Transcriptomics of immunity and disease in African Fruit Bats- important zoonotic reservoirs

摘要 根据美国国立卫生研究院的资助目标和R21研究计划的目标，该项目使用 转录学的力量，以了解蝙蝠的免疫能力与病毒感染的关系在自然， 可变的环境。该项目将在巴克内尔大学由共同PD/PIS博士DeeAnn Reeder共同领导 (国际公认的蝙蝠疾病和比较生理学专家)和肯·菲尔德博士(经典 训练有素的免疫学家，具有将转录转录方法应用于蝙蝠疾病生态学的专门知识)。目标 这项工作的目的是探索内在的(年龄、性别、生殖条件、目前的疾病状况)和外在的 (天气和食物可获得性的季节性变化)因素是非洲果蝠免疫变化的基础， 潜在大流行病毒(包括埃博拉)的蓄水池，这些病毒与 人类由于栖息地的改变。虽然近年来在理解上取得了重要进展 蝙蝠免疫，其中大部分来自细胞培养或有限的样本，主要来自东南亚和 澳大利亚蝙蝠；这项研究将填补分类和地理上的空白，并改变我们对 通过检查真实世界中的免疫过程来研究抗病毒免疫的变化。 为了执行这项工作，将在南苏丹的野外地点收集雄性和雌性觅食蝙蝠 无论是雨季还是旱季。对于特定的目的1，将使用脾组织样本来确定 免疫相关基因的差异表达(Illumina HiSeq 4000平台和三位一体分析管道) 功能，重点是抗病毒免疫。这些发现将在随后的定量聚合酶链式反应研究中得到证实， 将被用来检验最近提出的BAT抗病毒基因表达“始终开启”的假说，该假说 可能与水库库容有关。内因和外因与免疫之间的关系 基因表达将被用来描述抗病毒免疫力低下的时期，这可能会增加溢出风险。 对于特定的目标2，将分析与疾病状态相关的基因表达 疟疾寄生虫(肝孢子虫)载量高或病毒载量高(调查丝状病毒、冠状病毒、 副粘病毒和正粘病毒)，与匹配的对照组比较。对于有差异的基因 表达，qPCR将被用来寻找其他组织中类似的变化，与病毒发现相匹配(例如，高 口腔拭子中的病毒载量将促使唾液腺进行基因表达检查)。两国之间的关系 基因表达和疾病状态将在混合感染(疟疾)影响的背景下进行解释 以及病毒感染对抗病毒机制的影响。如果我们提出的具体目标得到实现，我们将显著 加深我们对蝙蝠免疫的认识，以及在自然条件下影响蝙蝠免疫的因素。这将是 提高我们预测病毒外溢何时更有可能以及如何改变环境的能力 条件，包括对自然景观的人为改变，可能会改变疾病过程。

ABSTRACT In line with the funding goals of the NIH and the objectives of the R21 research program, this project uses the power of transcriptomics to understand bat immune competence in relation to viral infection in a natural, variable environment. This project will be jointly led at Bucknell University by co-PD/PIs Dr. DeeAnn Reeder (internationally recognized expert in bat disease and comparative physiology) and Dr. Ken Field (classically trained immunologist with expertise in applying transcriptomic approaches to bat disease ecology). The goals of this work are to explore how intrinsic (age, sex, reproductive condition, current disease status) and extrinsic (seasonal shifts in weather and food availability) factors underlie immunological variation in African fruit bats, reservoirs for viruses of pandemic potential (including Ebola) that are becoming increasingly associated with people due to habitat modification. While important progress has been made in recent years in understanding bat immunity, much of this has been in cell culture or from limited sampling, largely from SE Asian and Australian bats; this study will fill this taxonomic and geographic gap and transform our understanding of variation in antiviral immunity by examining immune processes in the real world. To perform this work, male and female foraging bats will be collected at field sites in South Sudan during both the rainy and dry season. For Specific Aim 1, spleen tissue samples will be used to determine the differential expression (Illumina HiSeq 4000 platform and Trinity analysis pipeline) of genes involved in immune function, with an emphasis on antiviral immunity. Findings will be confirmed in subsequent qPCR studies and will be used to test the recently proposed hypothesis that bat antiviral gene expression is “always on”, which may be related to reservoir capacity. Relationships found between intrinsic and extrinsic factors and immune gene expression will be used to describe periods of low antiviral immunity, which may increase spillover risk. For Specific Aim 2, gene expression in relation to diseased state will be analyzed for bats with exceptionally high malarial parasite (Hepatocystis) loads or with high viral loads (surveying filoviruses, coronaviruses, paramyxoviruses and orthomyxoviruses), compared to matched controls. For genes with differential expression, qPCR will be used to look for similar changes in other tissues, matched to viral findings (e.g., high viral load from oral swabs will prompt gene expression examination in salivary glands). Relationships between gene expression and disease state will be interpreted in the context of the influence of co-infection (malaria) and of viral infection on antiviral mechanisms. If our proposed specific aims are achieved, we will significantly enhance our understanding of bat immunity and the factors that influence it under natural conditions. This will improve our ability to predict when viral spillovers may be more likely and how changing environmental conditions, including the anthropogenic alteration of natural landscapes, may alter disease processes.