A Novel Model for the Study of Lung Pathogenesis of SARS

描述（申请人提供）：严重急性呼吸综合征（SARS），由一种新型冠状病毒（SARS相关冠状病毒，SARS-CoV）引起，是一种严重的肺部疾病，具有高度的传播性和死亡率。在最近的全球流行期间，8098名感染者中有775人死于SARS。SARS流行的解决现在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以SARS冠状病毒感染的中国猕猴（Macaca mulatta）为动物模型，了解其肺部发病机制。我们的具体目标包括：（1）进一步研究SARS-CoV在中国猕猴中的肺部发病机制。(2)目的探讨中和抗体（Nabs）在SARS冠状病毒（SARS-CoV）肺致病机制中的作用。(3)目的探讨CD 8 + T细胞和B细胞在中国猕猴肺发病机制中的作用。在目标一，我们将集中在肺病理学的再现性，通过感染四只猕猴与SARS冠状病毒，并在规定的时间处死他们的病毒学，病理学和免疫学评价。一旦模型建立正确，我们将研究感染的早期事件。我们的假设是，病毒在呼吸系统中播种的早期事件将决定SARS疾病进展的过程。使用活病毒，在感染后第2天和第3天（p.i.）处死8只感染猴。另外四只猴子将被给予单轮假病毒。将采集一套完整的相关标本用于评价。通过确定最初的靶细胞，并通过将肺损伤的程度与肺隔室中的病毒载量和受感染细胞相关联，将更好地理解SARS的肺发病机制。在第二个目标中，14只猴子分为两组，在感染前接受高剂量和低剂量的Nabs。这些动物将被感染，随后在两个规定的时间点处死以进行分析。我们的假设是疫苗诱导的Nabs在决定疾病结果方面发挥积极作用。通过将肺损伤的程度与输注的Nab的量相关联，将定义Nab与肺发病机制的关系。在目标三中，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。在两项研究中，每项研究将对12只猴进行攻毒，在急性感染过程中，一项排除CD 8 + T细胞，另一项排除B细胞。在两个规定的时间点处死一半动物进行分析。这两个时间点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的肺部病理学的作用是有希望的。在整个拟定研究中，纳入了一些对照动物。病毒学方法包括病毒分离、用于检测病毒RNA的RT-PCR、用于定量病毒RNA的实时RT-PCR、用于病毒变异的DNA测序和用于病毒分布的原位杂交。病理学方法包括用于组织学评价的常规染色、用于细胞表型、蛋白共定位和病毒嗜性的免疫组织化学、原位杂交和共聚焦显微镜。免疫学方法包括用于细胞分型的流式细胞术、ELISA、IF、中和测定、用于体液应答的Western印迹分析和用于T细胞介导的应答的ELIspot。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.