One-month-old female Syrian hamsters (Japan SLC Inc.) and 7- to 8-mo-old female Syrian hamsters (Envigo) were used in this study. Baseline body weights were measured before infection. Under ketamine−xylazine anesthesia, four hamsters per group were inoculated with 105.6 PFU (in 110 μL) or with 103 PFU (in 110 μL) of UT-NCGM02 via a combination of the intranasal (100 μL) and ocular (10 μL) routes. Body weight was monitored daily for 14 d.
For virological and pathological examinations, two, four, or five hamsters per group were infected with 105.6 PFU (in 110 μL) or with 103 PFU (in 110 μL) of the virus via a combination of the intranasal and ocular routes; 3, 6, and 10 d postinfection, the animals were killed, and their organs (nasal turbinates, trachea, lungs, eyelids, brain, heart, liver, spleen, kidneys, jejunum, colon, and blood) were collected.
For the reinfection experiments, three hamsters per group were infected with 105.6 PFU (in 110 μL) or with 103 PFU (in 110 μL) of UT-NCGM02 or PBS (mock) via a combination of the intranasal and ocular routes. On day 20 postinfection, these animals were reinfected with 105.6 PFU of the virus via a combination of the intranasal and ocular routes. On day 4 after reinfection, the animals were killed, and the virus titers in the nasal turbinates, trachea, and lungs were determined by means of plaque assays in VeroE6/TMPRSS2 cells.
For the passive transfer experiments, eight hamsters were infected with 105.6 PFU (in 110 μL) or with 103 PFU (in 110 μL) of UT-NCGM02 via a combination of the intranasal and ocular routes. Serum samples were collected from these infected hamsters on day 38 or 39 postinfection, and were pooled. Control serum was obtained from uninfected age-matched hamsters. Three hamsters per group were inoculated intranasally with 103 PFU of UT-NCGM02. On day 1 or 2 postinfection, hamsters were injected intraperitoneally with the postinfection serum or control serum (2 mL per hamster). The animals were killed on day 4 postinfection, and the virus titers in the nasal turbinates and lungs were determined by means of plaque assays in VeroE6/TMPRSS2 cells. All experiments with hamsters were performed in accordance with the Science Council of Japan’s Guidelines for Proper Conduct of Animal Experiments and the guidelines set by the Institutional Animal Care and Use Committee at the University of Wisconsin–Madison. The protocol was approved by the Animal Experiment Committee of the Institute of Medical Science, the University of Tokyo (approval no. PA19-75) and the Animal Care and Use Committee of the University of Wisconsin–Madison (protocol no. V00806).
Detailed materials and methods for this study are described inSI Appendix .
For virological and pathological examinations, two, four, or five hamsters per group were infected with 105.6 PFU (in 110 μL) or with 103 PFU (in 110 μL) of the virus via a combination of the intranasal and ocular routes; 3, 6, and 10 d postinfection, the animals were killed, and their organs (nasal turbinates, trachea, lungs, eyelids, brain, heart, liver, spleen, kidneys, jejunum, colon, and blood) were collected.
For the reinfection experiments, three hamsters per group were infected with 105.6 PFU (in 110 μL) or with 103 PFU (in 110 μL) of UT-NCGM02 or PBS (mock) via a combination of the intranasal and ocular routes. On day 20 postinfection, these animals were reinfected with 105.6 PFU of the virus via a combination of the intranasal and ocular routes. On day 4 after reinfection, the animals were killed, and the virus titers in the nasal turbinates, trachea, and lungs were determined by means of plaque assays in VeroE6/TMPRSS2 cells.
For the passive transfer experiments, eight hamsters were infected with 105.6 PFU (in 110 μL) or with 103 PFU (in 110 μL) of UT-NCGM02 via a combination of the intranasal and ocular routes. Serum samples were collected from these infected hamsters on day 38 or 39 postinfection, and were pooled. Control serum was obtained from uninfected age-matched hamsters. Three hamsters per group were inoculated intranasally with 103 PFU of UT-NCGM02. On day 1 or 2 postinfection, hamsters were injected intraperitoneally with the postinfection serum or control serum (2 mL per hamster). The animals were killed on day 4 postinfection, and the virus titers in the nasal turbinates and lungs were determined by means of plaque assays in VeroE6/TMPRSS2 cells. All experiments with hamsters were performed in accordance with the Science Council of Japan’s Guidelines for Proper Conduct of Animal Experiments and the guidelines set by the Institutional Animal Care and Use Committee at the University of Wisconsin–Madison. The protocol was approved by the Animal Experiment Committee of the Institute of Medical Science, the University of Tokyo (approval no. PA19-75) and the Animal Care and Use Committee of the University of Wisconsin–Madison (protocol no. V00806).
Detailed materials and methods for this study are described in
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