Rapid, accurate diagnostics for arenaviral infections

描述(申请人提供)：Lassa、Junin、Machupo、Guanarito和Sabia病毒(Arenaviridae科)会在人类中引起严重疾病，特别是出血热。这5种病毒被归类为A类毒剂，因为它们被认为对美国的安全构成重大风险。对这些病毒引起的急性疾病进行快速、准确的诊断，对于公共卫生当局在平民故意接触传染病病毒、自然发生的人类疾病以及实验室人员意外接触传染病病毒的情况下做出及时和适当的反应至关重要。目前，我们及时准确诊断非典型性病毒感染的能力有限，而且在很大程度上未经证实。这项拟议工作的广泛目标是评估各种基于核酸的方法对快速、准确地检测ArenaVirus RNA的效用。有三个具体目标。目的#1建立代表A类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基于核酸的病毒RNA检测是必不可少的。目的#2建立检测LAS、JON、MAC、GTO和SAB病毒NP或GPC基因特异性RNA的常规和荧光探针RT-PCR方法。目的#3建立一种DNA芯片，用于检测LAS、JUN、MAC、GTO和SAB病毒的核转录因子基因特异性RNA，并从刻板印象或物种水平鉴定ARENAV。RT-PCR检测和基因芯片检测的敏感性将与病毒体外分离的成功与否进行比较，如空斑试验。将通过测试Lassa、Junin、Machupo和Guanarito病毒株以及未用于开发或测试第一代检测的其他阿拉伯病毒株(例如Ampari、Flexal)来进一步评估每种检测方法的准确性。

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.