Virology Core

点击翻译按钮获取中文摘要

PROJECT SUMMARY/ABSTRACT Respiratory infections induced by rhinoviruses (RV) play important roles in development, exacerbation and chronicity of asthma in both children and adults. This program is focused on identifying pathogenic mechanisms related to the virus biology (cellular receptor), host (genetics, immune response), and environment (farming lifestyle, microbiome) that lead to physiologic changes in airway function and determine the severity of RV-induced illnesses. Project I continues studies in a birth cohort involving farm and non-farm families, while the emphasis of Project II is on determining interactions between RV-C, associated with more severe wheezing illnesses in children, and host airway epithelial cells. Each of the two projects will use viral molecular diagnostic assays and require specialized preparations of purified RVs and other molecular reagents such as antibodies and cDNA clones that are essential for obtaining reproducible results. The Virology Core is established to provide Project I with a repertoire of sensitive and accurate molecular detection sampling strategies and assays to identify all common respiratory viruses (RVP) and bacteria (metagenomic sequencing). Tasks including using molecular techniques to assign RV species and types (RT-PCR and sequencing) for viruses detected in clinical samples. Comprehensive viral diagnostics are needed to evaluate the role of each virus species and RV type in pathogenesis of respiratory illnesses. The viral loads will be quantified by RT-qPCR in a subset of RV-positive nasal samples from human subjects of Project I for subsequent virus isolation, complete genome sequencing, cDNA cloning and use in Project II. The core will isolate, grow and characterize primary cultures of human airway epithelial cells for use in Project II, in which interactions between viruses and microbial isolates will be tested. In addition, this core will support the in vitro experiments of Projects I-II by supplying (1) purified stocks (infectious and inactivated) of selected RV serotypes/strains, (2) RV-specific antibodies, (3) cDNA clones of RV-A, RV-B and RV-C isolates for recombinant virus production, (4) performing viral RNA quantification (qRT-PCR) and RV infectivity (plaque and TCID50) assays, and determining anti-RV serum neutralizing antibody titers. The Core will also continue to provide expertise for the use of these reagents and viral diagnostic assays to the UW AADCRC program investigators, and to collaborate with other NIH-funded and international investigators.