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Theses and Dissertations

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Browsing Theses and Dissertations by Author "Akkina, Ramesh K., advisor"

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    Dengue viral pathogenesis and immune response in humanized Rag2-/-gamma c-/- (RAG-hu) mice
    (Colorado State University. Libraries, 2009) Kuruvilla, Jes G., author; Akkina, Ramesh K., advisor
    Dengue viral pathogenesis and vaccine studies are hampered by the lack of an ideal animal model mimicking human disease and eliciting an adaptive immune response. We utilized a novel mouse model that permits multi-lineage human hematopoiesis and immune response following transplantation with human hematopoietic stem cells. To generate immuno-competent humanized mice, we xenografted neonatal Rag2-/-γc-/- mice with human CD34+ hematopoietic stem cells, resulting in the de novo development of functional human adaptive immune system. To evaluate susceptibility to dengue viral infection, humanized mice were challenged with dengue virus (DENV). We observed viremia that lasted three weeks post infection with all four serotypes of dengue virus. We also demonstrated the presence of human anti-dengue antibodies. Anti-dengue IgM was first detected at 2 weeks post infection followed by IgG at 6 weeks. Our results demonstrated for the first time that humanized mice were capable of dengue viral primary immune responses, thus paving the way for further dengue immunopathogenesis studies. We challenged the mice that survived a primary infection with a secondary infection of dengue virus. We report increased viremia, severe dehydration, hemorrhage and morbidity in several mice during a secondary infection. We demonstrate the first instance of dengue hemorrhagic fever or dengue shock syndrome (DHF/DSS) like phenomenon reported in any humanized mouse model for dengue infections. We also report the identification of cells that are actively infected with DENV in the spleen and lymph nodes of these mice using in situ hybridization and immunofluoresence staining methods. We used a SCID-hu model with an engrafted human thymus to study the direct infection of T-cells by dengue virus. We demonstrate that T-cells and CD34+ hematopoietic stem cells do not support a dengue viral infection. We were also able to infect monocytes and dendritic cells that were isolated and developed from human peripheral blood in vitro. In order to develop a consistent assay for antibody dependent enhancement (ADE) of dengue infection we were able to infect macrophages that were differentiated in vitro from CD34+ hematopoietic stem cells. In summary, this model provides a new tool to further our understanding of a complex disease like dengue hemorrhagic fever and dengue shock syndrome as well as provide a new system to test new vaccines as well as therapeutics against dengue viruses in the human context.
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    Modeling dengue viral infection, insect transmission and multiplex PCR in humanized mice
    (Colorado State University. Libraries, 2012) Arab, Jennifer Suzanne, author; Akkina, Ramesh K., advisor; Callahan, Gerald N., committee member; Laybourn, Paul J., committee member
    Due to the restricted host specificity of dengue virus, up until now no ideal animal model has sufficiently mimicked key aspects of viral pathogenesis and immune response. In this regard, new advances with humanized mice(hu-mice), constructed by engrafting human tissues and cells and harboring a functioning human immune system, have been of great value in advancing research with human viruses Here we sought to utilize hu-mice to understand natural dengue pathogenesis and immune response. RAG1-/- γc-/- or RAG2-/- γc-/- mice are immune-compromised and do not reject foreign grafts. This allows engraftment of human hematopoietic stem cells via intrahepatic injection into newborn mice, forming the RAG-hu mouse model. Bone marrow, liver, thymus (BLT) mice are an improved version of these mice as they harbor a more robust human immune cell repertoire. BLT mice were prepared by transplanting seven week old RAG1-/- γc-/- or RAG2-/- γc-/- strain mice with fragments of human liver and thymus under the kidney capsule, followed by an intravenous injection of autologous hematopoietic stem cells. After construction, both models were injected subcutaneously and intraperitoneally with dengue virus and monitored for the presence of infection. Our results showed that RAG-hu and BLT mice were fully susceptible to dengue virus infection as evidenced by viremia, and generation of dengue specific human IgM and IgG antibodies. Additionally, the presence of IgM and IgG that are capable of virus neutralization illustrated the functionality of the human immune system reconstituted in these mice. To expand on the utility of this hu-mouse model, we also evaluated if dengue virus could be transmitted via mosquitoes to humanized mice, modeling the natural route of dengue transmission. Dengue infected mosquitoes were allowed to feed on BLT and RAG-hu mice and mice were monitored for the presence of dengue viremia. We achieved successful insect mediated transmission of dengue virus as evidenced by viremia and dengue specific antibody production in exposed mice. This achievement permits many novel experiments on vector competence and vector based viral intervention. Lastly, a multiplex quantitative real time PCR (qRT-PCR) assay capable of detecting and differentiating all four dengue viral serotypes was also established. This assay was able to detect each of the viral serotypes in a single reaction allowing for detection of virus in mixed infection studies. Taken together, these studies characterized a human immune competent hu-mouse model capable of supporting dengue viral infection with all four serotypes, as well as illustrating dengue-meditated disease and virus-specific immune responses.