Rapid Diagnostic Assays for Human Caliciviruses

本项目提出的研究重点是开发检测人类杯状病毒的快速诊断方法。在具体目标1中，我们将开发针对人类杯状病毒的广谱反应诊断试剂。人类杯状病毒分为诺如病毒和沙粒病毒两个属，在这些属中有许多在遗传和抗原性上截然不同的组。我们之前已经鉴定出识别诺如病毒共有表位的单抗。此外，我们和其他人已经确定了血型抗原 参与与诺沃克病毒颗粒的结合。我们最近证实了其中一种抗原(Lewis D)用于亲和纯化诺沃克病毒的特异性。这些和新开发的单调抗体和血型碳水化合物将用于开发快速、广泛反应的诊断分析。在具体目标2中，我们将探索新的快速检测人类杯状病毒的方法。以特定目标1为特征的高亲和力、交叉反应的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。待评估的格式包括那些已成功用于检测其他人类病毒的格式，包括传统的酶免疫分析法、层析免疫分析法、乳胶凝集分析法和斑点杂交免疫分析法。这些分析将与一组人和动物杯状病毒和临床标本进行测试，以确定敏感性、特异性、阳性和阴性预测值以及检测下限。开发的化验将通过向其他实验室(例如，疾控中心、国家卫生实验室)提供诊断测试来验证，使用RT-PCR化验来评估大型 人类杯状病毒的样本数量。在具体目标3中，将开发新的方法，将人类杯状病毒的检测与其潜在的传染性和灭活敏感性相关联。无法在体外培养人类杯状病毒，阻碍了对其传染性的评估。生物传感系统将利用绿色荧光蛋白作为报告信号，在转化的细胞系中启动病毒感染后表达。将对人类和动物杯状病毒的不同消毒措施进行评估。 这些结果将与可培养的动物杯状病毒的细胞培养结果相关联。

The focus of the studies proposed in this project is the development of rapid diagnostic assays for the detection of human caliciviruses. In Specific aim 1, we wilt develop broadly-reactive diagnostic reagents for human caliciviruses. Human caliciviruses are divided into two genera, Norovirus and Sapovirus, and within these genera are many genetically and antigenically distinct groups. We have previously identified monoclonal antibodies that recognize epitopes shared among the noroviruses. In addition, we and others have identified blood group antigens involved in binding to norovirus particles. We have recently confirmed the specificity of one of these antigens (Lewis d) for affinity purification of Norwalk virus. These and newly developed monoctonal antibodies and blood group carbohydrates will be characterized for use in the development of rapid, broadly-reactive diagnostic assays. In Specific aim 2, we will explore new rapid formats to detect human caliciviruses. High affinity, cross-reactive 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 include those that have been used successfully for the detection of other human viruses and include traditional enzyme immunoassays, chromatographic immunoassays, latex agglutination assays, and dot-blot immunoassays. The assays will be tested against a panel of both human and animal caliciviruses and clinical specimens to determine sensitivity, specificity, positive and negative predictive values, and limits of detection. Developed assays will be validated by providing the diagnostic tests to other laboratories (e.g., CDC, state health labs) using RT-PCR assays to evaluate large numbers of specimens for human caliciviruses. In Specific aim 3, new approaches will be developed to correlate detection of human caliciviruses with their potential infectivity and susceptibility to inactivation. The inability to cultivate human caliciviruses in vitro has hampered assessment of their infectivity. A biosensoring system will utilize green fluorescent protein as a reporter signal that is expressed following the initiation of a viral infection in a transformed cell line. Different disinfection measures will be evaluated for both human and animal caliciviruses and the results will be correlated with those obtained by cell culture for the animal caliciviruses that are cultivatable.