Virus. SARS-CoV–2 virus was isolated from the first confirmed COVID–19 patient in Hong Kong in Vera E6 cells at the BSL–3 core facility, LKS Faculty of Medicine, The University of Hong Kong. Stock virus at the titer of 107.25 TCID50/mL was prepared after three serial passages in Vero E6 cells in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 4.5 g/L D-glucose, 100 mg/L sodium pyruvate, 2% FBS, 100,000 U/L Penicillin- Streptomycin, and 25mM HEPES.
Animal experiments. Male golden Syrian hamsters at 4–5 weeks old were obtained from Laboratory Animal Services Centre, Chinese University of Hong Kong. All experiments were performed at the BSL–3 core facility, LKS Faculty of Medicine, The University of Hong Kong. The animals were acclimatized at the BSL3 facility for 6 days prior to the experiments. The study protocol have been reviewed and approved by the Committee on the Use of Live Animals in Teaching and Research, The University of Hong Kong (CULATR # 5323–20). For challenge studies, hamsters were anesthetized by ketamine(150mg/kg) and xylazine (10mg/kg) via intra-peritoneal injection and were intra-nasally inoculated with 8 x 104 TCID50 of SARS-CoV–2 virus in 80 µL DMEM. On days 2, 5, 7, three hamsters were euthanized by intra-peritoneal injection of pentobarbital at 200mg/kg. Lungs (left) and one kidney were collected for viral load determination and were homogenized in 1mL PBS. Brain, nasal turbinate, lungs (right, liver, heart, spleen, duodenum, and kidney were fixed in 4% paraformaldehyde for histopathological examination. On days 1, 4, 6 post-inoculation, hamsters were transferred to a new cage and fresh fecal samples (10 pieces) were collected on days 2, 5, 7 post-inoculation for quantitative real-time RT-PCR. For the transmission experiment, donor hamsters were anesthetized and inoculated with 8 x 104 TCID50 of SARS- CoV–2 virus. At 24 hours post-inoculation, each inoculated donor was transferred to co- house with one naïve hamster in a clean cage and co-housing of the animals continued for 13 days. For nasal wash collection, hamsters were anesthetized by ketamine(100mg/kg) and xylazine (10mg/kg) via intra-peritoneal injection and 160 µL of PBS was used to collect nasal washes from both nostrils of each animal. In each cage, the contact hamster was handled first followed by surface decontamination using 1% virkon and handling of the donor hamster. Body weight and clinical signs of the animals were monitored daily for 14 days.
Viral load determination by quantitative real-time RT-PCR. RNA was extracted from 140 µL tissue homogenate or nasal washes using QIAamp viral RNA mini kit (Qiagen) and eluted with 60 µL of water. The N gene of SARS-CoV–2 virus was detected and quantified using TaqMan™ Fast Virus 1-Step Master Mix as described28.
Serological assay. Serum samples were collected from donor hamsters on day 14 post- inoculation and from contact hamsters on day 13 post-contact. Serum were incubated at 56°C for 30 min before neutralization assay was performed to determine anti-SARS-CoV–2 virus in Vero E6 cells. Sera were firstly 1:5 diluted followed by serial 2 fold dilution and were incubated with 100 TCID50 of SARS-CoV–2 virus for 1 hour at 37°C. The mixture was added to Vero E6 cells and further incubated for 1 hour at 37°C. The inoculum was removed and replaced with fresh media, and the Vero E6 cells were observed for cytopathic effect on day 4 post-infection to determine the endpoint of neutralizing antibodies.
Histopathology and immunohistochemistry. Tissue (hearts, livers, spleens, duodenums, brains, right lungs and kidneys) were fixed in 4% paraformaldehyde and were processed for paraffin embedding. The 4-µm sections were stained with hematoxylin and eosin for histopathological examinations. For immunohistochemistry, SARS-CoV–2 N protein was detected using monoclonal antibody (4D11)29.
Statistical analyses. Kruskal-Wallis test and Dunn’s multiple comparisons test were used to compare viral loads in the lungs, kidney, or fresh faecal samples on days 2, 5, 7 post- inoculation. Area under the curve was calculated from the nasal washes of the donor and contact hamsters followed by Mann-Whiteny test. Data analyses were performed using Excel and GraphPad Prism 8.