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.

Extracellular vesicles (EVs), nanosized particles approximately 20-1000 nm in size, are rapidly garnering attention for their therapeutic potential. Specifically, human breast milk-derived extracellular vesicles (HBMDEVs) have been shown to confer protection in experimental models of necrotizing enterocolitis, an intestinal disease that primarily affects premature infants, resulting in mortality as high as 50%. However, traditional extracellular vesicle isolation techniques, such as differential ultracentrifugation, size exclusion chromatography, etc., are either time-consuming or require specialized instrumentation, neither of which is practical in the clinical setting. Hence, there is a need for a simple isolation method that will allow for rapid reactive administration of HBMDEVs to patients. Proposed here is a combination of centrifugation of human breast milk to remove fat and cellular debris, coupled with rapid ultrafiltration of the remaining skim milk that produces high yield and functional extracellular vesicles. Using this technique may allow for the use of HBMDEVs as a practical therapeutic option in the clinical setting.

Extracellular vesicles (EVs), nanosized particles approximately 20-1000 nm in size, are rapidly garnering attention for their therapeutic potential. Specifically, human breast milk-derived extracellular vesicles (HBMDEVs) have been shown to confer protection in experimental models of necrotizing enterocolitis, an intestinal disease that primarily affects premature infants, resulting in mortality as high as 50%. However, traditional extracellular vesicle isolation techniques, such as differential ultracentrifugation, size exclusion chromatography, etc., are either time-consuming or require specialized instrumentation, neither of which is practical in the clinical setting. Hence, there is a need for a simple isolation method that will allow for rapid reactive administration of HBMDEVs to patients. Proposed here is a combination of centrifugation of human breast milk to remove fat and cellular debris, coupled with rapid ultrafiltration of the remaining skim milk that produces high yield and functional extracellular vesicles. Using this technique may allow for the use of HBMDEVs as a practical therapeutic option in the clinical setting.