Overview
Epidemiologic studies imply that sequential infections with distinct dengue virus (DEN) serotypes are associated with the occurrence of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) (7). Understanding the basis for this phenomenon is crucial for developing therapies and a safe dengue vaccine. The lack of a small animal model has greatly hampered the elucidation of the mechanism of severe dengue. Our preliminary data suggests that the tropism of DEN early in infection may be similar in mice and humans. Therefore, we propose to use a murine model to study primary and secondary DEN infection, using cellular tropism and viral load/kinetics as endpoints. Since the cellular tropism of DEN in humans is not well defined, we will apply the techniques that are developed to study murine DEN infection to detect the cellular localization of replicating virus in humans through autopsy studies.
Specific Aim 1: Identify the cellular targets of DEN infection in mice and evaluate parameters that influence cellular tropism and viral load. In this Aim, we will define the cellular tropism of DEN in mice and determine how cellular tropism, viral load, and kinetics of DEN infection are affected by virus serotype and age and strain of mice during primary DEN infection. This knowledge will provide an alternative endpoint in the mouse model and will be used to assess alterations in secondary DEN infections.
Specific Aim 2: Develop a model for secondary DEN infection and evaluate changes in the in vivo cellular tropism, viral load, kinetics of DEN infection, and immune response. To develop a murine model for secondary DEN infection, several approaches will be used, including 1) sequential infections of a single mouse by different DEN serotypes, 2) passive transfer of DEN-immune sera to naive mice to mimic a primary infection followed by challenge with another DEN serotype, and 3) transfer of maternal antibodies, followed by DEN infection of offspring. After secondary infection, mice will be evaluated according to histopathologic, virologic, immunologic, and clinical parameters.
Specific Aim 3: Identify the cellular targets of DEN infection in fatal human cases via autopsy studies. In collaboration with the Ecuadorian Ministry of Health, the techniques developed in Aim 1 will be applied to determine the cellular localization of DEN antigen and RNA in human autopsies. Immuno-histochemistry will be performed on various tissues to identify cell type and presence of replicating DEN.
Summary
These studies will define the tissue and cellular tropism of DEN in a mouse model that should be relevant to early infection events in humans. Of significance for the development of a safe DEN vaccine, Aim 2 should begin to elucidate the mechanism of enhancement or protection in secondary DEN infections and to distinguish the role of T cells vs. Abs in the process. Importantly, the immunohistochemical techniques we opti-mize will be used in parallel to determine cellular targets of DEN in humans via autopsy studies. Together, these investigations will provide critical data regarding the cellular tropism of DEN in vivo and the effects of sequential DEN infections in a model system. Given that human studies of DEN pathogenesis are restricted to noninvasive procedures and that early events cannot be studied in autopsies, a small animal model of DEN infection will provide the critical link for studying early viral tropism and pathogenesis in vivo.
