Rapid, accurate diagnostics for arenaviral infections

描述（由申请人提供）：拉沙病毒、朱宁病毒、马丘波病毒、瓜纳里托病毒和萨比亚病毒（沙病毒科）在人类中引起严重疾病，特别是出血热。这5种沙粒病毒被列为A类病原体，因为它们被认为对美国的安全构成重大威胁。在平民故意接触传染性病毒、自然发生的人类疾病以及实验室人员意外接触传染性病毒的情况下，对这些病毒引起的急性疾病进行快速、准确的诊断，对于公共卫生当局及时作出适当反应至关重要。目前，我们及时准确诊断沙粒病毒感染的能力有限，而且在很大程度上未经证实。提出的工作的广泛目标是评估各种基于核酸的方法快速，准确地检测沙粒病毒RNA的效用。有3个具体目标。目的1：建立一个代表a类沙粒病毒遗传多样性的核衣壳蛋白（NP）和糖蛋白前体（GPC）基因序列数据库。了解这些病毒的遗传多样性对于合理设计寡核苷酸引物和沙粒病毒RNA核酸检测探针至关重要。目的2：开发传统的和基于荧光探针的RT-PCR检测LAS， JON， MAC， GTO和SAB病毒NP或GPC基因特异性RNA。目的3：开发一种DNA芯片，用于检测由LAS、JUN、MAC、GTO和SAB病毒NF基因特异性RNA产生的cDNA，并将沙粒病毒的特征表征到典型型或物种的水平。RT-PCR检测和cDNA微阵列检测的敏感性将与体外病毒分离的成功进行对比，如通过空斑测定。每项检测的准确性将通过检测拉沙、朱宁、马丘波和瓜纳里托病毒株以及未用于开发或测试第一代检测的其他沙粒病毒株（如Amapari、Flexal）进一步评估。

DESCRIPTION (provided by applicant): Lassa, Junin, Machupo, Guanarito, and Sabia viruses (family Arenaviridae) cause severe disease, particularly hemorrhagic fever, in humans. These 5 arenaviruses have been classified as Category A agents because they are considered to pose a significant risk to the security of the United States. Rapid, accurate diagnosis of acute disease caused by these viruses is critical to a timely and appropriate response by public health authorities in the event of an intentional exposure of civilians to infectious virus, naturally occurring human disease, and accidental exposure of laboratorians to infectious virus. At present, our ability to accurately diagnose arenaviral infections in a timely manner is limited and largely unproven. The broad objective of the proposed work is to assess the utility of various nucleic acid-based methodologies for rapid, accurate detection of arenaviral RNA. There are 3 specific aims. Aim #1 Generate a database of nucleocapsid protein (NP) and glycoprotein precursor (GPC) gene sequences representative of the genetic diversity of the Category A arenaviruses. Knowledge of the genetic diversity of these viruses is essential to rational design of oligonucleotide primers and probes for nucleic acid-based assays for arenaviral RNA. Aim #2 Develop conventional and fluorogenic probe-based RT-PCR assays for detection of LAS, JON, MAC, GTO, and SAB viral NP or GPC gene-specific RNA. Aim #3 Develop a DNA microarray for detection of cDNA generated from LAS, JUN, MAC, GTO, and SAB viral NF gene-specific RNA, and characterization of arenaviruses to the level of stereotype or species. The sensitivities of the RT-PCR assays and cDNA microarray will be measured against the success of virus isolation in vitro, as measured by plaque assay. The accuracy of each assay will be assessed further by testing strains of Lassa, Junin, Machupo, and Guanarito viruses, and strains of other arenaviruses (e.g., Amapari, Flexal) that were not used to develop or test the first generation assay.