Pathogen Discovery in Chronic Lung Disease by Mass Tag PCR and Microarray

描述（由申请人提供）： 该申请寻求支持，以调查病毒对三种可能与感染相关的慢性肺部疾病的发病机制的贡献：特发性肺纤维化（IFF），肺动脉高压（PAH）和闭塞性细支气管炎综合征（BOS）。我们将使用两种新技术：Mass Tag PCR和GreeneChips，调查新鲜冷冻的库存肺标本中是否存在病毒核酸。Mass Tag PCR是一种用于病原体监测的灵敏度高、高度多重的方法，其中UV可切割的特征标记与遗传靶标特异性引物结合，并通过质谱检测产物。GreeneChips是基于一个数据库的病毒阵列，该数据库独特地整合了国际病毒分类学大会（ICTV）系统发育数据库和GenBank序列数据库。目前的GreeneChip代表了一个包含超过100，000个脊椎动物病毒序列的数据库，并包含ICTV识别的所有1，710种脊椎动物病毒的至少3个遗传靶标。通过使用消减杂交和树枝状聚合物标记方法，灵敏度已经提高了3个数量级，达到10，000个RNA拷贝;预计额外的修饰将产生1，000个RNA拷贝或更好的灵敏度。随着病毒序列的确定，我们将建立真实的时间PCR检测方法，用于有效筛选病例和对照之间感染频率的差异。与干酪特异性相关的病原体将进一步研究，其目的是通过使用培养和免疫功能低下的动物模型系统，重点关注分子和系统发育分类、关联强度、解剖分布和病毒恢复。具体目标是：（1）通过使用Mass Tag PCR或GreeneChips，调查来自患有IPF、PAH和BOS的受试者的肺标本中是否存在病毒核酸序列;（2）分析代表通过使用Mass Tag PCR或GreeneChips鉴定的病毒特征的cPCR产物和/或cDNA文库的序列;（3）在IPF、PAH、BOS中确定感染的流行率并定量通过Mass Tag PCR或GreeneChip分析鉴定的疑似病原体的病毒负荷，通过真实的时间PCR和对照组织;（4）通过原位杂交和/或免疫组织化学确定疑似病原体在IPF、PAH或BOS组织中的解剖学分布;（5）确定对推定病毒病原体的免疫应答状态及其与疾病的关系;（6）尝试利用组织培养和动物模型系统分离目标1至5所涉病毒。

DESCRIPTION (provided by applicant): This application seeks support to investigate the contributions of viruses to the pathogenesis of three chronic lung diseases potentially linked to infection: idiopathic pulmonary fibrosis (IFF), pulmonary arterial hypertension (PAH), and bronchiolitis obliterans syndrome (BOS). We will survey fresh frozen, banked lung specimens for the presence of viral nucleic acids using two new technologies: Mass Tag PCR and GreeneChips. Mass Tag PCR is an exquisitely sensitive, highly multiplexed method for pathogen surveillance wherein UV-cleavable signatures are conjugated to primers specific for genetic targets and products are detected through mass spectroscopy. GreeneChips are viral arrays based on a database that uniquely integrates the International Congress for Taxonomy of Viruses (ICTV) phylogenetic database with the GenBank sequence database. The current GreeneChip represents a database of >100,000 vertebrate viral sequences and comprises a minimum of 3 genetic targets for all 1,710 vertebrate viruses recognized by ICTV. Sensitivity has already been enhanced 3 orders of magnitude to 10,000 RNA copies through use of subtractive hybridization and dendrimer labeling methods; additional modifications are expected to yield sensitivity of 1,000 RNA copies or better. As viral sequences are identified we will establish real time PCR assays to be used in efficiently screening for differences in frequency of infection between cases and controls. Pathogens specifically associated with caseness will be investigated further in aims that focus on molecular and phylogenetic classification, strength of association, anatomic distribution, and recovery of virus through use of culture and immunocompromised animal model systems. Specific aims are: (1) survey lung specimens from subjects with IPF, PAH, and BOS for the presence of viral nucleic acid sequences by using Mass Tag PCR or GreeneChips; (2) analyze the sequence of cPCR products and/or cDNA libraries representing viral signatures identified by using Mass Tag PCR or GreeneChips; (3) determine prevalence of infection and quantitate viral burden for the suspected pathogens identified by Mass Tag PCR or GreeneChip analyses in IPF, PAH, BOS, and control tissues by real time PCR; (4) determine the anatomic distribution of the suspected pathogens in IPF, PAH, or BOS tissues by in situ hybridization and/or immunohistochemistry; (5) determine the status of the immune response to the putative viral pathogen and its relationship to disease; (6) attempt to isolate viruses implicated in aims 1 through 5 using tissue culture and animal model systems.