found that the breadth of the cross-reactive MBC response was greater after a secondary DENV infection, with more MBCs recognizing multiple DENV serotypes as well as ZIKV. Overall, this shows that the breadth of DENV MBCs enables broader recognition of not-yet encountered DENV serotypes, as well as the related flavivirus ZIKV, in subsequent infections. Taken together, the work included in this dissertation has made significant contributions to the understanding of the development of B cell immunity after primary and secondary DENV and ZIKV infections in patients from a dengue-endemic setting, with important implications for vaccine development.

Diagnostic assays are designed to detect a unique analyte profile in a disease of interest. Nucleic acids contain an information-dense sequence, and thus are ideal candidates for unique analytes. The gold-standard of nucleic-acid-based detection is PCR which has high sensitivity, but involves time, expertise, and cost. DNA molecular logic technology holds much promise as an alternative molecular detection method due to the potential to save cost and expertise, while also achieving a high sensitivity. However, nucleic acid detection in biomedical applications carries with it the difficulty of choice of appropriate sequence and potential biological sample background. This work describes the development and testing of a DNA molecular logic implementation of a viral assay for dengue virus. This work initiated with an attempt at an implementation of a solution-based viral assay. The challenges arising from the solution-based implementation, suggested the need for additional tools and designs, and have given rise to the computational aims. Included in this work is the development of computational tools for aiding the design of DNA molecular logic bioassays, and the use of these tools in bioassay design. The tools developed are a continuous kinetic model of the DNA-based assay elements, a discrete stochastic model to characterize limit of detection, and an algorithm to search databases for assay target sequences. The result of this work are tools that may be used in the context of DNA-based bioassay design. Future directions suggest work on developments to the bioassay. These include an acoustophoretic microparticle-based assay for signal enrichment and a microarray-based host gene expression profile.

Several mosquito-borne diseases affecting humans are emerging or re-emerging in the United States. The early detection of pathogens in mosquito populations is essential to prevent and control the spread of these diseases. In this study, we tested the potential applicability of the Lawrence Livermore Microbial Detection Array (LLMDA) to enhance bio-surveillance by detecting microbes present in Aedes aegypti, Aedes albopictus and Culex mosquitoes that are major vector species globally, including in Texas. The sensitivity and reproducibility of the LLMDA was tested in mosquito samples spiked with different concentrations of dengue virus (DENV) revealing a detection limit of >100 but Ae. aegypti populations. Wolbachia was also detected in several of the field samples (Ae. albopictus and Culex spp.) by the LLMDA. Our findings demonstrated that the LLMDA can be used to detect multiple arboviruses of public health importance including viruses that belong to the Flavivirus, Alphavirus and Orthobunyavirus genera. Additionally, insect-specific viruses and bacteria were also detected from field-collected mosquitoes. Another strength of this array is its ability to detect multiple viruses in the same mosquito pool allowing for the detection of co-circulating pathogens in an area, and the identification of potential ecological associations between different viruses. This array can aid in the bio-surveillance of mosquito borne viruses circulating in specific geographical areas.

The spillover of emerging viruses into human populations continuously threatens public health. From 2013-2016, Ebola virus (EBOV) caused an explosive outbreak in West Africa with more than 28,000 cases, 11,000 of which were fatal. Simultaneously, from 2015-2016, Asian Zika virus (ZIKV) caused an unprecedented epidemic wave with an estimated 1.5 million infections and associated neuropathology throughout dengue virus (DENV) endemic regions of the Americas and the Caribbean. In response to these outbreaks, we employed seroepidemiological techniques and utilized a modified anthrax toxin delivery system to study the antibody and T cell responses in individuals infected or exposed to EBOV, African and Asian ZIKV, and DENV. Prior to the 2015-2016 outbreak, the incidence of ZIKV in Africa had not been evaluated and the prevalence estimates for DENV were scarce. We showed continued human transmission of ZIKV and DENV and phylogenetic analysis revealed evidence for distinct African ZIKV strains circulating in West Africa for decades. T cell analysis demonstrated that individuals mount sustained specific and cross-reactive responses to nonstructural protein 3 (NS3) during the acute and late convalescent phases. Development of a T cell diagnostic based on responses to NS3 distinguishes human infections by African ZIKV and DENV. We confirmed this finding in human immunodeficiency virus (HIV)-infected and uninfected individuals from Salvador, Brazil. We also showed that HIV-infection is associated with T cell responses that are lower in magnitude to ZIKV and DENV proteins. These findings fill a critical knowledge gap in the epidemiological research on ZIKV/DENV in Africa and have important implications for vaccine and diagnostic development. Serological surveys conducted after the 2013-2016 EBOV outbreak revealed that a significant portion of transmission events went undetected because some individuals contracted infection with few if any symptoms. While several hypotheses could explain this phenomenon, including properties of the infecting virus, low inoculum, route of transmission, or various host factors, a robust immune response is also a potential explanation. We identified EBOV antibody-positive individuals from Lagos, Nigeria and the Democratic Republic of Congo who had not experienced Ebola virus disease (EVD)-like illness. We showed that the seropositive asymptomatic individuals from Lagos had virus-specific T cell responses to the nucleoprotein, matrix protein, and glycoprotein that were greater in magnitude when compared to survivors of severe EVD. These findings suggest that T cell immunity may protect against severe EVD, which has important implications for understanding the immunopathogenesis of EVD and EBOV vaccine development.

This literature review will discuss Zika virus and the salient research on antibody dependent enhancement and how this mechanism may lead to congenital defects. Specific objectives include: the mechanism of antibody dependent enhancement, Zika and dengue virus pathogenesis, placenta pathophysiology, and how changes in viral virulence may play a role the pathogenesis of neurologic congenital defects seen in infants infected with Zika virus in utero. While some cohort studies have examined the relationship between prior dengue immunity, Zika virus infection in pregnancy, and effects on neonatal outcomes further prospective studies using large cohorts and more detailed lab testing and imaging is essential to better understand this relationship. A proposed study enrolling a large cohort of women in the 6th- 8th week of pregnancy from Northeastern Brazil will seek to further describe what additional risk dengue immunity may pose in the context of Zika virus. This risk is essential to understand, as Zika and Dengue viruses co-circulate in many regions of the world. Furthermore, participants in the proposed study will undergo bi-weekly screening for Zika virus through laboratory and ultrasound testing until their delivery. Infants will then have full neurologic testing and MRI scanning for the following year after birth to characterize any congenital defects. Neonates born to mothers with prior dengue immunity who contract Zika virus during pregnancy will be compared to neonates not exposed to Zika virus in utero. This experiment will illuminate the associated risk and evidence of ADE occurring with prior dengue immunity and Zika virus infection during pregnancy. Results from this study will not only help define risks of congenital defects with Zika virus, it will inform vaccine research and elucidate challenges in the administration of the current tetravalent dengue vaccine.

Dengue virus (DENV) is the most medically-important arbovirus worldwide, and infection with any of the four serotypes of the virus (DENV1-4) can lead to a range of outcomes, from inapparent infection to classical dengue fever (DF) to dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), severe disease manifestations characterized by endothelial barrier dysfunction and vascular leak. Non-structural protein 1 (NS1) is the only protein secreted from DENV-infected cells and is produced as a monomer, is found in association with cell membranes as a dimer, and is secreted as a hexamer. NS1 is a key component of the viral replication complex and has been implicated in immune evasion and pathogenesis. Recently, we and others described novel roles for NS1 in directly triggering endothelial barrier dysfunction and inducing inflammatory cytokine production from human immune cells, contributing to vascular leak in vivo. In this dissertation, the mechanisms behind NS1-induced pathogenesis of the endothelium are explored.Using in vitro techniques, we found that NS1 from all four DENV serotypes can induce endothelial hyperpermeability of human pulmonary microvascular endothelial cells (HPMEC) and showed that this effect is mediated by endothelial sialidases, cathepsin L, and heparanase. These enzymes contribute to degradation of the endothelial glycocalyx-like layer (EGL), an important determinant of endothelial barrier function, and inhibition of sialidases, cathepsin L, and heparanase prevented NS1-induced endothelial hyperpermeability. Next, we found that NS1 does not trigger the production of inflammatory cytokines from human dermal microvascular endothelial cells (HMEC-1) in vitro and showed that neither TLR4 nor TNF-α signaling contribute to NS1-mediated vascular leak in the dermis of mice in vivo. Further, we demonstrated that inhibition of sialidases, cathepsin L, and heparanase prevents NS1-induced hyperpermeability and EGL degradation of HMEC-1 in vitro and vascular leak in the mouse dermis in vivo. We then sought to elucidate the early events that occur immediately following NS1 binding to endothelial cells. Using a glycosylation mutant of NS1, N207Q, we found that the N207 glycosylation site is not required for binding of NS1 to HPMEC but is required for NS1-induced hyperpermeability and EGL degradation. Further, we observed that wild-type NS1 is internalized by endothelial cells via clathrin-mediated endocytosis and is trafficked to the endosome. While the N207Q mutant is internalized, it is not as efficient as wild-type, is not dependent on clathrin, and does not lead to endosomal localization.We then broadened our studies to include NS1 proteins from other flaviviruses, including Zika (ZIKV), West Nile (WNV), Japanese encephalitis (JEV), and yellow fever (YFV) viruses. We found that these proteins selectively bind to and alter the permeability of human endothelial cell monolayers from the lung, dermis, umbilical vein, brain, and liver in vitro and, remarkably, cause vascular leakage upon inoculation into mice in a tissue-dependent manner, reflecting the pathophysiology of each flavivirus. Mechanistically, each flavivirus NS1 protein leads to differential disruption of key endothelial glycocalyx components after activation of sialidases, cathepsin L, and heparanase.Finally, we sought to determine whether sera from individuals vaccinated with Takeda’s live-attenuated Tetravalent Dengue Vaccine candidate (TDV) could protect against NS1-induced hyperpermeability of HPMEC in vitro. We found that sera from DENV-naïve individuals at day 0 pre-vaccination did not protect against DENV2 NS1-induced hyperpermeability, while day 0 sera from DENV-pre-immune subjects provided varying levels of protection. However, all day 120 post-vaccination samples from both DENV-naïve and pre-immune subjects abrogated DENV2 NS1-induced endothelial hyperpermeability, and, across all serum samples, the magnitude of protection correlated with the respective anti-NS1 antibody concentration. We observed a similar pattern when evaluating protection against DENV2 NS1-induced EGL degradation. Lastly, we found that serum from vaccinees could cross-protect against NS1 from DENV1, DENV3, and DENV4, and this protection also correlated with anti-NS1 antibody concentration.Taken together, the work included in this dissertation represents a substantial advancement of the field’s understanding of NS1 and its pathogenesis and further supports a key role for NS1 in dengue disease. Although a number of novel findings are described here, additional important questions remain unanswered, specifically regarding the molecular pathways and players involved in NS1-mediated pathogenesis as well as broader topics such as the relative contributions of NS1 in the greater context of the pathogenesis of dengue and other flaviviral diseases. Nonetheless, this work provides considerable mechanistic insight into NS1 pathogenesis and establishes NS1 as a key target for therapeutic intervention and important component of dengue vaccines.

Dengue virus (DENV) is the most common mosquito-borne human viral disease worldwide, and infection with any of the four serotypes (DENV1-4) can result in a range of disease outcomes, from inapparent infection to classical dengue fever (DF) to the most severe forms, dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). The four DENV serotypes can cause repeat infections in a single individual, each time with a new serotype. While primary infection generates what is thought to be life-long neutralizing antibodies to the infecting serotype, secondary DENV infections elicit cross-neutralizing antibodies to two or more serotypes, as well as type-specific antibodies to the infecting serotype. While natural infection studies have provided valuable insights into the immune response to DENV infection, the complex antibody response needs further elucidation, especially in the light of recent vaccine trial results. In this dissertation, the functional properties and epitope specificity of the antibody response in primary and secondary natural DENV infections are explored. Recent advances in the characterization of human antibody responses to primary DENV infection indicate that highly neutralizing type-specific antibodies target quaternary epitopes on the DENV envelope protein. Among such type-specific antibodies is 5J7, a human monoclonal antibody (hmAb) isolated from a traveler infected with DENV3. Using two recombinant chimeric DENV4 viruses containing differently sized transplants of the 5J7 epitope, we measured the levels and kinetics of neutralizing antibodies targeting this epitope in post-primary DENV3 infection plasma in a large number of individuals in Nicaragua. We found that DENV3 neutralizing antibodies in post-primary DENV3 infections track with the 5J7 epitope to varying degrees and can be detected even years after infection. Some individuals, however, presented little to no recognition of this epitope even in the expanded transplant version, which suggested the existence of additional epitopes within the DENV3 repertoire that required further investigation. In addition to measuring the prevalence of antibodies directed to the 5J7 epitope in a population in a dengue-endemic area, this study validated and helped define the footprint of a quaternary highly neutralizing epitope. Having demonstrated that chimeric viruses are a powerful tool for mapping type-specific neutralizing antibody responses, we carried out a similar approach to measure type-specific neutralizing antibody responses to the epitope bound by 1F4, a DENV1 type-specific hmAb. This time, we analyzed post-primary DENV1 infection antibody response in individuals from two endemic regions -- Asia and the Americas -- where different genotypes of DENV1 are circulating. The 1F4 epitope was prevalent in both areas, although a higher proportion was found in the Asian population, supporting the hypothesis that intra-serotype diversity can affect neutralization potential. Our second finding was that both populations presented a bimodal pattern of recognition of the 1F4 epitope, suggesting that additional epitopes within the DENV1 repertoire may contribute to type-specific neutralization of the DENV1 serotype. After a secondary DENV infection, the neutralizing antibody response gains breadth, and potent broadly neutralizing antibodies may be elicited. In an attempt to investigate the molecular pathways that generate broadly neutralizing antibodies, together with collaborators at Vanderbilt University, we isolated and sequenced hmAbs from EBV-transformed PBMCs collected after secondary DENV infection. Subsequently, we performed deep sequencing of the immunoglobulin (Ig) genes of B cells in matched PBMCs after the first DENV infection of the same individual at time-points prior to the second infection. Although the limited sample size from our pediatric subjects did not allow the necessary depth for tracing the clonal lineages of broadly neutralizing antibodies, we gained substantial knowledge about the molecular and functional properties of hmAbs isolated from memory B cells (MBC) after secondary DENV infection. In our panel of hmAbs, we observed a predominance of serotype cross-neutralizing and somatically hypermutated clones. In fact, we observed a higher degree of somatic hypermutation (SHM) in cross-reactive clones compared to type-specific clones. Notably, we identified a number of potent DENV3 type-specific neutralizing hmAbs isolated after secondary DENV infection. In accordance with our previous study that predicted additional epitopes within the DENV3 repertoire, we mapped three additional novel antigenic sites for DENV3 neutralization, as well as one epitope that overlapped with the already characterized 5J7 site. Strikingly, the sequence of infecting serotypes may modulate the epitope and potency of DENV3 type-specific hmAbs. Moreover, the newly identified DENV3 hmAbs are protective against DENV3 challenge in a mouse model of DENV infection. As an increasing number of natural infection and immunization studies underscore the importance of the quality as well as the quantity of the antibody response, we investigated the contribution of type-specific versus cross-reactive antibodies to polyclonal neutralization after a first, second and third DENV infection in the same individual. Through depletion of antibody subpopulations in collaboration with collaborators at the University of North Carolina at Chapel Hill, we concluded that after the first infection, type-specific antibody populations drive a substantial proportion of the polyclonal neutralization. Following second and third infections, the cross-reactive populations dominate, although type-specific populations to each of the infecting serotypes can still be detected. Strikingly, in one case, we observed an increase in the proportion of the type-specific antibody population to the first infecting serotype and no evidence of a type-specific response to another serotype in a subsequent inapparent infection, suggesting homotypic reinfection. Depletion of antibody subpopulations enables detection of non-typical DENV infections and assessment of the frequency of heterotypic infections, homotypic reinfections and heterologous boosts, providing critical information regarding the epidemiology and long-term immunity to DENV.Taken together, the work included in this dissertation has made significant contributions to the field’s efforts to decipher the specificity and functional properties of the neutralizing antibody response following primary and secondary natural DENV infections.

Since 1970, human activities have permitted the spread of Dengue virus (DENV) and the primary mosquito vectors Aedes spp. to virtually every continent. Infection rates have increased more than 30-fold and it has become the most prevalent arboviral disease in the world. Every year, 3.6 billion people are at risk of infection and there are 390 million new infections, mostly among children. With no vaccine or specific treatment, early detection plays a significant role in decreasing fatality rates. Dengue infection has no pathognomonic clinical features, thus diagnostic tools are essential for diagnosis.In addition to a human transmission cycle, a variety of forest-dwelling non-human primates are hosts for DENV in a sylvatic cycle. Unfortunately, sylvatic cross-over events occur regularly and have resulted in disease outbreaks within humans. Vector surveillance plays a critical role in dengue detection and outbreak prevention. Current laboratory methods for detection and diagnosis of DENV require highly trained personnel and costly equipment that are impractical for regular surveillance and diagnostic use. Thus, new technologies to facilitate and enhance diagnostic and surveillance capabilities within each transmission cycle are urgently needed. This research describes the development of two novel biosensors using single-walled carbon nanotube transducers functionalized for the detection of whole DENV or DENV Non-Structural Protein 1 (NS1). Heparin, an analog of the heparan sulfate proteoglycans that are receptors for DENV, was used as a bioreceptor for detection of whole DENV virions within viral culture. This permits detection of DENV virions from a variety of viral culture-compatible samples; such as fluid or tissue samples from monkeys, vector mosquitos, and humans. Anti-dengue NS1 monoclonal antibodies were used to detect DENV NS1, a clinically accepted biomarker for DENV infection. This biosensor will allow early detection and diagnosis of the disease in Aedes mosquitos and human saliva. Both biosensors were selective and sensitive for their target analyte in a 10-μL sample over the clinically relevant concentration range with detection occurring in only 10-20 min. Each was constructed to be a portable, rapid, and inexpensive diagnostic tool suitable for use by minimally-trained personnel in the field, laboratory, or point-of-care location.

Dengue virus (DENV) infection is an important public health problem, being globally the most highly prevalent mosquito-borne viral illness. The World Health Organization (WHO) has estimated 2.5 billion people are at risk of DENV infection and each year 50- 100 million infections are reported, with over 500,000 hospitalisations and 25,000 deaths. DENV consist of four serotypes, each of which are capable of producing a wide spectrum of syndromes varying from mild dengue fever (DF) to more severe dengue haemorrhagic fever (DHF). Vascular leakage resulting from increased micro vessel endothelial permeability is one of the life-threatening complications in DHF. The relationship between DENV infection and the endothelium has previously been investigated both in vitro and in vivo experiments. Nevertheless, the mechanisms by which dengue infection leads to increased vascular permeability remain unclear. Understanding the underlying mechanisms of vascular leakage is required to identify better control strategies for DHF/DSS.

Dengue virus is a flavivirus of worldwide importance, with approximately 4 billion people across 128 countries at risk of dengue virus infection. Most cases present as classic dengue fever, a debilitating, but self-limited illness that manifests with high fever, retro-orbital pain, severe myalgia or arthralgia, and rash. However, in some cases, illness progresses to life-threatening dengue hemorrhagic fever or dengue shock syndrome. The identification of distinguishing clinical and laboratory features that occur during the early febrile phase of illness is important for developing a clinical prediction algorithm to differentiate dengue from other febrile illnesses, and severe dengue from mild dengue. In addition, the success of community-based programs for preventing dengue indicates that identifying environments that could benefit from intervention at a community level is critical in order to have the greatest impact and to target limited resources.This dissertation focuses on data from two ongoing studies in order to investigate the epidemiologic, clinical and laboratory features of pediatric dengue in Nicaragua. Chapter 1 reports on the clinical and laboratory features of dengue virus infection in Nicaraguan children. The aims of the study were to examine the frequency of clinical signs and symptoms by day of dengue illness and to analyze the association of signs and symptoms with dengue virus infection during the early febrile phase of illness and over the course of illness.Chapter 2 reports on the association of lower low-density lipoprotein cholesterol levels with severe dengue outcome. The aim of the study was to delineate the trajectories of cholesterol levels over time by dengue virus infection status in order to understand the effect of dengue virus infection on cholesterol metabolism. We also sought to delineate their trajectories by dengue severity and to assess the effect of cholesterol level at presentation on development of severe dengue.Chapter 3 reports on individual-, household- and neighborhood-level determinants of dengue virus seropositivity in a community-based cohort. The aim of the study was to identify risk factors for DENV infection among children living in urban neighborhoods in Managua, Nicaragua. We also sought to determine the seroprevalence of dengue virus infection and identify individual- and household-level risk factors for dengue virus infection in neighborhood groups categorized by similar socioeconomic, infrastructural and ecological characteristics.Together, these studies present important findings on the natural history of pediatric dengue in Nicaragua and provide the basis for future research to develop clinical prediction algorithms to discriminate dengue from other febrile illnesses, and severe dengue from mild dengue. They also reveal the importance of understanding neighborhood-level factors in targeting community-based programs to prevent dengue.

Background: Hepatic injury in dengue virus (DENV) infection is authenticated by hepatomegaly and an upsurge in transaminase levels. DENV replicates in hepatocytes and causes hepatocyte apoptosis both in vitro and in vivo. Understanding the molecular mechanisms of DENV-induced hepatic injury could facilitate the development of alternate chemotherapeutic agents and improved therapies. Findings: The p38 mitogen-activated protein kinase (MAPK) participates in both apoptosis-related signaling and pro- inflammatory cytokine production. The role of p38 MAPK in DENV-infected HepG2 cells was examined using RNA interference. The results showed that DENV infection activated p38 MAPK and induced apoptosis. The p38 MAPK activation and TNF-α production were controlled by p38 MAPK and CD137 signaling in DENV-infected HepG2 cells as activated p38 MAPK, TNF-α and apoptosis were significantly decreased in p38 MAPK and CD137 depleted DENV-infected HepG2 cells. Addition of exogenous TNF-α to p38 MAPK depleted DENV-infected HepG2 cells restored DENV-induced apoptosis in HepG2 cells. Conclusion: DENV induces CD137 signaling to enhance apoptosis by increasing TNF-α production via activation of p38 MAPK.

Dengue virus (DENV), a flavivirus with four serotypes (DENV1 to DENV4), is the leading cause of arboviral diseases worldwide. Despite decades of studies on dengue, no antiviral against DENV is currently available. The envelope (E) protein is involved in virus entry and assembly. The Cterminus of E protein contains two alpha-helices (EH1 and EH2) separated by conserved sequences (CS) in the stem region and two transmembrane domains in the anchor region. Previous cryo-electron microscopic (EM) study revealed that EH1 and EH2 are partially buried in the outer leaflet of viral membrane. The stem region contains 16 residues absolutely conserved among diverse flaviviruses. We hypothesize that the absolutely conserved stem residues are involved in critical steps of virus life cycle including assembly. In this study, we investigated the roles of the absolutely conserved stem residues on virus assembly and the mechanisms involved. Our long-term goal is to explore the stem region of DENV E protein as novel targets for the development of antivirals. In the first specific aim, we investigated the role of the absolutely conserved stem residues on virus assembly of DENV1 by examining the production of virus-like particles (VLPs). VLPs are similar to infectious virions in the structural, biochemical and antigenic properties, and can be generated by co-expression of precursor membrane (prM) and E proteins. Using a series of DENV1 prM/E expressing constructs containing a single proline or alanine substitution for each of the 16 highly conserved stem residues by site-directed mutagenesis we were able to examine the role that these play in assembly. After transfection to 293T cells, cell lysates and pellets derived from ultracentrifugation of cultural supernatants were subjected to Western blot analysis and a quantitative capture-ELISA using known concentrations of a recombinant E protein as standard. Compared with those of the wild type (WT), the amounts of E protein in pellets relative to cell lysates were reduced in 15 proline mutants at the EH1, CS and EH2 domains and greatly reduced in 9 mutants at the CS and EH2 domains, suggesting the importance of overall helical structure on assembly and the critical role of residues at the CS and EH2 domains, In the second specific aim, we investigated the mechanisms of impairment in assembly of DENV1 mutants. Immunoprecipitation assay revealed that alanine substitutions do not affect the prM-E interaction. Enzyme digestion experiment showed that these alanine substitutions do not affect the glycosylation of E protein and both WT and mutant prM/E proteins were primarily located in the ER. Subcellular fractionation experiment revealed that the amounts of prM/E proteins of the alanine mutants in the soluble fraction relative to membrane fraction were either less or greater than that of WT, suggesting that some alanine mutations affect the budding of VLPs from the ER membrane to the lumen of ER, whereas other alanine mutations affect the release of VLPs from ER lumen or other intracellular vesicles to outside of the cells. We also set up a transmission EM and immuno-gold EM to explore the morphological defects of these mutants. In summary, our findings indicate several absolutely conserved stem residues are involved in the assembly step of DENV life cycle; in particular, 9 residues in the CS and EH2 domains of the stem region are critical. While considerable efforts have been made to develop antivirals against DENV and other flaviviruses, there are no licensed antivirals against DENV currently available. The absolutely conserved nature of these residues may be potential targets for antiviral strategy to block virus assembly.

In the course of their life cycle, mosquitoes undergo an ontogenetic niche shift; immature larval development occurs within an aquatic habitat from which adult mosquitoes subsequently disperse into the terrestrial environment. While adult female mosquitoes transmit dengue to humans, the larval stage influences dengue virus transmission significantly in several ways. The biotic and abiotic conditions in which larvae develop influence epidemiologically important aspects of adult life history and influence the geographic distribution of mosquito species. Through vertical transmission, mosquitoes may serve as reservoir hosts for dengue during periods that are unfavorable for transmission to humans. For my dissertation, I conducted research that investigated how environmental conditions experienced by larvae influence dengue transmission both directly and indirectly. In chapter 1, I experimentally infected Aedes aegypti and Aedes albopictus larvae to determine their susceptibility to dengue and evaluate the potential for horizontal transmission within the larval habitat. I found that larvae are susceptible to dengue and that horizontal transmission could be possible. In chapter 2, I tested the hypothesis that dengue infection affected mosquito fitness. The effect of infection was sex dependent; infected males took significantly longer to develop and were significantly smaller, while infection in females was cost-free. This result indicates that female larvae infected through vertical transmission are likely to be able to transmit dengue to humans as adults. In chapter 3 and 4, I investigated how resources within the larval habitat influence development and competition. Using larval detritus as a resource, I studied its utilization within the habitat, and its influence on larval development and competition. I found that larval detritus was consumed rapidly and differently from another type of invertebrate detritus. When larval detritus was the main resource within the larval habitat, Ae. albopictus significantly outcompeted Ae. aegypti; Ae. aegypti were significantly smaller and took longer to develop when reared in containers with Ae. albopictus. This result indicates that interspecific competition with Ae. albopictus significantly decreases the population growth of Ae. aegypti and may influence its ability to transmit dengue. Overall, my results further demonstrate the influential role of mosquito larvae on dengue transmission and epidemiology.

The immunopathology of dengue virus (DENV) infection is associated with increased TNF-α production. In this study, small RNA-mediated regulation of TNF-α and the effect of TNF-α knockdown during DENV infection were analyzed. This provides insight into the role of TNF-α during DENV infection, both in terms of its contribution to immunopathogenesis and its regulatory mechanism by miRNAs. Utilizing a lentiviral expression system, human monocytic U937 cells that express short hairpin RNAs designed to target TNF-α mRNA were established. TNF-α expression was downregulated in these monocytes, and upon DENV infection they showed decreased endothelial cell activation ability. This demonstrates an overall decrease in the proinflammatory response upon TNF-α knockdown during DENV infection. To analyze the role of microRNAs (miRNAs) in the TNF-α response, miRNAs that potentially target the 3' UTR of TNF-α were predicted. Many of the miRNAs were differentially regulated during DENV infection. miR-320a and miR-592 were among those downregulated, and chosen for further analysis. TNF-α post-transcriptional regulation by miR-320a and miR-592 was confirmed utilizing a TNF-α 3'UTR luciferase reporter. U937 cells transfected with miR-320a and miR-592 mimics followed by DENV infection displayed decreased TNF-α expression and their culture supernatants demonstrated decreased ability to activate endothelial cells. It is concluded that one function of these miRNAs is to negatively regulate TNF-α, and their downregulation contributes to the immunopathogenesis of DENV infection.