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With a newly-obtained fiery red blood meal visible through her transparent abdomen, the now heavy female Aedes aegypti mosquito takes flight. Clostridium botulinum growing on egg yolk agar showing the lipase reaction.An atypical enlarged lymphocyte found in the blood smear from a HPS patient. (a) An atypical enlarged lymphocyte found in the blood smear from a HPS patient. (b)Burkholderia psudeomallei grown on sheep blood agar for 24 hours.


Roles of secreted NS1 and NS1 antibody in dengue virus infection and immunity

Eva Harris, UC Berkeley

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

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

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, California Academy of Science

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

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.

siRNA and miRNA pathways in pathogenesis of RNA viruses and cellular response

Andrew Fire, Stanford University

Cellular processes utilizing small RNAs as guides have been shown to function in potent and specific defense mechanisms protecting plant and invertebrate cells from pathogenesis by RNA viruses. Although vertebrate systems also use small RNA effectors and share much of the protein machinery for small-RNA-based regulation, and although disrupting this machinery affects replication of some vertebrate RNA viruses, the relevant RNA effectors and their roles in viral infection and host response remain to be elucidated. We describe recent data demonstrating that infection of vertebrate cells by several animal RNA viruses leads to production of virus-derived small RNA pools. The proposed experiments address roles for such small RNAs and characterize their place in host-virus interactions, in particular in infections in which there is evidence for a critical balance between viral replication and host response.

Identification of dominant drug targets in Dengue virus infections

Karla Kirkegaard, Stanford University

Positive-strand RNA viruses such as Dengue virus are major human pathogens that present daunting challenges to treatment design and implementation. There is no target-specific antiviral therapy approved by the Food and Drug Administration for any of the dozens of medically urgent positive-strand viral infection: hepatitis C viral infection is currently treated by the relatively non-specific combination of interferon and ribavirin, with limited efficacy and high toxicity. These difficulties are due to the very high error rate and rapid amplification of positive-strand RNA synthesis, leading to the rapid development of drug resistance during single antiviral therapy. We have discovered a method to suppress the growth of drug resistance during infection with RNA viruses, and propose herein to test the concept of identifying 'dominant drug targets' for Dengue virus. In model studies with poliovirus, we have found that certain viral proteins, when bound to drugs or otherwise defective, dominantly inhibit the growth of all viruses within the cell. These products tend to be those that either oligomerize or are required for a process that culminates in the oligomerization of another viral product. For poliovirus, specific examples of dominant drug targets are the capsid proteins, the RNA-dependent RNA polymerase, and an intramolecularly cleaving protease. For Dengue virus, the orthologs of these proteins are the core protein C, the envelope protein E, the polymerase NS5, and the protease NS2/3. Currently, antiviral compounds that in various stages of development are directed against each of these potential 'dominant drug targets'. For each of these compounds, and several that are directed against other viral products, we will select drug-resistant variants, reconstruct them into infectious cDNAs, and determine the relative growth of drug-sensitive and drug-resistant viruses during tissue-culture coinfections to develop a 'dominance index': the ratio of sensitive:resistant genomes at which the resistant genome is inhibited by 90%. The ability of drug-resistant viruses to grow during murine infection will be quantified and compared to these tissue-culture data. The development of logical strategies to combat viral drug resistance is crucial to combatting many pathogens, with Dengue virus as a currently pressing example.

Assessing Host B cell and Viral intra-host Diversity in Dengue virus infection

Poornima Parameswaran, UC Berkeley

Dengue is caused by four serotypes of dengue virus (DENV), a positive-sense RNA Flavivirus, and is a major global health problem that places three billion people at risk. Despite intense research efforts, there are no therapeutic agents or vaccines currently available for treatment or prevention. An intriguing aspect of DENV infections is the observed spectrum of disease severity, ranging from mild fever, to the debilitating but self-limited dengue fever, to a potentially fatal vascular leakage syndrome referred to as Dengue Hemorrhagic Fever/Dengue Shock Syndrome (DHF/DSS). The viral and host determinants of disease severity are not fully understood. Sequential infections by different DENV serotypes, virulence of the infecting strain, intra-serotype diversity, as well as host genetic and immune factors have been proposed as determinants of disease severity. We will investigate the complex tug-of-war between the intra-host evolutionary dynamics of viral populations and the host immune recognition of these viral populations, in an effort to study their contributions to DENV pathogenesis. We will use high-throughput sequencing to identify both genome-wide viral polymorphisms and diversity of infection-associated B cells that may have an impact on outcome of infection. This proposal takes advantage of long-term prospective studies in Nicaragua from which well-characterized samples are available (E. Harris, UC Berkeley; Nicaraguan Ministry of Health), and a mouse model of lethal dengue disease developed in the Harris laboratory, as well as access to sequencing technologies and bioinformatics support at the Broad Institute and at Stanford University. The goal of this work is to understand the course of DENV-induced pathogenesis by detailed characterization of viral genomes and elicited antibody repertoires. To accomplish this, we will integrate information on viral population diversity and its footprint on host antibody repertoires, with clinical and epidemiological information. First, mutation spectra in DENV populations in humans (during natural infection/disease) and mice (during experimental infections) will provide us with an understanding of the interplay between viral polymorphisms, and pathology/prognosis of DENV infection (Aim 1). Second, study of the human and mouse B-cell response during and after DENV infection will help define how viral genetics dictates specificity of the antibody response, and may help us distinguish between protective and enhancing roles for antibodies in DENV pathogenesis (Aim 2). This combinatorial approach will address critical unanswered questions in the dengue field.


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