Sample collection. Blood was obtained daily to assay for antiviral activity and
the presence of pIFN- and pIFN- by enzyme-linked immunosorbent assay
(ELISA) and to extract PBMC (see below). Blood and nasal swab specimens
were collected daily for the first 7 days after challenge for those animals that were
kept alive until the end of the experiment. After euthanasia and exsanguination
of the animals at set time points, tissue samples (three different anatomical
locations of skin, as well as inguinal and popliteal lymph nodes, and tonsils) were
taken and divided in two sets. One set was immersed in RNA later (Ambion,
Austin, TX) and stored at 70°C for RNA extraction, and the other was snapfrozen
in liquid nitrogen-chilled isopentane and placed in a cryomold embedded
in optimal cutting temperature compound (OCT; Sakura Finetek, Torrance, CA)
and stored at 70°C for analysis by histopathology and immunohistochemistry
(IHC).