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One of the most important clinical signs of dengue virus infection is the reduction of white blood cells and platelets in human peripheral blood (leukopenia and thrombocytopenia, respectively), which may significantly impair the clearance of dengue virus by the immune system. The cause of thrombocytopenia and leukopenia during dengue infection is still unknown, but may be related to severe suppression of bone marrow populations including hematopoietic stem cells and megakaryocytes, the progenitors of white blood cells and platelets respectively. Here, we explored the possibility that bone marrow suppression, including ablation of megakaryocyte populations, is caused by dengue virus infection of megakaryocytes. We used three different models to measure dengue virus infection and replication: in vitro, in a human megakaryocyte cell line with viral receptors, ex vivo, in primary human megakaryocytes, and in vivo, in humanized mice. All three systems support dengue virus infection and replication, including virus strains from serotypes 1, 2, and 3, and clinical signs, in vivo; all assays showed viral RNA and/or infectious viruses 7–14 days post-infection. Although we saw no significant decrease in cell viability in vitro, there was significant depletion of mature megakaryocytes in vivo. We conclude that megakaryocytes can produce dengue viruses in the bone marrow niche, and a reduction of cell numbers may affect bone marrow homeostasis. Author summary: Dengue virus is the most common cause of viral hemorrhagic fever in humans. Over half of the world's population lives in an at risk area for dengue virus infection, and this number will continue to grow as climate change allows the mosquito vectors of dengue virus to expand their breeding ranges to more temperate climates. Currently, there are no specific treatments for dengue virus infection. Understanding how dengue virus causes hemorrhagic fever could inform the development of these much needed treatments. Populations of important immune system mediators, such as white blood cells and platelets, are significantly dysregulated during dengue virus infection. These cells originate in the bone marrow, which experiences significant suppression, including a complete ablation of megakaryocytes (platelet progenitor cells), during DENV infection. Here, we add to the knowledge on how dengue virus induces bone marrow suppression by investigating whether dengue virus infects human megakaryocytes. We discovered that dengue virus infects human megakaryocytes in vitro, ex vivo, and in vivo models of dengue virus infection; however, dengue virus infection does not appear to directly affect viability of human megakaryocytes. Future studies will investigate whether infected megakaryocytes are still able to perform their functions of producing platelets and maintaining bone marrow homeostasis. [ABSTRACT FROM AUTHOR]

Mosquito saliva is a very complex concoction of >100 proteins, many of which have unknown functions. The effects of mosquito saliva proteins injected into our skin during blood feeding have been studied mainly in mouse models of injection or biting, with many of these systems producing results that may not be relevant to human disease. Here, we describe the numerous effects that mosquito bites have on human immune cells in mice engrafted with human hematopoietic stem cells. We used flow cytometry and multiplex cytokine bead array assays, with detailed statistical analyses, to detect small but significant variations in immune cell functions after 4 mosquitoes fed on humanized mice footpads. After preliminary analyses, at different early times after biting, we focused on assessing innate immune and subsequent cellular responses at 6 hours, 24 hours and 7 days after mosquito bites. We detected both Th1 and Th2 human immune responses, and delayed effects on cytokine levels in the blood, and immune cell compositions in the skin and bone marrow, up to 7 days post-bites. These are the first measurements of this kind, with human immune responses in whole animals, bitten by living mosquitoes, versus previous studies using incomplete mouse models and salivary gland extracts or needle injected saliva. The results have major implications for the study of hematophagous insect saliva, its effects on the human immune system, with or without pathogen transmission, and the possibility of determining which of these proteins to target for vaccination, in attempts to block transmission of numerous tropical diseases. [ABSTRACT FROM AUTHOR]