Infection of equine bronchial epithelial cells with a SARS-CoV-2 pseudovirus

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of COVID-19, can infect animals by binding to the angiotensin-converting enzyme 2 (ACE2). Equine infection appears possible due to high homology (≈97%) between human and equine ACE2, evidence of in vitro infection in cell lines expressing equine ACE2, and evidence of seroconversion in horses after exposure to persons infected with SARS-CoV-2. Our objective was to examine susceptibility of cultured primary equine bronchial epithelial cells (EBECs) to a SARS-CoV-2 pseudovirus relative to human bronchial epithelial cells (HBECs; positive control). ACE2 expression in EBECs detected by immunofluorescence, western immunoblotting, and flow cytometry was lower in EBECs than in HBECs. 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 co-cultivated with the pseudovirus at a multiplicity of infection of 0.1 for 6 hours, washed, and maintained in media. After 96 hours, eGFP expression in EBECs was demonstrated by fluorescence microscopy, and mean Δ Ct values from quantitative PCR were significantly (P < 0.0001) higher in HBECs (8.78) than HBECs (3.24) indicating lower infectivity in EBECs. Equine respiratory tract cells were susceptible to infection 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 infectious to horses. Serological and virological monitoring of horses in contact with persons shedding SARS-CoV-2 is warranted. IMPORTANCE This study provides the first published evidence for SARS-CoV-2 pseudovirus infection in equine airway epithelial cells, which were less susceptible to infection than cells of human origin. This was presumably due to lower ACE2 expression in equine cells, lower viral affinity for equine ACE2, or both. Our results are important considering recent evidence for asymptomatic seroconversion in horses following exposure to COVID-19 positive humans, despite this lower susceptibility, and increased affinity of viral variants of concern for equine ACE2 compared to ancestral strains. Thus, there is great need to better characterize SARS-CoV-2 susceptibility in horses for the benefit of veterinary and human health. ### Competing Interest Statement The authors have declared no competing interest.