SARS Coronavirus Interaction with Monocyte Macrophages

根据SARS患者的白细胞减少和实验感染猕猴的淋巴细胞减少的发现，已经表明SARS-CoV感染抑制免疫。本研究的目的是检测SARS-CoV对炎症细胞特别是单核巨噬细胞的感染易感性和先天免疫激活。为了确定单核巨噬细胞的易感性，我们打算用SARS CoV、SARS CoV-冠状病毒抗体混合物以及通过单核巨噬细胞与SARS CoV感染的VeroE 6细胞的共培养来进行感染实验。我们将通过TCID 50病毒输出试验、RT-PCR、免疫细胞化学（SARS-CoV山羊抗体和细胞类型特异性标记物CD 68）检测病毒复制，并通过TdT隧道试验检测细胞变圆、合胞体形成和凋亡的细胞病变。此外，我们还打算表征由以下物质诱导的先天性免疫应答： 通过微阵列（GEArray人炎性基因表达阵列，Superarray，Bethesda MD，Affyscore Human SARS基因芯片）和RT-PCR（Multiplex PCR for human inflammatory cytokines，Biosource Intl. Camarillo，CA，SARSCoV RT-PCR巢式方案）。分泌的和细胞内的细胞因子将通过ELIZA和FACS显示。结果将与229 E感染的单核细胞巨噬细胞进行比较，后者具有高水平的病毒输出并显示先天性应答。本研究将在BSL-3实验室进行，并将遵守标准操作规程（SOP）（正在审查中）。本提案中的实验解决了病毒直接感染单核细胞、抗体感染的免疫增强和细胞融合的间接感染以及控制诱导的免疫增强的病毒诱导的先天性应答的问题。 这些炎症反应反过来可能产生细胞因子或其他因子对炎症反应的次级作用。

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.