Rapid Diagnostics Assays and Antivirals for Noroviruses

项目总结(见说明)： 人类杯状病毒是引起胃肠炎的最重要原因之一。尽管人们越来越意识到 在这些作为腹泻疾病主要原因的病原体中，用于诊断人类杯状病毒感染的检测方法有 不适用于大多数临床实验室。也没有有效的抗病毒药物可供使用 用于治疗或预防这种感染。该项目将侧重于高通量方法，以开发检测人类杯状病毒的快速诊断分析方法，并开发高通量分析方法，以确定潜在有效的抗病毒药物。我们假设，在前一个项目期间开发的针对共享表位的试剂和策略可以被优化，并用于开发针对这些抗原性不同的病毒的广泛反应的诊断分析。在具体目标1中，我们将进一步优化可用于人类杯状病毒诊断分析的广谱反应试剂。这一目标的研究将优化具有一系列结合特异性的单链抗体(ScFv)和识别保守表位的单抗，这两种抗体都是在上一个资助期开发和表征的。在具体目标2中，我们将开发针对人类杯状病毒的广谱反应诊断试验。将确定可用于现场检测人类杯状病毒的快速格式。以特定目标1为特征的高亲和力、交叉反应的单链和单抗将用于只需最少的样品准备并在不到30分钟内产生结果的分析。待评估的格式是那些已成功用于检测其他人类病毒的格式，包括固相免疫分析法和乳胶凝集分析法。在Speciflc Aim 3中，我们建议开发高通量检测方法来筛选具有抗人类诺如病毒活性的药物。病毒蛋白水解酶抑制剂的高通量筛选将用于从小分子库中确定候选抑制剂。项目1研究的结果将导致新的检测方法可用于诊断这些重要的肠道病原体，并鉴定有可能减轻这些病原体引起的疾病负担的抗病毒药物。

PROJECT SUMMARY (See instructions): Human caliciviruses are among the most important causes of gastroenteritis. Despite the increasing awareness of these agents as a major cause of diarrheal illness, assays for the diagnosis of human calicivirus infection are not available to most clinical laboratories. There are also no effective antivirals available that can be used therapeutically or prophylactically for this infection. This project will focus on high throughput approaches to develop rapid diagnostic assays for the detection of human caliciviruses and high throughput assays to identify potentially effective antiviral agents. We hypothesize that the reagents and strategies developed in the previous project period to target shared epitopes can be optimized and used to develop broadly reactive diagnostic assays for these antigenically diverse viruses. In Specific Aim 1, we will further optimize broadly-reactive reagents that can be used to make diagnostic assays for human caliciviruses. Studies in this aim will optimize single chain (scFv) antibodies with a range of binding specificities and monoclonal antibodies that recognize conserved epitopes, both of which were developed and characterized in the previous funding period. In Specific Aim 2, we will develop broadly-reactive diagnostic assays for human caliciviruses. Rapid formats that will be useable in the field to detect human caliciviruses will be identified. High affinity, cross-reactive single chain and monoclonal antibodies characterized in specific aim 1 will be used in assays that require minimal sample preparation and yield a result in less than 30 minutes. Formats to be evaluated are those that have been used successfully for the detection of other human viruses and include solid-phase immunoassays, and latex agglutination assays. In Speciflc Aim 3, we propose to develop high throughput assays to screen agents for antiviral activity against human noroviruses. A high throughput screen for inhibitors ofthe viral protease will be used to identify inhibitor candidates from small molecule libraries. The results obtained from project 1 studies will lead to the availability of new assays for the diagnosis of these important enteric pathogens and the identification of antiviral agents with the potential to diminish the disease burden caused these agents.