A total of eight, 3- to 6-year-old, healthy, Cambodian-origin cynomolgus (Macaca fascicularis) and Chinese-origin rhesus (Macaca mulatta) macaques were equally selected and reared in indoor cages at the National Primate Research Center (Korea Research Institute of Bioscience and Biotechnology) (Table 1). All animals were fed fresh fruits and commercial monkey feed during the experimental procedures (Teklad, Global 20% Protein Primate Diet). They were transferred and reared in negative-pressure, NHP specific isolators in an Animal Biosafety Level 3 (ABSL-3) facility (Three-Shine Inc., Seoul, Korea). After 1 week, these animals were inoculated under anesthesia with 12.5 ml (2.1 × 106 TCID50/ml) of the same batch of virus via oral (5 ml), intratracheal (4 ml), nasal (1 ml), conjunctival (0.5 ml), and intravenous (2 ml) routes, as previously reported (Koo et al., 2020). At 3 dpi, all six lobes of the lungs of all animals were aseptically collected, homogenized with sterilized 10-fold (w/v) phosphate buffered solution (pH 7.4), and filtered with a 0.2-μm pore size syringe filter for virus isolation. The presence of viable viruses from lung tissues was examined by virus isolation using VERO cells, and the TCID50/ml was calculated according to the Reed–Muench method (Reed and Muench, 1938). Hematological analysis was performed from ethylenediaminetetraacetic acid (EDTA) blood samples with an auto-hematology analyzer (Mindray Inc., Shenzhen, China).

Information on the animals and SARS-CoV-2 used in this report.