Molecular Mechanisms of Sleep Responses to Viral Infection

描述(申请人提供)：流感是一种呼吸道病毒感染，通常伴随着严重的症状，如高烧和严重的睡眠需求。总而言之，这些症状是急性时相反应(APR)的一部分。对病毒APR的分子介体的了解还处于起步阶段。在破译中央管理的年鉴方面取得的一些进展源于这笔赠款支持的研究。我们的成就包括：显示了流感病毒感染过程中睡眠的变化；证明了在受感染的小鼠的肺部中发现了有毒的病毒副产物双链(DS)RNA，并证明了它会诱导APR；表征了小鼠的流感APR，并通过开发适当的感染模型和使用遗传缺陷的小鼠来研究APR的分子机制。最重要的是，我们最近证明，以前被认为不能进入老鼠大脑的人类流感毒株，实际上在鼻腔感染后的几个小时内就能进入。在大脑中，病毒RNA和病毒抗原伴随着细胞因子的诱导，具有介导APR的潜力。我们在AIMS 1和AIMS 2中对这些数据进行了扩展。AIMS 1解决了病毒、病毒蛋白和/或病毒RNA通过嗅神经从鼻腔传递到嗅球(OB)并诱导细胞因子产生的一般假设。我们将使用免疫组织化学、聚合酶链式反应技术和电子显微镜来确定病毒侵袭OB的时间进程及其与细胞因子表达的关系。双标记技术将用于确定病毒蛋白与细胞因子以及表达细胞因子和含有病毒蛋白的细胞类型的共存。目的2验证OB表达的细胞因子激活下丘脑(HT)细胞因子及其调节的APRs的假说。HT细胞因子表达相对于OB表达的时间将被确定。向OB内注射TNFa的效果以及切断OB-脑连接对HT细胞因子以及睡眠和发热反应的影响将被确定。目的3验证流感病毒侵袭中枢神经系统受宿主免疫状态影响的一般假设。我们确定对流感病毒的同源免疫是否提供了部分保护，防止病毒入侵大脑。目标1中的测量将与睡眠、体温、运动活动和体重测定一起用于目标2和目标3。AIM 4源于我们过去的工作，表明病毒dsRNA会导致流感样症状。因此，缺乏Toll样受体3(TLR3)(一种结合dsRNA的受体)的小鼠将受到病毒的攻击。包括睡眠和脑细胞因子表达在内的APR将被确定。上呼吸道病毒感染与婴儿猝死综合征之间存在关联；婴儿猝死综合征发生在睡眠期间，与睡眠异常有关。拟议的工作将提供对病毒如何导致疾病症状以及小岛屿发展中国家和病毒性脑炎/脑病等并发症的见解。

DESCRIPTION (provided by applicant): Influenza is a respiratory viral infection often accompanied by severe symptoms such as high fevers and a profound need to sleep. Collectively, these symptoms are part of the acute phase response (APR). The understanding of the molecular mediators of the viral APR is in its infancy. Some of the progress made in deciphering centrally regulated APRs resulted from research supported by this grant. Our accomplishments include; showing that sleep changes over the course of an influenza virus infection; demonstrating that a toxic viral by-product, double-stranded (ds) RNA, is found in lungs of infected mice and that it induces the APR; characterizing the influenza APR in mice, and investigating APR molecular mechanisms through development of appropriate infection models and using genetically-deficient mice. Most importantly, we recently demonstrated that strains of human influenza .previously considered incapable of accessing the mouse brain, in fact do so within a few hours following intranasal infection. In brain, viral RNA and viral antigen are accompanied by induction of cytokines with the potential for mediating the APR. We expand upon these data in Aims 1 and 2. Aim 1 addresses the general hypothesis that virus, viral proteins and/or viral RNA pass from the nasal cavities to the olfactory bulb (OB) via the olfactory nerve and induce cytokine production. We will use immunohistochemistry, polymerase chain reaction techniques, and electron microscopy to determine the time course of viral invasion of the OB and its relationship to cytokine expression. Double-labeling techniques will be used to determine co-localization of virus protein with cytokines and the cell types expressing cytokines and containing virus proteins. Aim 2 tests the hypothesis that OB-expressed cytokines activate hypothalamic (HT) cytokines and subsequent HT-regulated APRs. The timing of HT cytokine expression relative to OB expression will be determined. The effects of TNFa injection into the OB and the effects of cutting OB-brain connections on HT-cytokine and sleep and fever responses will be determined. Aim 3 tests the general hypothesis that invasion of the CNS by influenza virus is affected by immune status of the host. We determine whether homologous immunity to influenza virus offers partial protection against viral invasion of the brain. The measures made in Aim 1 will be used in Aims 2 and 3 along with sleep, body temperature, locomotor activity and body weight determinations. Aim 4 is derived from our past work showing that viral dsRNA induces flu-like symptoms. Thus mice deficient in toll- like receptor 3 (TLR3), a receptor that binds dsRNA, will be challenged with virus. APRs including sleep and brain cytokine expression will be determined. There is an association between upper respiratory viral infections and sudden infant death syndrome (SIDS); SIDS occurs during sleep and is associated with sleep abnormalities. The proposed work will provide insights into how viruses induce disease symptoms as well as such complications as SIDS and viral encephalitis/encephalopathy.