Abstract

   Porcine epidemic diarrhea virus (PEDV) infection causes severe diarrhea
   in pigs and can be fatal in newborn piglets. Exosomes are extracellular
   vesicles secreted by cells that transfer biologically active proteins,
   lipids, and RNA to neighboring or distant cells. Herein, the
   morphology, particle size, and secretion of exosomes derived from a
   control and PEDV-infected group are examined, followed by a proteomic
   analysis of the exosomes. The results show that the exosomes secreted
   from the Vero cells had a typical cup–shaped structure. The average
   particle size of the exosomes from the PEDV-infected group was 112.4
   nm, whereas that from the control group was 150.8 nm. The exosome
   density analysis and characteristic protein determination revealed that
   the content of exosomes in the PEDV-infected group was significantly
   higher than that in the control group. The quantitative proteomics
   assays revealed 544 differentially expressed proteins (DEPs) in the
   PEDV-infected group’s exosomes compared with those in the controls,
   with 236 upregulated and 308 downregulated proteins. The DEPs were
   closely associated with cellular regulatory pathways, such as the
   phosphatidylinositol–4,5–bisphosphate 3–kinase (PI3K)–protein kinase B
   (Akt) signaling pathway, extracellular matrix–receptor interaction,
   focal adhesion, and cytoskeletal regulation. These findings provide the
   basis for further investigation of the pathogenic mechanisms of PEDV
   and the discovery of novel antiviral targets.

   Keywords: PEDV, Vero cells, exosomes, proteomics, cellular regulatory
   pathway

1. Introduction

   Porcine epidemic diarrhea virus (PEDV) is the causative pathogen of
   porcine epidemic diarrhea (PED), and its infection of piglets causes
   severe diarrhea, vomiting, and dehydration, with mortality rates of up
   to 100% in newborn piglets [[44]1,[45]2]. PEDV, belonging to the genus
   Alphacoronavirus in the family Coronaviridae, is an enveloped virus
   with a single-stranded positive-strand RNA genome. The PEDV genome
   comprises approximately 28 kb and encodes four structural proteins,
   namely the spike glycoprotein, envelope protein, membrane glycoprotein,
   and nucleocapsid protein, as well as an accessory protein open reading
   frame 3 (ORF3), and sixteen nonstructural proteins [[46]3]. The first
   identification of PEDV occurred in Europe in 1978, followed by an
   outbreak of a mutant strain in China in 2010. Subsequently, North
   America experienced its first reported epidemic of PEDV in 2013.
   Presently, PEDV has become a global epidemic, resulting in substantial
   economic losses in the pig industry [[47]4].

   Exosomes are extracellular vesicles secreted by various eukaryotic
   cells, which are characterized by a single membrane and diameters
   ranging from 30 to 150 nm. The content, or cargo, of exosomes consists
   of lipids, nucleic acids, and proteins. Exosomes are especially
   enriched in transmembrane proteins, lipid-anchored membrane proteins,
   peripherally associated membrane proteins, and soluble proteins
   [[48]5]. Growing evidence suggests that exosomes play crucial roles in
   a range of physiological processes, such as intercellular
   communication, signal transduction, coagulation, and most importantly,
   immune responses [[49]6,[50]7]. Studies have found that exosomes have a
   critical function in the process of the viral infection of cells. On
   one hand, these vesicles can contain all or part of the genetic
   material and proteins of the virus, which promotes the infection of
   other cells [[51]8,[52]9]. On the other hand, exosomes have the
   capability to transfer antiviral molecules that are induced by host
   immune responses, which can subsequently inhibit virus replication
   [[53]10]. Bedford et al. found that during influenza virus infection,
   exosomes released into the respiratory tract carry virus antigens and
   trigger lung inflammation. They further demonstrated that the surface
   of these vesicles contains attachment factors for the influenza virus,
   which have the ability to neutralize the influenza virus and prevent
   virus binding and entry into target cells [[54]11]. The composition of
   cellular exosomes (nucleic acids or proteins) changes after the virus
   infects the cell. For instance, in the case of foot-and-mouth disease
   virus (FMDV), full-length genomic RNA and partial viral protein were
   detected in exosomes isolated from FMDV-infected PK–15 cells, which can
   transmit infection to naive PK–15 cells and suckling mice [[55]12].
   Zhang et al. found that the grass carp reovirus infection of CIK cells
   resulted in changes in the content of 761 proteins in the cellular
   exosomes, which are involved in various cellular metabolic processes
   such as translation, peptide biosynthesis, and metabolism [[56]13].

   Studies have found that exosomes are involved in the infection and
   proliferation process of PEDV. Chen et al. reported that the levels of
   complements C3, C6, and complement factor B (CFB) were significantly
   decreased in the serum exosomes of PEDV-infected piglets and that the
   exosomes from infected piglets restricted the PEDV infection of LLC–PK1
   cells [[57]14]. Yin et al. found that PEDV infection could alter the
   levels of microRNAs (miRNAs) involved in cyclic AMP (cAMP), Hippo,
   transforming growth factor beta (TGF–beta), hypoxia inducible factor 1
   (HIF–1), forkhead box O (FoxO), mitogen-activated protein kinase
   (MAPK), and Ras signaling pathways in host cell exosomes [[58]15]. Zhao
   et al. found that the abundance of miRNA-328-3p was significantly
   downregulated in the exosomes of PEDV–infected Vero E6 cells, and this
   miRNA was found to target and repress TJP3 (encoding tight junction
   protein 3, also known as ZO–3) to inhibit PEDV infection [[59]16].
   However, the role and mechanism of host–cell–derived exosomes during
   PEDV infection and proliferation are still not fully understood.

   In this study, we aim to isolate and characterize exosomes from
   PEDV–infected and uninfected Vero cells, and to examine the cellular
   exosome morphology, particle size, and secretory concentration.
   Furthermore, the proteomics of cellular exosomes are analyzed using
   tandem mass tag (TMT)–labeled liquid chromatography–mass spectrometry
   (LC–MS) to determine the differences between the exosomes from infected
   and uninfected cells. This study provides a foundation for further
   investigations of the role of exosomes in PEDV infection and
   proliferation and offers new insights for the prevention and control of
   PEDV.

2. Materials and Methods

2.1. Cell Culture and PEDV Strain

   Vero cells (CVCCCL28) were obtained from the National Center for
   Veterinary Culture Collection (Beijing, China). The cells were cultured
   in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10%
   fetal bovine serum (FBS) and 1% penicillin-streptomycin, obtained from
   Gibco BRL (Grand Island, NY, USA) and HyClone (Logan, UT, USA),
   respectively. PEDV CV777 strain (GenBank: [60]KT323979.1) was donated
   by the Institute of Veterinary Medicine, Jiangsu Academy of
   Agricultural Sciences.

2.2. Exosome Isolation and Purification

   Vero cells were grown in 25 cm^2 cell culture flasks and infected with
   CV777 at a multiplicity of infection (MOI) of 1.0 for 24 h
   (PEDV–infected group). Control cells (control group) were treated with
   an equivalent amount of culture medium. Each group was replicated three
   times. Cell supernatant exosome extraction was performed using an
   exosome concentration kit and an exosome purification kit (Rengen
   Biosciences Ltd., Liaoning, China). The brief procedure was as follows:
   First, the cell culture supernatant was added to a 50 mL centrifuge
   tube, followed by the addition of 4 mL of binding buffer. The mixture
   was inverted, and 1.6 mL of binding resin was added. The mixture was
   then inverted again and incubated at room temperature for 15 min. After
   incubation, the mixture was centrifuged at 1500× g for 3 min at room
   temperature. The collected resin was transferred to a 15 mL
   purification column and allowed to stand for 2 min. The column was then
   centrifuged at 2000× g for 2 min at room temperature, and the solution
   from the collection tube was discarded. The concentrated resin was
   washed twice with 2 mL of washing solution and centrifuged at 3000× g
   for 2 min. The exosome eluate was added to the concentrated resin and
   allowed to stand for 5 min. The mixture was then centrifuged at 3000× g
   for 2 min at room temperature to obtain the concentrated exosome
   solution. The purified Vero cell exosomes were obtained by adding the
   concentrated exosome solution to a Super EV purification column (an
   ultra–pure size exclusion column) and eluting stepwise using
   phosphate-buffered saline (PBS). The purified exosomes were stored at
   −80 °C.

2.3. Nanoparticle Tracking Analysis (NTA)

   The concentration and particle size of the extracted Vero cell exosomes
   were determined using a Zeta View Pmx110 Nanoparticle Tracking Analysis
   system (Particle Metrix, Inning am Ammersee, Germany), and the results
   were analyzed using ZetaView (version 8.05.14 SP7) software. The
   instrument was calibrated using polystyrene microspheres (100 nm).

2.4. Transmission Electron Microscopy (TEM)

   To visualize the morphology of exosomes, an exosome suspension was
   mixed with a 0.2% paraformaldehyde suspension and applied onto a
   formvar-coated copper grid. The sample was then stained with 1% uranyl
   acetate in aqueous water for 2 min, followed by filtering off the
   excess liquid. TEM was then performed using an FEI microscope (FEI,
   Hillsboro, OR, USA) to examine the sample.

2.5. Western Blot Analysis of Exosomal Proteins

   Exosome samples (20 μL) were placed in spiking buffer and denatured by
   heating at 97 °C for 10 min. The denatured proteins were separated
   using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis
   (SDS–PAGE) and transferred onto nitrocellulose membranes using a
   Bio-Rad dry blotting system (Bio–Rad, Hercules, CA, USA). The membranes
   were then blocked with 3% non-fat powdered milk in PBS for 1 h at 20~25
   °C. Then, monoclonal antibodies against CD63, CD9, tumor susceptibility
   101 (TSG101), and glyceraldehyde 3 phosphate dehydrogenase (GAPDH)
   (Abcam, Cambridge, UK) were diluted 1:1000 in 4% bovine serum albumin
   (BSA) buffer and incubated with the membranes overnight at 4 °C. The
   membranes were washed three times with PBST (PBS plus 0.1% Tween 20)
   and incubated with enzyme-labeled secondary antibodies at room
   temperature for 1 h. After washing the membranes with PBST three times,
   the chemiluminescence substrate was added, and the immunoreactive
   protein bands were detected using Tanon–5200Muiti fluorescence imaging
   system (Tanon, Shanghai, China).

2.6. Exosomal Protein Preparation

   Frozen exosome samples were transferred into 1.5 mL Eppendorf tubes and
   lysed using 300 µL of lysis buffer supplemented with 1 mM
   phenylmethylsulfonyl fluoride (PMSF). The samples were further lysed
   using sonication. The parameters were set as 1 s/1 s intervals, 3 min
   time, and 80 W power. After sonication, the samples were centrifuged at
   15,000× g for 15 min to remove insoluble particles, and this was
   repeated once to further exclude contaminants. The protein
   concentration was determined using a bicinchoninic acid (BCA) assay,
   and the protein solution was aliquoted and stored at −80 °C.

   Next, 10 µg of proteins from each sample were acquired and separated
   using 12% SDS-PAGE gel. The gel was then stained using Coomassie
   brilliant blue (CBB) as follows: Firstly, the gel was fixed for 2 h and
   stained for 12 h. After staining, the gel was washed with water until
   the bands were visualized. Finally, the stained gel was scanned using a
   Tanon 1600 automatic digital gel image analysis system (Tanon,
   Shanghai, China).

2.7. Proteome Sequencing

   The entire proteome was detected and analyzed via Shanghai Lu Ming
   Biological Technology Co. Ltd. (Shanghai, China). Briefly, the protein
   extract (100 µg) was mixed with the reducing buffer and incubated at 60
   °C for 1 h. Then, indole acetic acid (IAA) was added to the solution
   and incubated in the dark for 40 min. The solution was centrifuged at
   12,000 rpm for 20 min at 4 °C, and the flowthrough was discarded. After
   washing the samples twice using 100 µL of 300 mM triethylammonium
   bicarbonate (TEAB) buffer, 100 µL of TEAB containing 1 μg/μL trypsin
   was added for digestion at 37 °C for 12 h. The digested peptides were
   collected via centrifugation at 12,000 rpm for 20 min and lyophilized.
   For TMT labeling, the lyophilized sample was resuspended in 100 μL of
   200 mM TEAB. At room temperature, acetonitrile was added to dissolve
   the TMT reagent and mixed to obtain TMT labeling reagent. Then, 41 μL
   of the TMT labeling reagent was added to 40 μL of samples, mixed, and
   incubated for 1 h at room temperature. Finally, 8 µL of 5%
   hydroxylamine was added to each sample and incubated for 15 min to
   terminate the reaction. The labeled peptide solutions were lyophilized
   and stored at −80 °C.

   Reversed phase (RP) separation was performed on an 1100 HPLC System
   (Agilent, CA, USA) using an Agilent Zorbax Extend RP column (5 μm, 150
   mm × 2.1 mm). Mobile phases A (2% acetonitrile in high-performance
   liquid chromatography (HPLC) water) and B (98% acetonitrile in HPLC
   water) were used for the RP gradient. Tryptic peptides were separated
   at a fluent flow rate of 300 μL/min and monitored at 210 nm and 280 nm.
   The separated peptides were lyophilized for mass spectrometry. All
   analyses were performed using a Q-Exactive mass spectrometer (Thermo
   Fisher Scientific, Waltham, MA, USA) equipped with a Nanospray Flex
   source (Thermo Fisher Scientific). Samples were loaded onto and
   separated using a C18 column (15 cm × 75 µm) on an EASY–nLC TM 1200
   system (Thermo Fisher Scientific). Full MS scans were acquired in the
   mass range of 300~1600 m/z with a mass resolution of 70,000, and the
   automated gain control (AGC) target value was set at 1e6. The ten most
   intense peaks in the MS spectrum were fragmented with higher-energy
   collisional dissociation (HCD) with a normalized collision energy (NCE)
   of 32. MS/MS spectra were obtained with a resolution of 17,500, an AGC
   target of 2e5, and a maximum injection time of 80 ms. The Q–E dynamic
   exclusion was set at 30.0 s and run under positive mode.

2.8. Database Search

   Proteome Discoverer v.2.4 (Thermo Fisher Scientific) was used to
   thoroughly search all of the Q Exactive raw data against the sample
   protein database. The database search was performed using Trypsin
   digestion specificity. Alkylation on cysteine was considered as a fixed
   modification during the database searching. For the protein
   quantification, the TMT labeling method was selected. The global false
   discovery rate (FDR) was set to 0.01, and protein groups considered for
   quantification required at least 1 peptide. The differentially
   expressed proteins (DEPs) were screened using a standard of fold change
   (FC) > 1.5 or < 0.667, and p-value < 0.05. All DEPs were mapped to
   three main categories of Gene Ontology (GO) terms for annotation,
   namely cellular component, molecular function, and biological process,
   which were obtained from the GO consortium website
   ([61]http://www.ebi.ac.uk/GOA/, accessed on 17 December 2022). Kyoto
   Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis
   was also performed using DAVID 6.7 [[62]17,[63]18]. Protein–protein
   interaction (PPI) networks were constructed using the Search Tool for
   the Retrieval of Interacting Genes/Proteins (STRING) database [[64]19]
   ([65]http://string.embl.de/, accessed on 8 January 2023) and further
   visualized using Cytoscape software [[66]20].

2.9. Statistical Analysis

   All statistical analyses were performed using SPSS 16.0 statistical
   software (IBM Corp., NY, USA). Data are expressed as the mean ± SD. In
   this study, independent samples t-test was used for comparisons between
   groups. p < 0.05 was considered to be statistically significant.

3. Results

3.1. Characterization and Secretion of Exosomes from Control and
PEDV–Infected Vero Cells

   The morphology and size of the isolated Vero cell exosomes were
   characterized using NTA and TEM. The NTA measurements showed that the
   mean particle size of the exosomes of the Vero cells in the control
   group was 150.8 nm ([67]Figure 1A), and the mean particle size of the
   exosomes in the PEDV-infected group was 112.4 nm ([68]Figure 1B). The
   TEM analysis showed that the Vero cell exosomes from the control and
   PEDV-infected groups had a typical cup–shaped structure ([69]Figure
   1C,D).

Figure 1.

   [70]Figure 1
   [71]Open in a new tab

   Characterization of Vero–cell–derived exosomes from PEDV–infected group
   and control group. (A) Particle size and quantification from control
   cells. (B) Particle size and quantification of PEDV–infected cells. (C)
   Morphology of exosomes from control group observed via TEM. (D)
   Morphology of exosomes from PEDV–infected group. Magnification = ×60.0
   K; scale bars, 100 nm. PEDV, porcine epidemic diarrhea virus; TEM,
   transmission electron microscopy.

   The NTA assay data indicate that the average concentration of exosomes
   in the control group was 1.07 × 10^10 particles/mL, whereas in the
   PEDV–infected group, it was significantly higher at 9.37 × 10^10
   particles/mL (p < 0.001) ([72]Figure 2A). The Western blot analysis for
   the levels of exosomal signature proteins CD63, CD9, and TSG101
   ([73]Figure 2B) showed that the relative levels of CD63 was
   significantly higher in the PEDV-infected group than in the control
   group (p < 0.01) ([74]Figure 2C). These findings suggest that PEDV
   infection increases the content of exosomes in Vero cell culture
   medium.

Figure 2.

   [75]Figure 2
   [76]Open in a new tab

   Effect of PEDV infection on the secretion of exosomes from Vero cells.
   (A) The concentration of exosomes from control and PEDV–infected
   groups. (B) Western blot detection of exosomal signature proteins CD63,
   CD9, and TSG101. (C) Relative expression of exosomal protein CD63 from
   control and PEDV–infected groups. Note: ** p < 0.01, *** p < 0.001.
   TSG101, tumor susceptibility 101; GAPDH, glyceraldehyde–3–phosphate
   dehydrogenase.

3.2. Identification of Exosomal Proteins from Vero Cells

   To comprehensively assess the effect of PEDV infection on the levels of
   proteins in exosomes secreted by Vero cells, the proteomic profiles of
   the exosomes were characterized using TMT labeling LC–MS/MS. In this
   study, 32,635 peptides and 5001 protein groups were identified with an
   FDR of less than 0.01. These proteins were screened for confidence
   using a score sequest HT of > 0 and a unique peptide of ≥1. A total of
   4586 credible proteins were screened, and the information on the
   identified credible proteins is presented in [77]Supplementary Table
   S1. The principal component analysis (PCA) of the credible proteins
   showed significant differences in the PCA of the Vero cell exosome
   proteins between the control and PEDV–infected groups ([78]Figure S1A).
   The sample correlation analysis of the credible proteins showed a high
   inter–sample correlation and intra–group sample reproducibility
   ([79]Figure S1B). The sample-level clustering analysis of the credible
   proteins showed that the control and PEDV–infected groups clustered in
   different branches, respectively ([80]Figure S1C). The molecular
   weights of the identified credible proteins were mainly distributed in
   the range of 10 to100 kDa ([81]Figure S1D).

3.3. Analysis of Differentially Expressed Proteins in Exosomes from Control
and PEDV–Infected Groups

   According to the screening criteria (FC > 1.5 and p-value < 0.05), 544
   DEPs were identified from the exosomal protein analysis, among which
   236 proteins were upregulated and 308 proteins were downregulated
   ([82]Figure 3A; [83]Supplementary Table S2). The cluster heat map
   analysis showed significant clustering of the DEPs between the control
   and PEDV-infected groups ([84]Figure 3B). Among the DEPs, fibronectin 1
   (FN1), serine protease inhibitor (SERPIN) family proteins (SERPINA1,
   SERPINA5, SERPINE2, SERPINF2), and collagen family proteins (COL4A1,
   COL4A2, COL5A1, COL5A2, COL6A2, COL7A1) were significantly upregulated,
   while integrin beta-1 (ITGB1) protein, tetraspanin protein family
   (CD36, CD44, CD151, CD58), and solute carrier family proteins (SLC1A4,
   SLC4A4, SLC16A1, SLC34A1) were significantly downregulated in the
   exosomes from the PEDV-infected cells ([85]Figure 3C). These
   significant DEPs might be involved in the infection and proliferation
   processes of PEDV in host cells.

Figure 3.

   [86]Figure 3
   [87]Open in a new tab

   DEP analysis of exosomes from control and PEDV–infected groups. (A) The
   identified DEPs in the exosomes of PEDV–infected group compared with
   those in control group. (B) Heat map of comparative intergroup
   clustering analysis of DEPs. (C) Statistical analysis of protein
   expression levels of FN1, COL11A1, SERPINA1, ITGB1, CD36, and SLC1A4.
   Note: *** p < 0.001.

3.4. GO and KEGG Pathway Annotations of the DEPs

   The GO enrichment analysis was used to further analyze the cellular
   functions of the DEPs in the Vero cell exosomes. The results show that
   the GO terms were enriched into three categories involving the
   biological process, cellular component, and molecular function. Among
   the top 30 enriched GO terms, cell adhesion, extracellular matrix
   organization, and carbohydrate metabolic process were highly enriched
   in the biological process category. The cellular component category
   mainly focuses on extracellular exosome, extracellular space, and
   cell–cell adherens junction. Cadherin binding is involved in cell–cell
   adhesion, extracellular matrix structural constituent, and heparin
   binding, being particularly enriched in molecular function ([88]Figure
   4). From the GO enrichment analysis chord diagram, the DEPs with
   upregulated expression were mainly enriched in extracellular matrix
   organization, extracellular matrix, and collagen catabolic process,
   while those with downregulated expression were mainly enriched in
   cadherin binding involved in cell–cell adhesion, cell–cell adherens
   junction, and basement membrane ([89]Figure 5). These results suggest
   that the exosomal DEPs from the control and PEDV-infected groups are
   involved in cell adhesion, metabolism, and extracellular matrix
   composition.

Figure 4.

   [90]Figure 4
   [91]Open in a new tab

   GO annotation analysis of the DEPs identified in the exosomes isolated
   from control and PEDV–infected groups.

Figure 5.

   [92]Figure 5
   [93]Open in a new tab

   GO enrichment analysis chord diagram.

   The KEGG analysis of the DEPs showed that they are involved in a
   variety of life activities, and many of the proteins were annotated
   into the PI3K–Akt signaling pathway, focal adhesion, extracellular
   matrix (ECM)–receptor interaction pathway, and regulation of the actin
   cytoskeleton ([94]Figure 6). Thirty-five exosomal DEPs were enriched in
   the PI3K–Akt signaling pathway (e.g., ITGB1, YWHAE, CDC37, and ITGA3),
   26 DEPs were enriched in focal adhesion (e.g., LAMC1, RAP1B, FN1, and
   COL1A1), 25 DEPs were enriched in the ECM–receptor interaction pathway
   (e.g., CD36, HSPG2, and SPP1), and 20 DEPs were enriched in actin
   cytoskeleton regulation (e.g., MSN, EZR, and RDX). These results
   suggest that exosomes play a crucial role in cell signaling, and that
   many DEPs are associated with cellular immunity, cytoskeleton, and
   adhesion-related pathways.

Figure 6.

   [95]Figure 6
   [96]Open in a new tab

   KEGG pathway analysis of the DEPs identified in the exosomes isolated
   from control and PEDV–infected groups.

3.5. PPI Network Analysis of the DEPs

   In this study, the top 25 PPIs among the DEPs were analyzed to map the
   PPI interactions profile. The results show that 8 of the 25 DEPs were
   upregulated proteins, and 17 were downregulated proteins ([97]Figure
   7A). The DEPs associated with cellular immunity, cell adhesion,
   cytoskeleton, and cell death in the exosomes were analyzed for PPIs;
   those related to cellular immunity include 14 upregulated proteins
   (e.g., TLR4, APP, BMP4, CXCL12, CSF1, COL1A1) and 11 downregulated
   proteins (e.g., IL6, SYK, ITGB1, CALR, MSN, EZR) ([98]Figure 7B), and
   those related to cell adhesion include 16 upregulated proteins (e.g.,
   FN1, collagen family proteins, laminin family proteins, TNN, PIK3R3,
   TNC) and 8 downregulated proteins (e.g., ITGB1, ITGA3, CRK, GRB2,
   RAP1A, SPP1) ([99]Figure 7C). ITGB1, ITGA3, MSN, and ENAH are
   upregulated proteins associated with the cytoskeleton, while FN,
   PIK3R3, and CXCL12 are downregulated proteins ([100]Figure 7D); the
   levels of ITGB1, IL6, YWHAG, YWHAB, YWHAE, and YWHAH, which are
   involved in the regulation of cell apoptosis, were all decreased in the
   exosomes of the PEDV-infected group ([101]Figure 7E). It is worth
   noting that some proteins, such as ITGB1, ITGA3, IL6, and CD44
   (downregulated), and FN1, PIK3R3, TLR4, and COL1A1 (upregulated), are
   involved in various cell biological processes. These findings reveal
   that PEDV infection in Vero cells leads to significant alterations in
   the levels of several proteins with important biological functions in
   the exosomes.

Figure 7.

   [102]Figure 7
   [103]Open in a new tab

   PPI network analysis of the DEPs identified in the exosomes isolated
   from control and PEDV–infected groups. (A) PPI interaction network
   analysis of the top 25 protein interactions among the DEPs. (B) PPI
   interaction network analysis of DEPs enriched in cellular immune
   responses. (C) PPI interaction network analysis of DEPs enriched in
   cell adhesion. (D) PPI interaction network analysis of DEPs enriched in
   the cytoskeleton. (E) PPI interaction network analysis of DEPs enriched
   in apoptosis. The dots in the figure represent DEPs, red represents
   upregulated DEPs, and green represents downregulated DEPs; the size of
   the dots represents the level of connectivity, with larger dots
   indicating higher connectivity. The straight lines represent the
   proteins that are correlated with each other.

4. Discussion

   Although PEDV has been a global epidemic for many years and has caused
   serious economic losses in the pig industry, the pathogenic mechanism
   of PEDV infection in host cells still needs to be further investigated
   from different perspectives due to the high genetic heterogeneity of
   PEDV [[104]21]. Exosomes are extracellular vesicles that are secreted
   by cells and play a crucial role in intercellular communication. They
   transfer biologically active proteins, lipids, and RNAs to neighboring
   or distant cells, thereby performing an important biological function
   [[105]5]. However, the research on the biological functions of exosomes
   during the virus infection of host cells is still in its infancy
   [[106]22,[107]23]. Currently, an increasing number of researchers have
   focused on the role and mechanisms of host–cell–derived exosomes in
   PEDV infection. In this study, Vero cells are used as a research model
   due to their high susceptibility to PEDV infection, and are widely
   applied for PEDV virus isolation, vaccine preparation, and pathogenic
   mechanism studies [[108]15,[109]24,[110]25]. Porcine intestinal
   epithelial cells (IECs) are generally considered an ideal cellular
   model for studying PEDV pathogenic mechanisms since they are the host
   target cells for PEDV infection. However, the low replication
   efficiency of PEDV in porcine intestinal cell lines limits their
   application [[111]26]. It was found that PEDV infection exhibits
   similar mechanisms in both Vero cells and IECs. Cong et al. found that
   pAPN mediates the infection and release of PEDV in both Vero cells and
   IECs [[112]27]. PEDV N protein activates the P53–DREAM pathway,
   inducing Vero cell and IEC cycle arrest at the S phase [[113]26].
   Additionally, integrin avB3 promotes PEDV replication in Vero E6 cells
   and IECs [[114]28]. Therefore, it is representative to study the
   effects of PEDV infection on host cells’ exosome characteristics and
   proteomic profiles using Vero cells as model cells.

   Increasing evidence suggests that exosomes are involved in the cellular
   immune response to infection with viruses and are associated with virus
   infection, proliferation, and transmission
   [[115]29,[116]30,[117]31,[118]32]. Exosomes originate from late
   endosomes called multivesicular bodies (MVBs), which are a component of
   the endocytic pathway. MVBs contain intraluminal vesicles (ILVs) of
   varying sizes, which fuse with the cytoplasmic membrane and release
   ILVs into the extracellular environment, at which point they are
   referred to as exosomes [[119]7]. The ESCRT pathway is the most
   understood pathway for the mechanism of exosome biogenesis [[120]33].
   Additionally, the Rab GTPases family is also involved in exosome
   biogenesis [[121]22], whereby Rab11, Rab27, Rab5, Rab35, and Rab7 were
   identified as key regulators of exosome secretion [[122]33]. A study
   revealed that some viruses can manipulate the exosomal pathway for
   transmission, assembly, and excretion [[123]34]. The “Trojan” exosome
   hypothesis was first proposed by Gould et al. [[124]35], which suggests
   that the assembly and release of HIV share similarities with the
   biology of exosomes; HIV has evolved to exploit the exosome system and
   infect cells by packaging the virus [[125]35,[126]36]. HIV, Ebola
   virus, rabies virus, and herpes simplex virus 1 (HSV1) all employ
   distinct strategies to hijack members of the ESCRT pathway that are
   involved in exosome formation [[127]37,[128]38]. The studies revealed
   that HIV and HSV1 infection can induce alterations in the expression
   levels of host cell Rab27a, subsequently affecting exosome biogenesis
   [[129]39,[130]40]. Wang et al. found that rabies virus infection can
   increase the release of exosomes from host cells, and that the
   inhibitors of exosome secretion can significantly inhibit cellular
   exosome release and reduce viral proliferation [[131]41]. These studies
   suggest that viral infections can significantly affect exosome
   biogenesis. Notably, our results reveal that PEDV can significantly
   alter the size and number of host cell exosomes, suggesting that this
   might be related to the mechanism by which PEDV hijacks Vero cell
   exosome formation. A previous study showed that the use of the exosome
   formation inhibitor GW4869 could effectively inhibit virus
   proliferation [[132]13,[133]41]. However, in our preliminary research,
   we found that even at low concentrations (5 μM), the exosome inhibitor
   GW4869 significantly reduced the activity of Vero cells; therefore, we
   will try to use other approaches to verify the role of host exosomes in
   PEDV proliferation. However, the specific mechanism still requires
   further investigation.

   The proteomic analysis in this study showed that PEDV infection caused
   extensive changes to the protein levels in the Vero cell exosomes, and
   a total of 544 DEPs were identified. These exosomal DEPs might play
   important molecular signaling roles in cellular immunity and viral
   infection; e.g., ITGB1 is a transmembrane receptor that mediates the
   connection between the cell and its external environment [[134]42],
   which was recently found to be a co-receptor for angiotensin-converting
   enzyme 2 (ACE2), enhancing the binding of novel coronavirus proteins to
   their receptor ACE2 [[135]43]. The levels of SERPIN family proteins
   (SERPINA1, SERPINA5, SERPINE2, SERPINF2), which have a significant
   inhibitory effect on the proliferation of HIV, recombinant vesicular
   stomatitis virus (rVSV), and Zika virus (ZIKV) [[136]44], were
   significantly increased in the PEDV–infected Vero cell exosomes. There
   are two independent studies on the effect of PEDV infection on the
   miRNA and protein levels in exosomes. Yin et al. found that the levels
   of 115 miRNAs were altered in the exosomes of PEDV–infected cells
   compared with those in the controls, and these differentially abundant
   miRNAs were associated with a significant increase in the levels of
   miRNAs through association with cAMP, Hippo, TGF–beta, HIF–1, FoxO,
   MAPK, and Ras signaling pathways [[137]15]. Chen et al. found that PEDV
   infection significantly decreased the levels of complement proteins
   (C3, C6, CFB) in piglet serum exosomes, demonstrating that serum
   exosomes from newborn piglets can significantly inhibit PEDV infection,
   and that this inhibitory effect is closely related to complement
   [[138]14]. In addition, Zhao et al. discovered that miRNA–328–3p is
   significantly downregulated in the exosomes from PEDV-infected Vero E6
   cells. Furthermore, miRNA–328–3p can inhibit PEDV infection by
   targeting and suppressing TJP3 in receptor cells [[139]16]. These
   findings indicate that investigating the alterations in exosome
   composition during PEDV infection can provide clues to the pathogenic
   mechanisms of PEDV and identify potential antiviral targets.

   Exosomes act as mediators of intercellular crosstalk in metabolism,
   accurately reflect changes in the cellular microenvironment, and play
   crucial roles in information transmission, microenvironment modulation,
   and immune regulation [[140]34,[141]45,[142]46,[143]47]. The present
   study further analyzed the enriched GO categories of the DEPs and
   showed that many proteins were enriched in cellular biological
   processes such as cell adhesion, extracellular matrix organization, and
   carbohydrate metabolism, indicating that PEDV infection not only leads
   to abnormal levels of exosomal proteins associated with cellular
   material metabolism, but also has significant effects on the cell
   structure and extracellular matrix homeostasis. Moreover, the exosomal
   DEPs in the present study involve multiple cell signaling pathways such
   as the PI3K/Akt signaling pathway, focal adhesion, extracellular matrix
   (ECM)—receptor interaction pathway, and are also involved in the
   regulation of the actin cytoskeleton. The PI3K–Akt signaling pathway is
   a crucial intracellular signaling pathway that plays a significant role
   in promoting cell proliferation and metabolism, and certain viruses can
   exploit this signaling pathway to facilitate their own proliferation
   [[144]48,[145]49]. Herein, we revealed that 35 exosome-derived DEPs
   were enriched in the PI3K/Akt signaling pathway, which also represented
   the highest enrichment in the KEGG analysis of the DEPs. Previous
   studies on the effect of PEDV infection on the PI3K/Akt signaling
   pathway in host cells were conducted. Shen et al. found that PEDV
   infection significantly suppressed the expression of key molecules in
   the PI3K/Akt signaling pathway in IPEC–J2 cells [[146]50]. Lin et al.
   found that PEDV infection suppressed the protein synthesis of IPEC–J2
   cells via the downregulation of the PI3K–AKT/mTOR signaling pathways
   [[147]51]. Further studies revealed that non-structural protein 6 of
   PEDV induces autophagy through the PI3K/Akt/mTOR axis to promote virus
   replication [[148]52]. These findings indicate that PEDV infection
   induces significant alterations in the expression levels of regulatory
   molecules in the PI3K/Akt signaling pathway of the host cells.
   Therefore, we hypothesized that changes in the exosomal protein levels
   induced by PEDV infection might play an important role in intercellular
   transmission and regulation of the PI3K/Akt signaling pathway.

   Focal-adhesion-based cell–extracellular matrix interactions are
   essential for cell anchoring and cell migration, and plays a crucial
   role in development, immunity, and disease [[149]53,[150]54,[151]55].
   Research has revealed that herpes simplex virus 1 (HSV–1) envelop
   proteins (pUL7 and pUL51), localize to focal adhesions on the
   cytoplasmic membrane, and maintain the attachment of infected cells to
   their surrounding environment by modulating the activity of focal
   adhesion complexes [[152]56]. Ramsauer et al. found that the equine
   papillomavirus type 2 (EcPV2) infection of genital organs can cause
   changes in gene expression, with many DEGs being enriched in
   ECM–receptor interactions and focal adhesion pathways [[153]57]. In the
   present study, we found that many DEPs were enriched in the focal
   adhesion (26 DEPs) and ECM–receptor interaction (25 DEPs) pathways.
   Currently, no studies have reported the association of PEDV infection
   with host cell focal adhesion. However, there was an interesting study
   on ECM–related signaling and PEDV infection, which found extensive ECM
   remodeling in PEDV–infected intestinal epithelial cells. Moreover, the
   transcript levels of two genes closely related to the ECM pathway (CD44
   and SERPINE1) in PEDV-infected intestinal epithelial cells were
   significantly and positively correlated with the extent of PEDV
   replication. CD44 enhanced the expression of antiviral cytokines (such
   as IL–6, IL–18, and the antimicrobial peptide β-defensin 1) by
   activating NF–κB signaling. In contrast, PAI–1 (encoded by SERPINE1)
   promoted progeny virus release during PEDV infection [[154]58].
   Notably, we identified a significant decrease in the CD44 and IL6
   protein levels in the exosomes of the PEDV-infected Vero cells, while
   the levels of SERPIN family proteins (SERPINA1, SERPINA5, SERPINE2,
   SERPINF2) increased significantly. These findings suggest that PEDV
   might inhibit the antiviral response of cells and promote viral release
   through the ECM-related signaling pathway.

   The cytoskeleton includes microfilaments, microtubules, and
   intermediate fibers [[155]59]. Viral infection alters the normal
   composition of the cell, affecting the cytoskeletal structure to
   optimize virus entry, replication, and virion production
   [[156]60,[157]61]. Zhao et al. demonstrated that infection with PEDV
   can compromise the integrity of the IPEC–J2 cell barrier and also
   affect the remodeling of cellular microfilaments [[158]62]. Hou et al.
   demonstrated that microtubules are involved in PEDV infection and can
   influence PEDV fusion and accumulation in the perinuclear region, and
   they used single virus tracking techniques to dynamically observe
   different types of intracellular PEDV transport through microtubules
   [[159]63]. Our study revealed that PEDV infection led to changes in the
   levels of cytoskeleton regulatory proteins in the exosomes, further
   confirming the involvement of cytoskeletal structures in the PEDV
   infection of host cells.

   In particular, we identified two integrin proteins, ITGB1 and integrin
   subunit alpha 3 (ITGA3), which were significantly downregulated in the
   PEDV-infected cell-derived exosomes. These proteins are involved in
   various signaling pathways, including cell immune response, focal
   adhesion, and cellular cytoskeleton. However, their involvement in the
   PEDV infection of host cells has not been reported. ITGB1 acts as an
   ECM receptor, and recent studies have revealed that it interacts with
   the SARS-CoV-2 cell receptor ACE2 and assists ACE2 in mediating
   SARS-CoV-2 infection and entry into human cells; either reducing or
   blocking ITGB1 can inhibit the efficiency of SARS-CoV-2 infection
   [[160]43]. In addition, ITGB1 is a key receptor required for human
   cytomegalovirus (HCMV) proliferation [[161]64]. ITGA3 is an integrin
   located on the cell membrane that functions as a cell surface adhesion
   molecule, and its expression is associated with cancer metastasis
   [[162]65]. A study revealed that HIV-1 infection leads to significant
   alterations in the gene expression profile of macrophages. Among these
   changes, one of the most prominent altered genes is ITGA3, the
   upregulation of which facilitates viral budding and release [[163]66].
   Zeng et al. found that during the infection of Vero E6 cells with
   porcine acute diarrhea syndrome coronavirus (SADS–CoV), autophagy was
   induced through the regulation of the Akt/mTOR pathway, which promoted
   viral replication. Moreover, it was discovered that ITGA3 exerts an
   effective antiviral effect by inhibiting cellular autophagy [[164]67].
   Therefore, we speculated that ITGB1 and ITGA3 might play important
   regulatory roles in the process of PEDV infection in host cells.
   However, the specific functions and regulatory mechanisms still require
   further research.

5. Conclusions

   In summary, we observed significant alterations in the average particle
   size and secretion concentration of exosomes from the PEDV–infected
   group. Furthermore, using TMT-labeled LC-MS/MS, we quantitatively
   analyzed the differential protein compositions of the exosomes and
   identified 544 DEPs. These DEPs are involved in various cellular
   processes, such as cell adhesion, extracellular matrix organization,
   and carbohydrate metabolism, and are closely associated with cellular
   regulatory pathways including the PI3K–Akt signaling pathway,
   ECM–receptor interaction, focal adhesion, and cytoskeleton regulation.
   These findings will provide a basis for investigating the pathogenic
   mechanism of PEDV and discovering new antiviral targets.

Acknowledgments