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

项目摘要 哺乳动物肾病毒是世界范围内啮齿动物种群的地方病，其人畜共患传播可导致 严重危及生命的出血热（HF）综合征。在美洲，五种新大陆哺乳动物病毒 （NWA）已知会导致HF。在没有FDA许可的疫苗或抗病毒疗法的情况下，这些病毒 对公众健康构成重大风险，并对国家安全构成威胁。了解NWA发病机制的研究 并制定有效的对策是严重限制了缺乏小动物模型，忠实反映 人类疾病我们最近证明转基因表达人转铁蛋白受体1（huTfR 1）， 致病性NWA使用的已知细胞受体，赋予小鼠对致命疾病的易感性 在用阿根廷HF的病原体朱宁沙粒病毒（JUNV）攻击后。然而，老鼠没有 表现出出血性疾病的体征，这是人类NWA HF严重病例的突出特征。我们 和其他研究表明，感染了相关非致病性NWA的叙利亚金黄仓鼠， huTfR 1发展为严重的HF样综合征，具有出血性疾病的许多主要特征，包括 凝血病、大面积瘀点、口鼻粘膜区出血和血管渗漏。而且我们 最近显示，在仓鼠细胞系中huTfR 1的表达显著增强JUNV感染。因此，在本发明中， 我们假设，表达huTfR 1的仓鼠对JUNV感染易感，导致 一种更能代表人类疾病的类HF综合征。为了探索这一假设，我们将继续研究 具体目标：目标1。开发huTfR 1敲入（KI）金黄色叙利亚仓鼠系。我们将设计 并测试靶向仓鼠TfR 1基因3'末端的单向导（sg）RNA，用于插入huTfR 1开放的 阅读帧。通过将huTfR 1 cDNA经由T2 A肽接头紧接在 仓鼠TfR 1基因，我们将确保huTfR 1在生理水平和正常组织中表达 分布huTfR 1仓鼠系将通过完善的原核注射程序产生， Cas9/sgRNA核糖核蛋白复合物和huTfR 1 cDNA供体构建体。目标2.评价 huTfR 1仓鼠对JUNV感染的易感性。将对野生型和huTfR 1 KI仓鼠进行攻毒 与JUNV一起评估病毒复制和疾病发展的差异。我们预期这种表达 的huTfR 1将促进病毒复制，导致仓鼠中的HF样综合征。疾病参数 每日评估将包括体重、体温和临床疾病体征，包括瘀点和粘膜 流血了将从JUNV攻毒后一周采集的血液样本中测定病毒载量， 预期所述huTfR 1仓鼠会出现疾病迹象并具有可测量的病毒血症。最终 本项目的目标是产生一种新的NWA HF仓鼠模型，以支持宿主导向的 补充直接作用抗病毒药物的治疗，以改善患有严重 由致病性核武器引起的疾病。

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.