Rapid Diagnostics Assays and Antivirals for Noroviruses

项目总结（见说明）： 人类杯状病毒是引起肠胃炎的最重要原因之一。尽管人们越来越意识到 在这些作为结肠炎疾病的主要原因的试剂中，用于诊断人杯状病毒感染的测定是 大多数临床实验室都没有。也没有有效的抗病毒药物可以使用 治疗或预防这种感染。该项目将侧重于高通量方法，以开发用于检测人类杯状病毒的快速诊断方法和高通量方法，以识别潜在有效的抗病毒剂。我们假设，在前一个项目期间开发的靶向共享表位的试剂和策略可以优化，并用于开发这些抗原多样性病毒的广泛反应性诊断检测。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检测的广泛反应性试剂。这一目标的研究将优化具有一系列结合特异性的单链（scFv）抗体和识别保守表位的单克隆抗体，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泛反应性诊断检测。将确定可用于现场检测人类杯状病毒的快速格式。特异性目的1中表征的高亲和力、交叉反应性单链和单克隆抗体将用于需要最少样品制备并在不到30分钟内产生结果的测定。待评价的ELISA是那些已成功用于检测其他人类病毒的ELISA，包括固相免疫测定和乳胶凝集测定。在具体目标3中，我们建议开发高通量测定来筛选针对人诺如病毒的抗病毒活性剂。病毒蛋白酶抑制剂的高通量筛选将用于从小分子文库中鉴定抑制剂候选物。从项目1研究中获得的结果将导致用于诊断这些重要肠道病原体的新检测方法的可用性，并确定有可能减少这些药物引起的疾病负担的抗病毒剂。

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.