A Recombinant Cedar Virus-based Henipavirus Replication Platform for High-throughput Inhibitor Screening

基于重组雪松病毒的亨尼帕病毒复制平台，用于高通量抑制剂筛选

• 批准号: 9509144
• 负责人: CHRISTOPHER C BRODER
• 金额: $ 23.06万
• 依托单位: HENRY M. JACKSON FDN FOR THE ADV MIL/MED
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-12-20 至 2019-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9509144
• 关键词: Animals; Antiviral Agents; Australia; Binding; Biological Assay; Cell Communication; Cell Culture Techniques; Cell Line; Cell fusion; Cell membrane; Cells; Cellular biology; Chicago; Collaborations; Communicable Diseases; Containment; Eph Family Receptors; Ephrin-A1; Ephrin-B1; Far East; Glycoproteins; Growth; Hendra Virus; Henipavirus; Henipavirus Infections; Human; Illinois; Infection; Kinetics; Lead; Libraries; Livestock; Luc Gene; Luciferases; Lung diseases; Mammals; Measures; Mediating; Medical; Membrane; Membrane Fusion; Nipah Virus; Paramyxovirus; Pathogenicity; Public Health; RNA Viruses; Receptor Cell; Recombinants; Reporter; Reporter Genes; Research; Series; Signal Transduction; Surface Plasmon Resonance; System; Testing; Texas; Therapeutic; Time; Tropism; Universities; Vaccines; Viral; Virion; Virus; Virus Diseases; Virus Replication; Work; Zoonoses; base; biothreat; drug discovery; experimental study; genetic approach; glycoprotein G; high throughput screening; inhibitor/antagonist; insight; nervous system disorder; neutralizing antibody; pathogen; recombinant virus; reverse genetics; screening; small molecule; small molecule libraries; virology

Project Summary/Abstract The emergence of pathogenic viruses represents continuous infectious disease threats to public health. Among these, the paramyxoviruses, which include many important human and animal pathogens, also include two excellent examples of emerged, zoonotic pathogens of importance: the henipaviruses; Hendra virus (HeV) and Nipah virus (NiV). HeV and NiV have a uniquely broad host tropism capable of infecting at least 18 animal species across 6 orders of mammals. HeV and NiV can also cause a systemic and often fatal respiratory and/or neurological disease in 11 mammalian species including humans. These henipaviruses remain significant biothreats to humans and economically important livestock in Australia and throughout South East Asia, and there are no vaccines or antivirals approved for human use. The henipaviruses are single-stranded, negative sense, enveloped RNA viruses and possess two membrane anchored glycoproteins involved in virus entry, one for host cell receptor attachment (G glycoprotein) and the other a fusion (F) glycoprotein which facilitates virion and host cell membrane fusion. The G and F glycoproteins are the major antigenic targets of neutralizing antibodies and also the focus of several vaccine strategies. In contrast however, antiviral drug discovery for HeV and NiV has been significantly hampered because of the requirements of biosafety level-4 (BSL-4) containment, and presently there is a complete lack of any effective antiviral therapeutics against henipaviruses for human use. We have been extensively characterizing the recently identified non-pathogenic species of henipavirus, Cedar virus (CedPV). Using recombinant viral glycoprotein mediated cell-cell fusion assays, we have discovered that CedPV-mediated membrane fusion is similar to HeV and NiV, however, the ephrin receptor tropism of CedPV was found to be remarkably broad and fusion could be triggered by ephrin- B1 and -B2 as well as the glycophosphatidylinositol-anchored A subtype ephrins-A1, -A2, and -A5. The cell- cell fusion activity supported by each of these ephrin receptors also correlated with CedPV G binding ability as measured by surface plasmon resonance. Further, we have recently rescued recombinant CedPV encoding eGFP (rCedPV-GFP) using a reverse genetics approach. Our rCedPV platform represents a new virological system that can be used in a variety of applications to study various aspects of henipavirus cell biology and host cell interactions safely under BSL-2 containment. But of further importance, it is also an authentic henipavirus infection and replication reporter system now suitable for high-throughput screening (HTS) for the discovery of potentially pan-anti-henipavirus molecules. Using this new live-henipavirus platform, our objectives here will be to develop and optimize a rCedPV reporter system and utilize it in an HTS assay to discover antiviral molecules capable of inhibiting the infection of henipaviruses. Specifically, we will: 1) Adapt and develop CedPV reporters for the HTS assay; 2) Optimize the HTS parameters of recombinant virus infection and reporter activities; and 3) Pilot a HTS assay using the Prestwick Chemical Library.

项目概要/摘要 致病病毒的出现代表着传染病对公众健康的持续威胁。 其中，副粘病毒包括许多重要的人类和动物病原体，还包括 两个重要的人畜共患病病原体的绝佳例子：亨尼帕病毒；亨德拉病毒（HeV） 和尼帕病毒（NiV）。 HeV 和 NiV 具有独特的广泛宿主向性，能够感染至少 18 种动物 涵盖 6 个目哺乳动物的物种。 HeV 和 NiV 还可导致全身性且往往致命的呼吸道疾病 和/或包括人类在内的 11 种哺乳动物的神经系统疾病。这些亨尼帕病毒仍然存在 对澳大利亚和整个东南部的人类和具有重要经济意义的牲畜造成重大生物威胁 亚洲，目前还没有批准用于人类使用的疫苗或抗病毒药物。亨尼帕病毒是单链的， 负义、有包膜 RNA 病毒，并具有参与病毒的两种膜锚定糖蛋白 条目，一个用于宿主细胞受体附着（G 糖蛋白），另一个用于融合（F）糖蛋白， 促进病毒粒子和宿主细胞膜融合。 G和F糖蛋白是主要的抗原靶标 中和抗体也是几种疫苗策略的重点。但相比之下，抗病毒药物 由于生物安全4级的要求，HeV和NiV的发现受到了严重阻碍 （BSL-4）遏制，目前完全缺乏任何有效的抗病毒疗法 供人类使用的亨尼帕病毒。我们已经广泛地描述了最近发现的非致病性 亨尼帕病毒属，雪松病毒（CedPV）。使用重组病毒糖蛋白介导的细胞-细胞融合 分析中，我们发现 CedPV 介导的膜融合与 HeV 和 NiV 相似，然而， 发现 CedPV 的肝配蛋白受体趋向性非常广泛，并且可以通过肝配蛋白触发融合 B1 和 -B2 以及糖磷脂酰肌醇锚定的 A 亚型肝配蛋白 -A1、-A2 和 -A5。细胞- 这些肝配蛋白受体支持的细胞融合活性也与 CedPV G 结合能力相关，如 通过表面等离子体共振测量。此外，我们最近拯救了重组 CedPV 编码 eGFP (rCedPV-GFP) 使用反向遗传学方法。我们的 rCedPV 平台代表了一种新的病毒学 该系统可用于多种应用，以研究亨尼帕病毒细胞生物学的各个方面， BSL-2 遏制下的宿主细胞安全相互作用。但更重要的是，它也是一个真实的 亨尼帕病毒感染和复制报告系统现在适用于高通量筛选（HTS） 发现潜在的泛抗亨尼帕病毒分子。使用这个新的活亨尼帕病毒平台，我们 这里的目标是开发和优化 rCedPV 报告系统并将其用于 HTS 测定 发现能够抑制亨尼帕病毒感染的抗病毒分子。具体来说，我们将：1）适应 并开发用于 HTS 测定的 CedPV 报告基因； 2) 重组病毒HTS参数优化 感染和记者活动； 3) 使用 Prestwick 化学库试验 HTS 测定。