The amino acid sequences of ACE2 and TMPRSS2 from selected species were obtained from the ENSEMBL (mink) and UniProt (other species) databases and aligned using NCBI blastp. The overall sequence identity, the residues involved in the ACE2-virus binding interface, and the catalytic domain in TMPRSS2 were compared with the human sequence.

A fetal mink lung type II alveolar epithelial cell line, Mv1Lu (ATCC, Manassas, VA, USA) was grown in Eagle’s Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) and 1% penicillin-streptomycin. HEK 293T and its ACE2-overexpressing cell line (HEK 293T-ACE2) (ATCC, Manassas, VA, USA) were grown in DMEM with high glucose and 10% FBS. Both cell types were grown in T25 flasks until they reached approximately 90% confluency. For passaging, cells were washed once with phosphate-buffered saline (PBS), removed from the flask with 1.25 ml of 0.25% trypsin-EDTA for 3 min at 37°C, and then centrifuged to remove the supernatant. The pellet was resuspended in complete growth medium.

The following small molecule inhibitors were used: ambroxol (10 μM; TCI Chemicals, Portland, OR, USA), aprotinin (10 μM; Tocris, UK), Camostat (10 μM; Sigma-Aldrich, Burlington, MA, USA), Nafamostat (10 μM; TCI Chemicals, Portland, OR, USA), Dyngo4a (20 μM, Selleckchem, Houston, TX, USA), Chlorpromazine (CPZ) (10 μM; Sigma-Aldrich, Burlington, MA, USA), Pitstop2 (20 μM; Abcam, Cambridge, UK), mβCD (20 μM; Sigma-Aldrich, Burlington, MA, USA), genistein (20 μM; TCI Chemicals, Portland, OR, USA), nystatin (20 μM; Sigma-Aldrich, Burlington, MA, USA), and Filipin (20 μM; Sigma-Aldrich, Burlington, MA, USA).

The cells were seeded in 8-well chamber slides (Ibidi; Grafelfing, Germany). When they reached confluence, the cells were fixed at room temperature in 4% paraformaldehyde (PFA) in PBS for 15 min, washed with PBS, and permeabilized with 0.1% Triton X-100 for 10 min. The cells were then blocked with 10% donkey serum. Primary antibodies were diluted in blocking buffer. The following primary antibodies were used: goat anti-ACE2 (1:200; R&D Systems, Minneapolis, MN, USA), mouse anti-TMPRSS2 (1:200; Santa Cruz Biotechnology, Dallas, TX, USA), and mouse-EEA1 (1:200; BD Biosciences, San Jose, CA, USA).

Following overnight incubation with primary antibody at 4°C, the cells were washed three times with 0.2% Tween-20 in PBS. Next, the cells were incubated in the dark with Alexa Fluor-conjugated fluorescent secondary antibodies (1:500; Invitrogen, Carlsbad, CA, USA). After washing in PBS, the cells were mounted with diluted Vectashield and DAPI for nuclear staining. A Nikon Eclipse Ti microscope was used to image at 60× in water immersion. The NIS Elements Software and ImageJ software were used to image and analyze the data.

To determine if TMPRSS2 is localized in the plasma membrane, non-permeabilized (no Triton-X100, no Tween-20 was used) Mv1Lu cells were also labeled with a TMPRSS2 antibody. To establish the location of TMPRSS2, Mv1Lu cells were permeabilized with Tween-20 and labeled with the TMPRSS2 antibody and WGA (1:100; Vector Laboratories, Newton, CA, USA), a lectin that binds only to the cell surface. Antibody staining and mounting steps were performed as above. To obtain 3D Z-stack images, a Zeiss 880 laser scanning inverted confocal microscope (Zeiss, Oberkochen, Germany) was used with a 40× water immersion objective (0.5 μm optical thickness). Images were analyzed using Zen software (Zeiss) and ImageJ 3D viewer.

RNA was extracted from Mv1Lu cells using the RNeasy Mini Kit (Qiagen, Germantown, MD, USA) according to the manufacturer’s protocol. Primer sequences from Heller et al. (2006) are provided below:

RT-PCR was performed after generating first-strand cDNAs from 2 μg of total RNA using iScript Reverse Transcription Supermix (Bio-Rad, Hercules, CA, USA), as recommended by the manufacturer. Diluted cDNA (40 ng/μl) was amplified using Phusion Polymerase (Thermo Fisher Scientific, Waltham, MA, USA). The samples were analyzed using agarose gel electrophoresis (1%).

HEK293T cells were plated with antibiotic-free medium at a density of 7 × 10⁶ cells in a T75 flask and transfected via Lipofectamine3000 (Invitrogen, Carlsbad, CA, USA) using a total of 15 μg of BEI lentiviral plasmids (Table 1), 60 μl of Lipofectamine, and 20 μl of P3000 per T75 flask following the manufacturer’s protocol. After overnight incubation, fresh medium was added to cells supplemented with 1% BSA. Fluorescence microscopy was used to observe the transfection efficiency based on Zsgreen expression.

Conditioned medium was collected 48 h post-transfection. The conditioned medium was centrifuged, and the supernatant was filtered using a 0.45 μm syringe filter. The filtered supernatant was mixed with 5× PEG (Abcam, Cambridge, UK) and precipitated overnight at 4°C. The lentivirus was collected by centrifugation and resuspending the pellet in Viral Re-suspension Solution (Abcam, Cambridge, UK). Virus aliquots were stored at −80°C. Prior to the experiments, the transduction efficiency of each batch of viruses was tested via flow cytometry of infected HEK 293T-ACE2. Viral titers were calculated using the Poisson formula (Crawford et al., 2020; Song et al., 2023). The flow cytometry conditions were the same as those for the Mv1Lu cell protocol. The medium was collected from wells containing cells that were not transfected, and PEG-precipitated as described above, and then used for mock infection.

Mv1Lu cells were plated at a density of 30% and infected with SARS-CoV-2 viral pseudoparticles (multiplicity of infection, or MOI, of 0.1). The medium was replaced after overnight incubation of the infected cells. Fluorescence microscopy was used to observe transduction efficiency. Cells were trypsinized 48 h post-infection and washed three times with 0.5% BSA in PBS. After the final wash, the cells were resuspended in the same wash buffer and analyzed using flow cytometry. Novocyte flow cytometer was used to detect ZsGreen in the FITC channel. The resulting flow cytometer files were analyzed using NovoExpress software. Mock infection was used as a background control. Prior to infection, the cells were preincubated for 2 h with TMPRSS2 inhibitors or endocytosis inhibitors, which were kept in the medium until harvesting for flow cytometry.

Mv1Lu cells were seeded in 8-well chamber slides. The cells were incubated overnight in Dyngo4a and TRITC-conjugated Dextran (Invitrogen, Carlsbad, CA, USA). After fixation in 4% PFA, mounting was performed using diluted Vectashield with DAPI. Immunocytochemistry with an antibody against EEA1 was also performed to further validate dextran uptake, as described above for ACE2 and TMPRSS2.

The means and standard errors of the mean for three biological experiments were plotted using GraphPad Prism 7 software (GraphPad, San Diego, CA, USA). For infection data, the mean of the DMSO group was set to a value of 100, and the inhibitor groups were compared to this value. Statistical significance was determined using Minitab Statistics Software (Minitab, State College, PA, USA). When the data did not satisfy the assumptions of ANOVA (normal distribution and homogeneity of variances), they were subjected to a Box-Cox or logarithmic transformation. All infection analyses were performed using a one-way ANOVA, and TMPRSS2 cleavage analysis was performed using two-way ANOVA, in which the factors were time and treatment. When the ANOVA means were significant (p < 0.05), groups treated with inhibitors were compared to the DMSO control group using Dunnett’s post-hoc test.

Fifteen out of 16 mink ACE2 amino acid orthologs had more than 80% sequence similarity with human ACE2 (Table 2). Mink showed a high sequence identity (82.99%), like other known susceptible species, such as cats and dogs (Shi et al., 2020). Since ACE2 residues that directly bind to the RBD in the S protein have been identified (Lan et al., 2020), these residues in ACE2 were aligned in various vertebrates to predict their affinity to the viral S protein (Figure 2A). Lysine residues 31 and 353 are particularly critical for binding to the receptor binding domain of the S protein (Wan et al., 2020). In mink and other mammalian species that are readily infected by SARS-CoV-2, these critical residues were identical to those in humans. However, in species that are less susceptible, residues 31 (mouse, chicken, and bat) and 353 (mouse and rat) were different than human ACE2.

The substrate binding motifs and the catalytic domain of TMPRSS2 were aligned in various species to predict the protease activity (Figure 2B). While other species show high conservation of key residues, mink TMPRSS2 did not contain most of the critical motifs. For example, mink TMPRSS2 did not have S441, which is part of the catalytic triad (Fraser et al., 2022; Salleh and Deris, 2022). Also, all the substrate binding residues (D435, S460, and G462) were missing. Many of these residues are involved in inhibitor-binding: Camostat and Nafamostat (S441), aprotinin (S441 and S460), and ambroxol (Q438) (Fraser et al., 2022; Salleh and Deris, 2022).

To support high conservation and verify expression, the ACE2 receptor was examined in mink Mv1Lu cells using RT-PCR and immunocytochemistry. RT-PCR was performed using a human ACE2 primer set (hACE2) and mink ACE2 primer set (MvACE2) to amplify the ACE2 transcript (Figure 3A and Supplementary Figure 1). The HEK 293T-ACE2 and HEK 293T cell lines (primer controls) expressed the ACE2 gene, verifying the RT-PCR assay. Mink cDNA reverse-transcribed from Mv1Lu RNA showed robust amplicons for both primer sets, indicating the presence of the ACE2 transcript. Mv1Lu cells labeled with the ACE2 antibody had numerous puncta over their surfaces, demonstrating translation of the ACE2 transcript (Figure 3B). Cells labeled with donkey anti-goat secondary antibody alone (control) had no fluorescent labels (Figure 3C). The TMPRSS2 protease was expressed in Mv1Lu cells and appeared as numerous puncta (Figures 3D, E). Cells labeled with secondary donkey anti-mouse antibody-only control did not show fluorescent puncta (Figure 3E).

To determine if TMPRSS2 was localized in the plasma membrane, cells that were fixed but not permeabilized were labeled with TMPRSS2 antibody. No label was observed on the cell surface (Figures 4A–C). Then, cells were fixed and permeabilized with Tween-20, were labeled with WGA-FITC, a surface-binding lectin conjugate, and the TMPRSS2 antibody. In 3D reconstructions, WGA was localized on the cell surface and often seen aggregated into “hot spots” that were clearly elevated off the surface (Figure 4D). A top slice from the Z-stack further supported that WGA is a robust plasma membrane marker (Figures 4E–H) and that TMPRSS2 did not colocalize in these hot spots. In permeabilized cells, abundant aggregates or clusters of TMPRSS2 were observed inside the cells (Figure 4J), with very little to no colocalization of TMPRSS2 and WGA (Figures 4I, K). These data indicate that unlike other species, TMPRSS2 is not a plasma membrane protein in Mv1Lu cells.

Human SARS-CoV-2 pseudoparticles were produced using previously published procedures (Crawford et al., 2020; Song et al., 2023). Generated pseudoparticles were first titered with HEK293T-ACE2 cells using flow cytometry to detect the ZsGreen viral backbone (Figures 5A–C). High viral titers were obtained using 0.625 μl of pseudoparticles, resulting in a transduction efficiency of 53.44% (Figure 5A). Twofold serial dilutions of the pseudoparticles showed a gradual decrease in transduction: 29.99% with 0.3125 μl and 15.55% with 0.15625 μl (Figures 5B, C). The MOI was calculated using the Poisson equation to assess the SARS-CoV-2 pseudoparticle infection in Mv1Lu cells (Crawford et al., 2020). Viral infection in Mv1Lu cells showed 67.49% transduction efficiency, with an MOI of 0.3 (Figures 5D, E). The MOI was adjusted to 0.1 for all other infection assays. These results indicated that Mv1Lu cells are highly susceptible to human SARS-CoV-2 pseudoparticle infection.

TMPRSS2 inhibitors were used to determine if they could reduce infection in Mv1Lu cells (Figure 6A). The mean infection in DMSO was set to 100, and the inhibitor groups were compared to this value. Statistical analysis showed that none of the protease inhibitor groups were significantly different from the DMSO control.

To test the possibility of viral entry via endocytosis, Mv1Lu cells were treated with three endocytosis inhibitors (Dyngo4a, Pitstop2, and CPZ). Endocytosis was observed using dextran conjugated to TRITC, which fluoresces red upon uptake by endocytosis. In the DMSO control group, dextran-TRITC was taken up by the cells and appeared as small fluorescent puncta, consistent with its uptake by endocytosis (Figure 6B). Cells treated with endocytosis inhibitors (Dyngo4a, Pitstop2, and CPZ) showed a significant decrease in the uptake of dextran-TRITC (Figures 6C–E). Cells treated with Dyngo4a or CPZ showed little to no fluorescence, whereas those treated with Pitstop2 showed some evidence of endocytosis.

The EEA1 antibody was used to track early endosome formation. Endosomes were detected as small green puncta in the DMSO control group (Figure 6F). When cells were treated with Dyngo4a, Pitstop2, or CPZ, endosome formation was significantly reduced (Figures 6G–I). While endosomes were observed in the CPZ group, their numbers were far lower than in the DMSO control group.

Endocytosis inhibitors were next used to determine if they could reduce infection relative to the DMSO control (Figure 6J). Dyngo4a and Pitstop2 both reduced infection relative to the DMSO control, but only Dyngo4a was significant (p = 0.028). CPZ increased infection but was not significantly different from the control.

The TMPRSS2 and endocytosis pathways were simultaneously targeted using cocktails of inhibitors. When statistical significance was found, Dunnett’s post-hoc test was used to compare the groups to Dyngo4a alone. As seen before (Figure 6J), Dyngo4a alone significantly lowered infection compared with DMSO. None of the cocktails were significantly lower than Dyngo4a alone, and ambroxol/Dyngo4a was almost equivalent to DMSO, indicating that ambroxol negated the inhibitory effects of Dyngo4a (Figure 7A).

Pitstop2 alone lowered infection when compared to the DMSO control, while the addition of TMPRSS2 inhibitors increased infection above that observed with Pitstop2 (Figure 7B). The addition of aprotinin significantly increased infection, and the increase in ambroxol/Pitstop2 was close to significant (p = 0.08).

Caveolae-mediated endocytosis, another dynamin-dependent pathway, was targeted using cholesterol inhibitors, which included methyl-β-cyclodextrin (mβCD), nystatin, filipin, and genistein (Figure 8A). Nystatin significantly increased infection compared to the DMSO control. Other cholesterol inhibitors lowered infection by 3–20%, but none were significantly different from the DMSO control.

Cholesterol inhibitors were also mixed with Dyngo4a to block dynamin and caveolin simultaneously (Figure 8B). In Dunnett’s post-hoc test, Dyngo4a was used as the comparative group. Infection in the Dyngo4a group was significantly lower than the DMSO control. Cocktails containing mβCD, genistein, and filipin all lowered infection relative to Dyngo4a; however, only Dyngo4a/mβCD was significantly lower than Dyngo4a. In contrast, the addition of nystatin to Dyngo4a caused a significant increase in infection relative to Dyngo4a alone.

SARS-CoV-2 infection has devastated mink farms worldwide (Cahan, 2020; Molenaar et al., 2020), and the infection of mink has many similarities to SARS-CoV-2 infection of humans (Devaux et al., 2021). As examples, the symptoms displayed in mink are similar to those in humans, and the virus is rapidly transmitted in both mink and humans. Moreover, both spillover and spillback events have been reported in mink (Devaux et al., 2021).

Given the importance of mink as a carrier of SARS-CoV-2, it is desirable to have a mink cell line that allows in vitro experimentation and evaluation of potentially therapeutic drugs. As originally shown by Heller et al. (2006), Mv1Lu cells from mink express the ACE2 receptor. We extended this observation by showing that Mv1Lu cells are a valuable in vitro model for studying SARS-CoV-2 infection in mink, characterizing the SARS-CoV-2 entry mechanisms in these cells, and screening drugs that could inhibit infection. ACE2 was robustly expressed in Mv1Lu cells at both the RNA and protein levels. In mink, TMPRSS2 appeared to lack enzymatic activity unlike other mammalian species. Nevertheless, Mv1Lu cells showed abundant TMPRSS2 expression, which was internal to the plasma membrane and localized in clumps, suggesting that it may be sequestered in the endomembrane system in these cells. Mv1Lu cells efficiently take up SARS-CoV-2 pseudoparticles by endocytosis. Mv1Lu cells, which were originally isolated from fetal lungs and spontaneously immortalized, are easy to culture and readily available to any lab (Henderson et al., 1974). Mv1Lu cells allow convenient and rapid screening of drugs that can potentially prevent SARS-CoV-2 infection in mink. SARS-CoV-2 pseudoparticles, which cannot replicate, can be generated in a Biosafety Lab 2 cabinet and provide a safer alternative than using live virus. This infection system has been adapted to other cell types and species (Song et al., 2023). Pseudotyping has been used previously to study virus-host receptor binding (Wang et al., 2008; Lu et al., 2014; Phandthong et al., 2022, 2023) and can be applied to Mv1Lu cells, which are a robust in vitro model for studying coronavirus infection in mink.

The use of small molecule inhibitors with Mv1Lu cells enabled characterization of the viral entry pathway and identification of potential therapeutics for preventing infection (Figure 8). In humans, small molecule inhibitors have been used previously to show that SARS-CoV-2 enters host cells by both the TMPRSS2-based fusion and endocytosis pathways (Neerukonda et al., 2021; Peacock et al., 2022; Zhao et al., 2022). Our study showed that in mink some small molecule inhibitors were effective (Dyngo4a and mβCD), some were not (ambroxol, Camostat, Nafamostat, and aprotinin), and some worsened infection (CPZ and nystatin), demonstrating the importance of testing drugs before using them therapeutically (Figure 9). Given that mink TMPRSS2 was localized mainly inside the cells and lacked enzymatic activity, the minimal effects of TMPRSS2 inhibitors were not surprising. Moreover, S441 was reported to be necessary for proteolytic activity, as a substitution in this residue was sufficient to generate a catalytically null mutant in the type II transmembrane serine protease family (Afar et al., 2001). Mink TMPRSS2 sequence does not contain S441, suggesting minimal proteolytic activity. It is interesting that Mv1Lu cells have abundant TMPRSS2, which is localized in clusters within the cells, suggesting that it has another yet unrecognized function in mink.

Dyngo4a, which blocks the dynamin GTPase involved in various endocytic pathways (Macia et al., 2006), was the most effective small molecule inhibitor. Pitstop2, mβCD, and filipin, which block endocytosis (Vercauteren et al., 2010; von Kleist et al., 2011), were also effective in preventing pseudoparticle infection of Mv1Lu cells. Pitstop2 targets the clathrin heavy chain and prevents it from interacting with adaptor proteins required for clathrin-coated pit formation (Alkafaas et al., 2023). Other inhibitors blocked caveolae-mediated endocytosis by various mechanisms. For instance, mβCD inhibits endocytosis by depleting cholesterol (Rodal et al., 1999; Rejman et al., 2005; Zidovetzki and Levitan, 2007), which leads to a decrease in the number of caveolae in other cell types (Dreja et al., 2002). Although the mechanism of action of nystatin is similar to mβCD, it was cytotoxic in Mv1Lu cells, indicating it is not suitable for use in mink. Filipin interacts with membrane cholesterol and prevents the close packing of acyl chains (Alkafaas et al., 2023), and therefore, was effective in decreasing infection in mink. Genistein, an FDA-approved pharmacological inhibitor of Src kinase-dependent phosphorylation of caveolin-1 (Zhang et al., 2018), decreased infection in mink. Our results show that most endocytosis inhibitors were effective in preventing SARS-CoV-2 pseudoparticle infection in Mv1Lu cells; however, they varied in their efficacy. The results with nystatin demonstrate the importance of drug screening, as not all endocytosis inhibitors can be assumed to be effective. Overall, our data support the conclusion that the SARS-CoV-2 virus enters mink Mv1Lu cells mainly via dynamin-dependent endocytosis, which includes both clathrin-dependent and -independent pathways (Rennick et al., 2021).

Chlorpromazine, a FDA-approved drug for treating psychiatric disorders (Daniel et al., 2015; Wu et al., 2016), blocks clathrin-coated vesicle formation; however, it was not effective in inhibiting SARS-CoV-2 pseudoparticle infection and showed cytotoxicity in Mv1Lu cells. CPZ was effective in blocking live virus infection in human A549 cells (Weston et al., 2020; Plaze et al., 2021). Since psychiatric patients receiving CPZ were observed to show a lower prevalence of COVID-19 (Plaze et al., 2020), clinical trials are registered in Egypt and France to determine if administration of CPZ decreases the likelihood of contracting COVID-19 in humans (Stip et al., 2020). Our CPZ results suggest that species can differ in their sensitivity to this drug and that efficacy varies with species.

SARS-CoV-2 can also enter host cells by an alternate pathway that involves viral spike protein binding to the ACE2 receptor and subsequent cleavage of spike by the TMPRSS2 protease (Hoffmann et al., 2020). Most prior zoonotic studies on this pathway have focused on ACE2 conservation and ACE2 affinity to predict the host range of SARS-CoV-2 (Damas et al., 2020; Sun et al., 2021). However, Camostat and Nafamostat did reduce SARS-CoV-2 infection in human Calu-3 cells (Hoffmann et al., 2020; Wang et al., 2020), again demonstrating the importance of testing inhibitors on the species of interest. Together, our viral pseudoparticle infection assay and small molecule inhibitor data showed that Mv1Lu cells used endocytosis for SARS-CoV-2 entry, but not the TMPRSS2-based fusion pathway (Figure 9).

Our study provides a framework for future work on viral entry mechanisms and development of therapeutics for SARS-CoV-2 infections in zoonotic species. While our study focused on the wild-type spike strain (Wuhan-Hu-1), our protocol could be adapted for use with other variants and tested using other zoonotic species (Song et al., 2023). Finally, our approach could be used to explore an alternative endocytic entry pathway facilitated by soluble ACE2 (Yeung et al., 2021), highlighting the versatility of this infection system for drug screening.

Drug cocktails that target multiple viral proteins or multiple steps in the viral pathway are a promising type of therapeutics for treating viral diseases. Drug combinations can be more effective than single drugs in blocking viral entry by eliminating drug-resistant variants (Yang and Rao, 2021). It is possible that when one entry pathway is blocked, another pathway may be activated. Computational studies on SARS-CoV-2 infection predicted that targeting both TMPRSS2 and cathepsin lowered the infectability more than targeting either pathway alone (Padmanabhan et al., 2020). Similarly, in our study, infection was lowered more when Dyngo4a and mβCD were combined than when either was used independently. Dyngo4a ( Rennick et al., 2021) and mβCD (Rodal et al., 1999) were reported to block both clathrin and caveolin-mediated endocytosis, which appeared to be the case in our study. Adding mβCD may have acted by altering the fluidity of the plasma membrane, as shown by others (Dreja et al., 2002; Zidovetzki and Levitan, 2007).

Our study supports the conclusion that targeting dynamin and cholesterol with small molecule inhibitors, Dyngo4a and mβCD, can offer a new approach for the prevention of SARS-CoV-2 infection in mink and complement COVID-19 vaccines. mβCD is already approved as an additive to food by the Flavor Extract Manufacturers Association (FEMA) and is “generally recognized as safe” (FEMA GRAS 4028). mβCD is also currently used to increase the solubility of non-polar drugs for various routes of administration in humans (Uekama et al., 1998). Dyngo4a is not yet FDA-approved, but many preclinical reports propose Dyngo4a as a candidate therapeutic. For example, in an ex vivo perfused mouse heart model of ischemia/reperfusion injury, Dyngo4a inhibited mitochondrial fission by targeting mitochondrial dynamin (Drp1), leading to increased cardiomyocyte survival (Gao et al., 2013). Moreover, in the corneal epithelium (human in vitro and mouse ex vivo), oxidative stress and hyperosmolar stress (in the ER) can be alleviated by Dyngo4a (Webster et al., 2018; Martinez-Carrasco et al., 2020; Martinez-Carrasco and Fini, 2023). In our study, at Dyngo4a concentrations that were effective in blocking SARS-CoV-2 pseudoparticle entry, other cellular processes did not appear to be affected. For example, Mv1Lu cells continued to divide and appeared morphologically normal in the presence of Dyngo4a.

Nasal administration with nebulizers is one promising approach (Eedara et al., 2021) to effectively deliver therapeutic drugs to mink. The delivery of Dyngo4a and mβCD via nasal spray directly introduces the drugs to the desired target (respiratory system) in contrast to drugs that are injected or ingested. Given the ability of mβCD to solubilize various drugs, it is currently widely used as a vehicle for enhancing drug delivery via nebulization (Evrard et al., 2004; Chandrama Singh et al., 2022). Nebulization with Dyngo4a and mβCD could also be used in conjunction with other therapies, such as antisense oligos, which could be delivered simultaneously or in tandem (Zhu et al., 2022). Our results indicate that targeting the early infection steps of SARS-CoV-2 virus holds promise for the development of therapeutics that are effective in treating mink populations, a strategy that has been suggested for other animals by other virologists (Mercer et al., 2010; Mazzon and Marsh, 2019). Our data further show that when repurposing drugs to prevent SARS-CoV-2 infection in mink, focus should be on the endocytosis pathways.