Extra-pulmonary pathology caused by avian H5N1 and recombinant H1N1-1918 influenz

描述（由申请人提供）：持续的人类禽流感暴发强调需要加强研究工作，以充分表征感染期间的发病机制。患者的临床病程和死后病理学提示，要么呼吸道外组织中存在流感病毒，要么继发于呼吸道感染但仍无病毒的组织中存在流感病毒，要么两者兼而有之。动物研究也证明了禽流感病毒在一定程度上的肺外传播，并伴有器官受累，似乎与物种有关。评估非呼吸器官病理对细胞因子过度产生（人类疾病导致多器官衰竭的共同特征）的确切贡献，对于成功的临床管理至关重要，因为更好地了解导致脑病发展的因素也很常见。不幸的是，感染后的住院时间、英勇治疗努力的混淆效应，以及在已经严重受损的个体中排除主要器官活检的伦理考虑，需要使用动物模型来研究肺部和其他可能受感染的主要器官的早期感染。在过去的3年里，我们已经成功地优化了人类流感感染的猕猴模型，用于基因组应用。为此，我们使用了一种轻度致病性人流感病毒（a /Texas/36/91）来仔细表征其临床病程、病理和对病毒mRNA、病毒蛋白或活跃病毒复制的转录调控。接下来，我们将用德克萨斯流感病毒感染食蟹猴，该病毒含有来自1918年大流行性流感（A/Brevig Mission/1/18）的多达三种基因，据信源自一种禽流感病毒，以及高致病性禽流感（A/A/Vietnam/1203/04）。除了前几年所做的工作外，我们建议通过系统的显微镜检查、流感抗原染色、免疫细胞原位表型和炎症和免疫过程标志物的免疫组织化学染色，对呼吸道以外的组织中的任何病理进行深入表征，包括环加氧酶级联、巨噬细胞产生一氧化氮、诱导缺氧反应元件、血管生成、和其他人。该方法将通过激光捕获病变显微解剖，然后对相应mrna进行定量反转录PCR来补充和证实。我们认为，结合几年来对这一模型的直接经验和对已知或可能对人类具有高毒力的相关流感病毒的使用，提供了一个独特的机会来阐明人类禽流感感染的这一重要方面。禽流感在人类中的暴发已造成越来越高的发病率和死亡率。病毒对人类宿主的进一步适应很可能导致像1918年或“西班牙”流感那样的世界范围流行病，据信源自禽类，并在全世界造成2000万至4000万人死亡。由禽流感感染引起的人类疾病不仅限于呼吸道，而且似乎还会影响到其他器官，从而导致受感染者预后不良。我们建议对感染高致病性禽流感病毒和1918年流感基因重组的人类病毒的非人类灵长类动物进行深入的病理学研究，以充分表征感染对呼吸道外组织的影响，并以有助于减少感染后人类伤亡的知识体系为目标。

DESCRIPTION (provided by applicant): Ongoing outbreaks of avian influenza in humans stress the need for intensified research efforts to fully characterize the pathogenesis during infection. Clinical course in patients and post-mortem pathology have suggested either presence of influenza virus in tissues outside of the respiratory tract, disease secondary to respiratory infection but in tissues that remain virus-free, or both. Animal studies have also demonstrated extra- pulmonary spread of avian influenza viruses, to an extent and with organ-involvement that appear species- dependent. Assessing the exact contribution of non-respiratory organ pathology to cytokine overproduction, a common feature of the disease in humans leading to multi-organ failure, is critical to successful clinical management, as is a better understanding of the factors leading to development of encephalopathy, also commonly seen. Unfortunately, timing of hospitalization after infection, confounding effects of heroic therapeutic efforts, and ethical considerations precluding biopsy of major organs in already severely compromised individuals, necessitate the use of animal models to study early infection in lungs and other major organs likely affected by the infection. Over the past 3 years, we have successfully optimized a macaque model of human influenza infection for genomic applications. For this purpose, we have used a mildly pathogenic human influenza virus (A/Texas/36/91) to carefully characterize the clinical course, pathology, and transcriptional regulation in response to viral mRNA, viral proteins, or to active viral replication. Next, we will infect Cynomolgus macaques with Texas influenza virus containing up to three genes from the 1918 pandemic flu (A/Brevig Mission/1/18), believed to have originated from an avian virus, and with highly pathogenic avian influenza (A/A/Vietnam/1203/04). In addition to the work performed in previous years, we propose to do an in-depth characterization of any pathology in tissues other than the respiratory tract, through the use of systematic microscopic examination, influenza antigen staining, phenotyping of immune cells in situ, and immunohistochemistry staining for markers of inflammatory and immune processes, including the cyclooxygenase cascade, production of nitric oxide by macrophages, induction of hypoxia response elements, angiogenesis, and others. This approach will be supplemented and corroborated by laser capture microdissection of lesions followed by quantitative reverse transcription PCR for corresponding mRNAs. We believe that the combination of several years of direct experience with this model and the use of relevant influenza viruses, known to be or likely to be of high virulence in humans, provides a unique opportunity to elucidate this important aspect of avian influenza infection in humans. Bielefeldt Ohmann - R03 NARRATIVE Outbreaks of avian influenza in humans have caused increasingly high morbidity and mortality. Further adaptation of the virus to the human host is likely to result in a world-wide epidemic such as the 1918 or "Spanish" influenza, believed to have been of avian origin and to have caused 20 to 40 millions deaths worldwide. Human disease caused by infection with avian influenza is not limited to the respiratory tract, but appears to affect other organs in ways that result in poor prognosis in infected individuals. We propose to perform in-depth pathology work in non-human primates that are to be infected with a highly pathogenic avian virus and with a human virus recombined with genes from the 1918 influenza, in order to fully characterize the effect of infection on tissues outside of the respiratory tract and with the goal of contributing to the body of knowledge that will help minimize human casualties after infection.