Understanding viral-host interactions that are modulated during DENV infections
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.