B6 cg tg k18 ace2 2prlmn j

A total of 36 six-week-old hemizygous K18-hACE2 transgenic (B6.Cg-Tg(K18-ACE2)2Prlmn/J) female mice were used (The Jackson Laboratory, ME, USA). Animals were anesthetized under isoflurane and inoculated intranasally (i.n.) with 50 µl of DMEM containing 5×10⁴ PFU of SARS-CoV-2 isolate hCoV-19/Spain/SP-VHIR.02, D614G(S) from lineage B.1.610, kindly provided by Dr. Miguel Chillón (Universitat Pompeu i Fabra, Barcelona, Spain). Uninfected control mice were treated in parallel with DMEM. Six mice per group were inoculated intraperitoneally (i.p.) with 150 µg of each hcAb (equivalent to ~8 mg/kg for a mouse of ~18 g) in 150 µl of HBS at 24 h.p.i. Uninfected control mice were inoculated at the same time only with buffer. Animals received water and food ad libitum and were monitored daily for clinical signs and body weight. Euthanasia was applied when the animals exhibited irreversible disease signs. All surviving mice were anesthetized and euthanized at the end of the experiment.

All animal experiments involving SARS-CoV-2 were conducted in a biosafety level 3 (BSL3) facility. The animal handling was performed in accordance with the regulations outlined in the U.S. Department of Agriculture (USDA) Animal Welfare Act and the conditions specified in the Guide for Care and Use of Laboratory Animals (National Institutes of Health, 2011). The animal studies were approved by the ethical committee for animal experiments of the Israel Institute for Biological Research (IIBR) (protocol number M-77-20). Female K18-hACE2 transgenic mice (B6. Cg-Tg(K18-ACE2)2Prlmn/J; #034860) (Jackson Laboratory, Bar Harbor, USA) (6–8 weeks old) were maintained at 20–22 °C and a relative humidity of 50 ± 10% under a 12 h light/dark cycle. The animals were fed commercial rodent chow (Koffolk Inc. Ramat Hovav, Israel) and provided tap water ad libitum. Prior to infection, the mice were kept in groups of 10. The mice were randomly assigned to the experimental groups. For SARS-CoV-2 infection, the virus was diluted in phosphate-buffered saline (PBS) supplemented with 2% FBS (Biological Industries, Israel). Anesthetized animals (ketamine 75 mg/kg and xylazine 7.5 mg/kg in PBS) were infected by intranasal (i.n.) instillation of 20 µL SARS-CoV-2 500 PFU/mouse and injected i.p. with DTBN, 20 μg/mouse once every day for 5 days, in 0.1 mL of 10% saline: DMSO (vehicle).

Animal work was approved by the local University of Liverpool Animal Welfare and Ethical Review Body and performed under UK Home Office Project Licence PP4715265. Mice carrying the human ACE2 gene under the control of the keratin 18 promoter (K18-hACE2; formally B6.Cg-Tg (K18-ACE2) 2Prlmn/J) were purchased from Jackson Laboratories and Charles River. Mice were maintained under SPF barrier conditions in individually ventilated cages.
For each experiment, animals were randomly assigned into multiple groups. For SARS-CoV-2 infection, mice were anaesthetized lightly with isoflurane and inoculated intra-nasally with 50 µL containing 10³ PFU or 10⁴ PFU (cohort 3; Pango lineage B) SARS-CoV-2 in PBS (Table 1); control animals received PBS. For double infections (cohort 2), mice were anaesthetized lightly with KETASET i.m. and inoculated intranasally with 10² PFU IAV X31 in 50 µL sterile PBS. Three days later, they were infected with SARS-CoV-2 (Pango lineage B), as described above. Mock-infected mice served as controls.
Mice were monitored for any clinical signs and weighed. Animals were sacrificed at 3, 5, 6 or 7 days post-SARS-CoV-2 infection (Table 1) by an overdose of pentabarbitone. Mice were dissected and tissues collected immediately for downstream processing; the lungs were processed for other studies [43 (link),49 (link),50 (link)].

Transgenic mice with the human ACE2 gene under the control of K18 promoter (B6.Cg-Tg(K18-ACE2)2Prlmn/J, Jackson Labs, Strain #034860) were used in these studies. Prior to infection, the mice were implanted with a UID Temperature Microchip and continuously monitored for body temperature and activity using the UID Mouse Matrix (UIdevices). Male and female hACE2-K18 mice, ranging in age 6–9 months, were randomly assigned to receive either vehicle saline (mock controls) or SARS-CoV-2 at a dose of 1 × 10⁴ or 4 × 10³ 50% tissue culture infectivity dose (TCID50). Mice were anesthetized with 3–4% isoflurane, and 12.5 ml of either the vehicle or SARS-CoV-2 was pipetted into each naris (25 μL total volume). Following infection, mice were housed individually and managed in an ABSL3 facility (East Carolina University, Greenville, NC). Mice were weighed daily and monitored daily for symptoms for 10 days after infection. Mice that had rapid weight loss, lethargy, and became moribund between 6 and 10 days post-infection (dpi) were euthanized. Behavior and cognitive function tests were performed starting from 30 dpi. The mice were euthanized, and tissues were harvested at 45 dpi.