Rapid, accurate diagnostics for arenaviral infections

快速、准确地诊断沙粒病毒感染

• 批准号: 6561606
• 负责人: CHARLES F FULHORST
• 金额: $ 22.35万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-30 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6561606
• 关键词: Arenaviridae; Lassa virus; bioterrorism /chemical warfare; communicable disease diagnosis; computer system design /evaluation; diagnosis design /evaluation; diagnosis quality /standard; glycoproteins; method development; microarray technology; molecular biology information system; nucleic acid probes; nucleic acid sequence; nucleocapsid; polymerase chain reaction; rapid diagnosis; tissue /cell culture; virus RNA; virus diseases; virus genetics

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.

描述（由申请人提供）：Lassa，Junin，Machupo，Guanarito和Sabia病毒（家庭Arenaviridae）在人类中引起严重疾病，尤其是出血热。这5个体育症病毒已被归类为A类代理，因为它们被认为对美国的安全构成了重大风险。这些病毒引起的急性疾病的快速，准确的诊断对于公共卫生当局的及时和适当反应至关重要，如果平民有意暴露于传染病，自然发生的人类疾病以及实验室意外暴露于感染性病毒中。目前，我们及时及时诊断出体内病毒感染的能力是有限的，并且在很大程度上尚未得到证实。拟议的工作的广泛目标是评估基于核酸的各种方法的实用性，以快速，准确检测竞技病毒RNA。有3个具体目标。 AIM＃1生成一个核素蛋白（NP）和糖蛋白蛋白前体（GPC）基因序列的数据库，代表了A类体体的遗传多样性。这些病毒的遗传多样性的了解对于寡核苷酸引物的合理设计和基于核酸的竞技病毒RNA测定法的探针至关重要。 AIM＃2开发了常规和基于荧光探针的RT-PCR分析，以检测LAS，JON，MAC，GTO和SAB病毒NP或GPC基因特异性RNA。 AIM＃3开发了一个DNA微阵列，用于检测从LAS，JUN，MAC，GTO和SAB病毒NF基因特异性RNA产生的cDNA，以及将体ap体的表征到刻板型或物种水平。通过斑块测定法测量，将测量RT-PCR分析和cDNA微阵列的敏感性。每种测定法的准确性将通过测试LASSA，JUNIN，MACHUPO和GUANARITO病毒的菌株以及其他体育疾病病毒（例如Amapari，Flexal）的菌株进行进一步评估。