Abstract

Simple Summary

   Exosomes play a unique role in virus infection, antigen presentation,
   and suppression/promotion of body immunity. Porcine reproductive and
   respiratory syndrome virus (PRRSV) is one of the most damaging
   pathogens in the pig industry. Here, we used the PRRSV NADC30-like
   CHsx1401 strain to artificially infect 42-day-old pigs, isolate serum
   exosomes, and identify 33 significantly differentially expressed (DE)
   exosomal miRNAs between infection and control groups, and 18 DE miRNAs
   associated with PRRSV infection and immunity were screened as potential
   functional molecules involved in the regulation of PRRSV virus
   infection by exosomes.

Abstract

   Exosomes are biological vesicles secreted and released by cells that
   act as mediators of intercellular communication and play a unique role
   in virus infection, antigen presentation, and suppression/promotion of
   body immunity. Porcine reproductive and respiratory syndrome virus
   (PRRSV) is one of the most damaging pathogens in the pig industry and
   can cause reproductive disorders in sows, respiratory diseases in pigs,
   reduced growth performance, and other diseases leading to pig
   mortality. In this study, we used the PRRSV NADC30-like CHsx1401 strain
   to artificially infect 42-day-old pigs and isolate serum exosomes.
   Based on high-throughput sequencing technology, 305 miRNAs were
   identified in serum exosomes before and after infection, among which 33
   miRNAs were significantly differentially expressed between groups (13
   relatively upregulated and 20 relatively downregulated). Sequence
   conservation analysis of the CHsx1401 genome identified 8 conserved
   regions, of which a total of 16 differentially expressed (DE) miRNAs
   were predicted to bind to the conserved region closest to the 3′ UTR of
   the CHsx1401 genome, including 5 DE miRNAs capable of binding to the
   CHsx1401 3′ UTR (ssc-miR-34c, ssc-miR-375, ssc-miR-378, ssc-miR-486,
   ssc-miR-6529). Further analysis revealed that the target genes of
   differentially expressed miRNAs were widely involved in exosomal
   function-related and innate immunity-related signaling pathways, and 18
   DE miRNAs (ssc-miR-4331-3p, ssc-miR-744, ssc-miR-320, ssc-miR-10b,
   ssc-miR-124a, ssc-miR-128, etc.) associated with PRRSV infection and
   immunity were screened as potential functional molecules involved in
   the regulation of PRRSV virus infection by exosomes.

   Keywords: PRRSV, serum exosome, miRNAs

1. Introduction

   Porcine reproductive and respiratory syndrome virus (PRRSV) is a
   single-stranded positive-strand RNA virus with an envelope structure
   belonging to the order Nidovirales, family Arteriviridae, genus
   Betaarterivirus [[38]1,[39]2]. It is spherical or ellipsoidal with a
   diameter of 50–65 nm under a freezing electron microscope
   [[40]3,[41]4]. The PRRSV genome is about 15 kb in length with a 5′ cap
   and a 3′ polyA-tail and contains at least 10 open reading frames (ORFs)
   flanked by untranslated regions (UTRs) at both the 5′ and 3′ termini
   [[42]5,[43]6], and is wrapped by nucleocapsid protein, with lipid
   double-layer coating to form virus particles.

   Exosomes belong to vesicles with monolayer membrane structures and have
   the same topological structure as cells [[44]7]. The shape is
   “cup-shaped” or “disc-shaped” under an electron microscope
   [[45]8,[46]9]. Exosomes can exist in the circulatory system for a long
   time, and substances in exosomes can be absorbed by adjacent cells or
   distant receptor cells and then regulate the receptor cells to
   participate in the exchange of genetic materials between cells
   [[47]10,[48]11]. They are mainly composed of membrane surface
   substances and carried contents, including cell surface receptors,
   membrane proteins, soluble proteins, lipids, RNA (mRNA, miRNA, lncRNA,
   and viral RNA, etc.), genomic DNA, mitochondrial DNA
   [[49]12,[50]13,[51]14]. MicroRNAs (miRNAs) are a class of 18–25
   nucleotides (nt) evolutionarily conserved endogenous non-coding
   single-stranded small RNAs, which inhibit the translation process by
   inducing the degradation of target mRNA or by binding with 3′ UTR of
   target mRNA, leading to post-transcriptional gene silencing, then
   regulating the gene expression at the post-transcriptional level
   [[52]15,[53]16,[54]17]. It is estimated that miRNAs regulate more than
   60% of mammalian genes post-transcriptionally [[55]18,[56]19]. MiRNAs
   play an important role in intercellular communication and can also be
   used as a potential functional molecule for disease and virus
   infection, transmission, and defense [[57]20]. A growing number of
   studies have shown that miRNAs can be present in body fluids, such as
   saliva, urine, breast milk, and blood, and act through the body’s fluid
   circulatory system [[58]21,[59]22]. Exosomal miRNAs are considered to
   be endogenous regulators of gene expression and metabolism and can
   indicate various pathological conditions [[60]23,[61]24].

   Over the past two decades, it has been shown that miRNAs have crucial
   roles in the regulation of immune cell development, innate immune
   responses, and acquired immune responses. Some other miRNAs are
   reported to impair PRRSV infection through the following ways, directly
   target the PRRSV genome or PRRSV receptor, or play a role by regulating
   the host’s innate immune response. The miR-26 family can significantly
   damage virus replication, and miR-26a can inhibit the replication of
   type 1 and type 2 PRRSV strains in porcine alveolar macrophages (PAMs)
   by regulating the type I interferon (IFN) pathway, which is more
   efficient than miR-26b [[62]25,[63]26]. miR-30c and miR-125b are
   identified to modulate host innate immune response by targeting the
   type I IFN pathway and NF-κB pathway, respectively
   [[64]27,[65]28,[66]29]. MiR-23, miR-378, and miR-505 are antiviral host
   factors targeting PRRSV and have conservative target sites in type 2
   PRRSV strains [[67]30]. At the same time, host miR-506 has been
   identified to inhibit PRRSV replication by directly targeting PRRSV
   receptor CD151 in MARC-145 cells [[68]31]. miR-181 also can indirectly
   inhibit PRRSV replication by down-regulating PRRSV receptor CD163 in
   blood monocytes and PAMs [[69]32]. In addition, miRNAs can promote
   PRRSV replication by interfering with basic cell physiology. MiR-24-3p
   and miR-22 directly target 3′UTR of HO-1 during PRRSV infection to
   escape the inhibition of heme oxygenase-1 (HO-1), a heat shock protein
   (also known as HSP32) on PRRSV [[70]33,[71]34].

   Pigs are known to be more susceptible to PRRSV and less able to defend
   themselves against the entry of this pathogen into the organism
   [[72]35]. In the present study, the innate immunity and acquired
   immunity of pigs infected with this virus were studied at the molecular
   level using a strain prevalent in the field. A serum exosome isolation
   kit, transmission electron microscopy (TEM), nanoparticle tracking
   analysis (NTA), and Western blot (WB) were used to isolate and identify
   serum exosomes before and after infection with PRRSV, followed by small
   RNA sequencing analysis, identification, and analysis of differential
   expression results using bioinformatics methods to obtain a number of
   PRRSV-associated serum exosome miRNAs, followed by identification of
   data results using quantitative real-time PCR (qRT-PCR).

2. Materials and Methods

2.1. Animal Experiments

   Six PRRSV antigen and antibody double-negative healthy 42-day-old large
   white pigs were placed in the pig clean feeding system for isolation,
   healthcare, and environmental adaptation. All pigs were free to eat and
   drink without restrictions. When they were familiar with the conditions
   in the isolator, the pigs were nasally inoculated with 2 mL 10^5
   TCID[50]/mL PRRSV NADC30-like CHsx1401, which was mentioned by
   predecessors [[73]36,[74]37]. The blood of the pigs before (control
   group, n = 6) and 7 days after (treatment group, n = 6) virus
   inoculation was collected from the anterior vena cava for serum
   isolation. The cellular debris in the serum was removed by
   centrifugation at 3000 g for 15 min. All animal experiments in our
   study were approved by the Animal Ethics Committee of the Institute of
   Animal Science, Chinese Academy of Agricultural Sciences (CAAS)
   (Beijing, China), IAS2022-130.

2.2. Isolation and Purification of Serum Exosomes

   Exosome isolation and purification were carried out using the exoEasy
   Maxi kit (QIAGEN, Hilden, Germany, cat. no. 76064) according to the
   manufacturer’s protocol.

2.3. Transmission Electron Microscopy (TEM)

   Extracted exosome suspensions were spotted onto the formvar
   carbo-coated copper mesh, and the exosomes were rinsed with PBS and
   subjected to standard uranyl acetate staining for 3 min at room
   temperature. After drying for several minutes at room temperature, the
   grid was visualized and photographed at 100 kV by transmission electron
   microscope (HT-7700, Hitachi-High Tech, Tokyo, Japan).

2.4. Nanoparticle Tracking Analysis (NTA)

   Extracted exosomes were diluted with 1 × PBS by changing the volume
   from 10 to 30 μL. After the sample was tested, the concentration and
   size of serum exosomes were analyzed by an N30E flow nano-analyzer
   following the manufacturer’s instructions (NanoFCM, Xiamen, China).

2.5. Western Blot

   The extracted exosome samples were added to RIPA lysate mixed with
   protease inhibitor (Invitrogen, Waltham, MA, USA) and
   phenylmethylsulfonyl fluoride (PMSF) to extract the exosome protein,
   which was lysed on ice for 30 min. Then, according to the instructions
   of the Bradford kit, we quantified the concentration of serum exosome
   protein. Exosome proteins underwent thermal denaturation. The same
   amount of protein was separated on 12% SDS-PAGE gel and then
   transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore,
   Burlington, MA, USA). It was soaked in TBST containing 5% skimmed milk
   powder and sealed for 1 h at room temperature. We soaked the membrane
   in the diluted primary antibody (anti-CD9 antibody, Abcam, Boston, MA,
   USA, #ab92726; anti-CD81 antibody, Abcam, Boston, MA, USA, #ab109201)
   overnight at 4 °C, and recovered the primary antibody. We soaked the
   membrane in the diluted secondary antibody, incubated it at room
   temperature for 1 h, and recovered the secondary antibody. We laid the
   washed film of PBST on the fresh-keeping film, added equal volume mixed
   ECL a/b chromogenic solution, and placed it in the chemiluminescence
   imager.

2.6. Exosomal Small RNA Sequencing and Data Analyses

   Total RNA from the exosomes was extracted with Trizol according to the
   manufacturer’s instructions. We then detected the RNA concentration and
   optical density (OD) value and detected the degradation and purity of
   RNA with 1% agarose gel electrophoresis. Meanwhile, Agilent Bioanalyzer
   2100 was used to detect the integrity of RNA. We used the total RNA of
   exosomes after quality inspection. According to the manufacturer’s
   instructions, we used NEB NEXT multiplex small RNA library prep set for
   Illumina^® (Illumina, San Diego, CA, USA). The kit prepared a small RNA
   cDNA library and sequenced it to produce 50 nt single-end reads by the
   Illumina Novaseq 6000 platform. All the procedures for small RNA
   library preparation were accomplished by Novogene (Beijing, China).

   The data after quality control were aligned to the porcine reference
   genome (Sus scrofa 11.1) using bowtie. Known miRNAs were identified by
   the miRbase (v22.0) database [[75]38] ([76]https://www.mirbase.org,
   accessed on 14 January 2022), miRdeep2 (v0.0.5) [[77]39], and miRevo
   (v1.1) [[78]40] and were used to predict new miRNAs. At the same time,
   the differential expression analysis for miRNAs was performed by DESeq
   (v1.24.0) [[79]41], requiring |fold change| > 1.6 and p < 0.05.
   Alignment was performed using MEGA (V11) [[80]42] followed by single
   base scoring using PHAST (v1.6.9) [[81]43] and evaluation of the most
   conserved regions of 10 virus genes, including WUH3 (GenBank accession
   no. [82]HM853973), VR2332 (GenBank accession no. [83]U87392), JXA1
   (GenBank accession no. [84]EF112445), CH-1a (GenBank accession no.
   [85]AY032626), NADC30 (GenBank accession no. [86]HN654459), HUN4
   (GenBank accession no. [87]EF635006), HLJZD22-1812 (GenBank accession
   no. [88]MN648450), SC/DJY (GenBank accession no. [89]MT075480), and
   Lelystad (GenBank accession no. [90]M96262.2). RNAhybrid (V2.0)
   [[91]44] was used to predict the binding of the identified miRNA
   sequence to the 3′ UTR of the CHsx1401 virus genome. MiRanda (v3.3a)
   and RNAhybrid were used to target gene prediction. The clusterProfiler
   [[92]45] R package was used for GO (Gene Ontology) functional
   enrichment analysis of target genes and KEGG (Kyoto Encyclopedia of
   Genes and Genomes) pathway enrichment analysis.

2.7. Validation of miRNA Expression by RT-qPCR

   Total RNA was isolated from serum exosomes using Trizol (Invitrogen,
   Shanghai, China) according to the manufacturer’s protocol. The isolated
   RNA was verified by RT-qPCR on samples (n = 6 per group). cDNA was
   synthesized according to the instructions of miRNA 1st strand cDNA
   synthesis (by stem-loop) kit (Vazyme, Nanjing, China), and the
   fluorescence quantification was performed using ABI 7500 according to
   the instructions of miRNA universal SYBR qPCR master mix (Vazyme,
   Nanjing, China). The thermal cycle parameters used were as follows: the
   first stage: 95 °C for 30 s; Stage 2: 95 °C for 5 s, 60 °C for 34 s,
   and 40 cycles; Stage 3: 95 °C for 15 s, 60 °C for 1 min, and 95 °C for
   15 s. Primer sequences of miRNAs, the U6 gene, were used as a reference
   [[93]46] and listed in [94]Supplementary Table S1. All qRT-PCR
   verifications were performed using three biological replicates and with
   three replicates for each sample. The relative abundance of transcripts
   was calculated by the 2^−ΔΔCt method, and SPSS (v22.0) and GraphPad
   Prism (v8.0) were used for data analysis and mapping, respectively. p <
   0.05 means the difference is statistically significant.

3. Results

3.1. Relative Value of Antigen and Antibody after Virus Inoculation

   The results of PRRSV antigen and antibody tests before (day 0) and
   after the (day 7) challenge are shown in [95]Table 1. The serological
   detection of the PRRSV antigen and antibody before the challenge was
   negative, and the antigen was positive after the challenge, indicating
   that the pigs were successfully infected with CHsx1401.

Table 1.

   Antigen and antibody of (day 0) and (day 7) with challenge virus.
   Time Control/before Inoculation (Day 0) Treatment/after Inoculation
   (Day 7)
   Antigen (Ct) / 26.05
   / 25.31
   / 20.69
   / 24.70
   / 25.01
   / 22.55
   Antibody (S/P) 0.03 /
   0.12 /
   0.15 /
   0.06 /
   0.25 /
   0.00 /
   [96]Open in a new tab

   Note: antigen “/” means negative, antibody < 0.4 means negative, Ct
   means cycle threshold, S/P value = (sample value − negative control
   mean value)/(positive control mean value − negative control mean
   value).

3.2. Isolation and Identification of Serum Exosomes

   The vesicles isolated from serum were discovered by TEM. Most vesicles
   can clearly see the concave saucer- or disc-shaped exosomes in the
   middle. The membrane edge of exosomes is clearly visible, and the
   morphology is relatively complete ([97]Figure 1A,B). The nanoparticle
   tracking analysis showed that 95.73% of the exosomes had a diameter of
   30–150 nm, mainly around 72.25 nm, with an average diameter of 76.22
   nm, which was consistent with the size characteristics of exosomes
   ([98]Figure 1C). This size range was similar to that detected by TEM
   and further confirmed the identity of these vesicles as exosomes.
   Western blot analysis showed that the vesicles isolated from the serum
   samples were positive for CD9 and CD81 proteins ([99]Figure 1D). The
   above characteristics conform to the exosome identification standards
   formulated by the international society for extracellular vesicles
   (ISEV) in MISEV2018 [[100]47].

Figure 1.

   [101]Figure 1
   [102]Open in a new tab

   Main characteristics of serum exosomes. (A,B) show the morphological
   characteristics of vesicles by TEM. Scale bars are 500 nm and 100 nm,
   respectively. (C) NTA shows the diameter and concentration of most
   vesicles. (D) Western blot showed the presence of exosome markers CD81
   and CD9 in serum exosomes. Note: Mix in WB results is the sample mixed
   suspension isolated by the exoEasy Maxi kit.

3.3. Small RNA Sequencing of Serum Exosomes

   For each sample, the clean data reached 0.5 Gb, and the Q30 base
   percentage was above 96.20%. The clean reads of each sample were
   aligned with the pig reference genome. Among the 12 samples, the
   control group obtained 10,920,887, 10,248,696, 10,109,117, 10,655,494,
   9,217,285, and 9,782,523 reads, respectively. The treatment group
   obtained 11,889,518, 10,593,504, 10,593,504, 12,846,080, 10,105,325,
   11,729,451, and 9,789,542 reads, respectively. On average, 77.96% of
   the total clean reads comprised 19–22 nucleotides (nt) in length
   ([103]Figure 2A). The reads after quality control accounted for more
   than 92.59% of the total reads. The processed clean reads were aligned
   to the porcine reference genome, and the mapped rate of 12 libraries on
   the genome was more than 92.30%, and the mapped rate was 94.98%
   ([104]Figure 2B). It indicated that the constructed serum exosomal
   miRNA library was of high quality and suitable for further analysis.
   Details are listed in [105]Supplementary Table S2.

Figure 2.

   [106]Figure 2
   [107]Open in a new tab

   Overview of the small RNA transcriptome data. (A) Length distribution
   of read counts of serum exosome samples (nt = nucleotides); (B) rate of
   12 samples mapped to the reference genome.

3.4. Differentially Expression Analysis of miRNAs

   After quantitative analysis of the identified miRNA expression, miRNAs
   were screened by the thresholds described previously in [108]Section
   2.6. A total of 305 miRNAs were obtained before and after inoculation
   of the CHsx1401 strain (control, n = 6; treatment, n = 6). A total of
   33 differentially expressed (DE) miRNAs were identified between the two
   groups, 13 DE miRNAs were upregulated, and 20 DE miRNAs were
   downregulated in the treatment group ([109]Figure 3 and
   [110]Supplementary Table S3).

Figure 3.

   [111]Figure 3
   [112]Open in a new tab

   Differential expression of miRNAs in exosomes. (A) Volcano plot of
   miRNAs between control and treatment groups; (B) hierarchical
   clustering heatmap of DE miRNAs between control and treatment groups.

3.5. Functional Enrichment Analysis of miRNA Target Genes

   A total of 7283 target genes were predicted by 33 DE miRNAs, and the
   functions of target genes were mainly concentrated in the positive
   regulation of MAPK cascade, lipid metabolism process, regulation of
   intracellular signal transduction, ERK1 and ERK2 cascade, etc.
   ([113]Figure 4A). In terms of molecular functions, the differentially
   expressed miRNAs target genes mainly focus on GTP-enzyme regulatory
   activity, kinase activity, nucleoside triphosphatase regulatory
   activity, and other functions related to signal transduction and energy
   metabolism ([114]Figure 4B). In addition, among the cell components,
   the target genes mainly participate in the biological functions of
   supramolecular polymers, Golgi, autophagosomes, cell surface, early
   endosomes, etc. ([115]Figure 4C). The functions of these components are
   closely related to the formation of exosomes, which also explains the
   accuracy of the sequencing.

Figure 4.

   [116]Figure 4
   [117]Open in a new tab

   GO function enrichment analysis of DE miRNAs target genes. (A)
   Biological process of DE miRNAs target genes; (B) molecular functions
   of DE miRNAs target genes; (C) cellular components of DE miRNAs target
   genes.

   KEGG pathway enrichment analysis showed that the target genes were
   significantly enriched in endocytosis, the MAPK signaling pathway, the
   Rap1 signaling pathway, the sphingolipid signaling pathway, and the
   PI3K Akt signaling pathway (p < 0.05) ([118]Figure 5A). At the same
   time, the enriched pathways were classified and analyzed. The results
   showed that the KEGG pathway of the target gene was mainly enriched in
   environmental information processing, human diseases, and biological
   systems ([119]Figure 5B).

Figure 5.

   [120]Figure 5
   [121]Open in a new tab

   KEGG pathway enrichment analysis of target genes. (A) Significantly
   enriched KEGG pathway with target genes of DE miRNAs; (B)
   classification of significantly enriched KEGG pathways.

3.6. Targeting Prediction of Serum Exosomal miRNA and PRRSV CHsx1401 Genome

   According to the phastCons score of a single base after alignment by
   PHAST, a total of eight most conserved segments (black bands above the
   peak map) were obtained among the viral genomes ([122]Figure 6). A
   total of 31 DE miRNAs were found to bind to the conserved segment by
   predicting the miRNAs bound to the conserved segment. Among them, in
   the conserved region (14,644–15,020 nt) closest to the 3′ UTR
   (14,870–15,020) of CHsx1401 genome, 16 DE miRNAs are predicted to bind
   to it, including 5 miRNAs (ssc-miR-34c, ssc-miR-375, ssc-miR-378,
   ssc-miR-486, and ssc-miR-6529) that can bind to the 3′ UTR of CHsx1401.
   Among these miRNAs, only ssc-miR-223 was upregulated after infection,
   and other miRNAs were downregulated after infection. See
   [123]Supplementary Table S4 for details.

Figure 6.

   [124]Figure 6
   [125]Open in a new tab

   Conserved segments in the genome of CHsx1401 strain predicted by PHAST.

3.7. Screening DE miRNAs Related to Exosome Function and PRRSV

   A variety of differentially expressed miRNAs related to the function of
   exosomes and PRRSV were found by functional enrichment analysis of
   target genes. Among them, 11 DE miRNAs such as ssc-miR-4331-3p,
   ssc-miR-744, and ssc-miR-320 are involved in exosome uptake, and their
   target genes are mainly concentrated in the Ras gene family, annexin
   family, and ADP ribosylation gene family. Eighteen DE miRNAs, including
   ssc-miR-10b, ssc-miR-124a, and ssc-miR-128, participate in
   immune-related pathways, and their target genes are mainly concentrated
   in the MAPK gene family, PIK3 gene family, and protein phosphatase gene
   family. While 11 DE miRNAs are involved in virus invasion, the related
   target genes are mainly concentrated in the MAPK gene family and
   protein phosphatase gene family. Furthermore, multiple differentially
   expressed miRNAs, such as novel_102. Six DE miRNAs, including
   ssc-miR-320, ssc-miR-423-5p, ssc-miR-4331-3p, ssc-miR-7137-3p, and
   ssc-miR-744, are co-expressed in exosome function, PRRSV virus
   invasion, and immune-related pathways, as shown in [126]Figure 7.
   Details are shown in [127]Supplementary Table S5.

Figure 7.

   [128]Figure 7
   [129]Open in a new tab

   DE miRNAs related to exosome uptake, PRRSV invasion, and immunity.

3.8. QRT-PCR Assay of DE miRNAs between the Two Groups

   Five DE miRNAs were randomly selected for verification. According to
   the qRT-PCR results, the expression of ssc-miR-19a and ssc-miR-32
   increased in the treatment group, while ssc-miR-124a, ssc-miR-375, and
   ssc-miR-34c showed higher expression in the control group, consistent
   with the sequencing data ([130]Figure 8).

Figure 8.

   [131]Figure 8
   [132]Open in a new tab

   Five DE miRNAs validated by qRT-PCR.

4. Discussion

   PRRSV is still a stubborn pathogen in the global pig industry, causing
   huge economic losses in the world. At present, vaccination is mainly
   used to prevent and control PRRSV, among which the modified live (MLV)
   virus vaccine is the most widely used [[133]48]. Although this vaccine
   was effective in reducing PRRS outbreaks and incidence, it also greatly
   increased genetic variation and diversity of the virus and led to viral
   recombination between wild and live vaccine viruses in the field
   [[134]49,[135]50]. In recent years, the spread and prevalence of the
   recombinant virus NADC30-like PRRSV strain have caused multiple
   outbreaks of porcine reproductive and respiratory syndrome in China.
   The similarity between CHsx1401 and NADC30 used in this study remained
   at 92.2–99.1%. Since then, it has become an epidemic strain in China.
   Exosomes, as mediators of cell communication, are widely found in
   various body fluids and have unique advantages in disease diagnosis and
   treatment [[136]51,[137]52]. According to previous reports, exosomes
   play an important communication role in antigen presentation [[138]53],
   immune response [[139]53,[140]54], virus replication [[141]54], cancer
   [[142]55], neurodegenerative diseases [[143]56], angiogenesis
   [[144]57], tumor cell migration [[145]58] and invasion [[146]59], and
   have high research value.

   In this study, high-throughput sequencing technology was used to
   construct the miRNA expression profile of serum exosomes, and 33 DE
   miRNAs were identified. As we all know, the host-encoded miRNA can bind
   with the viral genome and then regulate the replication, synthesis, and
   release of the virus to limit infection and affect the pathological
   process [[147]15]. Studies of miRNAs targeting the viral genome have
   also been repeatedly reported in animals. gga-miR-454 and gga-miR-130b
   in chicken infectious bursal disease can target the viral genome to
   inhibit viral replication, while gga-miR-21 directly targets the viral
   protein VP1 to inhibit viral protein translation [[148]60,[149]61]. In
   PRRSV studies, ssc-miR-181 specifically binds to a highly conserved
   region downstream of the viral genome ORF4 and strongly inhibits PRRSV
   replication [[150]62]. In this study, the expression difference of
   ssc-miR-181 between the two groups did not reach a significant level.
   In our study, the genomes of nine different PRRSV viruses were compared
   with those of the CHsx1401 strain, and the eight most conserved
   segments were identified. It was predicted that 31 DE miRNAs could bind
   to the 8 most conserved segments of CHsx1401, and 16 DE miRNAs could
   bind to the conserved sequences close to the 3′ UTR of CHsx1401. Among
   them, 5 DE miRNAs (ssc-miR-34c, ssc-miR-375, ssc-miR-378, ssc-miR-486,
   and ssc-miR-6529) can simultaneously bind to the CHsx1401 3′ UTR. In
   addition, the upregulated expression of ssc-miR-223 was predicted to
   bind to the 3′UTR target of the PRRSV genome. The results showed that
   the conserved sequences of the virus genome might play a key role in
   its pathogenicity, and the miRNAs that can bind to the conserved
   sequences between the genomes of different PRRSV strains may have
   important significance in controlling the pathogenicity of the virus.
   Some differentially expressed miRNAs have been proven to be related to
   PRRSV by previous studies and even directly involved in the regulation
   of PRRSV, including ssc-miR-10b [[151]63], ssc-miR-378 [[152]30],
   ssc-miR-124a [[153]64], let-7f-5p [[154]65], ssc-miR-744 [[155]66], and
   ssc-miR-19a [[156]67].

   PRRSV can evade host defense by interfering with innate immune
   response. This process is regulated by many signaling pathways,
   including the MAPK signaling pathway, PI3K Akt signaling pathway,
   autophagy, chemokine, and TNF signaling pathway. At present, the MAPK
   signaling pathway includes three main pathways: ERK1/2, JNK, and p38
   pathway. Activation of the MAPK cascade can promote host cell
   apoptosis, assist the virus in escaping the host immune defense
   response and promote PRRSV replication [[157]68]. Moreover, the
   activation of c-Jun N-terminal kinases (JNKs) and p38 can also promote
   the release of the inflammatory factor IL-10 [[158]68,[159]69,[160]70]
   and enhance the inflammatory effect. In addition to inducing apoptosis,
   PRRSV can also induce autophagy, which can promote PRRSV replication.
   The activation of PI3K/Akt is necessary for virus entry and promotion
   of virus replication, and PRRSV-activated Akt inhibits host cell
   apoptosis by negatively regulating the JNK pathway [[161]71]. TNFα It
   can play an important role in the induction and regulation of
   inflammatory response together with other inflammatory factors, but TNF
   α Expression is affected by the negative regulation of PRRSV
   replication [[162]72]. In the present study, miRNAs (ssc-miR-10b,
   ssc-miR-122-5p, ssc-miR-124a, ssc-miR-128, ssc-miR-129a-5p, etc.)
   enriched in these pathways are involved in PRRSV-induced apoptosis,
   autophagy, and inflammation and are closely associated with viral
   immune response, immune evasion, and replication.

   The cell plasma membrane is rich in a variety of lipid rafts, and
   sphingolipid- and cholesterol-rich in sphingolipids (sphingomyelin and
   glycosphingolipids) are key molecules of lipid rafts. The recognition
   of lipids by some proteins of the virus may be a necessary condition
   for the entry of the virus [[163]73]. Envelope viruses insert viral
   envelope glycoproteins into lipid rafts at the stage of virus entry,
   interact with receptors located in lipid rafts, or change from their
   natural state to activated form to initiate or promote viral
   internalization/fusion, such as HSV, SARS coronavirus, and piglet
   epidemic diarrhea virus [[164]73,[165]74]. Previous studies found that
   the removal of cholesterol from the surface of MARC-145 cells
   significantly reduced PRRSV infection, demonstrating that inhibition of
   PRRSV infection was specifically mediated by the removal of cellular
   cholesterol. Depletion of cell membrane cholesterol significantly
   inhibited virus entry, particularly virus attachment, and release
   [[166]75]. Obviously, sphingolipid metabolism can regulate membrane
   structure and adhesion, which is of great significance in PRRSV virus
   invasion.

   Endocytosis was the most significant enrichment in this study.
   Endocytosis is an important mechanism of exosome uptake by target
   cells. Previous studies have shown that exosome uptake is an
   energy-demanding and cytoskeleton-dependent process, which highlights
   the potential role of endocytosis in this process [[167]76]. It has
   been proved that there are several pathways that can mediate this
   process, including phagocytosis, macropinocytosis, clathrin, etc.
   [[168]77,[169]78], which led to different classifications and roles of
   endocytosed substances. The enrichment of differentially expressed
   exosomal miRNAs in this pathway indicates that exosomes play an
   important role in PRRSV infection, and the regulation of content
   transport and uptake in exosomes may lead to pathophysiological changes
   in target cells and organs.

5. Conclusions

   Through the identification and bioinformatics analysis of serum
   exosomal miRNAs from PRRSV-infected pigs, a variety of PRRSV-related
   pathways and differentially expressed miRNAs were obtained in this
   study, such as ssc-miR-4331-3p, ssc-miR-744, ssc-miR-320, ssc-miR-10b,
   ssc-miR-124a, ssc-miR-128, etc., which play potential functional roles
   in PRRSV-induced immune response, invasion, and exosome uptake. In
   addition, because a single miRNA can target multiple genes and a single
   gene is also regulated by multiple miRNAs, there are a number of miRNAs
   that perform multiple functions in the above pathways. Some miRNAs have
   been verified to regulate PRRSV infection by acting on key receptors or
   directly targeting the virus genome, such as ssc-miR-10b, ssc-miR-378,
   miR-124a, let-7f-5p, ssc-miR-744, ssc-miR-19a, etc. Meanwhile, the
   present study also predicted a variety of miRNAs that can bind to the
   most conserved fragment of the 3′ UTR of the CHX1401 virus genome,
   including ssc-miR-34c, ssc-miR-375, ssc-miR-378, ssc-miR-486, and
   ssc-miR-6529, which may be important for regulating viral
   pathogenicity.

Acknowledgments