Study of Bone Marrow Failure Caused by B19 Virus Infection

描述（由申请方提供）：细小病毒B19（B19 V）感染通过破坏骨髓驻留的红系祖细胞（EPC）导致人骨髓衰竭，EPC支持病毒的自主复制。在由于高水平的红细胞破坏而对红细胞产生有高需求的患者中（例如，镰状细胞病患者），急性B19 V感染可引起短暂的再生障碍性危象。在免疫功能低下的患者中，持续的B19 V感染可能表现为纯红细胞再生障碍性贫血。在胎儿中，B19 V感染可导致胎儿水肿，一种严重的贫血形式。B19 V感染可引起EPCs的损伤，从而导致上述骨髓衰竭。B19 V感染的EPCs经历细胞死亡，具有明显的凋亡特征。这种细胞死亡主要由感染期间非结构性11 kDa蛋白（11 kDa）的高表达诱导，并涉及由caspase-10触发的caspase级联反应。在本研究中，我们的目标是，首先，揭示caspase-10介导的细胞凋亡的分子机制，响应于11 kDa。为此，我们将确定在B19 V感染诱导的细胞凋亡过程中，细胞凋亡途径的外在或内在激活启动子caspase-10。由于凋亡和抗凋亡通路在EPCs的红细胞生成和稳态中起着关键作用，我们建议鉴定与EPCs中11 kDa相互作用并抑制凋亡信号的细胞蛋白。其次，最近建立了模拟缺氧条件下骨髓中人EPCs的天然B19 V感染的B19 V培养系统，我们将对小分子文库（可在堪萨斯大学HTS核心实验室获得;包含超过100，000种药物样小化合物）进行高通量筛选（HTS）以获得抗B19 V感染候选药物。B19 V培养系统可以产生足够量的病毒用于这种筛选，并且细胞死亡标志物可以容易地应用于评估B19 V感染作为读数。我们将使用基于细胞的发光细胞毒性测定来筛选文库。由于B19 V疫苗目前不可用，免疫功能低下的患者将无法对疫苗作出反应，因此目前没有可行的治疗选择来减少急性或持续性B19 V感染患者中由B19 V感染引起的骨髓衰竭。本申请中提出的研究预计将确定开发能够从人EPCs中消除B19 V的药物的靶点，这将最终导致这些患者的骨髓衰竭减少。此外，我们对EPCs凋亡机制的分析有望揭示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。 公共卫生相关性：人类细小病毒B19对人类具有致病性，在某些严重情况下会导致骨髓衰竭。研究B19感染诱导的细胞死亡有助于从总体上了解B19导致骨髓衰竭的机制。因此，我们提出的工作将回答细小病毒B19感染的发病机制中的关键问题，并有可能确定用于治疗B19感染引起的骨髓衰竭的抗B19药物的候选者。

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.