Alan L. Rothman, M.D., Research Professor
Dengue is an acute febrile illness caused by any of four related flaviviruses (dengue virus [DENV] serotypes 1-4), small enveloped viruses containing a non-segmented positive-sense RNA genome. DENV infection is acquired through a transmission cycle between humans and mosquitoes of the genus Aedes, principally A. aegypti.
In humans, there is a wide spectrum of clinical manifestations of DENV infection. This ranges from an uncomplicated and self-limited febrile illness (dengue fever, DF) to a plasma leakage syndrome accompanied by bleeding (dengue hemorrhagic fever, DHF). Although DHF is observed in only a small percentage of DENV infection, it plays a large role in the public health problem of dengue because it can lead to shock and death.
Many factors contribute to the risk for DHF, but one of the most important factors is pre-existing immunity from an earlier DENV infection. Infection with one DENV only provides long-lasting protection against that serotype; sequential infection with multiple different DENV serotypes is therefore possible, and, because of the increasing global circulation of DENV, this has been occurring with increasing frequency in tropical areas of the world. The basis for the association of DHF with secondary DENV infection is a major subject of research in Dr. Rothman’s laboratory. We hypothesize that both the quality and timing of DENV-specific antibody and T lymphocyte responses influence whether their overall effect is beneficial (controlling DENV replication and reducing the severity of illness) or harmful (creating a ‘cytokine storm’ leading to plasma leakage). The results of our research will be applied to evaluation and management of patients with dengue and to development and testing of vaccines and therapeutics against dengue.
Anuja Mathew, Ph.D., Associate Research Professor
Dengue viruses are mosquito-borne viruses that infect individuals in tropical and subtropical countries. They comprise four closely related but distinct viruses termed serotypes 1 through 4. Studies in humans infected with dengue provide strong evidence for an immunologic basis for the pathogenesis of severe disease DHF. My research interests lie in assessing immune responses to acute viral infections with a focus on Natural Killer Cells and B lymphocytes.
Natural Killer cells are the primary effector cells of the innate immune response. NK cells are able to lyse virally infected cells and secrete large amounts of proinflammatory cytokines/chemokines without prior antigen sensitization. My laboratory recently found an interaction between a dengue peptide on the NS1 protein with KIR3DL1, a known inhibitory receptor on NK cells. We are interested in determining how dengue peptides modulate the function of subsets of NK cells. Furthermore we are interested in understanding whether NK cells are activated and contribute to protection or pathogenesis of severe dengue disease. Using clinical samples from a long standing cohort in Thailand we would like to evaluate NK cell activation using multi-parametric flow cytometry.
Antibodies generated in humans to DENV are of particular clinical relevance, because they have the potential during a subsequent infection to protect from or increase the risk of developing DHF. Although much is known regarding the specificity and efficacy of antibody responses, our knowledge of the function, phenotype, and specificity of the antigen-specific B cells that are responsible for antibody production, as well as for long-term humoral memory, is inadequate. We have recently developed reagents that enable the tracking of antigen-specific memory B cells. Using clinical samples we would like to create an immunoprofile of activated B cells and memory B cells that may provide prognostic markers for dengue immunity and/or immunopathology.
Carey L. Medin, Ph.D., Assistant Research Professor
Dengue virus (DENV) is a mosquito-borne human pathogen of global medical importance. DENV causes an acute febrile illness that, in some patients, is associated with a life-threatening plasma leakage syndrome, dengue hemorrhagic fever (DHF). It is thought that secondary heterotypic dengue virus infections are more likely to produce DHF. While there is ongoing debate regarding the contribution of different mechanisms in dengue illness, there is substantial evidence supporting both viral and host factors in disease pathogenesis. Several studies indicate that an increase in viremia during DENV infection correlates with severity of disease. In order for a virus to productively infect a cell it must be efficient at highjacking cellular mechanisms and avoid detection by the innate immune sensors. The research focus if this project is to investigate cellular organelle changes such as autophagosomes, lysosome, mitochondria, endoplasmic reticulum and peroxisomes during DENV infection in order to identify related cellular mechanisms that are modulated by DENV to enhance its own propagation. The significance of these studies will lead to a better understanding of cellular targets by DENV, which can identify new potential therapeutics to reduce viral levels during infection and lessen the severity of disease.
Construction of DENV reporter constructs for live cell analysis
Live-cell analysis of virus-infected cells by fluorescence microscopy or flow cytometry represents a promising approach to investigate virus-cell interactions. Current methods of identification of DENV-infected cells using antibody staining require permeabilization and fixation, which do not permit intact cell imaging. To this end, we have developed a reporter construct that generates a fluorescent signal specifically in cells infected with dengue (DENV). For several viruses, live cell imaging has revealed new mechanisms critical to viral replication or cell-to-cell spread and has been used for antiviral drug screening. We are currently using this reporter system to identify cellular changes during DENV infection using fluorescence microscopy see image above. To expand our repertoire of tools to investigate cellular changes that occur during DENV infection, we are creating additional reporter constructs for detection of DENV infection of living cells. The focus of this project will be to create constructs that will fluorescence only in DENV infected cells. These reporters will give us the ability to sort infected cultures to analyze changes in gene expression in directly infected cells and bystander cells and compare to uninfected cells.
Effects of IaIP on productive DENV infection in vitro
Inter alpha inhibitor protein, IaIP, is an endogenous protease inhibitor that blocks serine proteases such as thrombin and plasmin and proprotein convertases such as furin. DENV requires furin cleavage of the prM protein to M to produce infectious virions. Our research focus for this project is to characterize the effects of IaIP on DENV replication in vitro in collaboration with Yow-Pin Lim at Prothera Biologics, Inc. Since IaIP is an endogenous protein, it would potentially represent a significant advance over other potential therapeutics for DENV.