T5000 Diagnostic System in Ferret Model of Influenza

T5000雪貂流感模型诊断系统

基本信息

批准号：
7459912
负责人：
FREDERICK T KOSTER
金额：
$ 72.1万
依托单位：
LOVELACE BIOMEDICAL & ENVIRONMENTAL RESEARCH INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-07-15 至 2010-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7459912
关键词：
Aerosols Aliquot Animal Model Antiviral Agents Antiviral resistance Appearance Autopsy Base Composition Bioinformatics Breathing California Controlled Environment Detection Diagnosis Diagnostic Diagnostic tests Disease Dose Early Diagnosis Early treatment Environment Epithelial Cells Evaluation Exhalation Ferrets Forensic Medicine Funding Genetic Genetic Drift Genetic Recombination Genomic Segment Genotype Government Hour Human Individual Infection Infection Control Influenza Influenza A Virus, H5N1 Subtype Knowledge Laboratories Liquid substance Lung diseases Mass Spectrum Analysis Measurement Medical Surveillance Modeling Monitor Mutation Nosocomial Infections Numbers Organ Oseltamivir Persons Pharmacologic Substance Phenotype Plaque Assay Polymerase Chain Reaction Population Protocols documentation Quarantine RNA Range Relative (related person)Research Personnel Resistance Resolution Reverse Transcriptase Polymerase Chain Reaction Risk Robot Sampling Sensitivity and Specificity Serial Passage Shipping Ships Site Standards of Weights and Measures System Techniques Technology Testing Tigers Time Tissues Viral Viral Drug Resistance Virus Virus Diseases air filter computerized data processing day experience high throughput analysis high throughput screening improved in vivo influenzavirus pandemic disease pandemic influenza pathogen programs respiratory tool transmission process viral RNA viral detection

项目摘要

DESCRIPTION (provided by applicant): This proposal tests the T.I.G.E.R. platform (commercially referred to as the Ibis T5000) system in the ferret model of influenza and provides a high level of controlled detail not available from infected humans. The high resolution genotypic information derived from the mass spectrometrically-determined base compositions will provide unique capabilities with which to study and monitor the transmission and genetic drift of diverse influenza strains. This project will utilize the extensive experience of LRRI in animal models and aerosol technology in the ABSL-3 environment to test the Ibis T5000 system and associated influenza surveillance kit for sensitivity, specificity, early diagnosis capability, and aerosol exhalation in the ferret model of H3N2 and H5N1 influenza infection. Specific Aim #1: To identify the earliest time and respiratory secretion site of viral detection by TIGER, we will test secretions of ferrets infected by intranasal H3N2 and H5N1 strains in the ABSL-3 laboratory at LRRI. Sample RNA will be isolated by robot in the ABSL-3 and shipped to Ibis in California for RT-PCR and mass spectrometry analysis of amplicons on the T5000. To compare the sensitivity of T5000 system to standard viral culture and real-time RT-PCR we will test parallel aliquots of all samples. Specific Aim 2. To test the ability of TIGER to detect genetic drift through mutation, and shift through recombination and reassortment, we will infect ferrets with single strains (as part of Specific Aim #1) and with co-infections with H3N2 and H5N1 strains. During early infection and at necropsy secretions and organ samples will be analyzed for multiple genotypes detected by amplimers from six genomic segments. To test the in vivo emergence of resistant H5N1 strains during oseltamivir treatment we will monitor appearance and relative numbers of genotypically-defined resistant strains arising in vivo during 4 serial passages in ferrets. Specific Aim 3. To test the ability of TIGER to detect viral RNA in exhaled aerosols produced by infected ferrets throughout the course of disease, we will capture exhaled virus in our inhalation apparatus in the controlled environment of the bioBubble. We will compare the sensitivity of two aerosol sampling techniques to detect virus, high-volume air filters and liquid impingers and compare detection of exhaled virus with the standard quantitative real-time PCR and plaque assays. Finally we will compare the exhalation of different strains of H3N2 and H5N1 viruses. Relevance: The T5000 Diagnostic System provides a rapid (<8 hours) high-throughput sensitive diagnostic tool useful to direct early therapy in seasonal influenza virus infection, and increase the efficiency and efficacy of early therapy, isolation protocols and quarantine practices in pandemic influenza.

描述（由申请人提供）：该提案测试T.I.G.E.R.平台（商业上称为IBIS T5000）系统中的雪貂模型中的系统，并提供了高水平的受控细节，而受感染的人则无法获得。源自质谱标准的基础组成的高分辨率基因型信息将提供独特的能力，可通过研究和监测各种流感菌株的传播和遗传漂移。该项目将在ABSL-3环境中利用LRRI在动物模型和气溶胶技术中的丰富经验来测试IBIS T5000系统和相关的流感型损伤监测试剂盒，以实现敏感性，特异性，早期诊断能力以及在H3N2和H5N1 HIDN1 HIDN1 HIDENZA INFELECTION中的气溶胶振兴。具体目的1：为了确定老虎病毒检测的最早时间和呼吸道分泌部位，我们将测试LRRI的ABSL-3实验室中鼻内H3N2和H5N1菌株感染的雪貂的分泌物。样品RNA将通过ABSL-3中的机器人分离，并运送到加利福尼亚的宜必思省进行RT-PCR，并对T5000上的扩增子进行质谱分析。为了比较T5000系统与标准病毒培养和实时RT-PCR的灵敏度，我们将测试所有样品的平行等分试样。特定目标2。为了测试老虎通过突变检测遗传漂移的能力，并通过重组和保证进行转移，我们将以单菌株（作为特定目标＃1的一部分）以及与H3N2和H5N1菌株的共同感染感染雪貂。在早期感染期间，将分析尸检和器官样本，以分析由六个基因组段的助剂检测到的多种基因型。为了测试在Oseltamivir处理过程中抗性H5N1菌株的体内出现，我们将监测在雪貂4个串行通道中在体内产生的基因型定义抗性菌株的外观和相对数量。特定目的3。为了测试老虎在整个疾病过程中被感染雪貂产生的呼出气溶胶中的病毒RNA的能力，我们将在生物泡的受控环境中捕获吸入设备中的呼出病毒。我们将比较两种气溶胶采样技术的灵敏度，以检测病毒，大量空气过滤器和液体撞击器，并将呼出病毒的检测与标准定量实时PCR和斑块测定进行比较。最后，我们将比较不同菌株H3N2和H5N1病毒的呼气。相关性：T5000诊断系统提供了一种快速（<8小时）的高通量敏感诊断工具，可用于指导季节性流感病毒感染的早期治疗，并提高早期治疗，隔离方案和隔离疗法的效率和功效。