Human TfR1-expressing hamsters to model New World arenaviral hemorrhagic fever

项目总结 哺乳动物病毒在世界各地的啮齿动物种群中是地方性的，它们的人畜传播可导致 严重危及生命的出血热(HF)综合征。在美洲，五种新世界哺乳动物病毒 已知可引起心力衰竭。在没有FDA许可的疫苗或抗病毒疗法的情况下，这些病毒 对公共卫生构成重大风险，对国家安全构成威胁。了解NWA发病机制的研究 而制定有效的对策严重受限于缺乏真实反映的小动物模型 人类疾病。我们最近证实了人转铁蛋白受体1(HuTfR1)的转基因表达， 已知的致病核糖核酸使用的细胞受体，使小鼠对致死性疾病易感 在与阿根廷HF代理人Junin ArenaVirus(JUNV)挑战之后。然而，老鼠并不这样做 明显的出血性疾病迹象，这是人类严重病例的显著特征。我们 而其他人已经表明，感染了相关非致病性NWAs的金色叙利亚仓鼠不使用 HuTfR1发展为一种严重的类似心衰的综合征，具有出血性疾病的许多基本特征，包括 凝血障碍，广泛瘀点，口鼻粘膜出血和血管渗漏。此外，我们 最近的研究表明，在仓鼠细胞系中表达huTfR1可以显著增加JUNV的感染。因此， 我们假设，被设计成表达huTfR1的仓鼠将容易受到JUNV感染，导致 一种更能代表人类疾病的类心衰综合征。为了探索这一假设，我们将继续 以下具体目标：目标1.开发huTfR1敲入(Ki)金色叙利亚仓鼠品系。我们将设计 并测试针对仓鼠TfR1基因3‘端的单引导(Sg)RNA以插入huTfR1开放 阅读框。通过紧接在终止密码子之前的T2a肽连接子将huTfR1cDNA添加到 仓鼠TfR1基因，我们将确保huTfR1在生理水平和正常组织中表达 分发。HuTfR1仓鼠品系将通过成熟的原核注射程序产生， 构建了Cas9/sgRNA核糖核蛋白复合体和huTfR1cDNA供体。目标2.评估 HuTfR1仓鼠对JUNV感染的易感性野生型和huTfR1KI仓鼠将面临挑战 与JUNV合作评估病毒复制和疾病发展方面的差异。我们预料到了这一表达 HuTfR1的表达将促进病毒复制，导致仓鼠出现类似心衰的综合征。疾病参数 每日评估包括体重、体温和临床疾病体征，包括瘀点和粘膜。 在流血。病毒载量将从JUNV挑战赛后一周的血液样本中确定，时间点为 预计huTfR1仓鼠会出现疾病体征并出现可测量的病毒血症。终极的 本项目的目标是制造一种新的NWA HF仓鼠模型，以支持宿主定向的发育 补充直接作用抗病毒药物的治疗方法，以改善重症患者的预后 由致病的核糖核酸引起的疾病。

PROJECT SUMMARY Mammarenaviruses are endemic in rodent populations worldwide and their zoonotic transmission can lead to a severe life-threatening hemorrhagic fever (HF) syndrome. In the Americas, five New World mammarenaviruses (NWAs) are known to cause HF. In the absence of FDA-licensed vaccines or antiviral therapies, these viruses pose a significant public health risk and a threat to national security. Research to understand NWA pathogenesis and develop effective countermeasures is severely limited by the lack of small-animal models that faithfully mirror human disease. We recently demonstrated that transgenic expression of human transferrin receptor 1 (huTfR1), the known cellular receptor used by the pathogenic NWAs, confers susceptibility in mice to lethal disease following challenge with the Junin arenavirus (JUNV), the agent of Argentine HF. However, the mice do not manifest signs of hemorrhagic disease that are prominent features of severe cases of NWA HF in humans. We and others have shown that golden Syrian hamsters infected with related nonpathogenic NWAs that do not use huTfR1 develop a severe HF-like syndrome with many of the cardinal features of hemorrhagic disease, including coagulopathy, extensive petechia, bleeding from the oronasal mucosal region and vascular leak. Moreover, we recently showed that expression of huTfR1 in hamster cell lines significantly augments JUNV infection. Thus, we hypothesize that hamsters engineered to express huTfR1 will be susceptible to JUNV infection, resulting in a HF-like syndrome more representative of human disease. To explore this hypothesis, we will pursue the following Specific Aims: Aim 1. Develop a huTfR1 knock-in (KI) golden Syrian hamster line. We will design and test single guide (sg)RNAs targeting the 3’ end of the hamster TfR1 gene for insertion of the huTfR1 open reading frame. By appending the huTfR1 cDNA via a T2A peptide linker immediately before the stop codon of the hamster TfR1 gene, we will ensure expression of huTfR1 at physiological levels and with normal tissue distribution. The huTfR1 hamster line will be generated by a well-established pronuclear injection procedure with the Cas9/sgRNA ribonucleoprotein complex and the huTfR1 cDNA donor construct. Aim 2. Evaluate the susceptibility of the huTfR1 hamster to JUNV infection. Wild-type and huTfR1 KI hamsters will be challenged with JUNV to assess differences in viral replication and development of disease. We anticipate that expression of huTfR1 will boost viral replication leading to a HF-like syndrome in the hamsters. Disease parameters evaluated daily will include body weight, temperature and clinical disease signs including petechia and mucosal bleeding. Viral loads will be determined from blood samples taken one week after JUNV challenge, a point at which huTfR1 hamsters are expected to develop signs of disease and have measurable viremia. The ultimate goal of this project is to produce a novel hamster model of NWA HF to support development of host-directed therapies that would complement direct-acting antivirals to improve outcomes in patients suffering from severe disease caused by pathogenic NWAs.