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

描述（由申请人提供）：

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.