IMMUNOLOGICAL RESPONSE TO PRIMARY & SECONDARY INFLUENZA INFECTION IN NH PRIMATES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 该项目的目标是使用非人类灵长类动物感染模型，结合从受感染动物的血液和淋巴中分离出的特定细胞类型的基因组分析，来确定流感病毒与宿主的相互作用以及对感染的先天性和获得性免疫反应。在本报告所述期间，我们重点建立了通过流式细胞仪(使用新获得的FACS Aria仪器)分选细胞的技术和参数，并优化了从分离细胞中获取RNA和蛋白质的时间和方法，以便随后分析细胞基因表达和蛋白质丰度。不幸的是，在从分类的细胞群体中获得合适的RNA的努力中，发现了大量的技术障碍。尽管反复努力，但从分选的细胞中只能获得低质量的RNA。我们选择检查流感感染模型在很大程度上是基于免疫学核心对分类免疫细胞亚群进行详细表征的兴趣。卡策实验室已经对其他非人类灵长类流感感染模型进行了大致描述，包括与史密斯实验室正在进行的蛋白质组学研究。因此，获得适合于RNA分离的分类细胞的困难极大地降低了这项研究的潜在科学影响。出于这个原因，我们选择将注意力集中在一个不同的非人类灵长类动物模型上，在这个模型中，整个科学问题不是那么高度依赖细胞分选的性能。考虑到科学界未得到满足的需求，我们已将我们的资源重新用于进行一项急性SIV感染研究；详细情况载于进展报告的其他部分，在功能基因组学和传染病司的一般说明下。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The goal of this project is to use a nonhuman primate infection model, combined with genomic analyses of specific cell types isolated from the blood and lymph of infected animals, to define influenza virus-host interactions and the innate and adaptive immune responses to infection. During the current reporting period, we focused on establishing the techniques and parameters for sorting cells by flow cytometry (using a newly acquired FACS Aria instrument) and for optimizing the timing and methods for harvesting RNA and protein from isolated cells for subsequent analysis of cellular gene expression and protein abundance. Unfortunately, a large number of technical hurdles were uncovered in the effort to obtain suitable RNA from sorted cell populations. Despite repeated efforts, only low-quality RNA could be obtained from sorted cells. Our choice to examine an influenza infection model was largely premised on the interest of the Immunology Core to do a detailed characterization of sorted immune cell subsets. Other nonhuman primate influenza-infection models have already been generally characterized by the Katze Laboratory, including ongoing proteomics investigations with the Smith Laboratory. Consequently, the difficulty in obtaining sorted cells suitable for RNA isolation greatly diminished the potential scientific impact of this investigation. For this reason, we have chosen to focus attention on a different nonhuman primate model where the overall scientific questions are not so highly dependent on the performance of cell sorting. In consideration of unmet needs in the scientific community, we have redirected our resources to performing an acute SIV-infection study; details are given elsewhere in the progress report, under the general narrative for the Division of Functional Genomics and Infectious Disease.