A Novel Model for the Study of Lung Pathogenesis of SARS

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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.