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

描述(由申请人提供)： 这项应用寻求支持，以调查病毒在三种可能与感染有关的慢性肺部疾病的发病机制中的作用：特发性肺纤维化(IFF)、肺动脉高压(PAH)和毛细支气管炎闭塞性综合征(BOS)。我们将使用两项新技术调查新鲜的冷冻、储存的肺样本中是否存在病毒核酸：质量标签PCR和GreeneChips。质量标签聚合酶链式反应是一种高度敏感、高度多样化的病原体监测方法，将可紫外线切割的特征标记连接到针对遗传目标的特异引物上，并通过质谱学检测产物。GreeneChips是基于一个数据库的病毒阵列，该数据库唯一地整合了国际病毒分类大会(ICTV)系统发育数据库和GenBank序列数据库。目前的绿色芯片代表了一个包含100,000个脊椎动物病毒序列的数据库，并包含了ICTV识别的所有1,710种脊椎动物病毒的至少3个遗传目标。通过使用消减杂交和树状大分子标记方法，灵敏度已经提高了3个数量级，达到10,000个RNA拷贝；其他修改预计将产生1,000个RNA拷贝或更高的灵敏度。随着病毒序列的确定，我们将建立实时聚合酶链式反应分析，用于有效地筛查病例和对照之间感染频率的差异。将进一步研究与干酪病相关的病原体，重点是分子和系统发育分类、关联强度、解剖分布以及通过使用培养和免疫受损的动物模型系统恢复病毒。其具体目的是：(1)使用MASS Tag PCR或GreeneChips调查IPF、PAH和BOS患者的肺标本中是否存在病毒核酸序列；(2)分析通过MASS Tag PCR或GreeneChips鉴定的代表病毒特征的CPCR产物和/或cDNA文库的序列；(3)确定感染的患病率，并通过实时荧光定量PCR检测IPF、PAH、BOS和对照组织中疑似病原体的病毒载量；(4)通过原位杂交和/或免疫组织化学方法确定可疑病原体在IPF、PAH或BOS组织中的解剖分布；(5)确定对假定的病毒病原体的免疫反应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中涉及的病毒。

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.