K18 hace2 mice

All animal procedures were performed under the approval of the Committee on the Ethics of Animal Experimentation of the IGTP and the authorization of the Generalitat de Catalunya (code 10965).
Oropharyngeal swab and tissue samples from the lung, brain, and nasal turbinates were collected from 16 SARS-CoV-2-infected K18-hACE2 mice (50% male/50% female, 7 to 9 weeks old; Jackson Laboratory) for viral load (VL) determination. Mice were challenged with 1,000 50% tissue culture infective doses (TCID₅₀) of the SARS-CoV-2 D614G isolate, and tissue samples were collected on day 2, 4, or 7 after challenge or according to the humane endpoints defined in the supervision protocol (weight loss of >20%, drastic reduction in mobility, or significant reduction in the response to stimuli). Tissue samples were collected in 1.5-mL tubes containing Dulbecco’s modified Eagle’s medium (DMEM) supplemented with penicillin (100 U/mL) and streptomycin (100 μg/mL). Next, tissues were homogenized twice at 25 Hz for 30 s using a Tissue Lyser II instrument and a 1.5-mm Tungsten bead (Qiagen). After that, samples were centrifuged for 2 min at 2,000 × g, and the supernatants were collected and stored at −80°C until use.

To evaluate the effects of Disulfiram in vivo, we infected the K18-hACE2 humanized mice (B6.Cg-Tg(K18-ACE2)2Prlmn/J) (McCray et al. [25 (link)] Oladunni et al. [26 (link)], Bao et al. [27 (link)]). K18-hACE2 mice were obtained from The Jackson Laboratory and were bred in the Centro de Criação de Animais Especiais (Ribeirão Preto Medical School/University of São Paulo). For the experimental infection, animals were transferred to the BSL3 facility. Eight-week-old male K18-hACE2 mice were infected with 2 × 10⁴ PFU of SARS-CoV-2 (in 40 µL) by the intranasal route as previously described [26 (link)]. Twenty-four hours after the virus inoculation and once daily until day 5 post-infection (dpi), animals were treated with Disulfiram (50 mg/kg, i.p.) (n = 7) or vehicle (n = 7). Naive mice remained uninfected and untreated. On the 5 dpi, 6 h after the injection of Disulfiram or vehicle, animals were humanely euthanized for samples collection. All the experimental procedures were performed in accordance with the guide for the use of laboratory animals of the University of Sao Paulo and approved by the institutional ethics committee (066/2020).

Heterozygous K18-hACE2 mice [strain: JAX 034,860 B6.Cg-Tg(K18-hACE2)2Prlmn/J] were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Seven-week-old male K18-hACE2 mice were administrated 2.5 × 10⁴ TCID₅₀/mL SARS-CoV-2 via the intranasal route. The K18-hACE2 mice were monitored for changes in weight loss, lethality, and clinical symptoms every day after inoculation. At 6 dpi, SARS-CoV-2–infected K18-hACE2 mice were sacrificed by isoflurane and autopsied to assess the clinical lesions in several organs such as the brain, heart, lungs, spleen, and kidneys. The animal experiments were approved by the Institutional Animal Committee of the Jeonbuk National University (JBNU 2020-11-001) and performed in accordance with the guidelines of the Institutional Biosafety Committee. The study was conducted in compliance with the ARRIVE guidelines.

All animal procedures were performed under the approval of the Committee on the Ethics of Animal Experimentation of the IGTP and the authorization of the Generalitat de Catalunya (code 10965).
Oropharyngeal swab and tissue samples from the lung, brain, and nasal turbinates were collected from 16 SARS-CoV-2-infected K18-hACE2 mice (50% male/50% female, 7 to 9 weeks old; Jackson Laboratory) for viral load (VL) determination. Mice were challenged with 1,000 50% tissue culture infective doses (TCID₅₀) of the SARS-CoV-2 D614G isolate, and tissue samples were collected on day 2, 4, or 7 after challenge or according to the humane endpoints defined in the supervision protocol (weight loss of >20%, drastic reduction in mobility, or significant reduction in the response to stimuli). Tissue samples were collected in 1.5-mL tubes containing Dulbecco’s modified Eagle’s medium (DMEM) supplemented with penicillin (100 U/mL) and streptomycin (100 μg/mL). Next, tissues were homogenized twice at 25 Hz for 30 s using a Tissue Lyser II instrument and a 1.5-mm Tungsten bead (Qiagen). After that, samples were centrifuged for 2 min at 2,000 × g, and the supernatants were collected and stored at −80°C until use.