Rapid, accurate diagnostics for arenaviral infections

描述（由申请方提供）：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（沙粒病毒科）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，因为它们被认为对美国的安全构成重大风险。快速、准确地诊断由这些病毒引起的急性疾病对于公共卫生当局在平民故意暴露于传染性病毒、自然发生的人类疾病以及实验室人员意外暴露于传染性病毒的情况下及时、适当地做出反应至关重要。目前，我们及时准确诊断沙粒病毒感染的能力是有限的，而且在很大程度上未经证实。拟议工作的广泛目标是评估各种基于核酸的方法用于快速，准确检测沙粒病毒RNA的实用性。有三个具体目标。目的#1生成代表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探针用于沙粒病毒RNA的核酸检测是必不可少的。目的#2开发常规和基于荧光探针的RT-PCR检测方法，用于检测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微阵列，用于检测LAS、JUN、MAC、GTO和SAB病毒NF基因特异性RNA产生的cDNA，并在定型或种的水平上表征沙粒病毒。RT-PCR测定和cDNA微阵列的灵敏度将根据体外病毒分离的成功率进行测量，如通过空斑测定所测量的。将通过检测拉沙病毒、朱宁病毒、马丘波病毒和瓜纳里托病毒株以及其他沙粒病毒株（例如，Amapari，Flexal），未用于开发或检测第一代检测试剂盒。

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.