Little Brown Bats (Myotis lucifugus) Support the Binding of SARS-CoV-2 Spike and Are Likely Susceptible to SARS-CoV-2 Infection

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), believed to have originated from a bat species, can infect a wide range of non-human hosts. Bats are known to harbor hundreds of coronaviruses capable of spillover into human populations. Recent studies have shown a significant variation in the susceptibility among bat species to SARS-CoV-2 infection. We show that little brown bats (LBB) express angiotensin-converting enzyme 2 receptor and the transmembrane serine protease 2, which are accessible to and support SARS-CoV-2 binding. All-atom molecular dynamics (MD) simulations revealed that LBB ACE2 formed strong electrostatic interactions with the RBD similar to human and cat ACE2 proteins. In summary, LBBs, a widely distributed North American bat species, could be at risk of SARS-CoV-2 infection and potentially serve as a natural reservoir. Finally, our framework, combining in vitro and in silico methods, is a useful tool to assess the SARS-CoV-2 susceptibility of bats and other animal species.


Immunohistochemistry
The paraffin-embedded 5µm thick tissue sections were deparaffinized in xylene and rehydrated in ethanol. Following 10 min presoaking of the rehydrated sections in Trisbuffered saline (TBS), sections were subjected to heat-mediated antigen retrieval. Briefly, the slides were placed in 10 mM Tris 1mM EDTA buffer and heated at 90 • C for 10 min, following a cool-down period of 15 min. The antigen retrieved sections were blocked at 4 • C overnight using inactivated goat serum (diluted 25 µL/mL TBS), followed by a TBS wash. ACE2 protein was stained using the primary Anti-ACE2 antibody (Abcam catalog #ab15348), and TMPRSS2 was stained using a recombinant Anti-TMPRSS2 antibody (Abcam catalog #109131) for 1 h.
Following three washes with TBS, sections were further incubated with a secondary Anti-Rabbit IgG H&L antibody (Alexa Fluor 647) (Abcam catalog #ab150075) for 35 min. The sections were washed three times with TBS and mounted in ProLongGold antifade mountant with nuclear stain 4 ,6-diamino 2phenylindole, dihydro-chloride (DAPI). Negative controls were performed, omitting the primary antibody staining. Following 24 h of curing the sections at room temperature, the sections were imaged using Echo Revolve Fluorescent Microscope.

Preparation of Recombinant SARS-CoV-2/RBD Antigen
The recombinant SARS-CoV-2/RBD antigen was produced as detailed earlier [35] for an independent study [36]. Briefly, the plasmid pSL1510 containing SARS-CoV-2 Wuhan spike RBD (pCAGGS-RBD) was transfected in Expi293F cells using Expi293 Expression System (ThermoFisher Scientific catalog #14524) as per the manufacturer's instructions. The resultant protein from the cell culture supernatant was dialyzed in PBS and snap-frozen for storage at −80 • C. The recombinant SARS-CoV-2/RBD antigen was detected using Anti-6X His tag antibody (Abcam catalog #5000).

Virus Binding Assay
Virus binding assay with SARS-CoV-2 pseudovirus was performed as previously described with other RNA viruses such as influenza [37]. Briefly, deparaffinized and antigen-retrieved tissue sections were incubated with SARS-CoV-2 Wuhan pseudovirus for 2 h at 37 • C. Tissues incubated with TBS served as negative controls ( Figure S6). Following the virus incubation, the tissue sections were blocked overnight with inactivated goat serum. The sections were immunostained with primary anti-SARS-CoV-2 spike protein S1 monoclonal antibody (HL6) (ThermoFisher Scientific catalog #MA5-36247). Following 60 min of incubation with the primary antibody, sections were washed and incubated with a secondary Anti-Rabbit IgG H&L antibody (Alexa Fluor 647) (Abcam catalog #ab150075) for 35 min. The sections were washed three times with TBS and mounted in ProLongGold antifade mountant with DAPI. Following 24 h of curing the sections at room temperature, the sections were imaged using Echo Revolve Fluorescent Microscope.

Homology Modeling and Complex Structure Generation Using HADDOCK
ACE2 homology models of LBB and chicken were prepared in the SWISSMODEL [38] webserver using the templates with the highest sequence identities. The 6LZG [39] complex of human-ACE2-RBD was used to identify all polar contacts, and these contacts were specified as constraints for docking in HADDOCK [40] with the default protocol run on their webserver. The models with the least energy of the largest populated cluster were used for further binding energy and MD simulations.

Rosetta Simulations
Rosetta binding energy calculations were performed by first running the relax application with the ref2015 [41] energy function and extracting binding energy (dG_separated) using the InterfaceAnalyzer [42] application. Three hundred independent relax simulations were performed, and the top 10% output structures with the least Rosetta energy of complex were selected for computing the binding energy. Rosetta docking calculations were performed on the ROSIE2 online server [43].

Molecular Dynamics Simulations
Each protein complex was used to start four independent 100-nanosecond trajectories with random initial velocities. The ACE2-RBD (with and without RBD) complexes were prepared for molecular dynamics (MD) simulations using the protein preparation wizard of Maestro [44]. The hydrogen-bonding network was first optimized, and a heavy atomrestrained minimization with the OPLS3e [45] was carried out. Each complex was then solvated with water using the TIP3p [46] model in an orthorhombic box with 10-Å buffer distance in each dimension. The residual charges from the system were neutralized by adding Na + and Cl ions to maintain a 0.15 M salt concentration. MD simulations were performed using the Desmond [47] application within the Schrodinger software suite (v2019.4). The default relaxation protocol of Desmond was performed, followed by 100 ns of production simulations at 1 atm pressure and 300 K temperature, using the NPT ensemble under a periodic boundary condition using particle mesh Ewald [48]. A Time step of 2.0 fs and a nonbonded cut-off threshold of 9-Å were imposed. Finally, the SHAKE [49] algorithm was used to keep all bonds involving hydrogen atoms rigid. All MD trajectory analyses were performed using the Schrodinger suite's MD scripts.

Phylogenetic Analyses
The human, cat, chicken, little brown bat, big brown bat, and Mexican free-tailed bat ACE2 protein sequences were obtained from the GenBank database. The phylogenetic tree was constructed using Geneious prime 2022.1.1 software. The list of species and GenBank IDs is provided in Table S1.

Differential Distribution of ACE2 Receptor in LBB Upper and Lower Respiratory Tract
Abundant tissue distribution of ACE2 protein in the human and cat gastrointestinal tract (GIT) has been described previously [31,50,51]. These studies have demonstrated a higher expression of ACE2 receptors in the small intestine than in the lungs. In contrast, it has been suggested that the chicken ACE2 protein structure varies significantly from other domestic animals and primates and hence may not interact with SARS-CoV-2; consequently, this virus may not infect them [52]. Our results were consistent with these observations (Figure 1). Further, a phylogenetic analysis based on ACE2 protein sequences demonstrated close to 80% similarity in the human and bat ACE2 versus only 65% similarity in the human and chicken ACE2 (Figures 2 and S1). Based on the ACE2 receptor distribution studies and the computational analysis we performed, human and cat intestine tissues were selected to use as positive controls and chicken intestinal tissue as a negative control to validate our ACE2 immunohistochemistry staining in LBB gastrointestinal and respiratory tract.
The human, cat, chicken, little brown bat, big brown bat, and Mexican free-ta ACE2 protein sequences were obtained from the GenBank database. The phylogen was constructed using Geneious prime 2022.1.1 software. The list of species and G IDs is provided in Table S1.

Differential Distribution of ACE2 Receptor in LBB Upper and Lower Respiratory Tr
Abundant tissue distribution of ACE2 protein in the human and cat gastroin tract (GIT) has been described previously [31,50,51]. These studies have demons higher expression of ACE2 receptors in the small intestine than in the lungs. In c it has been suggested that the chicken ACE2 protein structure varies significant other domestic animals and primates and hence may not interact with SARS-CoV sequently, this virus may not infect them [52]. Our results were consistent with th servations (Figure 1). Further, a phylogenetic analysis based on ACE2 protein se demonstrated close to 80% similarity in the human and bat ACE2 versus only 65 larity in the human and chicken ACE2 (Figures 2 and S1). Based on the ACE2 r distribution studies and the computational analysis we performed, human and c tine tissues were selected to use as positive controls and chicken intestinal tissue a ative control to validate our ACE2 immunohistochemistry staining in LBB gastroin and respiratory tract.    Phylogenetic tree based on ACE2 protein sequences for selected species. The tree was constructed using the complete ACE2 protein sequences of North America's three most common bat species, Little brown bats (Myotis lucifugus), Big brown bats (Eptesicus fuscus), Mexican free-tailed bats (Tadarida brasiliensis), and other animal species used in this study (cat, chicken, and human). The Human ACE2 had higher sequence similarities with all the species except for chicken.
Owing to ~80% homology observed between LBB and human ACE2 proteins, the human anti-ACE2 antibody was used for the immunohistology staining. The relative abundance of ACE2 receptors was distinctly varied in the upper and lower respiratory tract of the LBB. While the ACE2-specific immunohistochemistry showed robust expression of ACE2 on the mucosal lining of the LBB trachea ( Figure 3A), the alveolar epithelium, alveolar duct, and visceral pleura of the lung revealed a relatively lower distribution of the ACE2 receptors ( Figure 3B). The lamina propria and submucosa of the tracheal tissue also showed a lesser distribution of the receptors. A total of ten adult and ten juvenile LBB respiratory tissues were investigated alongside the aforementioned positive and negative controls ( Figure S2). No observable difference was noticed in the respiratory tract ACE2 distribution of adult and juvenile LBBs.  Phylogenetic tree based on ACE2 protein sequences for selected species. The tree was constructed using the complete ACE2 protein sequences of North America's three most common bat species, Little brown bats (Myotis lucifugus), Big brown bats (Eptesicus fuscus), Mexican free-tailed bats (Tadarida brasiliensis), and other animal species used in this study (cat, chicken, and human). The Human ACE2 had higher sequence similarities with all the species except for chicken.
Owing to~80% homology observed between LBB and human ACE2 proteins, the human anti-ACE2 antibody was used for the immunohistology staining. The relative abundance of ACE2 receptors was distinctly varied in the upper and lower respiratory tract of the LBB. While the ACE2-specific immunohistochemistry showed robust expression of ACE2 on the mucosal lining of the LBB trachea ( Figure 3A), the alveolar epithelium, alveolar duct, and visceral pleura of the lung revealed a relatively lower distribution of the ACE2 receptors ( Figure 3B). The lamina propria and submucosa of the tracheal tissue also showed a lesser distribution of the receptors. A total of ten adult and ten juvenile LBB respiratory tissues were investigated alongside the aforementioned positive and negative controls ( Figure S2). No observable difference was noticed in the respiratory tract ACE2 distribution of adult and juvenile LBBs. Phylogenetic tree based on ACE2 protein sequences for selected species. The tree was constructed using the complete ACE2 protein sequences of North America's three most common bat species, Little brown bats (Myotis lucifugus), Big brown bats (Eptesicus fuscus), Mexican free-tailed bats (Tadarida brasiliensis), and other animal species used in this study (cat, chicken, and human). The Human ACE2 had higher sequence similarities with all the species except for chicken.
Owing to ~80% homology observed between LBB and human ACE2 proteins, the human anti-ACE2 antibody was used for the immunohistology staining. The relative abundance of ACE2 receptors was distinctly varied in the upper and lower respiratory tract of the LBB. While the ACE2-specific immunohistochemistry showed robust expression of ACE2 on the mucosal lining of the LBB trachea ( Figure 3A), the alveolar epithelium, alveolar duct, and visceral pleura of the lung revealed a relatively lower distribution of the ACE2 receptors ( Figure 3B). The lamina propria and submucosa of the tracheal tissue also showed a lesser distribution of the receptors. A total of ten adult and ten juvenile LBB respiratory tissues were investigated alongside the aforementioned positive and negative controls ( Figure S2). No observable difference was noticed in the respiratory tract ACE2 distribution of adult and juvenile LBBs.

Abundant Expression of ACE2 Receptors in the LBB GIT
The small intestinal tissue sections of LBB, subjected to ACE2-specific immunohistochemistry using the human anti-ACE2 antibody, showed abundant expression of the ACE2 receptor on the intestinal epithelial cells ( Figure 3C). The ACE2 immunolabeling was more prominent in epithelial cells distributed along the apical villi. Whereas the immunolabeling in other tissues of the small intestine was sporadic and low relative to the villus epithelium, including the crypt cells, lamina propria, muscularis, and intestine serosa. Moderate ACE2 was expressed by the secretory cells of the submucosal (Brunner's) glands in the duodenum.

Abundant Expression of TMPRSS2 in LBB Trachea and GIT
Similar to ACE2, previous studies have also shown that a plasma membrane-associated type II transmembrane serine protease (TMPRSS2) promotes SARS-CoV-2 infection [53,54]. TMPRSS2 was also found to be abundantly expressed in the LBB trachea and small intestine of LBB and moderate in the lungs ( Figure S3).

ACE2 Receptors in the LBB Trachea and GIT Support SARS-CoV-2 Binding
We used two alternative approaches to demonstrate the binding interactions between the ACE2 receptor and the SARS-CoV-2 in LBB tissues. In the first approach, virus-receptor binding assays were performed on LBB trachea and intestine tissue as well as the human, cat, and chicken intestine tissue using the SARS-CoV-2 spike pseudovirus (ps-SARS-CoV-2) generated in our laboratory. The LBB ACE2 receptors in the trachea and intestine were compatible with binding to the ps-SARS-CoV-2 ( Figure 4A-C). The virus binding pattern was consistent with the relative abundance of the ACE2 receptors, such that greater ACE2 expression correlated with more significant virus binding. The controls, human and cat intestinal tissue, also demonstrated high and moderate levels of ps-SARS-CoV-2 binding, respectively, to the ACE2 receptors compared to the negative control chicken intestinal tissue ( Figure 4D-F). The mock-treated tissues ( Figure S4) exhibited minimal background staining. In the second approach, trachea and intestinal tissue from bats and humans were also subjected to binding with recombinant SARS-CoV-2/RBD antigen that was produced as described previously [35]. The RBD binding pattern also followed the relative distribution of ACE2 in these tissues ( Figure 5), validating the potential ability of LBB ACE2 to function as an entry receptor for the virus. The negative control tissue for the recombinant SARS-CoV-2/RBD antigen-binding assay showed minimal background staining as no secondary antibody was used in the assay ( Figure S5).

Abundant Expression of ACE2 Receptors in the LBB GIT
The small intestinal tissue sections of LBB, subjected to ACE2-specific immunohistochemistry using the human anti-ACE2 antibody, showed abundant expression of the ACE2 receptor on the intestinal epithelial cells ( Figure 3C). The ACE2 immunolabeling was more prominent in epithelial cells distributed along the apical villi. Whereas the immunolabeling in other tissues of the small intestine was sporadic and low relative to the villus epithelium, including the crypt cells, lamina propria, muscularis, and intestine serosa. Moderate ACE2 was expressed by the secretory cells of the submucosal (Brunner's) glands in the duodenum.

Abundant Expression of TMPRSS2 in LBB Trachea and GIT
Similar to ACE2, previous studies have also shown that a plasma membrane-associated type II transmembrane serine protease (TMPRSS2) promotes SARS-CoV-2 infection [53,54]. TMPRSS2 was also found to be abundantly expressed in the LBB trachea and small intestine of LBB and moderate in the lungs ( Figure S3).

ACE2 Receptors in the LBB Trachea and GIT Support SARS-CoV-2 Binding
We used two alternative approaches to demonstrate the binding interactions between the ACE2 receptor and the SARS-CoV-2 in LBB tissues. In the first approach, virusreceptor binding assays were performed on LBB trachea and intestine tissue as well as the human, cat, and chicken intestine tissue using the SARS-CoV-2 spike pseudovirus (ps-SARS-CoV-2) generated in our laboratory. The LBB ACE2 receptors in the trachea and intestine were compatible with binding to the ps-SARS-CoV-2 ( Figure 4A-C). The virus binding pattern was consistent with the relative abundance of the ACE2 receptors, such that greater ACE2 expression correlated with more significant virus binding. The controls, human and cat intestinal tissue, also demonstrated high and moderate levels of ps-SARS-CoV-2 binding, respectively, to the ACE2 receptors compared to the negative control chicken intestinal tissue ( Figure 4D-F). The mock-treated tissues ( Figure S4) exhibited minimal background staining. In the second approach, trachea and intestinal tissue from bats and humans were also subjected to binding with recombinant SARS-CoV-2/RBD antigen that was produced as described previously [35]. The RBD binding pattern also followed the relative distribution of ACE2 in these tissues ( Figure 5), validating the potential ability of LBB ACE2 to function as an entry receptor for the virus. The negative control tissue for the recombinant SARS-CoV-2/RBD antigen-binding assay showed minimal background staining as no secondary antibody was used in the assay ( Figure S5).  the LBB trachea (A) and intestine (C). The LBB lung tissue (B) showed low-to-moderate binding of the virus. Human and cat jejunum (D,E), positive control tissues, showed preferential binding of the virus to the intestinal epithelium; however, chicken intestine (F), the negative control tissue exhibited minimal virus binding. The tissue sections were stained with rabbit anti-SARS-CoV-2 spike protein S1 monoclonal antibody (red) and DAPI nuclear stain (blue). Scale bar = 170 µm. the LBB trachea (A) and intestine (C). The LBB lung tissue (B) showed low-to-moderate binding of the virus. Human and cat jejunum (D,E), positive control tissues, showed preferential binding of the virus to the intestinal epithelium; however, chicken intestine (F), the negative control tissue exhibited minimal virus binding. The tissue sections were stained with rabbit anti-SARS-CoV-2 spike protein S1 monoclonal antibody (red) and DAPI nuclear stain (blue). Scale bar = 170 µm. The tissue sections were stained with rabbit polyclonal antibody to spike protein (red) and DAPI nuclear stain (blue). Scale bar = 170 µm.

Molecular Simulations Reveal Compatibility of SARS-CoV-2-Spike Receptor Binding Domain with LBB
The three-dimensional structures of the ACE2-RBD complexes for LBB and chicken were prepared using homology modeling [38] and docking refinement using HADDOCK [40] (see methods). The modeled docked complexes were validated for the accuracy of the binding location as described previously [55] based on whether RosettaDock [43] (see methods) produced docking funnels (Figures S7 and S8). The Rosetta binding energies were calculated to predict the strength of binding affinity of ACE2 from different species to SARS-CoV-2 spike RBD (see methods). The mean binding energies of human ACE2-RBD and cat ACE2-RBD complexes were significantly more negative than those of LBB ACE2 and chicken ACE2 ( Figure 6A), indicating comparatively stronger affinities of human and cat ACE2s ('higher' binding energy implies lower affinity and vice-versa). This observation is congruent with the in vitro studies that showed that the LBB ACE2 had approximately 15 times weaker binding affinity compared to that of human ACE2 [30]. However, the binding energy alone does not explain why chicken ACE2 had similar binding energy yet had negative binding in experiments ( Figure 6A). To investigate this further, we performed all-atom molecular dynamics (MD) simulations for all the respective protein complexes (human-ACE2, cat-ACE2, LBB-ACE2, and chicken-ACE2). Four independent replicate simulations starting from different random initial velocities for atoms were performed, yielding a 400 ns simulation trajectory for each complex. All replicate simulation results showed that ACE2 from human, cat, and LBB form stable complexes, while the chicken ACE2 protein shows an unstable complex with RBD. This was further quantified by calculating the root mean square deviation of RBD coordinates (keeping ACE2 coordinates fixed) from its initial position during the simulations ( Figure 6B). These The tissue sections were stained with rabbit polyclonal antibody to spike protein (red) and DAPI nuclear stain (blue). Scale bar = 170 µm.

Molecular Simulations Reveal Compatibility of SARS-CoV-2-Spike Receptor Binding Domain with LBB
The three-dimensional structures of the ACE2-RBD complexes for LBB and chicken were prepared using homology modeling [38] and docking refinement using HADDOCK [40] (see methods). The modeled docked complexes were validated for the accuracy of the binding location as described previously [55] based on whether RosettaDock [43] (see methods) produced docking funnels (Figures S7 and S8). The Rosetta binding energies were calculated to predict the strength of binding affinity of ACE2 from different species to SARS-CoV-2 spike RBD (see methods). The mean binding energies of human ACE2-RBD and cat ACE2-RBD complexes were significantly more negative than those of LBB ACE2 and chicken ACE2 ( Figure 6A), indicating comparatively stronger affinities of human and cat ACE2s ('higher' binding energy implies lower affinity and vice-versa). This observation is congruent with the in vitro studies that showed that the LBB ACE2 had approximately 15 times weaker binding affinity compared to that of human ACE2 [30]. However, the binding energy alone does not explain why chicken ACE2 had similar binding energy yet had negative binding in experiments ( Figure 6A). To investigate this further, we performed all-atom molecular dynamics (MD) simulations for all the respective protein complexes (human-ACE2, cat-ACE2, LBB-ACE2, and chicken-ACE2). Four independent replicate simulations starting from different random initial velocities for atoms were performed, yielding a 400 ns simulation trajectory for each complex. All replicate simulation results showed that ACE2 from human, cat, and LBB form stable complexes, while the chicken ACE2 protein shows an unstable complex with RBD. This was further quantified by calcu- lating the root mean square deviation of RBD coordinates (keeping ACE2 coordinates fixed) from its initial position during the simulations ( Figure 6B). These results suggest that the LBB-ACE2 is predicted to form a stable complex with RBD, which is comparatively weaker than human and cat-ACE2 proteins but is likely strong enough to initiate infection. While the chicken-ACE2 was predicted to have similar binding energy to LBB-ACE2 ( Figure 6A), it did not form a stable complex, suggesting a lack of binding to RBD (Figure 7), which is consistent with experimental data [30,32]. Having confirmed the formation of a stable LBB-ACE2 and RBD complex, we further characterized the functional interactions at the protein-protein interface.
Viruses 2023, 15, 1103 9 of 16 results suggest that the LBB-ACE2 is predicted to form a stable complex with RBD, which is comparatively weaker than human and cat-ACE2 proteins but is likely strong enough to initiate infection. While the chicken-ACE2 was predicted to have similar binding energy to LBB-ACE2 ( Figure 6A), it did not form a stable complex, suggesting a lack of binding to RBD (Figure 7), which is consistent with experimental data [30,32]. Having confirmed the formation of a stable LBB-ACE2 and RBD complex, we further characterized the functional interactions at the protein-protein interface. The human and cat-ACE2 proteins formed 20 and 19 interactions involving salt bridges and hydrogen bonds, respectively, while the LBB and chicken ACE2s formed 10 and 13 interactions, respectively (Figure 7). Interactions that were seen in less than 10% (<0.1 probability) of the frames during the simulations were not counted. The human and cat-ACE2s had ~70% common interactions with the RBD (Figure 7), also corroborating their similar binding energies. Interestingly, the strongest electrostatic interactions D30-K417 formed at the human-ACE2-RBD interface were also formed by cat-ACE2 and LBB-ACE2 as E30-K417 but not by chicken ACE2 (Figure 8), which has hydrophobic residues at the corresponding positions. Similar to cat-ACE2, LBB-ACE2 also forms a salt-bridge interaction with the E484 residue of the RBD (Figure 8B,C). Strong electrostatic interactions such as salt bridges provide dominant long-range forces and guide functional protein-protein association [56]. These results suggest that the lack of strong electrostatic interactions render the chicken ACE2 unable to bind to RBD by destabilizing the complex. The number of frames each interaction formed at the ACE2-RBD interfaces during 400 ns MD simulations is counted and is used to calculate the respective probability as the fraction of frames the interaction is present. The numbering of residues follows the PDB structures 6LZG and 7C8D for the Human ACE2-RBD and cat ACE2-RBD complexes.
For LBB and chicken, the numbering follows the aligned sequences as displayed in Figure S1. The number of frames each interaction formed at the ACE2-RBD interfaces during 400 ns MD simulations is counted and is used to calculate the respective probability as the fraction of frames the interaction is present. The numbering of residues follows the PDB structures 6LZG and 7C8D for the Human ACE2-RBD and cat ACE2-RBD complexes. For LBB and chicken, the numbering follows the aligned sequences as displayed in Figure S1.
The human and cat-ACE2 proteins formed 20 and 19 interactions involving salt bridges and hydrogen bonds, respectively, while the LBB and chicken ACE2s formed 10 and 13 interactions, respectively (Figure 7). Interactions that were seen in less than 10% (<0.1 probability) of the frames during the simulations were not counted. The human and cat-ACE2s had~70% common interactions with the RBD (Figure 7), also corroborating their similar binding energies. Interestingly, the strongest electrostatic interactions D30-K417 formed at the human-ACE2-RBD interface were also formed by cat-ACE2 and LBB-ACE2 as E30-K417 but not by chicken ACE2 (Figure 8), which has hydrophobic residues at the corresponding positions. Similar to cat-ACE2, LBB-ACE2 also forms a salt-bridge interaction with the E484 residue of the RBD (Figure 8B,C). Strong electrostatic interactions such as salt bridges provide dominant long-range forces and guide functional protein-protein association [56]. These results suggest that the lack of strong electrostatic interactions render the chicken ACE2 unable to bind to RBD by destabilizing the complex. The number of frames each interaction formed at the ACE2-RBD interfaces during 400 ns MD simulations is counted and is used to calculate the respective probability as the fraction of frames the interaction is present. The numbering of residues follows the PDB structures 6LZG and 7C8D for the Human ACE2-RBD and cat ACE2-RBD complexes. For LBB and chicken, the numbering follows the aligned sequences as displayed in Figure S1.

Discussion
Bats serve as reservoirs of hundreds of coronaviruses capable of spillover into human populations. Previous studies reported the presence of SARSr-CoVs in various Old-World bat species, including horseshoe bats in the genus Rhinolophus [57]. SARS-CoV-2 is known to infect a wide range of non-human animal hosts. While it is shown that SARS-CoV2 uses ACE2 for entry and TMPRSS2 for S protein priming, other host factors such as neuropilin-1 and receptor tyrosine kinase AXL are also found to facilitate viral entry [54,58,59]. Further, proteases such as cathepsin L and cathepsin B can play an important role in coronavirus entry by cleaving and activating spike protein for the entry [60]. In vitro experimental studies using human cells expressing bat ACE2 orthologues suggest a significant variation in susceptibility among bat species to SARS-CoV-2 infection [61]. Consequently, recent experimental studies show that big brown bats (Eptesicus fuscus) are resistant to SARS-CoV-2 infection. In contrast, Mexican free-tailed bats (Tadarida brasiliensis) [26] and Egyptian Rousette bats (Rousettus aegyptiacus) [25] are found to be susceptible to experimental SARS-CoV-2 infection. However, the susceptibility of several North American bat species to SARS-CoV-2 infection has yet to be fully understood. A key determinant of host susceptibility is the expression of ACE2 receptors on target tissues that are compatible and accessible for virus binding. Using immunohistochemistry, virus binding assays, Rosetta binding energy calculations, and all-atom molecular dynamics (MD) simulations, we show that LBB, a widely distributed bat species in North America, express ACE2 in their

Discussion
Bats serve as reservoirs of hundreds of coronaviruses capable of spillover into human populations. Previous studies reported the presence of SARSr-CoVs in various Old-World bat species, including horseshoe bats in the genus Rhinolophus [57]. SARS-CoV-2 is known to infect a wide range of non-human animal hosts. While it is shown that SARS-CoV2 uses ACE2 for entry and TMPRSS2 for S protein priming, other host factors such as neuropilin-1 and receptor tyrosine kinase AXL are also found to facilitate viral entry [54,58,59]. Further, proteases such as cathepsin L and cathepsin B can play an important role in coronavirus entry by cleaving and activating spike protein for the entry [60]. In vitro experimental studies using human cells expressing bat ACE2 orthologues suggest a significant variation in susceptibility among bat species to SARS-CoV-2 infection [61]. Consequently, recent experimental studies show that big brown bats (Eptesicus fuscus) are resistant to SARS-CoV-2 infection. In contrast, Mexican free-tailed bats (Tadarida brasiliensis) [26] and Egyptian Rousette bats (Rousettus aegyptiacus) [25] are found to be susceptible to experimental SARS-CoV-2 infection. However, the susceptibility of several North American bat species to SARS-CoV-2 infection has yet to be fully understood. A key determinant of host susceptibility is the expression of ACE2 receptors on target tissues that are compatible and accessible for virus binding. Using immunohistochemistry, virus binding assays, Rosetta binding energy calculations, and all-atom molecular dynamics (MD) simulations, we show that LBB, a widely distributed bat species in North America, express ACE2 in their respiratory and GI tracts that support SARS-CoV-2 binding and therefore, could be susceptible to SARS-CoV-2 infection.
We found that LBBs express abundant levels of the ACE2 receptors in their upper airway as well as small intestinal villus epithelium and can functionally bind SARS-CoV-2, indicating the possibility that, should LBBs be exposed to the virus, they could become infected. This raises the possibility that if the infection did occur in wild LBBs, these bats could contribute to the transmission of the virus in North America. The tracheal epithelium in LBB showed prominent expression of ACE2 protein and was observed to bind to both ps-SARS-CoV-2 and recombinant SARS-CoV-2/RBD antigen. Nevertheless, the lower respiratory tract, particularly the lung tissue, only had a low-to-moderate expression of the ACE2 receptors.
With regards to ACE2 protein localization, various human tissues, such as the nasopharynx, lung, stomach, and small intestine, have demonstrated prominent receptor expression [50]. Similarly, ACE2 protein expression has been demonstrated in the gastrointestinal tract of domestic cats [31], whereas independent experimental studies have shown that species such as chickens and ducks are not susceptible to SARS-CoV-2 infection [62]. Based on this evidence, in this study, human and cat intestinal tissues were used as positive control tissues to demonstrate ACE2 protein expression and SARS-CoV-2 binding. In contrast, chicken intestinal tissue was utilized as a negative control.
Molecular simulations are being extensively used to model the ACE2-RBD interactions to predict the susceptibilities of different species to the wild-type SARS-CoV-2 and its different variants [63,64]. Such studies are valuable for large-scale surveillance studies to draw inferences on zoonotic origins [65], host susceptibilities [66], and viral adaptabilities [67]. In this study, we used molecular simulations to predict the binding affinity and the interactions involved in binding the LBB-ACE2 protein with the SARS-CoV-2 RBD. Based on previous observations and reports, corresponding known simulations of the human-ACE2 and cat-ACE2 complexes with RBD were used as positive controls, and those of the chicken ACE2-RBD modeled complex structure were used as negative controls for comparison.
Previously, it has been shown that a kidney cell line derived from the big brown bat [68] and lung cell lines derived from Lander's horseshoe bat (Rhinolophus landeri) and Daubenton's bat (Myotis daubentonii) could not be infected with SARS-CoV-2 [54]. Further, an experimental challenge of big brown bats with SARS-CoV-2 via nasal and oropharyngeal route showed that these bats are resistant to infection with the virus [24]. This could be due to either the overall low ACE2 expression in the respiratory tract or the presence of structurally different ACE2 protein which limits the binding to SARS-CoV-2 [69]. Considering the distinct bat species studied in this regard, the previous observations cannot be directly extended to LBBs or other bat species; nonetheless, our study indicates that there is only a minimal expression of ACE2 in LBB bat lungs and that the virus binding was not as prominent in the lung tissues when compared to that in the upper respiratory tract or parts of the intestines.
Additionally, similar to humans, the ACE2 protein was highly expressed in intestinal epithelial cells lining the villi in LBB and observed to bind to SARS-CoV-2, indicating the functional competence of LBB ACE2 as a SARS-CoV-2 receptor to promote entry into the LBB GIT. In line with our observations, infection of bat intestinal organoids by SARS-CoV-2 has also been demonstrated recently in an organoid culture of Chinese rufous horseshoe bats (Rhinolophus sinicus) [33]. Considering the lack of complete information regarding the ecological, behavioral, and genetic diversity of the bat species under the order Chiroptera, extrapolation of any results needs to be cautiously executed since observations made in one bat species may not hold true for others. Therefore, the potential SARS-CoV-2 infection susceptibility of other North American and, indeed, global bat species in addition to LBBs remains largely unknown. Our limited understanding of the role of bats in SARS-CoV-2 biology raises a critical need for comprehensive surveillance of different bat species for their role in maintaining SARSr-CoVs in the wild.
SARS-CoV-2 continues to evolve and the emerging variants could have altered host tropism and potentially gain the ability to infect new host species. For example, the mouse ACE2 did not bind to the original SARS-CoV-2 spike protein but was bound with higher affinity to spike proteins of the BA.1 and BA.2 variants [47]. Experimentally determined structures of mouse ACE2 bound to BA.1 RBD revealed an interaction network similar to that seen in the human ACE2-RBD complex [70]. Both molecular dynamics-based and forcefield-based methods are well suited for simulating the interactions of protein complexes and predicting mutations in proteins responsible for increasing or decreasing binding affini-ties [71]. However, such methods are less accurate for predicting actual protein-protein binding affinities [72]. Recently emerging machine-learning algorithms are combining structure and force-field calculations to achieve better accuracies at reproducing experimental binding affinities [73,74]. Such methods rely on the presence of high-throughput mutational datasets, which are currently limited to human protein complexes [74,75]. The development of computational surveillance methods focused on animal species is still lacking and is crucial to detect potential spillovers to combat current and emerging pandemics such as COVID-19.
In conclusion, we observed prominent expression of ACE2 in the LBB respiratory and gastrointestinal tract that are accessible for SARS-CoV-2 viruses to bind. We also observed a stable complex formation of LBB-ACE2 with the SARS-CoV-2 RBD but with a weaker binding affinity when compared to the cat-and human-ACE2 proteins in silico.
Our results indicate that LBB, a widely distributed North American bat species, could be at risk of SARS-CoV-2 spillover infection and potentially serve as a natural reservoir and warrant targeted surveillance of LBB. Given the wide variety of bats (1456 species), it is not practically feasible to perform experimental infections to determine their susceptibility to SARS-CoV-2. Therefore, our study provides a practical framework comprising a combination of in vitro and in silico methods to establish the ability of a given animal species to support SARS-CoV-2 binding. Further investigation can be performed to determine the susceptibility of LBB species to SARS-CoV-2 infection using LBB respiratory and gastrointestinal cells. These experiments will also allow a detailed understanding of the disease pathology in this species. The accuracy of molecular simulation models can further be improved by incorporating data from new experiments to validate predictions. Continued use of molecular simulations will allow the prediction of the susceptibility of different species to SARS-CoV-2 and its variants.

Conclusions
Our study utilized a molecular simulation model to predict the binding affinity and interactions involved in LBB ACE2 and SARS-CoV-2 RBD. Further, these observations were validated with experimental studies showing that LBBs express ACE2 in their respiratory and gastrointestinal tracts, indicating their susceptibility to SARS-CoV-2 infection. Additional in vitro and in vivo studies are warranted to determine the susceptibility of other North American and global bat species to SARS-CoV-2 infection, as well as the role of little brown bats in the virus transmission.