Abstract
OBJECTIVE
To examine the susceptibility of cultured primary equine bronchial epithelial cells (EBECs) to a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus relative to human bronchial epithelial cells (HBECs).
SAMPLE
Primary EBEC cultures established from healthy adult horses and commercially sourced human bronchial epithelial cells (HBECs) were used as a positive control.
METHODS
Angiotensin-converting enzyme 2 (ACE2) expression by EBECs was demonstrated using immunofluorescence, western immunoblot, and flow cytometry. EBECs were transduced with a lentivirus pseudotyped with the SARS-CoV-2 spike protein that binds to ACE2 and expresses the enhanced green fluorescent protein (eGFP) as a reporter. Cells were transduced with the pseudovirus at a multiplicity of infection of 0.1 for 6 hours, washed, and maintained in media for 96 hours. After 96 hours, eGFP expression in EBECs was assessed by fluorescence microscopy of cell cultures and quantitative PCR.
RESULTS
ACE2 expression in EBECs detected by immunofluorescence, western immunoblotting, and flow cytometry was lower in EBECs than in HBECs. After 96 hours, eGFP expression in EBECs was demonstrated by fluorescence microscopy, and mean ΔCt values from quantitative PCR were significantly (P < .0001) higher in EBECs (8.78) than HBECs (3.24) indicating lower infectivity in EBECs.
CLINICAL RELEVANCE
Equine respiratory tract cells were susceptible to cell entry with a SARS-CoV-2 pseudovirus. Lower replication efficiency in EBECs suggests that horses are unlikely to be an important zoonotic host of SARS-CoV-2, but viral mutations could render some strains more infective to horses. Serological and virological monitoring of horses in contact with persons shedding SARS-CoV-2 is warranted.
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of the disease known as coronavirus disease 2019 (COVID-19). A wide range of species of domestic animals and wildlife have been infected by SARS-CoV-2.1–4 Human handling of horses results in considerable direct contact between species, including proximity to mucosal surfaces of the mouth and nose during activities such as haltering, placing bits, and feeding. Serological evidence of exposure to SARS-CoV-2 in horses has been documented after exposure to infected humans,5,6 but clinical signs of infection in horses have not been reported to date.
Infection with SARS-CoV-2 primarily occurs by binding of the virus to the angiotensin-converting enzyme 2 (ACE2). The equine and human ACE2 receptors (eACE2 and huACE2, respectively) have high homology (estimated to be 97.3%; Figure 1), including the domain that binds the SARS-CoV-2 spike protein.7–9 Evidence of infectivity of SARS-CoV-2 for horses is conflicting. The virus could neither be isolated nor detected by reverse transcription PCR (RT-PCR) from nasal swabs, rectal swabs, or various tissues collected from a horse after intranasal infection with SARS-CoV-2.10 However, SARS-CoV-2 can infect equine dermal fibroblasts and human cells expressing eACE2 in vitro, although to a lesser extent than cell lines expressing huACE2.8,11–13 Furthermore, seroconversion has been documented in large populations of horses exposed to handlers with COVID-19, although there were no clinical signs of disease or virus recovered from nasal swabs.5 To the authors’ knowledge, infection of equine respiratory tract cells with SARS-CoV-2 or a SARS-CoV-2 pseudovirus has not been reported. We hypothesized that equine bronchial epithelial cells (EBECs) express ACE2 and would be susceptible to transduction with a lentivirus construct expressing the spike protein of SARS-CoV-2 that binds to ACE2. Our objectives were to characterize the expression of ACE2 by EBECs using western immunoblotting, flow cytometry, and immunofluorescence and to qualitatively and semi-quantitatively characterize susceptibility to pseudovirus transduction of EBECs relative to human respiratory tract cells.
Angiotensin-converting enzyme 2 (ACE2) protein sequences for various species and horses, and their similarity to human ACE2 (huACE2). (A) ACE2 protein sequence alignment of species based on likely human contact and exposure (ie, pets and livestock) or wild animals documented as susceptible to infection (eg, bats and whitetail deer), using a scheme adapted from a previous report14 and updated to include horses (Supplementary Table S1). Overall sequence homology to huACE2, and specific residues identified as critical for interaction between the huACE2 protein and the receptor binding domain of SARS-CoV-2 (using a scheme adapted from Gultom et al14), were compared and ranked vertically by overall % homology of the ACE2 sequence to huACE2; amino acid changes are denoted by colored blocks, and * denotes a predicted sequence. (B) Ribbon representation of the huACE2–Wuhan receptor binding domain (RBD, pale green and green) and eqACE2–Wuhan RBD (pale blue and green) complex structures. The ACE2 regions Ser19–Ala614 were used to fit the complexes. The very conserved H-bonds between ACE2 Lys353 atom O and RBD Gly502 atom HN are shown as dotted black lines. The interface stabilizing ionic interactions between Asp/Glu 30 and Lys417 are indicated as blue bonds. (C) ACE2 side of the human/equine ACE2–Wuhan RBD interface. Human and equine ribbon representations are shown in pale green and pale blue, respectively. Sidechains of the contact amino acids are shown in bond representations. Bonds present in both complexes are shown in turquoise. The bonds of Gly354, which is part of the interface in the huACE2–Wuhan RBD complex alone, are shown in pale green while the bonds of Thr82, Arg357, and Arg393, which are part of the interface in the eqACE2–Wuhan RBD complex alone, are shown in sky blue. (D) RBD side of the human/equine ACE2–Wuhan RBD interface. The ribbons for the Wuhan RBD in both complexes are shown in green. Side-chains of the contact amino acids are shown in green bond representations. Bonds of Ala465 and Phe486 are part of the interface in the equine ACE2–Wuhan RBD complex alone. They are shown in thinner green bond representations. (E) Ribbon representations of the complexes of human/equine ACE2 and Wuhan/delta/omicron RBD. In this representation, the regions of Thr333–GLY526 were used to fit the complexes. HuACE2 and eqACE2 are shown in pale green and pale blue ribbons. Wuhan, delta, and omicron RBD’s are shown in green, brown, and light gold ribbons, respectively. The interface stabilizing ionic interactions in the Wuhan and Delta RBD complexes are indicated with the blue number 1. The corresponding ionic interactions in the huACE2–omicron RBD and eqACE2–omicron RBD are indicated with the blue numbers 2 and 3.
Citation: American Journal of Veterinary Research 84, 9; 10.2460/ajvr.23.06.0132
Methods
Ethics statement
All methods were performed in accordance with relevant guidelines and regulations for animal use and for laboratory practices including environmental health, occupational safety, and biosafety (Texas A&M University Infectious Biohazard Committee IBC# 2017-105). Bronchial epithelial cells were harvested from horses euthanized for humane reasons unrelated to disease of the respiratory tract in accordance with practices approved by the Texas A&M University Institutional Animal Care and Use Committee.
eACE2 protein sequence modeling
The ACE2 protein sequences for mammalian and avian species, selected based on likely human contact and exposure (ie, pets and livestock) or wild animals documented as susceptible to infection (eg, bats and whitetail deer), using a scheme adapted from a previous report14 and updated to include horses, were retrieved from the National Center for Biotechnology Information Protein Database (NCBI, https://www.ncbi.nlm.nih.gov/, accession data; Supplementary Table S1). Sequence alignment was performed using the Clustal Omega plugin (http://www.clustal.org/omega) in Geneious Prime (Version 2021.2.2; https://www.geneious.com/) with default settings. Overall sequence homology to huACE2 (NCBI accession, #BDH16358.1), and specific amino acid residues identified as critical for the interaction between the huACE2 protein and the receptor binding domain (RBD) of SARS-CoV-2 (adapted from the work of Gultom et al14) were compared amongst these species.
Next, complex crystal structures for ACE2 peptidase domains (ACE2-PD) and SARS-CoV-2 RBD’s were downloaded from the PDB-databank (https://www.rcsb.org). For human ACE2-PD, complexes were found with the Wuhan (6m0j), Delta (7wbq), and Omicron (7wbl) RBD variants. For equine ACE2-PD, complexes were found only with Wuhan (7cf5) and Omicron (7xby) RBD variants. Structural fits, visualizations, H-bond, and ionic interaction analyses, and determinations of the interface amino acids were performed using the software MOLMOL.15
Culture of EBECs
EBECs were harvested postmortem from 5 adult horses (aged 11–19 years; 3 geldings and 2 mares) with no history of respiratory disease, absence of abnormal findings referent to the upper or lower respiratory tract from physical examination at the time of euthanasia, results of complete blood count, and gross findings of the lungs postmortem. EBECs from 2 horses were used for western immunoblotting, flow cytometry, and immunofluorescence to detect ACE2 and from 3 horses for pseudovirus infection studies. Primary cultures were established as previously described.16,17 The lungs were removed en bloc within 1 hour of euthanasia, infused with 1 liter of ice-cold Hank’s buffered saline solution (HBSS) with penicillin (200 U/mL), streptomycin (200 µg/mL), and amphotericin B (2.5 µg/mL) into the trachea, and transported to the laboratory on ice. Lung parenchyma was bluntly removed to isolate primary and secondary bronchi, which were then sectioned in roughly 5-cm tubular segments and submerged for 30 minutes in ice-cold HBSS with penicillin (200 U/mL), streptomycin (200 µg/mL), and amphotericin B (2.5 µg/mL). The bronchial epithelium was sharply dissected from the underlying submucosa and then minced into approximately 1-mm2 segments and divided into batches of roughly 500 mg minced tissue. Each batch of tissue was placed into a Petri dish with 12 mL of 0.25% trypsin–0.6 mM EDTA solution and incubated at 37 °C for 2 hours in 5% CO2 under gentle agitation. Digestion was stopped by adding 5 mL ice-cold 20% fetal bovine serum (FBS) in HBSS. The suspension was filtered through sterile double-layered gauze, then a sterile cell strainer (pore size 40 µm). The cellular suspension was centrifuged at 250 X g for 10 minutes at 4 °C, the supernatant was discarded, and cells were resuspended in 12 mL warm supplemented airway culture media (Airway Epithelial Cell Growth Basal Media, Promocell; supplemented with Growth Medium SupplementPack [Promocell], 10% FBS, penicillin [200 U/mL], streptomycin [200 µg/mL], and amphotericin B [2.5 µg/mL]). This suspension was placed into a treated T75 cell culture flask and incubated for 30 minutes at 37 °C in a humidified, 5% CO2 environment. The media was gently removed from the T75 flasks, allowing adhered fibroblasts to remain within the flask, and the cellular suspension containing EBECs was centrifuged at 200 X g for 10 minutes at 4 °C. The supernatant was discarded, and cells were resuspended in 1 mL of supplemented airway culture media. Cells were counted using a cell counter (CellometerAuto T4, Nexcelom Bioscience) with trypan blue for viability assessment, and plated at a concentration of 2.5 X 105 live cells/milliliter on a collagen-coated tissue-culture-treated 24-well plate. Media was changed at 24 hours, and then every 2 days afterward.
Culture of HBECs
Human bronchial epithelial cells (HBECs, Promocell) were acquired as a cryopreserved positive control cell line. Primary cultures were established by thawing the cryovial in a 37 °C water bath for 2 minutes and resuspending cells into warmed airway culture media (Airway Epithelial Cell Growth Basal Media, Promocell; supplemented with Growth Medium SupplementPack, Promocell), and plated at a density of 1.5 X 103 cells/cm2 into a T25 flask. Media was changed at 24 hours, and then every 2 days afterward. Subsequent passages were plated into 24-well plates in parallel with EBECs to serve as a positive control primary cell line.
Detection of ACE2 expression via western immunoblotting
EBECs were lysed with cold radioimmunoprecipitation assay (RIPA) lysis buffer and protease inhibitor (Sigma Aldrich), then separated on 12% SDS-PAGE gels under reducing conditions. Membranes were transferred to polyvinylidene difluoride (PVDF) membranes, blocked with 5% dried nonfat milk in 1% Tween-20 and phosphate-buffered saline (PBS), and probed with a polyclonal rabbit anti-huACE2 antibody targeting the C-terminus (ProSci; Cat #3217; 1:1,000 dilution) and a peroxidase-conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories; Cat #711-035-152; 1:6,000 dilution). Lysate of human lung adenocarcinoma A549 cells (ATCC; Cat #CCL-185) was used as a positive control as they have been demonstrated to express ACE2 and can be infected with SARS-CoV-2.18–21 For loading control, a separate aliquot of each sample during each immunoblot was probed with a monoclonal mouse anti-human GAPDH antibody (Invitrogen; #43700; 1:500 dilution) and a peroxidase-conjugated goat anti-mouse secondary antibody (Abcam; Cat #6789; 1:5,000 dilution). Protein bands were developed using Radiance Plus® Femtogram HRP Substrate (Azure Biosystems) and visualized using the Bio-Rad Chemidoc Touch imaging system.
Detection of ACE2 expression via flow cytometry
EBECs and HBECs were washed in PBS, lifted with Accutase (StemCell Technologies), counted, and divided into individual aliquots containing 1 X 106 cells in 100 µL PBS containing 2% FBS. Cells were incubated with affinity-purified polyclonal goat anti-huACE2 antibody recognizing the extracellular domain (R&D Systems; Cat #AF933; 1:40 dilution) for 30 minutes, washed twice, and resuspended with 300 µL of PBS containing 2% FBS. Cells were then stained with a secondary rabbit anti-goat Alexa Fluor 555 antibody (Invitrogen; Cat #A32732; 1:400 dilution) for 30 minutes at room temperature, washed twice, and resuspended in 100 µL PBS containing 2% FBS. The cells were then fixed with 2% paraformaldehyde for 30 minutes. The ACE2 Alexa Fluor 555 labeled fixed EBECs and HBECs were run on a Luminex/Amnis Image Stream X Mark II imaging flow cytometer to measure the amount of ACE2 expressed in the cells. Settings for cytometry using the INSPIRE (Luminex/Amnis) acquisition software were as follows: (1) the 40X objective was used to collect the images and light from the cells interrogated by the laser; (2) the 488-nm laser was set at 100 mW of power; (3) the side-scatter laser (785 nm) was set at 2 mW of power; (4) the core diameter was set at 10 μm; and, (5) the fluid velocity was set at 132 mm/sec. At least 10,000 events were collected for each sample. IDEAS software (version 6.3, Luminex/Amnis) was used to analyze the data collected from the Image Stream. The gating strategy (Supplementary Figure S1) was set up with singlets being selected with the y-axis being the aspect ratio of channel 1 bright-field and the x-axis being the area of channel 1 bright-field. Cells with an aspect ratio >0.6 were selected as single cells. The single cells were then gated for in-focus cells by the cells with the greater gradient RMS values in channel 1 bright-field. The single, focused cells were then observed for AlexaFluor 555-positive cells by plotting the intensity of channel 2 (FITC channel) on the y-axis and the intensity of channel 3 (Alexa Fluor 555) on the x-axis. The ACE2 Alexa Fluor 555-positive cells were gated as the population shifted toward the Alexa Fluor 555 direction away from the autofluorescence background of the cells. The proportions of ACE2-positive EBECs and HBECs were compared using a χ2 test.
Detection of ACE2 expression using immunofluorescence
EBECs were plated at a density of 250,000 cells/chamber in 0.1% collagen-coated ibiTreat µ-Slide 2-well chambered coverslips (Ibidi) and allowed to grow over 72 hours, changing media daily. Cells were then washed with PBS, fixed with PBS-buffered 4% paraformaldehyde solution, and blocked with 10% goat serum in PBS for 2 hours. Slides were then incubated with primary polyclonal rabbit anti-huACE2 antibody (ProSci; Cat #3217) at 1:240 dilution in 1% BSA overnight. After incubation, cells were washed 3 times with PBS and incubated with secondary goat antirabbit Alexa Fluor 488 antibody (Invitrogen; Cat #A11008) at 1:500 dilution in 1% BSA for 2 hours, followed by 2 µg/mL of Hoechst 33342 (Invitrogen) in 1% BSA for 1 minute. After incubation, cells were washed 3 times with PBS, treated with 1 drop of Prolong Gold Antifade Reagent (Invitrogen), and covered with a micro-coverslip. An additional set of fixed EBECs were permeabilized with 0.1% Triton-100X in PBS for 12 minutes at room temperature, washed 3 times with PBS, and blocked with 10% goat serum in PBS for 2 hours. To confirm epithelial cell type, permeabilized EBECs were incubated with murine AE1/AE3 pan-cytokeratin antibody (Biocare Medical; Cat #CM011A) at 1:100 dilution in 1% BSA in PBS at 4 °C overnight, followed by a secondary goat anti-mouse Alexa Fluor 594 antibody (Cell Signaling Technologies; Cat #8890; 1:500 dilution) for 2 hours at 4 °C before applying a coverslip as previously described. Slides were imaged immediately after staining using a Zeiss 780 confocal microscope with laser excitation of 405 nm and 594 nm or 405 nam and 594 nm, and emission was collected using wavelengths of 410–480 nm and 596–700 nm or 410–480 and 596–700 nm, respectively. In a subsequent experiment, EBECs and HBECs were plated at the same density and under similar conditions, and processed as previously described, with the exception of secondary staining with secondary goat anti-rabbit Alexa Fluor 555 antibody (Invitrogen; Cat #A32732) at 1:500 dilution in 1% BSA for 2 hours, followed by 2 µg/mL of Hoechst 33342 in 1% BSA for 1 minute. Laser excitation wavelengths were 405 nm and 561 m and emission wavelengths of 410–480 nm and 575–700 nm, respectively.
Pseudovirus transduction of EBECs
A commercially acquired lentiviral construct pseudotyped with the spike (S) protein of SARS-CoV-2 (SARS-CoV-2 isolate Wuhan-Hu-1; GenBank accession no. NC_045512.2) and expressing enhanced green fluorescence protein (eGFP) (VectorBuilder; vector VB900088-2229upx) as a reporter was used. Transduction using approximately 132,000 transduction units (TU) of pseudovirus and 1 µg/mL polybrene (VectorBuilder) was performed in cultured EBECs derived from 3 horses; HBECs obtained commercially were included as positive controls. Transduced and nontransduced EBECs and HBECs were examined after 96 hours by fluorescence microscopy for evidence of green fluorescence. Images were obtained with an Evos FL microscope, using the EVOS GFP light cube (ThermoFisher Scientific), with excitation of 482/25 nm and emission of 524/24 nm. RNA was extracted from bronchial cells treated with TRIzol reagent (ThermoFisher) using a commercial kit (Zymo Research; Direct-zol DNA/RNA Miniprep Kit) according to the manufacturer’s instructions. DNA was reverse-transcribed from 0.5 µg of RNA using a commercial kit (Quiagen; RT2 First Strand Kit). Quantification of transduction of EBECs and HBECs with the SARS-CoV-2 pseudovirus was determined by real-time qPCR (rt-PCR) for gene expression of the enhanced green fluorescence protein gene (egfp); expression of the β-2 microglobulin gene (B2M) was used as a comparative reference. A no-template preparation (negative control) and a plasmid encoding egfp (positive control, pClneoEGFP human RASSF6b, Addgene plasmid #37021) along with nontransduced EBECs and HBECs were included. The rt-PCR reaction used 1 μL containing 50 ng of cDNA, added to 5 μL of a commercial master mix (Applied Biosystems; TaqMan Fast Advanced), 0.5 μL of a primer/probe premix against either eGFP, equine, or human B2M (Applied Biosystems; TaqMan Gene Expression Assays; for YFP∼YFP (eGFP): FAM-MGB, assay# Mr04097229_mr; for equine B2M: FAM-MGB, assay# Ec03468699_m1; for human B2M: FAM-MGB, assay# Hs00187842_m1), and 3.5 μL of buffered nuclease-free water. Each condition was repeated in triplicate, and each sample from the 3 experiments was tested in triplicate by qPCR. Samples were processed using commercial software for rt-PCR (QuantStudio Real-Time PCR Software v1.3, Applied Biosystems). Ct values of egfp expression were compared among treatments using generalized linear modeling, with post hoc comparisons made using the method of Tukey. The ΔCt values (ie, Ct[egfp] – Ct[B2M]) were determined for EBECs and HBECs and compared using the Wilcoxon rank sum test.
Results
eACE2 sequence analysis
To characterize the equine ACE2 sequence in silico, the ACE2 protein sequences for various species, selected based on likely human contact and exposure (ie, pets and livestock) or wild animals documented as susceptible to infection (eg, bats and whitetail deer), using a scheme adapted from a previous report14 and updated to include horses, were retrieved from the National Center for Biotechnology Information Protein Database (NCBI, accession data; Supplementary Table S1). When these sequences were aligned for overall sequence homology to huACE2 and compared at specific residues identified as critical for the interaction between the huACE2 protein and the receptor binding domain of SARS-CoV-2 (methods adapted from Gultom et al14), horses had greater overall homology than species known to be capable of becoming infected and shedding virus (Figure 1), although amino acids at critical residues varied among species.
We compared the linear amino acid sequences of human and equine ACE2-PD (Ser19 – Ala614). Both ACE2-PD’s differed only at 87 positions (= 14.7% of positions) and this number decreased to 16 positions (= 2.7%) when we excluded conservative substitutions such as Asp into Glu. Given this very high sequence identity (85.3%) and homology (97.3%), we expected very similar 3D structures for both proteins as reflected by our 3D-structural fits of the ACE2-PD backbone atoms (Figure 1; Supplementary Table S2). The pairwise root mean square displacements (RMSD) between the ACE2-PD backbone atoms were calculated using the crystal structure complexes of Wuhan RBD - human ACE2 (6m0j), Wuhan RBD - equine ACE2 (7fc5), delta RBD - human ACE2 (7wbq), omicron RBD - human ACE2 (7wbl), and omicron RBD - equine ACE2 (7xby) were between 0.59Å and 1.25Å (Supplementary Table S2). Similarly, the pairwise RMSD’s of the RBD’s in these 3D structures were between 0.47Å and 1.73Å (Supplementary Table S3). More importantly, the pairwise RMSD’s of the RBD’s were still only between 1.37Å and 3.62Å when we used the ACE2-PD parts for fitting and moved the RBD’s along (Supplementary Table S4). Note that the resolutions of these crystal structures were in the same range (2.45Å–3.40Å). These results showed that the 3D structures of the human and equine ACE2 had overall the same structure. The 3D structure of the different SARS-CoV-2 variant RBD’s were also the same. Furthermore, the orientations of the ACE2-PD’s relative to the RBD’s in these complexes were the same. In all 5 ACE2-RBD complexes, there was a very strongly conserved H-bond bridging the ACE2 and RBD backbones at Lys353 and Gly502. The distances between the donor on the RBD side (HN atom) and the acceptor on the ACE2 side (O atom) were 1.56Å–2.15Å. Also, the angles between the line from the atom connected to the donor and the donor and the line from the atom connected to the donor and the acceptor were 4.4°–9.2°. There were also conserved ionic interactions between human/equine ACE2 and Wuhan/delta RBD’s (Asp/Glu30 on the ACE2 side and Lys417 on the RBD side). Similar ionic interactions were present also within ACE2-Omicron RBD further towards the C-terminus on the central interface helix on the ACE2 side. The contact in the human ACE2-omicron RBD was between Glu35 and Arg493 and in equine ACE2-omicron RBD between Glu38 and Arg498. We defined the overall ACE2-RBD interface as contacts between any nonhydrogen atom pairs within 3.5Å across the interface. The interfaces between human/equine ACE2 and Wuhan RBD were conserved with 14 amino acid contacts on each side of the interface (Figure 1; Supplementary Tables S4 and S5). On the ACE2 side, the human ACE2-Wuhan complex had 1 more contact at Gly354, and the equine ACE2-Wuhan RBD 3 more contacts at Thr82, Arg 357, and Arg393. On the RBD side, only the equine ACE2-Wuhan RBD had 2 more contacts at Ala475 and Phe486. The human ACE2–delta RDB interface featured the same core contacts as the human/equine ACE2–Wuhan RBD interface; however, the contacts to Thr27 and Glu35 on the ACE2 side were missing (Supplementary Tables S5 and S6). The human/equine ACE2 – omicron RBD interfaces were different from the human/equine ACE2–Wuhan RBD interface. They still shared 7/14 and 10/14 of the human/equine ACE2–Wuhan RBD core contacts on the ACE2 and RBD side.
ACE2 expression
To demonstrate ACE2 expression in equine airway tissues, primary EBEC cultures were established according to previously described methods,16,17 and human bronchial epithelial cells (HBECs, Promocell) were cultured in parallel as a positive control. Expression of ACE2 was detected in EBECs by western immunoblot (Figure 2), with strongest bands detected in EBECs for the 90-kDa isoform, weakly at the 130-kDa isoform (strongest in human lung tissues22), and not detected at the 55-kDa isoform. ACE2 expression was also detected in EBECs by fluorescence immunocytochemistry (Figure 3) after confirmation of epithelial cell type by cytokeratin staining. Flow cytometry of ACE2 indicated significantly (P < .0001) fewer EBECs (13.4%) were positive for ACE2 compared with HBECs (35.3%; Figure 4).
Western immunoblot for ACE2 expression in equine bronchial epithelial cells (EBECs). Lysates of EBECs and human lung adenocarcinoma cells (A549, positive control) were probed with rabbit anti-huACE2 antibody targeting the C-terminus. Bands were strongest in EBECs for the 90-kDa isotype, weak for the 130-kDa isotype (strongest in human lung tissues21), and not detected at the 55-kDa isotype. Experiments were performed with EBECs from 2 unrelated horses. An additional aliquot of each sample was probed concurrently with mouse anti-human GAPDH antibody as loading control.
Citation: American Journal of Veterinary Research 84, 9; 10.2460/ajvr.23.06.0132
Fluorescent immunocytochemistry for expression of ACE2 and cytokeratin in equine bronchial epithelial cells (EBECs) and human bronchial epithelial cells (HBECs). Permeabilized EBECs were stained to confirm epithelial cell type (A, C) with monoclonal murine anti-cytokeratin AE1/AE3 antibody. Nonpermeabilized EBECs were stained for ACE2 expression (B, D) with rabbit anti-huACE2 antibody. Cells were imaged with a 10X lens (A, B; line scale = 200 µm) and with 20X lens (C, D; line scale = 50 µm). In a subsequent experiment, EBECs and HBECs were stained for ACE2 (E, F) with huACE2 antibody. Both were imaged with 40X lens, but EBECs were shown at higher magnification (F) than HBECs (E) to demonstrate staining detail (line scale = 50 µm in both images).
Citation: American Journal of Veterinary Research 84, 9; 10.2460/ajvr.23.06.0132
Imaging flow cytometry of ACE2 expression in equine bronchial epithelial cells (EBECs) and human bronchial epithelial cells (HBECs). Cultured EBECs and HBECs were fixed and stained with an affinity-purified polyclonal goat anti-huACE2 antibody targeting the extracellular domain. Gating of ACE2 positive cells indicated significantly (P < .0001) fewer ACE-2 positive cells in EBECs (13.4%) than in HBECs (35.3%).
Citation: American Journal of Veterinary Research 84, 9; 10.2460/ajvr.23.06.0132
Pseudovirus transduction
To demonstrate susceptibility to SARS-CoV-2, the primary cultures of EBECs and HBECs were treated with a lentivirus pseudotyped with the SARS-CoV-2 spike protein and expressing enhanced green fluorescent protein (eGFP) as a reporter at an MOI of 0.1 for 6 hours. After 96 hours, fluorescence microscopy demonstrated green fluorescence in EBECs and HBECs transduced with the pseudovirus construct encoding eGFP (Figure 5); no green fluorescence was observed in nontransduced cells. To compare susceptibility between equine and human cells, qPCR was performed to quantify the eGFP expression within transduced cells, relative to expression of a housekeeping gene (β-2-microglobulin, B2M). eGFP Ct values were significantly (P < .0001) lower in transduced than nontransduced EBECs and HBECs, indicating significantly higher expression of egfp in transduced cells (Figure 6). The ΔCt (ie, Ct[egfp] – Ct[B2M]) values were significantly smaller for HBECs (mean = 3.24; SD = 0.11) than EBECs (mean = 8.78; SD = 0.88) indicating greater relative expression, and greater transduction, of egfp in HBECs than EBECs.
eGFP expression of equine bronchial epithelial cells (EBECs) and human bronchial epithelial cells (HBECs) imaged by fluorescent microscopy, after transduction with lentivirus pseudotyped with SARS-CoV-2 spike protein and expressing eGFP as a reporter. Cultured cells were transduced and imaged after 96 hours in brightfield and GFP channels. Fluorescence-positive cells indicate successful pseudovirus cell entry and subsequent expression of eGFP within epithelial cells competent to transduction. Representative images from experiments successfully repeated in 2 unrelated horses.
Citation: American Journal of Veterinary Research 84, 9; 10.2460/ajvr.23.06.0132
Quantitative PCR for expression of egfp and β-2-microglobulin (B2M, housekeeping gene) in EBECs and HBECs, compared with a nontemplate control (NTC) and a DNA plasmid expressing egfp as positive control. Each condition was tested in triplicate, and each sample also was tested in triplicate. (A) Lower Ct values indicate higher egfp expression in infected wells compared with uninfected wells, but no difference between EBECs and HBECs. (B) The ΔCt values (Ct[egfp] – Ct[B2M]) indicate higher egfp expression in infected HBEC cells compared with infected EBECs. Error bars indicate standard deviations.
Citation: American Journal of Veterinary Research 84, 9; 10.2460/ajvr.23.06.0132
Discussion
Our findings indicate that EBECs can be transduced with a pseudovirus expressing the SARS-CoV-2 S protein, suggesting that horses can be infected with SARS-CoV-2. These findings are consistent with recent reports of seroconversion to SARS-CoV-2 in horses.5,6,23 Our results also concur with in vitro evidence of SARS-CoV-2 infection in cells expressing eACE2, both in human cell lines transfected to express the protein8,11,13 and in an equine dermal fibroblast cell line expressing ACE2.12 Our results are plausible given the high homology between the eACE2 and huACE2, and similarity in the critical regions of the interaction site between the RBD of the S protein and eACE2 (Figure 1).9,11,24 Human and equine ACE2-PD amino acid sequences had high homology (97.3%) and overall the same 3D structure. The interface to the Wuhan RBD featured 14/18 identical contacts. The RBD orientation and mode in the complexes were also the same. Using a primary culture of EBECs isolated from healthy horses to represent a site of primary aerosol exposure to inhaled viruses in horses, we demonstrated susceptibility to cell entry with pseudovirus in all 3 horses tested.
The lower efficiency of transduction for EBECs than HBECs indicates that horses might be less susceptible to infection than humans. Our results regarding the relative efficiency of cell entry into EBECs and HBECs should be interpreted with caution because of the small number of horses tested and the differences between EBECs harvested from horses and commercially sourced HBECs. However, our findings of lower infectivity are consistent with reports suggesting that horses are not highly susceptible to infection with SARS-CoV-2. Experimental infection with SARS-CoV-2 was unsuccessful in a single horse10 and the virus could not be detected in horses with exposure to persons testing positive for SARS-CoV-2 by rt-PCR25 or serological testing.5 Lower susceptibility of horses to infection with SARS-CoV-2 was predicted by crystallographic modeling of eACE226 and was reported for nonrespiratory cell lines expressing eACE2. The probability of SARS-CoV-2 entry via eACE2 has been predicted using crystallography modeling but reported estimates have ranged from >90%27 to 48%; the latter value was comparable with that estimated for mice (viz., predicted entry probability of 50%), a species not used as a research model because they are considered to be at low risk of infection with SARS-CoV-2.26 In another study, binding affinity between eACE2 and the RBD of the S protein of wild-type SARS-CoV-2 was ∼32 times lower than that of huACE2.9 Notably, however, this difference was halved by a single amino acid substitution in eACE2; this finding highlights the potential for some viral variants with altered binding affinity to be more infectious in horses. Human cell lines expressing eACE2 have been demonstrated to be less susceptible to infection with SARS-CoV-2 than cells expressing huACE2.8,11,13 An equine dermal fibroblast cell line was successfully infected in vitro with SARS-CoV-2 with lower efficiency than human cell lines12; however, dermal fibroblasts may not be representative of the respiratory epithelium (the in vivo target tissue for aerosol infection). The reasons why equine respiratory epithelial cells appear less susceptible to the SARS-CoV-2 pseudovirus than human respiratory epithelial cells despite the high homology of the ACE2 receptor is unknown. Variations as small as a single amino acid in the sequence of eACE2 can lead to the markedly increased binding affinity of the S protein of SARS-CoV-2.9 As depicted (Figure 1), some species that are highly susceptible to SARS-CoV-2 such as mink and ferrets have lower homology with human ACE2 than horses.14 Transmembrane protease, serine 2 (TMPRSS2) plays a critical role in mediating cell entry of SARS-CoV-2,18,20,24 and differences in the sequence or expression of this protein between horses and humans could explain the difference. Alternatively, differences in survival in the endosomal compartments of horses and humans could explain differences in infectivity between horses and humans. Conceivably, horses might be susceptible to infection with SARS-CoV-2 but with less efficient respiratory tract cell entry and replication than humans, resulting in transient, subclinical infection.
Several SARS-CoV-2 pseudovirus constructs expressing the S protein of SARS-CoV-2 variants of concern were able to enter cells expressing eACE2 more readily than that of pseudovirus with the wild-type S protein, and the efficiency of cell entry by the pseudovirus of the delta strain was equivalent to that in huACE2-expressing cells.8 This increased susceptibility with more variants of concern may also be the case in EBECs, based on our 3D models demonstrating similar binding between the human ACE2-RBD and equine ACE2-RBD of delta and omicron (Figure 1). However, our investigation was constrained to a lentivirus pseudotyped with the S protein of the wild-type strain (SARS-CoV-2 isolate Wuhan-Hu-1) as a corollary of infection, which may have limited cell entry into EBECs. Thus, it is possible that variants of SARS-CoV-2 might emerge that can more readily infect equine airways.
Our study has several limitations. We used a pseudovirus to demonstrate infectivity rather than the SARS-CoV-2 virus. The extent to which cell entry by pseudovirus represents infection with strains of SARS-CoV-2 and whether the virus can persist within infected EBECs remain unknown. The use of a pseudovirus had the advantage of enabling us to work at biosafety level 2 conditions to demonstrate the potential for infection of equine respiratory epithelial cells expressing ACE2. This pseudovirus reporter system has been used as a corollary of infection in cells expressing eACE213 and is a standard assay for rapidly evaluating the efficiency of cell entry and neutralization with SARS-CoV-2 for vaccine development.28,29 The concordance of our findings of lower efficiency of cell entry of EBECS than HBECs with the pseudovirus with results of lower infectivity of SARS-CoV-2 of cells expressing eACE2 than hACE23,8,11 indicates that the pseudovirus is a valid surrogate for SARS-CoV-2. We detected ACE2 in equine cells using antibodies against huACE2. This might have lowered sensitivity for detecting eACE2 in equine tissues. Our cell culture model was maintained in submerged conditions, leading to partial differentiation and fewer ciliated cells than what would be achieved either by an air-liquid interface culture system or what might be expected in vivo. Submerged conditions can decrease ACE2 expression in human respiratory epithelial cells, thereby decreasing infection with SARS-CoV-2 virus30; however, submerged conditions have been used successfully for SARS-CoV-2 pseudovirus transductions in other cell lines8,11 and for SARS-CoV-2 infection of respiratory cell lines.7 Nevertheless, the use of an air-liquid interface format to enable more complete differentiation of respiratory epithelial cells might more accurately reflect the susceptibility of equine airways to SARS-CoV-2 infection. We did not examine the extent to which eACE2 is expressed by other regions of the equine respiratory tract. Ciliated cells have higher ACE2 expression and tropism for SARS-CoV-2 infection in humans and other species,31–33 but this has not yet been determined for horses. Expression of ACE2 varies among regions of the respiratory tract of humans and several domestic animal species, with the greatest levels apically in the nasopharynx, decreasing levels in the conducting airways and lower airways, and then higher expression in alveolar cells.28–29 This pattern of ACE2 expression appears to be a limiting factor for susceptibility to infection in vitro and in vivo.34–37 Differences in the expression of eACE2 among regions of the equine airway have not been well characterized. Expression of eACE2 in the alveoli and bronchi was not detected by immunohistochemistry in a single horse.33 However, ACE2 mRNA expression was detected in equine tracheal and lung tissue lysates.35 We have detected ACE2 expression using immunohistochemistry in equine alveoli and bronchi (unpublished data). Further investigation of variation in eACE2 expression and susceptibility to SARS-CoV-2 among epithelial cells of the equine airway is warranted.
This experiment was performed in cells isolated from 5 healthy adult horses (2 for flow cytometry, immunofluorescence, and western blotting and 3 for pseudovirus infection studies). Expression of ACE2 in airway epithelium as well as susceptibility to SARS-CoV-2 infection is correlated with age in humans.38–40 This relationship has not been well-documented in veterinary species. Adult cats and dogs were more susceptible than animals less than 1 year of age41 and aged ferrets were more susceptible to infection than juvenile ferrets in an experimental challenge model,42 but there was no correlation between age and seroprevalence in a study of nearly 1,200 horses.23 This relationship has been associated with increased co-expression of ACE2 with proteases that cleave spike protein and permit viral cell entry, such as TMPRSS2.42–44 Gene expression of TMPRSS2 has been documented in bronchial epithelium of adult horses;45 however, TMPRSS2 protein expression within the tissues nor its relation to age has not been well documented in horses. Sequence analysis of equine TMPRSS2 is sufficiently different from the human homolog that it has been suggested that other proteases and cofactors are likely more important for SARS-CoV-2 cell entry.24 A nonpermissive cell line transfected with equine ACE2 and equine TMPRSS2 was not successfully infected by an early strain of SARS-CoV-2 and this was largely attributed to differences in TMPRSS2 structure.46 Thus, we did not pursue the investigation into TMPRSS2 expression in equine cells, but this may be worthy of further study, particularly in larger sample sizes and in aged horses, and could contribute to the lower efficiency of pseudovirus transduction in EBECs relative to HBECs.
Despite these limitations and the absence of evidence of SARS-CoV-2 infecting horses either experimentally10 or naturally,5,6,25,47 we demonstrated that EBECs are susceptible to cell entry with a SARS-CoV-2 pseudovirus. These findings suggest that SARS-CoV-2 can infect equine respiratory tract cells. If horses are susceptible to subclinical aerosol infection due to close contact with humans, they might serve as a potential reservoir for viral transmission to humans and for the selection of viral mutations that render some strains more infective to horses (and possibly other animals). Horses in pasture might be exposed to wildlife infected with SARS-CoV-2, such as white-tailed deer that might have a relatively high prevalence of infection with SARS-CoV-2.2,48 Serological and virological monitoring of horses in contact with persons shedding SARS-CoV-2 is warranted in light of seroconversion of a horse exposed to a person infected with SARS-CoV-2,6 and contact with horses by humans infected with SARS-CoV-2 should be avoided due to potential zoonotic transmission. The lower infectivity for EBECs than HBECs of the SARS-CoV-2 pseudovirus construct used in this study indicates that the risk of zoonotic transmission from horses to humans of the Wuhan strain of SARS-CoV-2 is likely relatively low. Nevertheless, further investigation of the susceptibility of horses to emerging strains of SARS-CoV-2 infection is warranted because the infectivity of horses and the risk of zoonotic transmission might be higher for emerging strains of SARS-CoV-2.
Acknowledgments
The authors acknowledge the assistance of the Image Analysis Laboratory (RRID: SCR_022479) and Flow Cytometry Facility (RRID: SCR_022169), School of Veterinary Medicine & Biomedical Sciences, Texas A&M University. The authors thank Dr. Jeannine Ott and Mrs. Jocelyne Bray for technical assistance.
Disclosures
The authors have nothing to disclose. No AI-assisted technologies were used in the generation of this manuscript.
Funding
The project was funded by the Link Equine Research Endowment, Texas A&M University, the Department of Large Animal Clinical Sciences, School of Veterinary Medicine & Biomedical Sciences, Texas A&M University, and the Judy Calder Foundation.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org.
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