Rapid Diagnostic Assays for Human Caliciviruses

该项目的研究重点是开发用于检测人类杯状病毒的快速诊断方法。在具体目标1中，我们将开发用于人类杯状病毒的广泛反应性诊断试剂。人类杯状病毒分为两个属，诺如病毒和Sapovirus，并且在这些属内有许多遗传和抗原性不同的组。我们先前已经鉴定了识别诺如病毒之间共享的表位的单克隆抗体。此外，我们和其他人已经确定了血型抗原 参与与诺如病毒颗粒的结合。我们最近已经证实了这些抗原之一（刘易斯d）的特异性亲和纯化诺瓦克病毒。这些和新开发的单色调抗体和血型碳水化合物将被表征用于开发快速，广泛反应的诊断测定。在具体目标2中，我们将探索新的快速模式来检测人类杯状病毒。特异性目的1中表征的高亲和力、交叉反应性单克隆抗体将用于需要最少样品制备并在30分钟内产生结果的测定。待评价的抗体包括已成功用于检测其他人类病毒的抗体，包括传统的酶免疫测定法、色谱免疫测定法、乳胶凝集测定法和斑点免疫测定法。将针对一组人和动物杯状病毒以及临床标本对检测试剂盒进行检测，以确定灵敏度、特异性、阳性和阴性预测值以及检测限。将通过向其他实验室（例如，CDC，州卫生实验室）使用RT-PCR测定来评估大 人类杯状病毒的标本数量。在具体目标3中，将开发新的方法来将人类杯状病毒的检测与其潜在的感染性和对灭活的敏感性相关联。由于不能在体外培养人类杯状病毒，因此阻碍了对其感染性的评估。生物传感系统将利用绿色荧光蛋白作为在转化细胞系中病毒感染开始后表达的报道信号。将对人类和动物杯状病毒的不同消毒措施进行评价 并且该结果将与通过可培养的动物杯状病毒的细胞培养获得的结果相关联。

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.