SARS Coronavirus Interaction with Monocytes Macrophages

根据SARS患者的白细胞减少和实验感染猕猴的淋巴衰竭的研究结果，有人认为SARS冠状病毒感染会抑制免疫。本研究旨在探讨SARS冠状病毒对炎性细胞，特别是单核巨噬细胞感染的易感性和天然免疫激活作用。为了确定单核巨噬细胞对SARSCoV的敏感性，我们打算用SARS CoV、SARSCoV-冠状病毒抗体混合物以及通过单核巨噬细胞与感染SARSCoV的VeroE6细胞共同培养来进行感染实验。我们将通过TCID50病毒产量试验、RT-PCR、免疫细胞化学(抗SARS-CoV的山羊抗体和细胞类型特异性标志物CD68)检测病毒复制，并通过TDT隧道试验通过细胞圆形、合体形成和细胞凋亡来检测细胞致病性。此外，我们还打算描述以下物质诱导的先天免疫反应 SARSCoV在单核巨噬细胞中的基因表达水平的基因表达谱芯片(基因芯片)和逆转录聚合酶链式反应(RT-PCR)。CAMARILEO，CA，SARSCoV RT-PCR套式协议)。ELIZA和FACS将显示分泌和细胞内的细胞因子。结果将与感染229E的单核巨噬细胞进行比较，后者具有高水平的病毒输出并显示出天生的反应。这项研究将在BSL-3实验室进行，并将遵守标准操作程序(SOP)(正在审查中)。该方案中的实验涉及病毒对单个核细胞的直接感染、抗体感染的免疫增强、细胞融合的间接感染以及控制诱导免疫的病毒诱导的先天反应。 继而可能产生细胞因子或其他因素对炎症反应的次要作用的反应。

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.