Intranasal BALB/c Mouse Model for Influenza

Five-week-old female BALB/c mice (Japan SLC), under mixed anesthesia (medetomidine-butorphanol-midazolam), were intranasally inoculated with 50-μl samples of 1 to 10⁵ FFU virus in PBS. The body weights and survival of the mice were monitored daily for 14 days. Mice that lost more than 30% of their original weight were humanely euthanized. Lungs and brains of mice infected with 10 FFU virus were collected 3 and 6 dpi, and virus titers were assayed by focus-forming assays. Enzyme-linked immunosorbent assays (ELISAs) were performed on lung homogenates using CCL2 (MCP1), IL-6, IL-10, TNF-α, IFN-γ, and IFN-β Quantikine kits (R&D Systems). For histopathology analysis, the lungs of mice infected with 10 FFU of virus were collected at 3 dpi, fixed in 4% buffered paraformaldehyde, embedded in paraffin, cut into 5-μm sections, stained with hematoxylin and eosin, and examined by light microscopy. Immunohistochemical staining of the viral antigen was performed, as described previously (17 (link), 41 (link)), using a monoclonal antibody (C43) specific for influenza A virus nucleoprotein and a Mouse-on-Mouse (MOM) peroxidase kit (Vector Laboratories) with diaminobenzidine as the chromogen and hematoxylin as the counterstain. Unrelated antibodies were used in place of the primary antibody as controls.