Abstract

   The pathogenesis of porcine circovirus type 2b (PCV2b) and swine
   influenza A virus (SwIV) during co-infection in swine respiratory cells
   is poorly understood. To elucidate the impact of PCV2b/SwIV
   co-infection, newborn porcine tracheal epithelial cells (NPTr) and
   immortalized porcine alveolar macrophages (iPAM 3D4/21) were
   co-infected with PCV2b and SwIV (H1N1 or H3N2 genotype). Viral
   replication, cell viability and cytokine mRNA expression were
   determined and compared between single-infected and co-infected cells.
   Finally, 3′mRNA sequencing was performed to identify the modulation of
   gene expression and cellular pathways in co-infected cells. It was
   found that PCV2b significantly decreased or improved SwIV replication
   in co-infected NPTr and iPAM 3D4/21 cells, respectively, compared to
   single-infected cells. Interestingly, PCV2b/SwIV co-infection
   synergistically up-regulated IFN expression in NPTr cells, whereas in
   iPAM 3D4/21 cells, PCV2b impaired the SwIV IFN induced response, both
   correlating with SwIV replication modulation. RNA-sequencing analyses
   revealed that the modulation of gene expression and enriched cellular
   pathways during PCV2b/SwIV H1N1 co-infection is regulated in a
   cell-type-dependent manner. This study revealed different outcomes of
   PCV2b/SwIV co-infection in porcine epithelial cells and macrophages and
   provides new insights on porcine viral co-infections pathogenesis.

   Keywords: porcine circovirus, swine influenza A virus, epithelial
   cells, macrophages, co-infection, viral pathogenesis, virus replication

1. Introduction

   The porcine respiratory disease complex (PRDC) causes enormous economic
   loss to the swine industry worldwide [[30]1]. Porcine reproductive and
   respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2)
   and swine influenza A virus (SwIV) are the main viruses associated with
   PRDC and are involved in costly outbreaks in swine production sites
   [[31]2].

   PCV2 is a non-enveloped single-stranded circular DNA virus that belongs
   to the Circoviridae viral family, genus Circovirus [[32]3]. PCV2 is the
   etiological agent of several diseases and syndromes such as
   post-weaning multisystemic wasting syndrome (PMWS), porcine dermatitis
   and nephropathy syndrome (PDNS), among others which are collectively
   named porcine circovirus-associated disease (PCVAD) [[33]4]. The
   prevalence of PCV2 infection is high in swine farms all over the world
   [[34]5]. However, most PCV2 infections are reported to be subclinical
   [[35]5]. It is noteworthy that the virus can alter the innate immune
   response and cause immunosuppression, which favors co-infection and/or
   secondary infection with other pathogens [[36]6]. Eight genotypes of
   PCV2 have been described to date (PCV2a-PCV2h) [[37]7,[38]8,[39]9]. The
   emergence of the PCV2b genotype in Canada was associated with a
   significant death rate increase during the post-weaning multisystemic
   wasting syndrome (PMWS) outbreak in late 2004 [[40]10] and was reported
   as the predominant genotype identified during the same period in
   samples submitted to the diagnostic service of the Faculty of
   Veterinary Medicine (Faculté de médecine vétérinaire) of the University
   of Montreal [[41]10,[42]11].

   Swine influenza A viruses (SwIV) are enveloped single-stranded negative
   sense RNA viruses belonging to the Orthomyxoviridae viral family, genus
   Alphainfluenzavirus [[43]3]. The subtypes of influenza A virus (IAV)
   are determined by the hemagglutinin and neuraminidase proteins which
   are embedded into the envelope of the virion. Eighteen HA subtypes
   (H1–H18) and eleven NA subtypes (N1–N11) have been reported to date
   [[44]12,[45]13]. SwIV subtypes H1N1, H1N2, H3N2, and A(H1N1)pdm09 have
   been the most frequently reported influenza A virus subtypes in swine
   worldwide [[46]14]. In Canada, SwIV subtypes H1N1, H1N2 and H3N2 have
   been circulating in swine herds since at least the 1980s
   [[47]15,[48]16,[49]17,[50]18,[51]19,[52]20,[53]21]. In 2009,
   A(H1N1)pdm09 was reported in swine in the Canadian provinces Manitoba,
   Alberta, Saskatchewan and Quebec [[54]22]. Infections caused by SwIV
   are characterized by high morbidity and very low mortality [[55]23]. It
   is known that IAV induces high levels of cytokine release very often
   associated with viral loads and severity of the disease [[56]24].

   PCV2 co-infections with SwIV or PRRSV have been extensively studied in
   vivo [[57]25,[58]26,[59]27,[60]28,[61]29,[62]30,[63]31,[64]32,[65]33]
   and in vitro [[66]34,[67]35,[68]36,[69]37]. Even though PCV2 and SwIV
   co-infections are also prevalent in pigs [[70]1], pathogenesis studies
   concerning this co-infection are scarce. Interestingly, an in vivo
   epidemiologic assessment showed that PCV2-positive pigs were more
   likely to be infected with SwIV than PCV2-negative pigs [[71]38]. Other
   authors have reported that PCV2/SwIV H1N1 co-infection did not affect
   PCV2 replication in experimentally infected pigs, whereas PCV2
   infection did increase SwIV-related clinical disease in the infected
   animals [[72]39].

   PCV mostly targets cells of monocyte/macrophage lineage in vivo but can
   infect other cell types [[73]40,[74]41]. SwIV preferentially targets
   epithelial cells of the respiratory tract, but alveolar macrophages can
   also be infected [[75]42,[76]43,[77]44]. Epithelial cells detect viral
   pathogens via pattern recognition receptors (PRRs) and release
   anti/proinflammatory cytokines to recruit and activate innate immune
   cells, which finally trigger innate and adaptive immune responses
   [[78]45,[79]46]. On the other hand, the importance of macrophages for
   host defense against invading pathogens is well-known [[80]47]. They
   are professional phagocytic cells that clear infectious particles and
   apoptotic cells and participate in the adaptive immune response by T
   cells activation via antigen presentation [[81]47]. It is well-known
   that concomitant infections modulate viral pathogenesis and affect host
   immune cells’ responses [[82]1]. However, there is a lack of
   information regarding the effect of PCV2b/SwIV co-infections at the
   cellular and molecular level. The objective of the present study was to
   evaluate the effects of PCV2b/SwIV co-infections on virus-targeted host
   cells such as swine respiratory epithelial cells and porcine alveolar
   macrophages. The results of the present study revealed that PCV2b
   modulates SwIV replication during co-infection, meanwhile affecting
   SwIV mRNA cytokine expression and cellular genes modulation in infected
   cells, in a cell-type-dependent manner.

2. Materials and Methods

2.1. Cells

   Newborn pig tracheal epithelial cell line (NPTr) and immortalized
   porcine alveolar macrophage (iPAM 3D4/21) cell line were used for all
   PCV2b and SwIV single and co-infections. Madin–Darby Canine Kidney
   (MDCK) cell line was used for SwIV titration and propagation. The NPTr
   cell line was kindly provided by Dr. M. Ferrari (Instituto
   Zooprofilattico Sperimental, Brescia, Italy) [[83]48]. The NPTr and the
   MDCK (ATCC CCL-34) cells were cultured in Eagle’s Minimum Essential
   Medium (EMEM) (Wisent Bioproducts, Saint-Jean-Baptiste, QC, Canada)
   supplemented with 10% fetal bovine serum (FBS) (Wisent Bioproducts,
   Saint-Jean-Baptiste, QC, Canada), 1 mM sodium pyruvate, 10 I.U./mL of
   penicillin, 10 μg/mL of streptomycin and 250 g/L amphotericin B
   solution (Wisent Bioproducts, Saint-Jean-Baptiste, QC, Canada)
   [[84]48]. The iPAM 3D4/21 cell line (ATCC CRL-2843) was maintained in
   RPMI 1640 medium (Invitrogen Corporation, GibcoBRL, Burlington, ON,
   Canada) with 10% FBS and adjusted to contain 2 mM L-glutamine
   (Invitrogen), 10 mM HEPES (Invitrogen), 1 mM sodium pyruvate, 1 mM
   non-essential amino acids (Invitrogen) and 0.1 mg/mL streptomycin/100U
   penicillin solution (Invitrogen). All cells were cultivated in a
   humidified incubator in 5% CO[2] atmosphere at 37 °C.

2.2. Viruses

   The PCV2b strain (FMV-06-0732) was isolated from a clinical case in
   Quebec in 2006 (GenBank accession number: [85]JQ994270) [[86]10]. It
   was serially propagated in NPTr cells and then purified and
   concentrated following ultracentrifugation on a 30% sucrose cushion
   using the SW28 Beckman Coulter rotor (Beckman Coulter Canada Inc.,
   Mississauga, ON, Canada) at 25,000 rpm for 4 h.

   The SwIV H1N1 (A/swine/St-Hyacinthe/148/1990) was isolated from pigs
   with respiratory symptoms during a 1990/91 outbreak of respiratory
   disease in Quebec (GenBank accession number: [87]U11703) [[88]16]. The
   SwIV H3N2 (A/swine/Quebec/1708732/2015(H3N2) was isolated from a pig
   with acute respiratory disease (GenBank accession number: [89]KX571068,
   [90]KX571087, [91]KX571114, [92]KX571138, [93]KX571161, [94]KX571164,
   [95]KX571200, [96]KX571217). The sample was submitted by a veterinarian
   practitioner to Diagnostic Veterinary Virology Laboratory (DVVL) of the
   University of Montreal in 2015 as part of routine swine flu diagnostic
   testing. Viral titers of PCV2b and SwIV stocks were determined in NPTr
   cells and MDCK, respectively, via the Spearman–Kärber method
   [[97]49,[98]50]. Viral titers were expressed in tissue culture
   infectious dose 50% per mL (TCID[50]/mL).

2.3. Immunofluorescence Assay

   Single infection and co-infection were confirmed using an
   immunofluorescence assay (IFA) as previously described [[99]51].
   Briefly, the cell medium was removed and infected cells were fixed with
   a mixture of acetone–methanol (50/50, v/v) and incubated for a 20 min
   period. Thereafter, fixed infected cells were washed three times with a
   PBS solution and then permeabilized with a solution containing 0.1%
   triton X-100 in PBS and incubated for 10 min. After incubation with a
   blocking solution (1% bovine serum in PBS-Tween for 20 min), cells were
   incubated with a 1/200 dilution of the polyclonal PCV2 porcine
   antiserum [[100]51] at 37 °C for 90 min and/or with 1/200 diluted
   monoclonal mouse anti-NP SwIV primary antibody (Bio-Rad, Hercules, CA,
   USA). Then, the cells were washed three times with a solution
   containing 1% bovine serum in PBS-T and incubated with a 1/75 dilution
   of a goat anti-swine rhodamine conjugated secondary antibody (Jackson
   ImmunoResearch, West Grove, PA, USA) and/or a 1/200 dilution of goat
   anti-mouse FITC conjugated secondary antibody (Invitrogen Corporation,
   GibcoBRL, Burlington, ON, Canada ), at 37 °C for 60 min. After three
   washing steps, the cells were visualized using a Leica DMI 4000
   inverted widefield fluorescence microscope (Leica Microsystems Inc.,
   Richmond Hill, ON, Canada). Pictures were acquired with a DFC 490
   digital camera (Leica Microsystems Inc.) and images were analyzed using
   Leica Application Suite Software, version 2.4.0 (Leica Microsystems
   Inc.) and ImageJ software (Laboratory for Optical and Computational
   Instrumentation, LOCI, University of Wisconsin, Madison, WI, USA)
   [[101]52]. Mock-infected cells were included in the immunofluorescence
   assay to verify the specificity of the antibodies ([102]Supplemental
   Figure S1).

2.4. PCV2b/SwIV Co-Infection

   To obtain PCV2b/SwIV co-infected cells, NPTr or iPAM 3D4/21 cells were
   firstly infected with PCV2b at an MOI of 0.05. Afterwards,
   PCV2b-infected cells were passaged at least three times in the presence
   of the virus to achieve a stable persistent infection. PCV2b-infected
   cells were thereafter dispensed into 24-well plates (1 × 10^5
   cells/well) and then co-infected with SwIV H1N1 or H3N2 subtypes at a
   MOI of 1 for 1 h in the presence of trypsine (1ug/mL). Cells were then
   washed three times with PBS. Fresh medium with 2% FBS was added to the
   wells and the plates were incubated in a humidified incubator with 5%
   CO[2] at 37 °C. After 4, 12, 24, 48 and 72 h SwIV following infection,
   the cells were subjected to 2–3 rounds of freeze–thaw cycles to release
   virus particles and cell debris, which were then removed via
   centrifugation at 8000 rpm at 4 °C for 15 min. The supernatant was kept
   at −80 °C until virus titer determination. SwIV titers were determined
   via the Spearman–Kärber method [[103]49,[104]50] in MDCK cells and
   expressed as tissue culture infectious dose 50% per mL (TCID[50]/mL).
   PCV2b quantification was performed via qPCR assay as described by
   Gagnon et al. (2008), using the primers and probe presented in
   [105]Supplemental Table S1 [[106]11]. PCV2b concentrations were
   expressed as DNA copy amount per mL.

2.5. Cell Viability Assay

   Cell viability assay was performed at 24 h PCV2b/SwIV post-infection
   with the Celltiter 96 Aqueous One Solution Cell Proliferation Assay kit
   (Promega, Madison, WI, USA), according to manufacturer’s instruction.
   Briefly, 20 µL of the reagent were added to each well of the
   microplate, the cells were then incubated for 2 h at 37 °C and the
   absorbance was measured at 490 nm (Biotek^® Synergy HT plaque reader,
   Winooski, VT, USA). The infected cells’ viability percentages were
   calculated using the non-infected cells as control. The experiments
   were carried out in triplicate and repeated at least two times.

2.6. Cytokine mRNAs Expression in PCV2b- and/or SwIV-Infected Cells

   The modulation of mRNAs expression of the following interleukins (ILs),
   IL-6, IL-8, IL-10 and interferons (IFNs), IFN-α, IFN-β and IFN-γ in
   co-infected cells versus single-infected cells was assessed via
   RT-qPCR. Total cellular RNA was extracted from infected and
   mock-infected NPTr or iPAM 3D4/21 cells and purified with the RNeasy
   Mini Kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s
   instructions. Total RNA concentration was measured with a Qubit
   fluorometer (Thermo Fisher, Walthman, MA, USA). Then, 1 μg of total
   cellular RNA was reverse transcribed using M-MLV reverse transcriptase
   (Invitrogen, Burlington, ON, CA), according to the manufacturer’s
   protocol. After the reverse transcription reaction, the cDNA was used
   in qPCR reactions with PowerTrack SYBR Green Master Mix kit (Thermo
   Fisher Scientific, Waltham, MA, USA) on a QuantStudio 3 Real-Time PCR
   System (Thermo Fisher Scientific). Quantification of differences
   between groups was carried out using the 2^−ΔΔCt method.
   β[2]-microglobulin (B2M), β-actin (ACTB) and peptidylprolyl isomerase A
   (PPIA) were used as normalizing genes to compensate for potential
   differences in cDNA amounts. Mock-infected cells were used as a
   calibrator reference in the analysis. The primers used for the specific
   amplification of the targeted cDNA are described in [107]Supplemental
   Table S1 [[108]53,[109]54].

2.7. 3′mRNA-Seq Library Preparation and Sequencing

   Total RNA was purified for RNA-seq analysis from infected cells as
   described in the previous section. RNA concentration and quality were
   assessed using Agilent 2100 Bioanalyzer apparatus with the RNA 6000
   Nano Kit (Agilent Technologies, Santa Clara, CA, USA). Only RNA samples
   with acceptable RNA integrity number (RIN > 7) were submitted to
   library preparation. The cDNA libraries were constructed using
   Lexogen’s QuantSeq^TM 3′mRNA-Seq Kit (Lexogen GmbH, Vienna, Austria)
   according to the manufacturer’s recommendations. A total of 12
   libraries, including 3 libraries from PCV2b-infected cells, 3 from SwIV
   H1N1-infected cells, 3 from PCV2b/SwIV H1N1-infected cells and 3 from
   mock-infected cells, were synthesized. Concentration and quality of the
   purified libraries were assessed using the Qubit fluorometer (Thermo
   Fisher, Walthman, MA, USA) and an Agilent high-sensitivity DNA kit with
   a Bioanalyzer (Agilent, CA, USA), respectively. All libraries were
   sequenced with a MiSeq high-throughput apparatus (Illumina, San Diego,
   CA, USA) using v3 cartridges (150-cycles) according to the
   manufacturer’s instructions.

2.8. Bioinformatic Analysis

   Raw sequencing data were imported in FASTQ format into CLC Genomics
   Workbench (version 22.0.1, Qiagen, CA, USA). Reads were trimmed for
   quality and adaptors using the CLC Genomics Workbench software and
   trimmed reads were mapped to Sus scrofa rRNA 12S, 16S and 18S. After
   removing the rRNA mapped reads, the remaining unmapped reads were
   mapped to the reference genome (Sscrofa11.1). The mapped reads were
   then used for differential expression calculations. Differentially
   expressed genes (DEGs) with a false discovery rate (FDR) threshold <
   0.05 and a fold change >1.5 were selected and used for volcano plots
   and Venn diagram visualization in CLC Genomics workbench. Pathway
   enrichment analysis was performed with the Database for Annotation,
   Visualization and Integrated Discovery (DAVID, 2021) [[110]55,[111]56].
   Additionally, the list of DEGs from the co-infected cells was analyzed
   with ClueGo application (version 2.5.5) available in Cytoscape (version
   3.9.1) to identify protein–protein interaction networks and associated
   cellular pathways [[112]41,[113]42,[114]43,[115]44]. All sequences were
   deposited in GEO repository (accession number [116]GSE229215).

2.9. Statistical Analyses

   All statistical analyses were performed using GraphPad Prism software
   (GraphPad Prism 7.0.0). The different statistical tests used within
   each analysis are described in figure legends. Differences were
   considered significant at p-value < 0.05.

3. Results

3.1. Modulation of Swine Influenza A Virus Replication in Co-Infected
PCV2b/SwIV H1N1 Cells

   The immunofluorescence assay (IFA) performed on co-infected NPTr and
   iPAM 3D4/21 cells confirmed the simultaneous presence of PCV2b and SwIV
   H1N1 antigens in the cells ([117]Figure 1). Similar results were
   observed in PCV2b/SwIV H3N2 co-infected cells. PCV2b internalized with
   similar efficiency in NPTr and iPAM 3D4/21 cells as the percentage of
   PCV2b-positive cells determined via IFA was similar for both types of
   cells. However, the number of SwIV H1N1-positive cells was
   significantly higher in infected NPTr cells than in infected iPAM
   3D4/21 cells (p < 0.001).

Figure 1.

   [118]Figure 1
   [119]Open in a new tab

   PCV2b and SwIV H1N1 co-localization in both NPTr and iPAM 3D4/21
   co-infected cells. IFA was performed at 24 h post-infection to detect
   PCV2b (red) and SwIV H1N1 (green) in co-infected cells. The cells were
   infected with 0.05 MOI of PCV2b and passaged 2–3 times. Then,
   PCV2b-infected cells were infected with 1 MOI of SwIV H1N1. Nuclear
   staining with DAPI is shown in blue. Co-localization pictures were
   realized with ImageJ. The red arrows point to single cells expressing
   both PCV2b and SwIV H1N1 antigens. Statistical analyses were carried
   out using a two-way ANOVA followed by Sidak’s multiple comparison test
   (GraphPad Prism software, version 7.00). *** p < 0.001.

   The infectious titer of SwIV H1N1/H3N2 subtypes and the PCV2b viral
   genome quantity in co-infected cells was determined and compared to
   single-infected cells at different times post-infection ([120]Figure
   2). The SwIV H1N1 and H3N2 infectious titer at 24 h post-infection
   (hpi) was significantly higher (p < 0.0001) in single-infected NPTr
   cells compared to single-infected iPAM 3D4/21 cells. These results were
   expected considering that SwIV preferentially infects epithelial cells
   of the respiratory tract [[121]42]. However, PCV2b qPCR quantification
   results were similar in both infected NPTr and infected iPAM 3D4/21
   cells ([122]Figure 3), confirming the IFA results ([123]Figure 1).

Figure 2.

   [124]Figure 2
   [125]Open in a new tab

   PCV2b co-infection effects on SwIV replication in infected NPTr and
   iPAM 3D4/21 cells. The SwIV titer was determined in MDCK cells using
   the Spearman–Kärber method and expressed in tissue culture infectious
   dose 50% per mL (TCID[50]/mL). The PCV2b/SwIV H1N1 (A) and PCV2b/SwIV
   H3N2 (C) co-infection experiment in NPTr cells, and PCV2b/SwIV H1N1 (B)
   and PCV2b/SwIV H3N2 (D) co-infection experiment in iPAM 3D4/21 cells
   were repeated 3 times. Data are presented with standard deviation (SD)
   values. Statistical analyses were carried out using two-way
   repeated-measures ANOVA (GraphPad Prism software, version 7.00). * p <
   0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 3.

   [126]Figure 3
   [127]Open in a new tab

   SwIV co-infection effects on PCV2b replication in infected NPTr and
   iPAM 3D4/21 cells. PCV2b quantification in co-infected NPTr (A) and
   iPAM 3D4/21 (B) cells was performed using qPCR assay and expressed as
   PCV2b genome copies per mL of sample. The experiments were repeated 3
   times. Mock-infected cells were PCV2 qPCR-negative (with Ct > 36) and
   the results are not illustrated in the figure. Data are presented with
   standard deviation (SD) values. Statistical analyses were carried out
   using two-way repeated-measures ANOVA (GraphPad Prism software, version
   7.0.0).

   During co-infection, it was found that PCV2b modulated the replication
   kinetics of both subtypes of SwIV, H1N1 and H3N2, in the infected
   cells. In fact, the SwIV infectious titer decreased significantly after
   24 hpi in PCV2b/SwIV co-infected NPTr cells compared to cells infected
   only with SwIV ([128]Figure 2A,C). Inversely, in infected iPAM 3D4/21
   cells the infectious titers of SwIV H1N1 and H3N2 were significantly
   higher in co-infected cells versus single-infected cells ([129]Figure
   2B,D). However, the presence of SwIV did not seem to influence the
   PCV2b replication during co-infection. ([130]Figure 3).

3.2. Cell Viability Assay

   To evaluate whether the modulation of SwIV replication during
   co-infection could affect cell viability compared to single infection,
   a cell viability assay was performed at different times post-infection
   in single-infected and co-infected NPTr and iPAM 3D4/21 cells. As
   expected, the cell viability decreased from 24 hpi to 72 hpi in
   co-infected as well as in single-infected cells compared to
   mock-infected cells ([131]Figure 4). Moreover, the dual infection of
   PCV2b and SwIV in both NPTr and iPAM 3D4/21 cells significantly
   decreased the cell viability over time compared to SwIV single-infected
   cells. Nevertheless, no significant differences were found between
   PCV2b co-infected and single-infected cells.

Figure 4.

   [132]Figure 4
   [133]Open in a new tab

   Cell viability was determined in single-infected and co-infected
   PCV2b/SwIV cells at 24 hpi (A,B), 48 hpi (C,D) and 72 hpi (E,F) in NPTr
   (A,C,E) and iPAM 3D4/21 cells (B,D,F). The experiments were repeated
   three times. The data represent percentage of cell viability in
   infected cells with respect to mock-infected cells and are presented
   with standard deviation (SD) values. Statistical analyses were carried
   out using one-way ANOVA followed by Tukey’s multiple comparison test
   (GraphPad Prism software, version 7.0.0). Different superscripts
   indicate a statistically significant difference (p < 0.05) between
   groups.

3.3. Modulation of Cytokines’ mRNAs Expression in Co-Infected Cells

   The levels of mRNA expression of pro-inflammatory (IL-6 and IL-8) and
   anti-inflammatory (IL-10) cytokines, as well as of type I IFN (IFN-α/β)
   and type II IFN (IFN-γ), were measured at 24 h post-infection in
   single-infected and co-infected cells. The results obtained with
   infected NPTr cells revealed that IL-6, IL-8, and IL-10 mRNA
   expressions were higher in PCV2b/SwIV H1N1 and PCV2b/SwIV H3N2
   co-infected cells compared to SwIV H1N1 and SwIV H3N2 single-infected
   cells, respectively ([134]Figure 5A). However, no difference was found
   for mRNA expression of interleukins tested between PCV2b/SwIV H1N1- and
   PCV2b-infected cells. In PCV2b/SwIV H3N2-infected NPTr cells, IL-6 and
   IL-10, but not IL-8, mRNA expression was reduced compared to PCV2b
   single-infected cells ([135]Figure 5A).

Figure 5.

   [136]Figure 5
   [137]Open in a new tab

   PCV2b/SwIV co-infection effects on the modulation of cytokines’ mRNA
   expression. The mRNA expressions of IL-6, IL-8 and IL-10 in NPTr cells
   (A) and iPAM 3D4/21 (B) as well as of IFN-α, IFN-β and IFN-γ in NPTr
   cells (C) and iPAM 3D4/21 (D) were determined using RT-qPCR assays. The
   2^−ΔΔCt method was used to calculate the fold change of cytokine mRNA
   expression in infected cells with respect to mock-infected cells at 24
   h post-infection. The experiments were repeated at least three times.
   All data are presented with standard deviation (SD) values. Statistical
   analyses were carried out using an ordinary two-way ANOVA followed by
   Tukey’s multiple comparison test (GraphPad Prism software, version
   7.0.0). Different superscripts indicate a statistically significant
   difference (p < 0.01) between groups within the same tested cytokine
   mRNA.

   As shown in [138]Figure 5B, in iPAM 3D4/21 cells, the co-infection with
   PCV2b and SwIV H1N1 did not significantly impact the IL-6 mRNA
   expression. However, in co-infected PCV2b/SwIV H3N2 cells, IL-6
   expression was reduced compared to SwIV H3N2-infected cells, but not
   when comparing to PCV2b single-infected cells. IL-10 expression
   decreased in PCV2b/SwIV H1N1 co-infected iPAM 3D4/21 cells compared to
   PCV2b- or SwIV H1N1-infected cells. The dual infection of PCV2b and
   SwIV H3N2 increased the expression of IL-10 regarding to SwIV H3N2
   alone, however, the transcriptional level of this cytokine was
   significantly reduced in these infections compared to PCV2b
   single-infected cells.

   In the case of IFN type I response, a clear synergistic effect was
   observed in co-infected NPTr cells as IFN-α and IFN-β mRNAs expression
   was significantly higher in co-infected cells compared to in PCV2b or
   SwIV (H1N1 or H3N2) single-infected cells ([139]Figure 5C). Similar
   results were obtained for IFN-γ in PCV2b/SwIV H1N1 co-infected cells
   compared to single-infected cells. However, although significant
   differences were observed in the mRNA expression of IFN-γ between
   PCV2b/SwIV H3N2 and SwIV H3N2 infections, no difference was found when
   compared to PCV2b single-infected cells ([140]Figure 5C).

   Regarding the IFN response in co-infected iPAM 3D4/21 cells
   ([141]Figure 5D), no difference was observed on the mRNA expression of
   the IFNs tested compared to PCV2b-infected cells. Nevertheless, IFN-β
   and IFN-γ mRNA expressions were significantly higher in SwIV (H1N1 or
   H3N2) single-infected cells compared to PCV2b/SwIV co-infected cells.
   Additionally, IFN-α mRNA expression was higher in SwIV H3N2-infected
   than in co-infected cells. It is interesting to note that the cell type
   influenced the modulation of IFN mRNA expression. For example, the mRNA
   expression levels of IFN-α, IFN-β and IFN- γ were up-regulated in NPTr
   cells following PCV2b infection ([142]Figure 5C) compared to
   mock-infected cells; however, in PCV2b-infected iPAM 3D4/21, there was
   a trend toward down-regulation of those mRNAs ([143]Figure 5D). This
   difference in IFNs’ transcriptional response was also observed between
   co-infected NPTr and iPAM 3D4/21 cells ([144]Figure 5C,D). Overall,
   these results at least revealed that dual PCV2b and SwIV infection
   modulates cytokine transcriptional responses in a cell-type-dependent
   manner.

3.4. Differentially Expressed Genes (DEGs) and Pathway Enrichment Analysis in
PCV2b-, SwIV H1N1- and PCV2b/SwIV H1N1-Infected Cells

   RNA-seq analysis was performed to characterize and compare the
   transcriptomic response in NPTr and iPAM 3D4/21 co-infected and
   single-infected cells. The transcriptomic analyses were performed at 24
   hpi to ensure there was a significant amount of suitable viable cells
   following viral infection. SwIV H1N1 was chosen for the present
   experiment because during SwIV H1N1/PCV2b co-infection, the modulation
   of SwIV replication was greater at 24 hpi compared to that with SwIV
   H3N2/PCV2b co-infection ([145]Figure 2).

   First, 3′mRNA sequencing was used to identify the DEGs in infected
   cells compared to mock-infected cells. In the NPTr cells, 365, 98 and
   627 DEGs were identified in PCV2b-, SwIV H1N1- and PCV2b/SwIV
   H1N1-infected cells, respectively, when compared to mock-infected
   cells. ([146]Figure 6A). The number of up-regulated and down-regulated
   genes in each infection is listed in [147]Supplemental Tables S2 and
   S3. In iPAM 3D4/21 cells, 316, 64 and 164 DEGs were identified in
   PCV2b-, SwIV H1N1- and PCV2b/SwIV H1N1-infected cells, respectively,
   compared to mock-infected cells. ([148]Figure 6B). The number of
   up-regulated and down-regulated genes in each infection is shown in
   [149]Supplemental Tables S4 and S5.

Figure 6.

   [150]Figure 6
   [151]Open in a new tab

   Venn diagram of differentially expressed genes (DEGs) in PCV2b/SwIV
   co-infected cells. Mock-infected cells were used as control to identify
   DEGs in NPTr (A) and iPAM 3D4/21 (B) single-infected and co-infected
   cells. The numbers in overlapping areas represent the number of DEGs
   shared among the different virus infection experimental groups. A false
   discovery rate (FDR) < 0.05 and a fold change cut-off of 1.5 was used
   to identify the DEGs.

   The identified DEGs were used to perform pathway enrichment analysis to
   determine the cellular pathways impacted during viral infection in
   single- and co-infected cells. After the enrichment analysis, the top
   10 over-represented pathways with an FDR < 0.05 were retained. Only
   pathways with a fold enrichment > 2 are illustrated in infected NPTr
   cells ([152]Figure 6). In the case of iPAM 3D4/21 cells, the number of
   enriched pathways with the same FDR ≥ 2 was extremely high. That is why
   only pathways with a fold enrichment > 4 are depicted in iPAM 3D4/21
   cells.

   The pathway enrichment analysis in NPTr cells revealed the following
   cellular pathways: ECM–receptor interaction, protein processing in
   endoplasmic reticulum, focal adhesion and regulation of actin
   cytoskeleton; these were among the most enriched pathways in
   PCV2b-infected cells as well as in co-infected cells ([153]Figure
   7A,C). Other over-represented pathways in NPTr co-infected cells
   included mRNA surveillance, HIF-1 signaling pathway, spliceosome,
   phagosome, NOD-like receptor signaling pathway and influenza A pathway
   ([154]Figure 7E). In SwIV H1N1-infected NPTr cells, the most enriched
   pathways were related to the protein synthesis machinery and included
   the following pathways: translation, ribosome, metabolism of proteins,
   among others involved in similar functions ([155]Figure 7C).
   Additionally, nonsense-mediated decay (NMD) pathways were identified in
   SwIV H1N1-infected NPTr cells. The pathway enrichment analysis
   performed with iPAM 3D4/21 cells showed that in single-infected as well
   as in co-infected cells, NF-Kappa B and TNF signaling pathways were the
   most enriched pathways in the set of DEGs analyzed. However, in
   co-infected cells, additional enriched cellular pathways were found;
   these included apoptosis, influenza A, HIF-1 signaling pathway and MAPK
   signaling pathways ([156]Figure 7B,D–F).

Figure 7.

   [157]Figure 7
   [158]Open in a new tab

   KEGG pathway enrichment analysis of the DEGs identified in
   single-infected and co-infected cells. Enriched KEGG pathways after
   PCV2b, SwIV H1N1 and PCV2b/SwIV H1N1 infection in NPTr cells ((A,C,E),
   respectively) and in iPAM 3D4/21 cells ((B,D,F), respectively) are
   illustrated. The FDR value < 0.05 was considered for statistically
   significant enriched pathways.

   An additional analysis was performed using the Cytoscape’s ClueGo
   application with the list of DEGs from the co-infected cells to
   identify protein–protein interaction networks and to confirm the
   cellular pathways modulated during PCV2b/SwIV H1N1 co-infection. This
   analysis confirmed the enrichment of cellular pathways such as mRNA
   surveillance, spliceosome, protein processing in endoplasmic reticulum,
   phagosome and regulation of actin cytoskeleton in NPTr co-infected
   cells ([159]Supplemental Figure S2). In co-infected iPAM 3D4/21 cells,
   HIF-1 signaling pathway, TNF signaling, regulation of innate immune
   response, IL-17 signaling, adaptive immune response based on somatic
   recombination of immune receptors built from immunoglobulin superfamily
   domains, apoptosis and influenza A pathways were identified
   ([160]Supplemental Figure S3). The [161]Supplemental Figures S2 and S3
   illustrate several DEGs that are involved in more than one cellular
   pathway in co-infected NPTr and iPAM 3D4/21 cells, respectively.

4. Discussion

   In the present study, PCV2b replication was not modulated in the
   presence of both SwIV strains (H1N1 or H3N2) nor in both infected cell
   lines being tested ([162]Figure 3). These results are in accordance
   with those of previous studies. For example, Wei et al. 2010 concluded
   that infection of caesarean-derived colostrum-deprived pigs with SIV
   H1N1, one week after a previous infection with PCV2, did not influence
   PCV2 replication in dually infected pigs, while PCV2 infection
   increases SwIV-related clinical disease [[163]39]. Unfortunately, this
   previous study did not provide any information regarding the effect of
   PCV2/SwIV co-infection on SwIV replication. Interestingly, our results
   demonstrated that PCV2b modulated SwIV replication in a
   cell-type-dependent manner ([164]Figure 2). In fact, PCV2b decreased
   the replication of SwIV in NPTr cells, whereas the SwIV titer was
   enhanced in iPAM 3D4/21 co-infected cells. Productive replication of
   SwIV in macrophages can alter antiviral macrophage functions such as
   phagocytosis, resulting in enhanced disease severity and impaired
   bacterial clearance during secondary bacterial infection
   [[165]47,[166]57].

   It is noteworthy that at 24 hpi, both PCV2b/SwIV co-infected NPTr and
   iPAM 3D4/21 cells had a significant decrease in cell viability compared
   to SwIV single-infected cells. Previous studies have reported that a
   decreased cell viability significantly reduces the production of
   infectious viruses from pig respiratory epithelial cells [[167]58].
   This would explain why SwIV replication was significantly reduced in
   PCV2b/SwIV co-infected NPTr cells in the present study. However, in
   co-infected iPAM 3D4/21 cells, the SwIV replication was rather
   enhanced, even when the cell viability was decreasing over time in
   dual-infected cells compared to SwIV single-infected cells. Previous
   studies revealed that PCV2b infection in PAMs activates NF-κB,
   phospho-Akt and MAPK signaling pathways in infected cells. The
   activation of these signaling pathways can be detected in the first 30
   min after PCV2 infection [[168]59]. It is known that influenza virus
   replication starts early after virus entry in the cells. Viral mRNA
   starts to accumulate within the first hour post-infection and viral
   genome replication starts 1.5 to 2 hpi [[169]60]. The modulation of
   cytokines and the activation of NF-κB and MAPK signaling pathways,
   among others, have been shown to be important for a successful
   influenza virus infection [[170]61]. It is possible that the modulation
   of cytokines’ expression and the activation of signaling pathways in
   iPAM 3D4/21 cells in the first 24 h after PCV2b infection could lead to
   an enhanced SwIV replication in co-infected cells in the present study,
   even when the cell viability decreased throughout the viral infection.

   PCV2b has been previously reported to induce up-regulation of cytokines
   such as IL-6, IL-8 and IL-10 in vitro [[171]59,[172]62,[173]63]. In the
   present study, IL-6 and IL-10 mRNA expressions were increased via PCV2b
   infection in NPTr and iPAM 3D4/21 cells in accordance with previous
   studies reporting up-regulation of IL-6 in epithelial cells [[174]63]
   and increased levels of IL-6 and IL-10 in porcine alveolar macrophages
   (PAMs) [[175]64,[176]65]. In the present study, IL-8 mRNA was
   upregulated in PCV2b-infected NPTr cells, as expected, whereas it was
   not in PCV2b-infected iPAM 3D4/21 cells ([177]Figure 5). These results
   do not correlate with those of previous reports showing a significant
   up-regulation of IL-8 mRNA expression and/or protein production in
   PCV2-inoculated swine alveolar macrophages [[178]66,[179]67]. The
   contradictory results obtained in the present study may be due to the
   time points selected to measure the expression of this cytokine in
   macrophages after PCV2b infection or the use of an immortalized PAM
   cell line (3D4/21) instead of primary porcine alveolar macrophages. It
   is noteworthy that cytokine mRNA expression was determined in
   persistently PCV2b-infected cells to mimic the superinfection events
   that occurs in PCV2b previously infected pigs. Therefore, a persistent
   PCV2b infection in iPAM 3D4/21 cells could have a differential effect
   on mRNA expression compared to an acute PCV2b cell infection.

   Induction of pro-inflammatory cytokines during SwIV infection has been
   correlated with viral replication and clinical signs [[180]68]. In the
   current study, IL-6, IL-8, and IL-10 mRNA expressions were higher in
   PCV2b/SwIV (H1N1 or H3N2) co-infected NPTr cells compared to SwIV H1N1
   and SwIV H3N2 single-infected cells. However, no difference was
   observed regarding the tested interleukin mRNA expression levels
   between PCV2b/SwIV H1N1- and PCV2b-infected cells. Nevertheless, in
   PCV2b/SwIV H3N2-infected cells, IL-6 and especially IL-10 mRNA
   expression levels were significantly reduced compared to PCV2b
   single-infected NPTr cells ([181]Figure 5A). These results reveal that
   the modulation of interleukin expression via PCV2b/SwIV co-infection on
   NPTr cells might be influenced by the SwIV genotype being investigated.

   Regarding the mRNA expression of antiviral cytokines such as IFNs,
   their expression level modulations in co-infected cells were found to
   be cell-type-dependent. In NPTr cells, the IFN type I (α/β) mRNA
   expression levels were synergistically up-regulated in co-infected
   cells compared to single-infected cells, whereas in iPAM 3D4/21 cells,
   a trend towards a reduced IFN type I and II mRNA expression was
   observed in PCV2b single-infected and co-infected cells ([182]Figure
   5). Several researchers have previously reported contradictory data in
   regard to the modulation of IFN response following infection with
   PCV2b. Wang et al. (2022) reported that PCV2 infection interferes with
   the activation of type I IFNs signaling pathway and inhibits the
   IFN-induced ISGs expression in vivo and in vitro [[183]69]. Others have
   reported the inhibition of IFN-α in porcine peripheral blood
   mononuclear cells (PBMCs) [[184]70,[185]71] and the inhibition of type
   I IFN (IFN-α /IFN-β) in PK-15 cells [[186]72,[187]73] following PCV2
   infection or in the presence of PCV2 viral protein or DNA. In addition,
   according to Gao et al. (2014), PCV2 significantly inhibited
   pseudorabies virus (PRV)-induced IFN-γ mRNA expression in swine PBMC in
   vitro [[188]74]. However, other authors have reported that porcine
   circovirus type 2 induces type I interferon production in porcine
   alveolar macrophages [[189]75] and IFN-β in PK-15-infected cells
   [[190]76]. Interestingly, it is known that the PCV2 genome contains CpG
   motifs with both IFN-α inhibitory and stimulatory properties [[191]77].
   Kekarainen et al. (2008b) suggested that PCV2 viral elements can
   distinctly regulate cytokine production according to the cell
   population [[192]78]. Overall, it is easy to conclude that IFN response
   modulation following PCV2 infection is complex and could vary depending
   on the experimental models being studied (in the present study:
   tracheal epithelial cells versus macrophages).

   It is known that type I and III IFNs are rapidly induced after
   influenza virus sensing via pattern recognition receptors (Toll-like
   receptors, RIG-I-like receptors, NOD-like receptors) on respiratory
   epithelial cells. The released IFNs bind to their cognate receptors and
   activate the JAK-STAT signaling pathway. This triggers the
   transcription of hundreds of IFN-stimulatory genes (ISGs) that induce
   an “antiviral state” in infected and nearby cells to ultimately
   restrict viral replication and propagation
   [[193]79,[194]80,[195]81,[196]82,[197]83]. In a study performed by Wu
   et al. (2022), the authors overexpressed microRNA let-7 in A549 cells,
   which resulted in an increase of interferon type I mRNA expression and
   consequently inhibited influenza virus infection [[198]84]. Moreover,
   Fong et al. (2022) demonstrated that IFN-γ inhibits influenza virus
   replication in respiratory epithelial cells by reducing viral binding
   onto the cells [[199]85]. The results obtained during the present study
   suggest that the increased expression of IFN mRNA in
   PCV2b/SwIV-infected NPTr cells could lead to a reduction in SwIV viral
   replication compared to single SwIV-infected cells. It is also
   well-known that the influenza virus possesses several strategies to
   counteract IFN response [[200]86,[201]87]. That would explain why the
   increased IFN mRNA expression in co-infected NPTr cells could be
   responsible for a partial reduction in, and not a complete shutdown of,
   SwIV replication ([202]Figure 2). Interestingly, Czerkies et al. (2022)
   have reported interference between respiratory syncytial virus (RSV)
   and influenza A virus H1N1 in human alveolar epithelial cells (A549).
   The authors of the report found that previous infection of cells with
   RSV does not prevent a subsequent influenza virus infection, whereas
   RSV indeed protects bystander cells against influenza virus infection
   by triggering secretion of type I and type III IFNs [[203]88]. A
   similar effect in PCV2b/SwIV co-infected NPTr cells could be involved
   and explain the PCV2b impact on the apparent inhibition of SwIV
   replication. However, this PCV2b bystander phenomenon effect on SwIV
   replication is highly plausible, though it still needs to be
   demonstrated. In iPAM 3D4/21 co-infected cells, PCV2b completely
   impaired the modulation of the transcriptional expression of IFN-β and
   IFN-γ by SwIV. Gao et al. (2014) have reported a similar PCV2 effect on
   IFN expression during dual infection with pseudorabies virus (PRV) in
   PBMC [[204]74]. In this previous study, PCV2 significantly inhibited
   the ability of inactivated PRV to induce IFN-γ expression. The authors
   suggested that PCV2 could affect the cell immune response to PRV
   [[205]74]. In the context of the present study, it is believed that the
   impaired IFN response observed in PCV2b persistently infected iPAM
   3D4/21 cells could favor SwIV infection and replication. It is
   important to point out that although a modulation of the expression of
   IFNs’ mRNA was revealed in the infected cells, these results require
   confirmation using other methods, such as ELISA.

   Differential transcriptome analysis of PCV2b/SwIV H1N1 and
   single-infected porcine cells revealed distinct signatures on host gene
   expression that may have impacted SwIV H1N1 replication during the
   co-infection. Several of the most enriched pathways identified in
   co-infected cells in this study are known to be involved in the host
   antiviral response and influenza virus pathogenesis and propagation
   [[206]61] such as NOD-like receptor signaling pathway in co-infected
   NPTr cells and apoptosis and MAPK signaling pathways in co-infected
   iPAM 3D4/21 cells ([207]Figure 7). NOD-like receptors (NLR) are a
   specific family of cytosolic pattern-recognition receptors (PRRs) that
   contains more than 20 members in mammals and plays a pivotal role in
   the recognition of intracellular ligands [[208]89]. A group of NLRs are
   associated with the formation of the inflammasome, a cytosolic
   multiprotein complex that, once activated, induces the production of
   IL-1β and IL-18 and the recruitment of immune cells to the site of
   infection [[209]90,[210]91]. The NLRP3 inflammasome has an important
   role in the immune response against influenza A virus infection. Its
   role in both protective and detrimental immune responses during
   influenza A virus infection has been studied and extensively reviewed
   [[211]91,[212]92,[213]93,[214]94]. Interestingly, NLRP3 was found to be
   up-regulated in PCV2b/SwIV H1N1-infected NPTr cells (FC 6.16, FDR =
   0.04) but not in SwIV H1N1 and PCV2b single-infected cells. Moreover,
   DDX3X, a host protein which has been implicated in the coordination of
   host defense against influenza virus by activating the NLRP3
   inflammasome and type I interferon response [[215]95,[216]96] was
   upregulated in co-infected NPTr cells (FC 3, FDR = 5.84 × 10^−4) and in
   PCV2b-infected cells (FC 2.42, FDR = 0.01), but not in SwIV
   H1N1-infected cells.

   Apoptosis and MAPK pathways can be modulated during the replication
   cycle of the influenza virus to favor viral infection. Wurzer et al.
   (2003) have found that caspase 3 activation during the onset of
   apoptosis is important for efficient influenza virus propagation
   [[217]97]. The MAPK pathway, on the other hand, has been shown to
   positively regulate multiple steps of influenza virus replication such
   as RNA synthesis, vRNP export and release (budding) of virus,
   clathrin-independent endocytosis and viral internalization, nuclear
   export and protein synthesis, (see the review of Kumar et al., 2018
   [[218]98] for information and references regarding the role of MAPK