Microarray-based detection and typing of rhinoviruses

描述(由研究人员提供)：本申请的目标是进一步开发一种新的DNA微阵列来检测呼吸道分泌物中的鼻病毒(RV)，并进行一项研究，以检验引起下呼吸道症状的RV菌株与仅引起上呼吸道症状的RV菌株在基因上是否不同。这些目标将通过三个目标来实现。目的1：通过培养每个RV血清型的原型并确定其在微阵列上的杂交模式，来确定所有102个RV血清型的特定杂交模式。微阵列现在有204个RV的检测寡核苷酸，由仅有的5个全序列血清型(RV1B、RV2、RV14、RV16、RV89)的基因组设计。在应用微阵列确定所有102个血清型的杂交模式之前，我们将对所有另外97个尚未测序的血清型进行全序列测定，以设计新的检测寡核苷酸并扩大微阵列。目的2：开发生物信息学软件，分析微阵列结果，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)每个RV血清型的杂交数据(来自AIM 1)；(B)从现场样本中分离的多达10个单一血清型的菌株的杂交数据(以评估单一血清型的杂交变异性)；以及(C)基于RV基因组和寡核苷酸序列的计算机模型杂交模式预测。目的3：验证芯片和生物信息学软件在现场样本的急性呼吸道感染和良好特征的临床表现。此外，还将比较导致下呼吸道疾病(如支气管炎、哮喘加重和慢性阻塞性肺病)的RV的杂交模式与只导致上呼吸道症状的RV的模式，以确定微阵列基因分型是否可以识别毒力变异。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。我们已经从呼吸道感染患者身上收集了75份样本。PPG的病毒学核心(加利福尼亚州病毒学实验室，VRDL)进行RV血清分型，拥有大多数RV原型和针对特定血清型的多个分离株。一旦得到验证，微阵列将允许在一个步骤中对所有轮状病毒进行检测、血清分型和基因分型，这些病毒不仅会导致普通感冒，还会导致严重发病率的疾病，如哮喘和慢性阻塞性肺病的恶化。

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.