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）：该提案测试了 T.I.G.E.R.该平台（商业上称为 Ibis T5000）系统应用于雪貂流感模型，并提供受感染人类无法获得的高水平受控细节。从质谱测定的基础成分中获得的高分辨率基因型信息将为研究和监测不同流感毒株的传播和遗传漂变提供独特的能力。该项目将利用LRRI在ABSL-3环境中动物模型和气溶胶技术方面的丰富经验，在H3N2和H5N1流感感染的雪貂模型中测试Ibis T5000系统和相关流感监测套件的敏感性、特异性、早期诊断能力和气溶胶呼出量。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 实验室测试鼻内 H3N2 和 H5N1 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到加利福尼亚州的 Ibis，在 T5000 上对扩增子进行 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 的敏感性，我们将测试所有样品的平行等分试样。具体目标 2. 为了测试 TIGER 通过突变检测遗传漂移以及通过重组和重配进行转变的能力，我们将用单一毒株（作为具体目标 #1 的一部分）感染雪貂，并用 H3N2 和 H5N1 毒株同时感染。在早期感染期间和尸检时，将对分泌物和器官样本进行分析，以检测来自六个基因组片段的放大器检测到的多种基因型。为了测试奥司他韦治疗期间体内耐药 H5N1 菌株的出现情况，我们将监测雪貂 4 次连续传代期间体内出现的基因型定义的耐药菌株的出现和相对数量。具体目标 3. 为了测试 TIGER 检测受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的病毒。我们将比较两种检测病毒的气溶胶采样技术、大容量空气过滤器和液体撞击器的灵敏度，并将呼出病毒的检测与标准定量实时 PCR 和噬斑检测进行比较。最后我们将比较不同毒株的H3N2和H5N1病毒的呼出量。相关性：T5000 诊断系统提供了一种快速（<8 小时）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，并提高大流行性流感的早期治疗、隔离方案和检疫实践的效率和功效。