Dengue
Roles of secreted NS1 and NS1 antibody in dengue virus infection and immunity
Eva Harris, UC Berkeley
Abstract:
Dengue virus (DV) infection causes a spectrum of disease ranging from self-limited Dengue Fever to life threatening Dengue Hemorrhagic Fever/Dengue Shock Syndrome. The mechanisms of immune protection and pathogenesis are poorly understood, but DV nonstructural protein 1 (NS1) likely plays a key role. NS1 is present in the cytoplasm and on the surface of infected cells and is secreted in a soluble form (sNS1) that circulates in blood and correlates with increased disease severity. In vitro, sNS1 can activate complement, bind to infected and uninfected cells, and increase viral output from infected hepatoma cells but it is unclear if or how sNS1 affects viral dissemination or disease progression in vivo. Similarly, the role of antibodies (Abs) against NS1 is unclear due to the lack of appropriate in vivo models. We will use a mouse model to examine the functions of sNS1 and the effects of anti-NS1 Abs on DV infection. Sera obtained from mice, prospective studies of dengue in Nicaragua, and dengue vaccine trials will be used to generate in vitro assays to assess the repertoire of anti-NS1 Abs in infected mice, primary and secondary natural DV infections, and vaccine recipients. A specific aim of this project is to assess the localization and function of sNS1 during DV infection in mice and fatal human dengue cases. Another aim is to evaluate the effects of anti-NS1 polyclonal and monoclonal Abs on peripheral DV infection in mice. Finally, we will characterize the repertoire of anti-NS1 Abs generated by DV infection and vaccination and will develop in vitro tests to detect anti-NS1 Abs in human sera likely to have protective or pathogenic effects during subsequent DV infection. Together, these results will both elucidate mechanisms of DV pathogenesis and contribute to the development of a safe and effective dengue vaccine.
Transcriptional profile of the host response to dengue fever
David Relman, Stanford University
Abstract:
Dengue fever is one of the most important emerging infections across the globe. The goal of this project is to characterize the transcriptional responses of children with dengue fever to identify critical features of the protective and pathogenic features of the host response, and to provide a basis for developing novel diagnostic and prognostic tools in humans with dengue fever and other similar illnesses. In the first specific aim, the relationship between transcriptional response and disease evolution will be characterized by genome-wide transcript abundance levels measured in peripheral blood mononuclear cell (PBMC) samples collected from children diagnosed with dengue fever in Managua, Nicaragua. We will evaluate the clinical and laboratory features associated with inter-individual variation in gene expression, and determine the relationship between transcriptional profiles and WHO staging criteria. Analysis of sequential samples will provide important information about the relative stability of clinically relevant expression signatures, and help to identify temporal trends in associated biological processes. In a second specific aim, we will identify a gene set whose corresponding transcript abundance predicts the clinical evolution of acute dengue infection leading to dengue hemorrhagic fever / dengue shock syndrome. A third specific aim will use transcription profiles to compare dengue fever and dengue-like illnesses to identify host response features that are dengue-specific, and those that are conserved among many infections.
Detection and discovery of viral pathogens associated with dengue-like symptoms
Joseph DeRisi, UC San Francisco
Abstract:
Large-scale viral surveillance studies are critical to monitoring the global emergence of infectious disease and yield important information about the prevalence and seasonality of circulating virus species and the possible emergence of novel species. Dengue virus (DV) infects an estimated 50-100 million people annually in a rapidly expanding geographic range and is the most common vector-borne viral disease of humans. Currently available laboratory diagnostic methods, however, fail to detect DV infection in 30-70% of patients presenting with hallmark dengue symptoms. The purpose of this study is to use the best available diagnostic technologies to delineate the spectrum of viral pathogens that cause dengue-like illnesses in a tropical environment and to utilize the data as a platform for the discovery of novel or divergent human viral pathogens. This study will use the Virochip to characterize human sera and PBMC samples from Nicaraguans enrolled in a hospital-based study of patients presenting with dengue-like symptoms over multiple seasons to capture the prevalence, seasonality and dual infection rates for all known viruses in suspected dengue cases over time. We also propose to utilize ultra-deep sequencing for a select number of samples to discover previously uncharacterized agents. We will pursue full genome cloning and molecular characterization for promising leads, including the development of PCR-based rapid diagnostics and serological reagents for subsequent studies.
Genetic signatures of dengue virus emergence
Shannon Bennett, University of Hawaii
Abstract:
Dengue (DENV) has re-emerged in the last 25 years as one of the most significant emerging infectious diseases worldwide, posing a significant category A biodefense threat to the United States. Its global resurgence, while partly explained by changing human demographics, increasing hyperendemicity, and mosquito vector expansions, remains poorly understood, especially with respect to the importance of virus evolution. We fill this critical gap by conducting a comparative evolutionary examination of DENV strains undergoing epidemic expansion, from replicate populations across the Asia-Pacific arena, paired with directed tissue and animal model assays to assess changes in virus phenotype related to epidemics. Our long-term goal is to resolve how viral genetic change drives disease emergence. We will study dengue viruses isolated from epidemic and endemic forms to confirm the impact of genetic changes in experimental models to assess dengue epidemic potential and pathogenesis. This goal will be accomplished by: (i) determining the evolution of DENV between endemic and epidemic transmission phases to identify synapomorphies of higher epidemic potential; (ii) studying the relationship between genetic changes in DENV epidemic strains and their phenotypic effects in in vitro models; and (iii) confirming the relationship between genetic changes in DENV epidemic strains and their phenotypic effects in in vivo models.
Mosquito innate immune responses to arbovirus infections
Anthony James, UC Irvine
Abstract:
The genes involved in mosquito immune responses to parasitic and bacterial infections have been studied extensively, however similar knowledge of vector responses to viruses is limited. Our long-term goal is to use knowledge of mosquito responses to viruses to design novel disease-control methods. This research focuses on the development and usage of whole-genome tiling arrays for analyses of gene expression in the mosquito vector Aedes aegypti following infection with arboviruses. The information generated by this study will reveal global and specific responses of mosquitoes to a number of viral pathogens, and provide materials for the development of genetics-based disease control strategies. This project will: (i) design whole-genome tiling arrays containing interrogation probes for the genomes of Ae. aegypti, and the arboviruses, Dengue, Yellow Fever and Chikungunya; (ii) identify mosquito protein-coding and noncoding RNAs with altered transcription patterns following infection with viruses and validate their expression profiles by quantitative gene amplification; and (iii) design and test transgenes expressing reporter molecules under the regulation of virus infection-induced mosquito promoters.
Flavivirus RNA replication proteins: novel targets for drug discovery
Bert L. Semler, UC Irvine
Abstract:
Dengue virus and West Nile virus are mosquito-borne emerging flaviviruses with considerable potential as agents for bioterrorism that are involved in natural disease outbreaks across the world. In addition to vaccine development and anti-viral therapeutics aimed at viral structural proteins, there is a need to identify steps in the intracellular replication cycles of these flaviviruses that can be targeted by small molecule inhibitors. One such step is the replication of viral RNA, a process mediated by a viral encoded RNA polymerase, in conjunction with viral and host cell proteins. Thus, the assembly of viral RNA replication complexes provides unique protein-protein interfaces that can be utilized as targets for anti-viral therapeutics. This project will develop cell-free assays to study dengue virus and West Nile virus RNA replication in vitro. Once established, these assays would be suitable for screening small molecule inhibitors of viral RNA synthesis. The aims are to: (i) develop reagents and cell-free extracts for flavivirus RNA replication studies and (ii) carry out structure-based mutagenesis of key flavivirus RNA replication proteins. The ability to assay molecular interactions and flavivirus RNA replication using the proposed in vitro assays will greatly aid structure-activity relationship studies for lead compound optimization using structure-guided rational drug design.
Structure and function of dengue protease
Peter Kuhn, The Scripps Research Institute
Abstract:
The overarching goal of this project is to identify compounds that specifically inhibit Dengue proteases by producing 3D crystals of all Dengue proteases, a critical step for successful structure-aided drug design efforts. Building upon proof of concept, we will also co-crystallize already existing peptidic inhibitors and a fragment-based library to effectively design and optimize high-throughput inhibitor screening assays. The production of these complex structures, which can be used immediately by currently ongoing initiatives, will be important deliverables to the infectious disease community, as they will enable a number of groups to work on proteases produced by Dengue virus as well as other flaviviruses.
