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 i nclude: (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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL080211-01
Application #
6905863
Study Section
Lung Cellular, Molecular, and Immunobiology Study Section (LCMI)
Program Officer
Peavy, Hannah H
Project Start
2005-05-01
Project End
2009-04-30
Budget Start
2005-05-01
Budget End
2006-04-30
Support Year
1
Fiscal Year
2005
Total Cost
$636,721
Indirect Cost
Name
Aaron Diamond AIDS Research Center
Department
Type
DUNS #
786658872
City
New York
State
NY
Country
United States
Zip Code
10016
Liu, L; Wei, Q; Nishiura, K et al. (2016) Spatiotemporal interplay of severe acute respiratory syndrome coronavirus and respiratory mucosal cells drives viral dissemination in rhesus macaques. Mucosal Immunol 9:1089-101
Liu, Li; Wei, Qiang; Alvarez, Xavier et al. (2011) Epithelial cells lining salivary gland ducts are early target cells of severe acute respiratory syndrome coronavirus infection in the upper respiratory tracts of rhesus macaques. J Virol 85:4025-30
Luo, Minqi; Liu, Huan; Zhuang, Ke et al. (2009) Prevalence of drug-resistant HIV-1 in rural areas of Hubei province in the People's Republic of China. J Acquir Immune Defic Syndr 50:1-8
Chen, Zhiwei; Huang, Yaoxing; Zhao, Xiuqing et al. (2008) Design, construction, and characterization of a multigenic modified vaccinia Ankara candidate vaccine against human immunodeficiency virus type 1 subtype C/B'. J Acquir Immune Defic Syndr 47:412-21
Chen, Yunxin; Liu, Li; Wei, Qiang et al. (2008) Rhesus angiotensin converting enzyme 2 supports entry of severe acute respiratory syndrome coronavirus in Chinese macaques. Virology 381:89-97
Ba, Lei; Yi, Christopher E; Zhang, Linqi et al. (2007) Heterologous MVA-S prime Ad5-S boost regimen induces high and persistent levels of neutralizing antibody response against SARS coronavirus. Appl Microbiol Biotechnol 76:1131-6
Zhu, Weijun; Fang, Qing; Zhuang, Ke et al. (2007) The attenuation of vaccinia Tian Tan strain by the removal of the viral M1L-K2L genes. J Virol Methods 144:17-26
Liu, Li; Fang, Qing; Deng, Fei et al. (2007) Natural mutations in the receptor binding domain of spike glycoprotein determine the reactivity of cross-neutralization between palm civet coronavirus and severe acute respiratory syndrome coronavirus. J Virol 81:4694-700
Zhang, Yong; Lu, Lin; Ba, Lei et al. (2006) Dominance of HIV-1 subtype CRF01_AE in sexually acquired cases leads to a new epidemic in Yunnan province of China. PLoS Med 3:e443
Yi, Christopher E; Ba, Lei; Zhang, Linqi et al. (2005) Single amino acid substitutions in the severe acute respiratory syndrome coronavirus spike glycoprotein determine viral entry and immunogenicity of a major neutralizing domain. J Virol 79:11638-46