Microarray-based detection and typing of rhinoviruses

描述（由研究者提供）：本申请的目的是进一步开发一种新型DNA微阵列，用于检测呼吸道分泌物中的鼻病毒（RV）的血清型和基因型，并开展一项研究，检查引起下呼吸道症状的RV菌株与仅引起上呼吸道症状的RV菌株是否在遗传上有所不同。这些目标将通过三个目标来实现。目的1：通过培养每种血清型的原型并在微阵列上确定其杂交模式，确定所有102种RV血清型的特异性杂交模式。该微阵列目前有204个检测RV的寡核苷酸，它们是从5种完全测序的血清型（RV1B、RV2、RV14、RV16、RV89）的基因组中设计出来的。在应用微阵列确定所有102种血清型的杂交模式之前，我们将对所有尚未测序的97种血清型进行完全测序，以设计新的检测寡核苷酸并扩展微阵列。目标2：开发生物信息学软件来分析微阵列结果并确定血清型匹配。我们将使用来自3个来源的数据开发该软件：(a)每种RV血清型的杂交数据（来自Aim 1）；(b)从野外样本中分离的最多10株单一血清型的杂交数据（以评估单一血清型的杂交变异性）；(c)基于RV基因组序列和寡核苷酸序列的杂交模式的计算机模型预测。目的3：在急性呼吸道感染患者的现场样本中验证微阵列和生物信息学软件，并确定其临床表现。此外，将引起下呼吸道疾病（如支气管炎、哮喘加重和慢性阻塞性肺病）的RV的杂交模式与仅引起上呼吸道症状的RV的杂交模式进行比较，以确定微阵列基因分型是否可以识别出毒性变异。这些研究将与PPG-1PO1-AIS0496的目标相结合：检查鼻病毒株之间的差异是否决定了感染引发哮喘恶化的可能性。我们从呼吸道感染患者身上收集了75份样本。PPG的病毒学核心（加州州立病毒学实验室，VRDL）进行RV血清分型，并拥有大多数RV原型和针对特定血清型的多种分离株。一旦得到验证，微阵列将允许在一个步骤中检测、血清分型和基因分型所有RV，这些RV不仅导致普通感冒，而且还导致哮喘和慢性阻塞性肺病加重等严重发病率疾病。

DESCRIPTION (provided by investigator): The goals of this application are to further develop a novel DNA microarray to detect, serotype, and genotype rhinoviruses (RV) in airway secretions, and to conduct a study examining whether RV strains causing lower respiratory symptoms differ genetically from those causing only upper respiratory symptoms. These goals will be achieved through 3 aims. Aim 1: To define specific hybridization patterns for all 102 RV serotypes by growing a prototype of each and determining its hybridization pattern on the microarray. The microarray now has 204 detection oligonucleotides for RV's, designed from the genomes of the only 5 fully sequenced serotypes (RV1B, RV2, RV14, RV16, RV89). Before applying the microarray to determine the hybridization pattern for all 102 serotypes, we will fully sequence all additional 97 serotypes not yet sequenced to design new detection oligonucleotides and expand the microarray. Aim 2: To develop bioinformatics software to analyze microarray results and determine a serotype match. We will develop this software using data from 3 sources: (a) hybridization data for each RV serotype (from Aim 1); (b) hybridization data from up to 10 strains of a single serotype isolated from field samples (to assess the hybridization variability for a single serotype); and (c) from computer model prediction of hybridization patterns based on the sequences of RV genomes and of oligonucleotides. Aim 3: To validate the microarray and bioinformatics software in field samples from subjects with acute respiratory infections and well-characterized clinical presentations. In addition, hybridization patterns of RV's that cause lower airway disease (e.g. bronchitis, exacerbations of asthma and COPD) will be compared with the patterns of RV's that cause only upper airway symptoms to determine whether microarray genotyping can identify virulent variants. These studies will interface with a goal of PPG-1PO1-AIS0496: To examine whether differences among strains of rhinovirus determine the likelihood of infection provoking asthma exacerbations. We have collected >75 samples from subjects with respiratory infections. The PPG's Virology Core (California State Virology Laboratory, VRDL) performs RV serotyping, and has most RV prototypes and multiple isolates for specific serotypes. Once validated, the microarray will allow, in a single step, detection, serotyping and genotyping of all RV's, which cause not only common colds, but also illnesses with significant morbidity such as exacerbations of asthma and COPD.