Serial culture of murine primary airway epithelial cells and ex vivo replication of human rhinoviruses |
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Authors: | Brockman-Schneider Rebecca A Amineva Svetlana P Bulat Maria V Gern James E |
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Affiliation: | Department of Pediatrics and Medicine, University of Wisconsin-Madison, Madison, WI, USA. rabrockm@wisc.edu |
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Abstract: | Human rhinoviruses (HRV) are the primary etiological agents in cold infections, and represent a serious risk to individuals with chronic respiratory disease such as asthma. In order to develop treatment options for HRV infections, murine models are a crucial component in the study of infection mechanisms due to the wide array of reagents and techniques available to study murine immunology. We present here a cell culture system for studying isolated murine epithelial cell responses to HRV. Monolayers of primary mouse airway epithelial cells were maintained in a serial culture system, and the identity and purity of the cell population was confirmed via immunostaining (positive for cytokeratin, negative for vimentin). Infection of these cells with a minor group rhinovirus (HRV-1A) was evidenced by increases in viral RNA, de novo synthesis of viral proteins, and production of infectious virus. This model will be useful in experiments to define mechanisms of viral replication and host/virus interactions within airway epithelial cells. |
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Keywords: | HRV, human rhinovirus COPD, chronic obstructive pulmonary disease LDLr, low density lipoprotein receptor DMEM, Dulbecco's minimal essential medium F12, Ham's F12 medium MEM, minimal essential medium FCS, fetal calf serum PCT, progenitor cell targeted DMSO, dimethyl sulfoxide qPCR, quantitative polymerase chain reaction HEPES, N-2-Hydroxyetheylpiperazine-N'-2-ethane sulfonic acid MOI, multiplicity of infection SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis ICAM-1, intercellular adhesion molecule 1 CPE, cytopathic effect RNA, ribonucleic acid PFU, plaque forming unit FITC, fluorescein isothiocyanate ALI, air liquid interface DAPI, 4',6-diamidino-2-phenylindole |
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