Study of Bone Marrow Failure Caused by B19 Virus Infection

描述（由申请人提供）：细小病毒B19 （B19V）感染通过破坏支持病毒自主复制的骨髓常驻红细胞祖细胞（EPCs）导致人骨髓衰竭。由于红细胞破坏水平高，对红细胞产生需求高的患者（如镰状细胞病患者），急性B19V感染可引起短暂的再生危象。在免疫功能低下的患者中，持续的B19V感染可能表现为纯粹的红细胞发育不全。在胎儿中，B19V感染可引起胎儿水肿，这是一种严重的贫血。B19V感染诱导内皮祖细胞损伤，导致上述骨髓衰竭。b19v感染的EPCs发生细胞死亡，具有明显的凋亡特征。这种细胞死亡主要是由感染期间非结构性11kDa蛋白（11kDa）的高表达引起的，并涉及由caspase-10触发的caspase级联反应。在这项研究中，我们的目标是，首先揭示caspase-10介导的细胞凋亡响应11kDa的分子机制。为此，我们将确定在B19V感染诱导的凋亡过程中，外源性或内源性哪种凋亡途径激活了启动物caspase-10。由于凋亡和抗凋亡通路都在红细胞生成和EPCs的稳态中发挥关键作用，我们提出在EPCs中鉴定与11kDa相互作用并转导凋亡信号的细胞蛋白。其次，我们最近建立了一个B19V培养系统，模拟在缺氧条件下骨髓中人EPCs的天然B19V感染，我们将对一个小分子文库（可在堪萨斯大学HTS核心实验室获得，包含超过10万种药物样小化合物）进行高通量筛选（HTS），以寻找抗B19V感染的候选药物。B19V培养系统可以产生足够数量的病毒进行这种筛选，并且细胞死亡标记可以很容易地用作评估B19V感染的读数。我们将使用基于细胞的发光细胞毒性试验筛选文库。由于目前无法获得B19V疫苗，并且免疫功能低下的患者无法对疫苗产生反应，因此目前没有可行的治疗方案来减少急性或持续性B19V感染患者因B19V感染引起的骨髓衰竭。本申请中提出的研究有望确定能够从人类EPCs中消除B19V的药物开发靶点，这将最终减少这些患者的骨髓衰竭。此外，我们对EPCs凋亡机制的分析有望揭示在某些疾病条件下可能参与红细胞生成或红细胞生成中断的途径。

DESCRIPTION (provided by applicant): Parvovirus B19 (B19V) infection causes human bone marrow failure by destroying the bone marrow resident erythroid progenitor cells (EPCs), which support autonomous replication of the virus. In patients with a high demand for erythrocyte production due to high levels of erythrocyte destruction (e.g., sickle-cell disease patients), acute B19V infection can cause transient aplastic crisis. In immunocompromised patients, persistent B19V infection may manifest as pure red-cell aplasia. In the fetus, B19V infection can cause hydrops fetalis, a severe form of anemia. B19V infection induces damage of EPCs, which results in the above bone marrow failures. B19V-infected EPCs undergo cell death with clear apoptotic features. This cell death is induced mainly by high expression of the non-structural 11kDa protein (11kDa) during infection, and involves a caspase cascade triggered by caspase-10. In the proposed research, we aim, firstly, to reveal the molecular mechanism underlying caspase-10- mediated apoptosis in response to 11kDa. To this end, we will determine which apoptotic pathway the extrinsic or the intrinsic activates the initiator caspase-10 during B19V infection-induced apoptosis. As both the apoptotic and anti-apoptotic pathways play key roles in erythropoiesis and the homeostasis of EPCs, we propose to identify the cellular proteins that interact 11kDa and transduce the apoptotic signal in EPCs. Secondly, having recently established a B19V culture system that mimics native B19V infection of human EPCs in bone marrow under hypoxic conditions, we will carry out high throughput screening (HTS) of a library of small molecules (available at the University of Kansas HTS Core Laboratory; contains over 100,000 druglike small compounds) for anti-B19V infection drug candidates. The B19V culture system can generate virus in sufficient quantity for such screening, and the cell death marker can be readily applied to assess B19V infection as a readout. We will use a cell-based luminescent cytotoxicity assay to screen the library. Because B19V vaccine is currently unavailable, and immunocompromised patients would be unable to respond to a vaccine, there are currently no viable treatment options for diminishing the bone marrow failure caused by B19V infection in patients with acute or persistent B19V infection. The study proposed in this application is expected to identify targets for the development of drugs capable of eliminating B19V from human EPCs, and this will ultimately lead to diminished bone marrow failures in these patients. In addition, our analysis of the mechanism underlying apoptosis of EPCs is expected to reveal pathways that may be involved in erythropoiesis or the disruption of erythropoiesis under certain disease conditions. PUBLIC HEALTH RELEVANCE: Human parvovirus B19 is pathogenic to humans, causes bone marrow failure in some severe circumstances. Study of B19 infection-induced cell death will help us to understand the mechanism of B19 caused bone marrow failure in general. Our proposed work will thus answer critical questions in the pathogenesis of parvovirus B19 infection, and have the potential to identify candidates of anti-B19 drugs for treating B19 infection-caused bone marrow failure.