Virus preparation, AAV production, infection, and tissue harvest

The influenza virus strain A/PR/8/34 (PR8, H1N1) was used in this study. Viral stocks were obtained by transfecting MDCK cells, as provided by Pr. YK Choi’s laboratory (Chungbuk National University, Chungbuk, South Korea). The virus was harvested 24 h after transfection and stored at − 80 °C until use. For infection, mice were anesthetized with isoflurane and oxygen. Intranasal infection was performed by administering 50 μl of phosphate buffered saline (PBS) containing IAV at a dose of 3.5 × 10⁵ PFU/ml, ensuring deposition of the virus in the lower respiratory tract. AAV9-EGFP and AAV9-EGFP-mCXCL14 vectors were purchased from Vector Builder (Chicago, Illinois, USA) as 2 × 10¹³ stocks and administered to mice via oropharyngeal delivery under anesthesia. A bolus inoculation of 50 µl of AAV9-EGFP at a dose of 10¹¹ GC (genome copies) per mouse was given using a pipette. Infection procedures were performed in a biosafety level 2 laboratory in accordance with the animal facility guidelines of the Biomedical Research Institute of Seoul National University Hospital. Lung tissues were harvested with PBS and cut using a razor. Dissociation medium was prepared by adding 50 μl of collagenase type IV (0.1 g/ml, Worthington Biochemical Corporation, NJ, USA) and 50 μl of DNase I solution (1 mg/ml, Sigma, St. Louis, Missouri, USA) to 50 ml of RPMI 1640 medium and incubated at 37 °C for 2 h on a shaking platform.