Pandit Rachana, Ipinmoroti Ayodeji O, Crenshaw Brennetta J, Li Ting, Matthews Qiana L
Microbiology Program, Department of Biological Sciences, College of Science, Technology, Engineering and Mathematics, Alabama State University, Montgomery, AL 36104, USA.
Department of Biological Sciences, College of Science, Technology, Engineering and Mathematics, Alabama State University, Montgomery, AL 36104, USA.
Biomedicines. 2023 Mar 21;11(3):976. doi: 10.3390/biomedicines11030976.
Coronavirus (CoV) has persistently become a global health concern causing various diseases in a wide variety of hosts, including humans, birds, and companion animals. However, the virus-mediated responses in animal hosts have not been studied extensively due to pathogenesis complexity and disease developments. Extracellular vesicles (EVs) are widely explored in viral infections for their intercellular communication, nanocarrier, and immunomodulatory properties. We proposed that coronavirus hijacks the host exosomal pathway and modulates the EV biogenesis, composition, and protein trafficking in the host. In the present study, Crandell-Rees feline kidney (CRFK) cells were infected with canine coronavirus (CCoV) in an exosome-free medium at the multiplicity of infection (MOI) of 400 infectious units (IFU) at various time points. The cell viability was significantly decreased over time, as determined by the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Post-infection EVs were isolated, and transmission electron microscopy (TEM) showed the presence of small EVs (sEVs) after infection. NanoSight particle tracking analysis (NTA) revealed that EV sizes averaged between 100 and 200 nm at both incubation times; however, the mean size of infection-derived EVs was significantly decreased at 48 h when compared to uninfected control EVs. Quantitative analysis of protein levels performed by dot blot scanning showed that the expression levels of ACE-2, annexin-V, flotillin-1, TLR-7, LAMP, TNF-α, caspase-1, caspase-8, and others were altered in EVs after infection. Our findings suggested that coronavirus infection impacts cell viability, modulates EV biogenesis, and alters cargo composition and protein trafficking in the host, which could impact viral progression and disease development. Future experiments with different animal CoVs will provide a detailed understanding of host EV biology in infection pathogenesis and progression. Hence, EVs could offer a diagnostic and therapeutic tool to study virus-mediated host responses that could be extended to study the interspecies jump of animal CoVs to cause infection in humans.
冠状病毒（CoV）一直是全球健康关注的焦点，它在包括人类、鸟类和伴侣动物在内的多种宿主中引发各种疾病。然而，由于发病机制的复杂性和疾病的发展，病毒在动物宿主中的介导反应尚未得到广泛研究。细胞外囊泡（EVs）因其细胞间通讯、纳米载体和免疫调节特性而在病毒感染中得到广泛探索。我们提出冠状病毒劫持宿主外泌体途径并调节宿主中的EV生物发生、组成和蛋白质运输。在本研究中，将克兰德尔-里斯猫肾（CRFK）细胞在无外泌体培养基中以400个感染单位（IFU）的感染复数（MOI）在不同时间点感染犬冠状病毒（CCoV）。通过3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四氮唑溴盐（MTT）试验确定，细胞活力随时间显著降低。感染后分离出EVs，透射电子显微镜（TEM）显示感染后存在小EVs（sEVs）。纳米视场颗粒跟踪分析（NTA）显示，在两个孵育时间点，EVs大小平均在100至200nm之间；然而，与未感染的对照EVs相比，感染衍生的EVs在48小时时的平均大小显著减小。通过斑点印迹扫描进行的蛋白质水平定量分析表明，感染后EVs中血管紧张素转换酶2（ACE-2）、膜联蛋白-V、小窝蛋白-1、Toll样受体7（TLR-7）、溶酶体相关膜蛋白（LAMP）、肿瘤坏死因子-α（TNF-α）、半胱天冬酶-1、半胱天冬酶-8等的表达水平发生了改变。我们的研究结果表明，冠状病毒感染会影响细胞活力，调节EV生物发生，并改变宿主中的货物组成和蛋白质运输，这可能会影响病毒的进展和疾病的发展。未来对不同动物CoV的实验将提供对感染发病机制和进展中宿主EV生物学的详细了解。因此，EVs可以提供一种诊断和治疗工具，用于研究病毒介导的宿主反应，这可能扩展到研究动物CoV跨物种传播导致人类感染的情况。
