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Modeling dengue viral infection, insect transmission and multiplex PCR in humanized mice




Arab, Jennifer Suzanne, author
Akkina, Ramesh K., advisor
Callahan, Gerald N., committee member
Laybourn, Paul J., committee member

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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.


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