A Novel Model for the Study of Lung Pathogenesis of SARS

描述（由申请人提供）：由一种新型冠状病毒（SARS相关冠状病毒，SARS- cov）引起的严重急性呼吸系统综合征（SARS）是一种具有高度传染性和致死率的严重肺部疾病。在最近的全球流行病中，8098名感染者中有775人死于SARS。现在解决SARS疫情对了解肺损伤的原因具有重要意义，这在很大程度上依赖于建立有效的动物模型。我们的长期总体目标是利用sars冠状病毒感染的中国猕猴（Macaca mulatta）作为动物模型，了解肺部发病机制。我们的具体目的包括：(1)进一步表征sars冠状病毒在中国猕猴中的肺部发病机制。(2)探讨中和抗体（nab）在中国猕猴SARS-CoV肺发病机制中的作用。(3)确定CD8+ T细胞和B细胞在中国猕猴肺部发病机制中的作用。在第一个目标中，我们将重点关注肺部病理的可重复性，方法是用sars冠状病毒感染四只猕猴，并在规定的时间牺牲它们，进行病毒学、病理学和免疫学评估。一旦模型正确建立，我们将研究感染的早期事件。我们的假设是，病毒在呼吸系统的早期播种事件将决定SARS疾病的进展过程。使用活病毒，8只受感染的猴子将在感染后的第2天和第3天（p.i.）被处死。另外四只猴子将被注射单轮假病毒。将收集一套完整的相关标本进行评估。通过确定初始靶细胞，并将肺损伤程度与病毒载量和肺室感染细胞相关联，将更好地了解SARS的肺发病机制。在第二个实验中，14只猴子被分成两组，在感染前分别注射高剂量和低剂量的Nabs。这些动物将被感染，随后在两个确定的时间点被处死以供分析。我们的假设是疫苗诱导的抗体在决定疾病结果方面发挥积极作用。通过将肺损伤程度与nab输注量相关联，可以明确nab与肺发病机制的关系。在第三个目标中，我们的假设是，通过特异性免疫反应对病毒复制的早期控制决定了SARS的结果。这两项研究将分别对12只猴子进行挑战，在急性感染过程中，一只猴子的CD8+ T细胞被耗尽，另一只猴子的B细胞被耗尽。一半的动物将在两个指定的时间点牺牲进行分析。这两个时间点被定义为急性期和恢复期。通过这样做，特异性免疫反应在确定SARS肺病理中的作用有望获得。在整个拟议的研究中，包括一些对照动物。病毒学方法包括病毒分离、检测病毒RNA的RT-PCR、定量病毒RNA的实时RT-PCR、病毒变异的DNA测序和病毒分布的原位杂交。病理学方法包括用于组织学评估的常规染色，免疫组织化学，原位杂交和共聚焦显微镜用于细胞表型，蛋白质共定位和病毒趋向性。免疫学方法包括流式细胞术进行细胞分型，ELISA， IF，中和试验，Western Blot分析体液反应，ELIspot检测T细胞介导的反应。这些方法被反复使用以支持每一个目标。

DESCRIPTION (PROVIDED BY APPLICANT): Severe acute respiratory syndrome (SARS), caused by a novel coronavirus (SARS-associated coronavirus, SARS-CoV), is a severe pulmonary disease with a high degree of transmissibility and mortality. During the recent global epidemic, 775 out of 8098 infected people died of SARS. The resolution of the SARS epidemic now places great significance on the understanding of the cause of pulmonary damage, which will largely rely on the establishment of a valid animal model. Our long term overall objective is to understand the pulmonary pathogenesis using SARS-CoV infected Chinese macaque (Macaca mulatta) as an animal model. Our specific aims include: (1) to further characterize the lung pathogenesis of SARS-CoV in Chinese macaques. (2) To determine the role of neutralizing antibodies (Nabs) in modulating the lung pathogenesis of SARS-CoV in Chinese macaques. (3) To determine the role of CD8+ T cells and B cells in the lung pathogenesis in Chinese macaques. In aim one, we will focus on the reproducibility of lung pathology, by infecting four macaques with SARS-CoV and sacrificing them at defined times for virological, pathological and immunological evaluation. Once the model is properly established, we will study the early events of infection. Our hypothesis is that the early events of viral seeding in the respiratory system will determine the course of SARS disease progression. Using live virus, eight infected monkeys will be sacrificed on days 2 and 3 post infection (p.i.). Another four monkeys will be given a single-round pseudovirus. A complete set of relevant specimens will be collected for the evaluation. By defining the initial target cells and by correlating the extent of lung damage with the viral load and infected cells in lung compartments, a better understanding of the pulmonary pathogenesis of SARS will be obtained. In aim two, 14 monkeys divided into two groups will receive high and low doses of Nabs before infection. These animals will be infected and subsequently sacrificed at two defined time points for analysis. Our hypothesis is that vaccine-induced Nabs play an active role in determining the disease outcome. By correlating the extent of lung damage with the quantities of Nabs infused, the relationship of the Nabs to lung pathogenesis will be defined. In aim three, our hypothesis is that the early control of viral replication by specific immune responses determines the outcomes of SARS. 12 monkeys will be challenged in each of the two studies, one depleted of CD8+ T cells and the other depleted of B cells during the course of acute infection. Half of the animals will be sacrificed at two defined time points for analysis. These two time points are defined as the acute phase and the recovery phase. By doing so, the role of specific immune responses in determining the lung pathology of SARS is hopefully obtained. Throughout the proposed study, some control animals are included. The virological methods include viral isolation, RT-PCR for detecting viral RNA, real-time RT-PCR for quantifying viral RNA, DNA sequencing for viral variation and in situ hybridization for viral distribution. The pathological methods include conventional staining for histological evaluation, immunohistochemistry, in situ hybridization and con-focal microscopy for cell phenotyping, protein co-localization and viral tropism. The immunological methods include flow cytometry for cell typing, ELISA, IF, neutralization assay, Western Blot analysis for humoral response, and ELIspot for T cell-mediated response. These methods are repeatedly used to support each of the aims.