SARS Coronavirus Interaction with Monocyte Macrophages

根据SARS患者白细胞减少和实验感染猕猴淋巴细胞耗竭的发现，提示SARS冠状病毒感染抑制免疫。本研究的目的是研究sars冠状病毒对感染的易感性和炎症细胞特别是单核巨噬细胞的先天免疫激活。为了确定单核巨噬细胞的易感性，我们打算用SARS冠状病毒、SARSCoV-冠状病毒抗体混合物以及单核巨噬细胞与SARSCoV感染的VeroE6细胞共培养进行感染实验。我们将通过TCID50病毒输出试验、RT-PCR、免疫细胞化学（山羊抗SARS-CoV抗体和细胞类型特异性标记CD68）检测病毒复制，通过TdT隧道试验检测细胞圆切、合胞体形成和凋亡的细胞致病性。此外，我们打算表征先天免疫反应诱导

Based on findings of leucopenia in SARS patients and lymphoid depletion in experimentally infected macaques, it has been suggested that SARS-CoV infection suppresses immunity. The intent of this research is to examine the susceptibility to infection and innate immune activation of inflammatory cells specifically monocyte macrophages by SARS-CoV. To determine monocyte macrophage susceptibility, we intend to conduct infection experiments with SARS CoV, with SARSCoV- coronavirus antibody mixtures and by coculture of monocyte macrophages with SARSCoV-infected VeroE6 cells. We will detect viral replication by TCID50 virus output assay, by RT-PCR, by immunocytochemistry (goat antibody to SARS-CoV and cell type specific markers CD68) and cytopathicity by cell rounding, syncytium formation and apoptosis by TdT tunnel assay. Also we intend to characterize the innate immune responses induced by SARSCoV in monocyte macrophages at the gene expression level by microarray (GEArray human inflammatory gene expression arrays, Superarray,Bethesda MD, Affymetrix Human SARS gene chip)and RT-PCR ( Multiplex PCR for human inflammatory cytokines, Biosource Intl. Camarillo, CA, SARSCoV RT-PCR nested protocol ). Secreted and intracellular cytokines will be shown by ELIZA and FACS. Results will be compared to 229E-infected monocyte macrophages which have high levels of virus output and show innate responses. This research will be done in the BSL-3 laboratory and will adhere to the standard operating procedures (SOPS) (under review). The experiments in this proposal address the issues of direct infection of mononuclear cells by the virus, of immune enhancement of infection by antibody and of indirect infection by cell fusion and the virus-induced innate responses which govern the induced immune responses that in turn may produce the secondary effect of cytokines or other factors on inflammatory responses.