Equine Infectious Disease Laboratory, Department of Large Animal Clinical Sciences, School of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, TX.
Department of Microbial Pathogenesis & Immunology, School of Medicine, Texas A&M University, College Station, TX.
Am J Vet Res. 2023 Jul 18;84(9). doi: 10.2460/ajvr.23.06.0132. Print 2023 Sep 1.
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).
Primary EBEC cultures established from healthy adult horses and commercially sourced human bronchial epithelial cells (HBECs) were used as a positive control.
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.
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.
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.
研究培养的原代马支气管上皮细胞（EBEC）相对于人支气管上皮细胞（HBEC）对严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）假病毒的易感性。
本研究使用从健康成年马建立的原代 EBEC 培养物和商业来源的人支气管上皮细胞（HBEC）作为阳性对照。
通过免疫荧光、western 免疫印迹和流式细胞术证实 EBEC 中血管紧张素转换酶 2（ACE2）的表达。用携带 SARS-CoV-2 刺突蛋白的慢病毒假型转导 EBEC，该蛋白与 ACE2 结合并表达增强型绿色荧光蛋白（eGFP）作为报告基因。将细胞以 0.1 的感染复数转导假病毒 6 小时，洗涤并在培养基中维持 96 小时。96 小时后，通过细胞培养物的荧光显微镜观察和定量 PCR 评估 EBEC 中的 eGFP 表达。
免疫荧光、western 免疫印迹和流式细胞术检测到 EBEC 中 ACE2 的表达低于 HBEC。96 小时后，通过荧光显微镜观察到 EBEC 中 eGFP 的表达，定量 PCR 的平均 ΔCt 值在 EBEC 中显著（P <.0001）高于 HBEC（8.78 对 3.24），表明 EBEC 中的感染性较低。
马呼吸道细胞易受 SARS-CoV-2 假病毒的细胞进入。EBEC 中较低的复制效率表明马不太可能成为 SARS-CoV-2 的重要人畜共患病宿主，但病毒突变可能使某些毒株对马更具感染力。有必要对与 SARS-CoV-2 感染者接触的马进行血清学和病毒学监测。
