Fig. 1: Schematic diagrams illustrating ferret study design.

Experimental layout of age-dependent pathogenesis of SARS-CoV-2 in ferret animal model. a Timetable showing time points of virus inoculation, observation and monitoring of clinical manifestation and sample collection in infected ferrets by age groups. Orange circle denotes for time points of temperature and weight measurement, green circle for nasal washes and rectal swab sample collection, and blue circle indicates nasal turbinate and lung tissue harvest for viral titration. b Timetable for ferret-to-ferret transmission study and blood sample collection for serum neutralizing and IgG antibody titration: at 24 h post-infection of the infected ferret groups, 12–24 month-old naive ferrets (n = 3/group) were introduced in direct contact with infected ferrets and was co-housed with each group of infected ferrets in the same cage up to 12 days. Several time points are indicated for monitoring and sample collection. Orange circle denotes time point for temperature and body weight measurement, while purple circle indicates points for collection of nasal washes for viral titration. Red water drop shape indicates euthanasia after whole blood collection. c Timetable for RNA-seq analysis: groups of ferrets (n = 6/group) were SARS-CoV-2 inoculated at 0 dpi with exception to the mock infection group. PBS-treated ferrets (n = 9) were intranasally inoculated with phosphate-buffered saline (PBS) for the mock-infection group. Ferrets of the same ages as the test groups (n = 9 (Juvenile = 3, young adult = 3, aged = 3)) were then euthanized at 2 dpi, and lungs were harvested for RNA-seq analysis. SARS-CoV-2 infected ferrets by age (n = 3/group) were euthanized at 2 dpi and 5 dpi respectively, and lungs were harvested for RNA-seq analysis.