CRFK cells were used in this study as a host model for FCoV infection. They were obtained from the American Type Culture Collection (ATCC). CRFK cells were cultured in Eagle’s Minimum growth media (EMEM) (Fisher Scientific), including L-glutamine supplemented with 10% horse serum (HS) (Fisher Scientific), which is important as a source of growth factors and necessary nutrients needed for cell growth, 1% penicillin/streptomycin (Fisher Scientific), and 0.2% (0.5 μg/mL) amphotericin B (Fisher Scientific). Importantly, exosome-free media was made with 2% exosome-depleted horse serum in compliance with the laboratory procedure for virus infection. CRFK cells were placed in a 37°C incubator enriched with 5% CO₂ and allowed to reach about 70–80% confluency.

Feline coronavirus [Enteric; Strain: WSU791683 (3)] stock was utilized in this study, which was acquired from ATCC. The viral stock concentration (titer) was 8.9 × 10⁶ TCID₅₀/mL. The cytopathic effect (CPE) experiment was performed over a 10-day infection cycle, and the cytopathic effects caused by FCoV on the CRFK cells were determined to obtain the necessary multiplicity of infection (MOI). CRFK cells were exposed to several MOIs (50 IFU, 500 IFU, 2500 IFU, 4000 IFU) of FCoV and noted for CPE at 24-h intervals. CPE describes the alterations in the morphology of host cells following viral intrusion. Examples of these changes include circularizing virus-infected cells and merging with neighboring cells. CPE was not identified with an MOI of 50 IFU and 500 IFU for the duration of 10 days and 8 days after infection, correspondingly. Nevertheless, at a MOI of 2,500 IFU and 4,000 IFU in CRFK cells exhibited CPE following 3 days and 1 day of infection, correspondingly. Therefore, we chose a MOI of 2,500 IFU for further experimentation at 48 h and 72 h incubation time points based on CPE assay.

After cell densities reached approximately 70–80% confluency, cells were trypsinized and counted using a countess cell counter. Approximately 5.0 × 10⁵ cells were seeded per cell culture dish and then incubated throughout the night at 37°C and 5% CO₂. Subsequently, the cell-free medium was removed, and a 2% EMEM medium devoid of exosomes was added per cell culture dish. The EMEM medium was made with exosome-depleted HS, EMEM including L-glutamine, 1% penicillin/streptomycin, and 0.2% (0.5 μg/mL) amphotericin B. Uninfected dishes were used as controls, and infected dishes were infected with feline CoV at a MOI of 2,500 IFU. Both uninfected and FCoV-infected dishes were incubated for 48 h and 72 h time points (37°C and 5% CO₂). The cell supernatant was separately gathered from FCoV-infected and uninfected dishes and kept at −80°C for later isolation of EVs.

MTT assay (colorimetric assay) evaluates the cell viability and cytotoxicity that measures metabolic activities. In 96 well plates, 1 × 10⁴ CRFK cells were added independently in triplicates and incubated throughout the night, maintaining 37°C and 5% CO₂ conditions. The growth medium was discarded on the subsequent day, and a 2% exosome-free medium was added to each well. The following day, the cells were infected with FCoV at MOI of 2,500 IFU, and the infected cells were incubated for 48 h and 72 h while the control wells remained in the exosome-free medium. Cells were exposed to 50 μL of 5 mg/mL MTT in 1X PBS and incubated for a duration of 4 h, maintaining 37°C and 5% CO₂ conditions. Following incubation, a 100 μL stop solution was introduced to each well. Finally, the absorbance was measured at 570 nm, and every sample was analyzed in triplicates. The number of viable cells was investigated via a bright field microscope and compared to the CRFK cell viability at 48 and 72 h.

Previously, collected control and infected exosome-free cell supernatant were centrifuged at 1,300 Revolutions Per Minute (rpm) for a duration of 10 min at 4°C utilizing an Allegra X-14R Centrifuge. Then, the pellets were discarded, and the supernatant was collected again. Subsequently, the media was subject to centrifugation at 3900 rpm for 10 min using an Allegra X-14R Centrifuge, and then the supernatant was filtered using a 0.22 μm porosity filter. Subsequently, the supernatant was moved into an ultracentrifuge tube, the volume was prepped with 1X PBS and centrifuged at 10,800 rpm for 45 min at 4°C using a Beckman Coulter Optima L-70 K ultracentrifuge. The supernatant was once more gathered, and centrifugation was performed at 32,000 rpm for 70 min at 4°C. The supernatant was removed, and roughly 500 μL of purified EV pellets were saved from each control and infected tube. To inhibit the protein degradation, a protease inhibitor (10 μL/mL) was introduced to the isolated EVs and stored at −80°C until further experimentation (43).

TRIzol reagent was used for the extraction and purification of DNA and RNA of isolated EVs. EV samples weighing 5 μg were treated with 1 unit (U) of RNase-free DNAase I for DNA extraction and 1 U of micrococcal nuclease (MNase) for RNA extraction. For total DNA, RNase-free DNAase I treated with 5 μg control and infected CRFK-derived isolated EV samples were incubated in a water bath for a duration of 30 min at 37°C. Subsequently, they were processed with 50 mM Ethylenediaminetetraacetic acid (EDTA) treatment for a duration of 10 min at 65°C. DNA isolation was followed through the TRIzol extraction method (44). Total RNA was extracted by incubating EVs with 1% Triton-X-100 on ice for 30 min and exposed to MNase at 37°C for 15 min. Subsequently, the RNA isolation was followed by the TRIzol extraction method. Finally, total DNA and RNA in isolated EV samples were analyzed using Nanodrop (Thermo Scientific).

The quantitation of the total protein of CRFK-derived EVs was analyzed using a BCA assay. Five μL of 0, 0.2, 0.4, 0.8, and 1.6 μg/μL standards [bovine serum albumin (BSA)], CRFK-derived control, and infected EVs were introduced in triplicates in a 96-well plate. Subsequently, BSA protein assay reagents A and B were added, 25 and 200 μL, respectively, to each well. The 96-well tissue culture plate was covered with aluminum foil and positioned on a shaker for a duration of 10 min. Then, the plate was read at 595 nm absorbance. The standard curve was graphed to discover the accurate protein concentration in CRFK-derived control and infected EVs.

NanoSight tracking analysis (NTA) was performed to characterize the nanoparticles as well as to analyze the concentration (particles per mL) and nano-size distribution of the CRFK-derived control and infected EVs based on the rate of Brownian motion and light scattering using a Zeta View R Particle Matrix Tracking Analyzer instrument. For analysis, a 20 μL volume of EV samples was diluted at 1:75 in microbial cell culture-grade water before loading the prepared EV samples into the chamber of the Zeta View instrument. The mean values were investigated at 11 distinct positions.

Scanning electron microscope (SEM) was performed to examine the surface morphology of the CRFK-derived EVs. The CRFK-isolated EVs were vortexed and stabilized with 2.5% EMS-quality glutaraldehyde at a 1:1 ratio. 30 μL of exosome samples were added in vesicle mixtures to a clean carbon disc-SEM mounting stud. The vesicles were immobilized subsequent to drying and left overnight. Then, the EV samples on a carbon-SEM mounting stud were affixed to an SEM stage utilizing carbon paste. Prior to imaging using Phenom XL G2 Desktop SEM, a 5 nm coating of gold–palladium alloy was implemented by sputtering to improve surface conductivity. SEM was executed within the reduced beam energies. To achieve the best results in vesicle surface morphology under SEM, freshly isolated exosomes were fixed and attached to a conductive, adhesive carbon substrate immediately following isolation and were imaged within a week. An examination of exosome size was performed utilizing the SEM images with the assistance of Image-J software.

Dot blot experiment examines the expression of particular protein markers such as exosomal markers, immune response markers, pathogenic markers, apoptotic proteins, and stress-specific proteins (Hsps) in CRFK-derived EVs. Five μL of CRFK-derived EVs were added to the reducing buffer (1: 1) and were boiled for a duration of 10 min at 95°C. Prepared control and infected EVs were dotted on the nitrocellulose membrane and blocked for 30–45 min with 5% nonfat dry milk to prevent nonspecific bonding at room temperature (RT). Then, the membrane was washed three times for 10 min each with 1× Tris-buffered saline containing Tween-20 (0.2%) buffer solution (TBST) and incubated with primary antibodies including Alix (Fisher Scientific), CD63 (Santa Cruz Biotechnology), TSG101 (Fisher Scientific), ACE2 (DHSB), TMPRSS2 (DHSB), anti-flotillin-1 (BD Bioscience), Clathrin (BD Bioscience), cadherin (DSHB), CD29 (DSHB), anti-TLR3 (Abnova), TLR6 and 7 (Invitrogen), IRF4 (DSHB), mCCL22 (RD Systems), TGFβ-3(DHSB), TNF-α (Bioss Antibodies Inc.), CD47(Bioss Antibodies Inc.), LAMP-1 (human) (DSHB), ATPase (DSHB), TSPAN8, HSPB8-13B6 (Hsp22) (Invitrogen), HSPB1-1 (Hsp27) (DSHB), Hsp100 (DSHB), DIS3-1D7 (DSHB), and cleaved caspase-3 (RD Systems). The nitrocellulose membrane was washed three times on a subsequent day using a TBST buffer solution. Horseradish peroxidase (HRP)-conjugated secondary antibody, goat anti-mouse (Fisher Scientific), goat anti-rat (Fisher Scientific), or goat anti-rabbit (Novus Biologicals LLC) were added to the blocking buffer and incubated with membranes for a duration of 60–120 min. The target protein signals were identified utilizing the Super Signal West Femto Maximum Sensitivity Substrate (Invitrogen). Subsequently, the image was developed through the Bio-Rad ChemiDoc™ XRS+ System (Bio-Rad Laboratories).

Protein biomarkers were further analyzed using a western blot. Here, approximately 32 μL of isolated EVs were mixed with reducing buffer at a 1:1 ratio and were boiled (95°C) for a duration of 10 min. Prepared samples were placed in a 4–20% 1.5 mm Bio-Rad precast gel and run at 100 V. The procedure continued overnight until proteins were transferred to the Polyvinylidene difluoride (PVDF) membrane in a transfer chamber at 45 mA. Then, the PVDF membrane was blocked for 35 min using the blocking solution (5% nonfat dry milk) at RT and washed three times for 10 min each using 1× TBS containing Tween-20 (0.2%) buffer solution. Then, the PVDF membrane was incubated with primary antibodies, including ACE2 (DHSB) and TMPRSS2 (DHSB). On a subsequent day, the membrane was washed three times for 10 min with TBST buffer solution. It was incubated with secondary antibodies, which can be either HRP-conjugated goat anti-mouse, goat anti-rat, or goat anti-rabbit, and was added to the blocking buffer for 1–2 h at RT. Finally, signals were developed using Super Signal West Femto Maximum Sensitivity Substrate (Invitrogen).

Statistical analysis was conducted using a t-test on the acquired data employing the GraphPad Version 5 software and Bio-Rad imaging program. The statistical significance was calculated by mean value ± standard deviation, and the statistical significance of the p-value was described as p ≤ 0.05(*), p ≤ 0.01(**), p ≤ 0.001(***), p ≤ 0.0001(****).

The cellular morphology was analyzed using bright-field microscopy, which indicated a reduced CRFK cell count with an increased incubation time after FCoV infection (Figure 1A). The cell viability was assessed utilizing an MTT assay, which exhibited that the cell viability of FCoV-infected CRFK cells was reduced with increased incubation time (Figure 1B). The cell viability of FCoV-infected cells was significantly reduced by nearly 9% (* p ≤ 0.05) and 15% (* p ≤ 0.05) at 48 h and 72 h, respectively, contrasted to the control CRFK cells. The decrease in cell viability with prolonged incubation times after FCoV infections indicates that FCoV infection significantly induces cell demise in CRFK cells.

To examine the impact of FCoV on EVs, CRFK cells were infected with FCoV at 48 h and 72 h time points. Cell supernatants were ultracentrifuged through a series of high-speed centrifugation steps to isolate and purify EVs produced from CRFK cells. Isolated EVs were examined using SEM and NTA to identify the surface morphology, particle size in nanometers (nm), and concentration (particles per mL). The SEM image revealed the surface morphology of CRFK-derived control and FCoV-infected EVs in both time references. Figure 2A represents an SEM image, which indicates control CRFK-derived EVs at the 72 h-time point at 5 μm, verifying the presence of EVs in the sample. NTA was performed to characterize EVs by determining the mean particle size (nm) and concentration (particles/mL). The mean particle size was slightly increased (143.4 nm) in FCoV-infected EVs compared to the control EVs (131.9 nm) at 48 h (Figure 2B). The mean particle size was slightly decreased (120.9 nm) in FCoV-infected EVs compared to the control EVs (126.6 nm) at 72 h (Figure 2B). Therefore, NTA analysis confirmed that the average size of EVs ranges from 100 to 200 nm at 48 and 72 h times points. The particle concentration (particles/mL) of FCoV-infected EVs exhibited a negligible decrease compared to the control EVs at both time points (Figure 2C). At 48 h, the mean concentration of control and infected EVs were 2.5 × 10⁷ and 2.0 × 10⁷ particles/mL, respectively. In addition, at 72 h, the mean concentration of control and infected EVs were 2.2 × 10⁷ and 1.8 × 10⁷ particles/mL, respectively. The levels of total DNA, RNA, and protein were analyzed in control and FCoV-derived EVs at 48 h and 72 h times points. Figure 3A indicates that there was a gradually increasing trend in total DNA with increased incubation time. Figure 3B indicates that there was a negligible increase in total RNA levels with increased incubation time. However, the total protein level of FCoV-infected EVs was significantly increased as compared to the control EVs at 48 h (**** p ≤ 0.0001) (Figure 3C).

Classical exosome proteins are used to characterize EVs; they are found on the surface of the exosome membrane and the inner space (45). Alix and TSG101 are multivesicular body (MVB) related proteins engaged in the endosomal-sorting complex, which is necessary for transportation (ESCRT) (45). TSG101 protein marker plays a significant role in the control of growth and differentiation (46). CD63 are tetraspanins that play a crucial function in sorting cargo-like premelanosome protein (PMEL) onto intraluminal vesicles (ILVs) within MVBs (47, 48). In the present investigation, we conducted a dot blot to assess the expression of classical biomarkers. The dot blots of Alix (Supplementary Figure S1A), TSG101 (Supplementary Figure S1B), and CD63 (Supplementary Figure S1C) in control and infected derived EVs were analyzed via the Bio-Rad imaging program and GraphPad. FCoV-infected CRFK-derived EVs presented significantly elevated expression of Alix (∗∗p ≤ 0.01), TSG101 (∗∗∗p ≤ 0.001), and CD63 (∗∗p ≤ 0.01) at 48 h infection and significantly increased expression of TSG101 (∗p ≤ 0.05) at 72 h infection relative to the control EVs, respectively, (Figures 4A–C). Our results indicate that FCoV infection affects the abundance of tetraspanins and regulates the ESCRT pathway.

ACE2 has been determined to be a cellular receptor on the cell surface for both SARS-CoV and SARS-CoV-2 (5). ACE2 is a carboxypeptidase, mainly detected in the heart, lungs, and kidneys. It is recognized as a counter-regulator of the renin-angiotensin system (RAS) and substantially influences the cardiovascular system (5, 49). Transmembrane protease, Serine 2 (TMPRSS2), plays a pivotal role as a cofactor in SARS-CoV-2 entry and is able to activate glycoproteins of respiratory viruses (50, 51).TMPRSS2 is found in epithelial cells at different locations, such as respiratory, genitourinary, and gastrointestinal systems (52). ACE2 attaches with the spike protein of SARS-CoV-2 to obtain access to the host cell and serine protease (53). TMPRSS2 is able to activate the S protein and release subunit S2, which facilitates the merging of the viral and cellular membranes. Consequently, viral genes enter the host cell and spread (54, 55). Therefore, in SARS-CoV-2 infection, ACE and serine protease TMPRSS2 play critical roles in viral entry and S protein priming, respectively (54, 56). In our investigation, we evaluated the presence of ACE2 and TMPRSS2 in isolated CRFK-derived control and FCoV-derived EVs and both time points via western blot and dot blot analysis (Figures 5A,B). The western blot analysis of ACE2 and TMPRSS2 indicated gel bands, approximately 130 kilodaltons (kDa) and 60 kDa, respectively. The ACE2 protein marker was significantly increased at 48 h (∗p ≤ 0.05) and 72 h (∗p ≤ 0.05) in FCoV-derived EVs relative to the control-derived EVs (Figure 5C). TMPRSS2 was found to significantly increase in FCoV infection-derived EVs relative to the uninfected control-derived EVs at 48 h (∗p ≤ 0.05) and 72 h (∗∗p ≤ 0.01) (Figure 5D). These results indicated the presence of CoV host receptor (ACE2) and TMPRSS2 in the control and FCoV-derived EVs produced from CRFK cells.

We evaluated the expression of Flotillin-1 and Clathrin membrane trafficking protein markers in isolated CRFK-derived control EVs and FCoV-derived EVs by utilizing a dot blot assay. Flotillin-1 is a membrane-associated lipid raft protein engaged in endocytosis, cell signaling, protein trafficking, protein sorting, and gene expression (57). Levels of Flotillin-1 (Supplementary Figure S1D) in FCoV infection-derived EVs were significantly upregulated at 48 h and 72 h (∗p ≤ 0.05 and ∗∗p ≤ 0.01, respectively) contrasted with control-derived EVs (Figure 6A). Another membrane trafficking molecule, Clathrin, acts as a prototype self-assembling protein and is able to coat transport vesicles (58). Clathrin is involved in receptor-mediated endocytosis, which plays a significant role in membrane trafficking and mitosis (58, 59). Clathrin expression (Supplementary Figure S1E) in EVs was significantly elevated after FCoV infection at the 72 h (∗∗p ≤ 0.01) time point (Figure 6B). Hence, our finding suggested that FCoV infection of CRFK regulates membrane protein trafficking and EV formation.

The expressions of several adhesion molecules were analyzed in CRFK-derived EV post-infection via dot blot analysis. Cadherin is a transmembrane cell–cell adhesion molecule and plays an important role in tissue morphogenesis by regulating cell signaling (60). Cadherin (Supplementary Figure S1F) was slightly reduced at the 48 h infection time point and significantly increased at 72 h (∗p ≤ 0.05) in FCoV-derived EVs relative to the control-derived EVs (Figure 6C). Moreover, we determined the impact of FCoV exposure on CD29 expression (Supplementary Figure S1G and Figure 6D, respectively). CD29 is also known as integrin β-1 (61). Integrins are large proteins composed of α/β-chain subunit cell adhesion molecules and are able to regulate cell adhesion and signaling (62). CD29 was significantly decreased at 48 h (∗p ≤ 0.05) and significantly elevated at 72 h (∗∗∗∗p ≤ 0.0001) in infected EVs as compared to the uninfected. Therefore, these results indicated that FCoV infection of CRFK cells modulates the process of internalization and encapsulation as well as membrane protein presence within EVs.

We evaluated the expression of several toll-like receptors (TLRs) in EVs obtained from CRFK cells after FCoV infection. TLRs are known as pattern recognition receptors, which are accountable for stimulating innate immune responses and pathogen recognition (63). In this study, we investigated TLR3, 6, and 7 expressions via dot blot analysis (Supplementary Figures S1H–J, respectively). TLR3 is a double-stranded RNA (dsRNA), which is found on the endosome membrane and plays a significant role in innate immune responses against viral infections (64). TLR3 is able to activate the transcriptional factors of Interferon Regulatory Factors (IRFs), Nuclear Factor-Kappa β (NF-κβ, and Activating Transcription Factor 1(ATF1) (65). It is also capable of inducing the formation of Interferon-Beta (IFN-β) proinflammatory cytokines (65). We found that TLR3 was significantly increased at 72 h in FCoV-derived EVs relative to the uninfected control EVs (∗∗∗p ≤ 0.001) (Figure 7A). Similarly, Figure 7B indicates that the expression of TLR6 was significantly increased at 72 h in FCoV-derived EVs relative to the uninfected EVs (∗p ≤ 0.05). TLR2 is capable of forming heterodimers with TLR6, which leads to activating the myeloid differentiation primary response 88 (MyD88)-dependent signaling pathway to induce the production of proinflammatory cytokines (66). TLR6 also activates NF-κB (67). We further analyzed the expression of TLR7, which recognizes single-stranded RNA (SSRNA) viruses (68). It also activates the generation of Tumor Necrosis Factor (TNF) and interleukin-6 (IL-6) proinflammatory cytokines (69). FCoV infection induced a significant upregulation in TLR7 in infected CRFK-derived EVs contrasted with uninfected control EVs at 72 h (∗p ≤ 0.05) (Figure 7C). Hence, these findings indicate that TLRs modulate the presence of protein markers associated with proinflammatory and immune activation within the EV’s response to FCoV infection.

We examined the level of immune proteins, including interferon regulatory factor 4 (IRF4) (Supplementary Figure S1K), cluster of differentiation 47 (CD47) (Supplementary Figure S1L), transforming growth factor-beta-3 (TGF-β-3) (Supplementary Figure S2A), and mouse c-c motif chemokine ligand 22 (mCCL22) (Supplementary Figure S2B), and inflammatory markers such as tumor necrosis factor-alpha (TNF-α) (Supplementary Figure S2C) in CRFK-derived EVs after FCoV infection. FCoV infection induced a slight upregulation and downregulation in IRF4 protein levels in FCoV-derived EVs as compared to the control-derived EVs at the 48 h and 72 h time points, respectively (Figure 8A). IRF4 functions as a transcription factor for interferons that play a significant regulatory role in the immune system (70). To further analyze immune proteins, we examined the expression of CD47, a transmembrane immunoglobulin Ig superfamily protein that plays a significant role in inhibiting phagocytosis (71), TGF-β-3 which plays a pivotal role in tissue fibrosis (72), and mCCL22. CD47 was found to have significantly increased in CRFK-derived infected EVs relative to the uninfected control EVs at both 48 h (∗p ≤ 0.05) and 72 h (∗p ≤ 0.05) (Figure 8B). TGF-β-3 was significantly downreglated in EVs after FCoV infection at 48 h (∗p ≤ 0.05) and significantly upregulated 72 h (∗p ≤ 0.05) (Figure 8C). Figure 8D indicates that the presence of the mCCL22 protein marker was significantly increased in FCoV-infected EVs contrasted with the uninfected EVs at 72 h (∗p ≤ 0.05). We conducted further analysis to examine TNF-α, a cytokine that acts as a key regulator of inflammatory responses (73). Levels of TNF-α were significantly lower in CRFK-derived infected EVs relative to the uninfected control EVs at 48 h (∗∗∗p ≤ 0.001) and significantly higher at the 72 h (∗∗∗p ≤ 0.001) than that in the control-derived EVs at 72 h time point (Figure 8E). Therefore, these results demonstrated the expression of immune and inflammatory response-associated biomarkers in EVs obtained from CRFK cells following FCoV infection.

We investigated the expression of several transmembrane molecules, stress-specific heat shock proteins (Hsps), and varying caspases in control and infection-derived EVs after FCoV infection. We measured the expression of LAMP-1 (human) (Supplementary Figure S2D), a transmembrane glycoprotein that can be found in lysosomes (74), ATPase (Supplementary Figure S2E), and TSPAN8 (Supplementary Figure S2F) transmembrane protein in CRFK-derived EVs’ post-infection. We detected that LAMP-1 was significantly increased at 48 h and 72 h, respectively (∗p ≤ 0.05 and ∗p ≤ 0.05). ATPase was significantly upregulated at 72 h (∗p ≤ 0.05) after FCoV infection (Table 1). Additionally, TSPAN8 was significantly downregulated and significantly upregulated at the 48 h (∗p ≤ 0.05) and 72 h (∗p ≤ 0.05) time points, respectively (Table 1) in FCoV-derived EVs when compared to the control-derived EVs. Therefore, these results indicated that FCoV infection modulates the transmembrane protein expression in CRFK-derived EVs and the packaging of EVs.

Hsps are recognized as molecular chaperones involved in unfolding cellular proteins due to stress or high-temperature (75). We analyzed the FCoV-infected EVs for the presence of Hsp22, Hsp100, Hsp27, and DIS3 (Supplementary Figures S2G–J, respectively) (Table 1). At the 48 h infection time point, Hsp22, Hsp100, Hsp27, and DIS3 were confirmed to be significantly downregulated (∗p ≤ 0.05), significantly upregulated (∗p ≤ 0.05), slightly downregulated, and significantly upregulated (∗p ≤ 0.05), respectively in FCoV-derived EVs in contrast with the uninfected control EVs. Additionally, at the 72 h time point, Hsp22 (∗p ≤ 0.05), Hsp100 (∗p ≤ 0.05), and DIS3 (∗∗∗p ≤ 0.001) were significantly upregulated, and Hsp27 was slightly upregulated after FCoV infection. These findings confirmed that these stress-specific protein markers were regulated during FCoV infection.

Caspases play a significant role in cell death and inflammation responses (76). The expression of cleaved caspase-3 (Supplementary Figure S2K) was significantly increased at 48 h (∗p ≤ 0.05) and 72 h (∗∗∗p ≤ 0.001) in FCoV-derived EVs relative to the uninfected control EVs (Table 1). Hence, our results show that FCoV infection influences the caspase protein in EVs produced by CRFK cells.