Animals were anesthetized as previously described [32] with isoflurane inhalation and intranasally inoculated with a fluid volume of 100μL. For bronchoalveolar lavage studies, cotton rats were inoculated with either 5μg of purified G protein, or 5μg or 20μg recombinant mouse CX3CL1 (458-MF, R&D Systems, Minneapolis, MN, USA). To determine the best serotype of AAV for studies in the cotton rat respiratory tract, cotton rats were inoculated with 109−10 DNAase resistant particles (DRP) with different serotypes of AAV expressing GFP for flow cytometry studies. Blood samples were obtained by retro-orbital bleed in isofluorane narcosis. For the studies of inflammation and immunogenicity, cotton rats were inoculated with 2x1010 DRP of various AAV constructs and challenged with 105 TCID50 of RSV A2. 100μg house dust mite (HDM, Dermatophagoides pteronyssinus) antigen was absorbed to aluminum phosphate (AdjuPhos, Brenntag, Ballerup, Denmark) at a 1:1 ratio for 30 minutes at room temperature and injected into cotton rats intraperitoneally (IP). Sensitization was followed 8 days later with intranasal administration of 100μg HDM in a volume of 100μL PBS. Four days after HDM administration, cotton rats were euthanized through CO2 inhalation and lungs were collected for histologic examination.

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