T5000 Diagnostic System in Ferret Model of Influenza

T5000雪貂流感模型诊断系统

• 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
• 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

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）：本提案测试T.I.G.E.R.平台（商业上称为Ibis T5000）系统在雪貂模型的流感，并提供了一个高水平的控制细节不可从受感染的人。从质谱测定的基础组成中获得的高分辨率基因型信息将提供独特的能力，用于研究和监测不同流感毒株的传播和遗传漂移。该项目将利用LRRI在动物模型和ABSL-3环境中的气溶胶技术方面的丰富经验，在H3 N2和H5 N1流感感染的雪貂模型中测试Ibis T5000系统和相关流感监测试剂盒的灵敏度、特异性、早期诊断能力和气溶胶呼出。具体目标1：为了确定TIGER检测病毒的最早时间和呼吸道分泌部位，我们将在LRRI的ABSL-3实验室检测鼻内H3 N2和H5 N1毒株感染的雪貂的分泌物。样本RNA将由ABSL-3中的机器人分离，并运送至加州的Ibis，在T5000上进行扩增子的RT-PCR和质谱分析。为了比较T5000系统对标准病毒培养和实时RT-PCR的灵敏度，我们将对所有样本的平行等分试样进行检测。具体目标2。为了测试TIGER通过突变检测遗传漂变的能力，以及通过重组和重配检测遗传漂变的能力，我们将用单一菌株感染雪貂（作为特定目标#1的一部分），并用H3 N2和H5 N1菌株共感染雪貂。在早期感染期间和尸检时，将分析分泌物和器官样本中的多种基因型，这些基因型由来自六个基因组区段的扩增物检测到。为了测试在奥司他韦治疗期间耐药H5 N1菌株的体内出现，我们将监测在雪貂中连续传代4次期间体内出现的基因型定义的耐药菌株的外观和相对数量。具体目标3。为了测试TIGER检测感染雪貂在整个疾病过程中产生的呼出气溶胶中病毒RNA的能力，我们将在bioBubble的受控环境中用吸入装置捕获呼出病毒。我们将比较两种气溶胶采样技术检测病毒的灵敏度，高容量空气过滤器和液体撞击器，并比较呼出病毒的检测与标准的定量实时PCR和空斑检测。最后，我们将比较不同株H3 N2和H5 N1病毒的呼气。相关性：T5000诊断系统提供了一种快速（<8小时）的高通量灵敏诊断工具，可用于指导季节性流感病毒感染的早期治疗，并提高大流行性流感的早期治疗、隔离方案和检疫措施的效率和功效。