Lethal H5N1 Virus Infection and Respiratory Disorders

描述（由申请人提供）：甲型H5N1禽流感病毒感染可导致60%的患者死亡率，这些患者在感染后最初6天内出现呼吸困难和肺部炎症等呼吸异常，直至呼吸衰竭伴低氧血症，数天后死亡。导致感染导致死亡的呼吸衰竭的机制尚不清楚，也没有有效的疫苗/治疗方法。动物研究主要集中在感染的免疫学和病毒学方面，没有研究呼吸病理生理学。此外，这些研究表明，在肺部感染时，致死性H5N1病毒与非致死性H5N1病毒不同，最初（感染后2-3天）侵入迷走神经，然后侵入大脑，导致感染后8天死亡，这表明在H5N1病理学中可能涉及迷走神经。在迷走神经内运动的肺感觉纤维主要由支气管肺c纤维（PCFs）组成。外周刺激pcf可引发呼吸困难和肺部炎症，中枢可诱导低氧和高碳酸血症通气反应（dHVR和dHCVR）。这些外周和中枢效应是通过PCFs将SP释放到肺部和孤束核（mNTS）中部分别作用于局部神经激肽1受体（NK1R）来实现的。由于dHVR和dHCVR负责产生呼吸衰竭，我们最近在病毒感染的早期阶段测试了这些化学反射。我们的初步数据显示，HK483 （H5N1致死性毒株）而HK486病毒（非致死性毒株）在感染后2-3天导致显著的dHVR和dHCVR，无病毒血症，并在感染后8天杀死小鼠。有趣的是，这种死亡在pcf退化或sp敲除小鼠中不存在。因此，在本项目中，我们将首先通过测量感染期间小鼠的心肺活动、肺部变化和化学反射来表征HK483病毒诱导的心肺疾病，并将这些疾病与呼吸衰竭（死亡）联系起来，从而为进一步的机制研究奠定基础。其次，我们将定义HK483病毒侵入PCF以增加其活性和敏感性，PCF变性可减少或防止病毒引起的呼吸系统疾病（死亡）。第三，我们将揭示HK483病毒促进PCF依赖性SP释放到肺部和mNTS，以上调PCF和mNTS神经元中接受PCF输入的NK1R表达。此外，将确定全身或外周阻断NK1Rs和选择性损伤mNTS NK1R神经元对HK483病毒诱导的心肺疾病和死亡的影响。在这个项目中，将使用电生理、生化、药理学和免疫细胞化学方法。我们的上述预测结果将：1)形成一种新的神经病毒学概念，即致命的H5N1病毒侵入PCFs，诱导其形态和功能变化；2)对致死性病毒感染致呼吸衰竭的发病机制有了新的认识；3)促进预防策略和药物治疗的发展，以防止呼吸衰竭和死亡。

DESCRIPTION (provided by applicant): Avian influenza A (H5N1) virus infection results in ~60% mortality in patients who present respiratory abnormalities from dyspnea and pulmonary inflammation during the first 6 days post-infection to respiratory failure with hypoxemia, leading to death several days later. The mechanisms underlying the respiratory failure responsible for the infection-induced death are unclear and no effective vaccine/treatment is available. Animal studies have focused on immunology and virology of the infection without studying respiratory pathophysiology. Additionally, these studies showed that upon pulmonary infection, the lethal H5N1 viruses, differing from nonlethal ones, initially invaded (2-3 days post-infection) the vagus nerve and then brain, resulting in death 8 days post-infection, pointing to a possible vagal involvement in H5N1 pathology. Pulmonary sensory fibers traveling within the vagus nerve are composed primarily of bronchopulmonary C-fibers (PCFs). Stimulation of PCFs peripherally triggers dyspnea and pulmonary inflammation and centrally induces depressed hypoxic and hypercapnic ventilatory responses (dHVR and dHCVR). These peripheral and central effects are achieved by PCFs releasing SP into the lungs and the middle region of the nucleus tractus solitarius (mNTS) to act on local neurokinin 1 receptor (NK1R), respectively. Because dHVR and dHCVR are responsible for generating respiratory failure, we recently tested these chemoreflexes at the early stage of the viral infection. Our preliminary data showed that HK483 (a lethal H5N1 strain) but not HK486 virus (a nonlethal one) led to remarkable dHVR and dHCVR 2-3 days post-infection without viremia and killed the mice 8 days post-infection. Interestingly, this death was absent in PCF-degenerated or SP-knockout mice. Therefore, in this project, we will first characterize the HK483 virus-induced cardiorespiratory disorders by measuring cardiorespiratory activities, pulmonary changes, and chemoreflexes in mice over the infection period and correlate the disorders to respiratory failure (death), thereby building a bas for further mechanistic studies. Second, we will define that HK483 virus invades PCFs to increase their activity and sensitivity and that PCF degeneration diminishes or prevents the virus-induced respiratory disorders (death). Third, we will reveal that HK483 virus promotes PCF-dependent SP release into the lungs and mNTS to upregulate NK1R expression in PCFs and mNTS neurons receiving PCF inputs. Moreover, the effects of systemic or peripheral blockade of NK1Rs and selective lesion of mNTS NK1R neurons on the HK483 virus-induced cardiorespiratory disorders and death will be determined. In this project, electrophysiological, biochemical, pharmacological, and immunocytochemical approaches will be used. Our predicted results as described above will: 1) form a novel neurovirological concept that lethal H5N1 virus invades PCFs to induce their morphological and functional changes; 2) gain new insight into the mechanisms underlying the pathogenesis of the lethal viral infection-induced respiratory failure; and 3) catalyze the development of preventive strategies and pharmacological therapies to protect against respiratory failure and death.