T5000 Diagnostic System in Ferret Model of Influenza

描述（由申请人提供）：本提案在雪貂流感模型中测试T.I.G.E.R.平台（商业上称为Ibis T5000）系统，并提供受感染人类无法获得的高水平控制细节。从质谱测定的碱基组成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在动物模型和气溶胶技术方面的丰富经验，在ABSL-3环境中测试Ibis T5000系统和相关流感监测试剂盒在H3N2和H5N1流感感染雪貂模型中的敏感性、特异性、早期诊断能力和气溶胶呼出。具体目的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实验室检测经鼻内H3N2和H5N1株感染的雪貂的分泌物。样本RNA将由机器人在ABSL-3中分离，并运往加州Ibis公司，用于T5000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的敏感性，我们将测试所有样品的平行等比。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（作为Specific Aim #1的一部分）感染雪貂，并同时感染H3N2和H5N1菌株。在早期感染和尸检时，将对分泌物和器官样本进行分析，以确定6个基因组片段的扩增物检测到的多种基因型。为了测试在奥司他韦治疗期间体内出现的耐药H5N1毒株，我们将监测在雪貂体内连续4次传代期间出现的基因典型定义耐药毒株的外观和相对数量。具体目标3。为了测试TIGER在整个疾病过程中检测被感染雪貂呼出的气溶胶中病毒RNA的能力，我们将在生物气泡控制的环境中，在吸入装置中捕获呼出的病毒。我们将比较两种气溶胶采样技术检测病毒的灵敏度，即大容量空气过滤器和液体撞击器，并将呼出病毒的检测与标准的实时定量PCR和空斑测定进行比较。最后，我们将比较不同H3N2和H5N1病毒株的呼出情况。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，并提高大流行性流感早期治疗、分离方案和检疫实践的效率和疗效。

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.