Swine Influenza A virus infection sets the local immunological landscape in subsequent infection with Porcine Reproductive and Respiratory Syndrome virus

Swine Influenza A virus infection sets the local immunological landscape in subsequent infection with Porcine Reproductive and Respiratory Syndrome virus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Swine Influenza A virus infection sets the local immunological landscape in subsequent infection with Porcine Reproductive and Respiratory Syndrome virus Janaïna Grevelinger, Olivier Bourry, Selma Schmidt, François Meurens, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5928429/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Jun, 2025 Read the published version in Veterinary Research → Version 1 posted You are reading this latest preprint version Abstract Farmed pigs are frequently exposed to respiratory infections, with swine influenza A virus (swIAV) and porcine reproductive and respiratory syndrome virus (PRRSV) being key drivers. Most co-infection studies with these viruses have focused on PRRSV infection followed by swIAV. However, the reverse scenario, where swIAV is given first and then PRRSV, has not been explored. This infection sequence is plausible under natural conditions and warrants further study, especially given that influenza A virus has been shown in mice to impair alveolar macrophages, which are the target cells for PRRSV. This study aimed to evaluate the impact of swIAV infection on the alveolar macrophage population, clinical signs, immune responses, and viral loads during a secondary infection with PRRSV initiated seven days after the initial swIAV exposure. Results demonstrated that primary swIAV infection did not exacerbate the clinical progression of PRRSV infection, nor did it result in significant differences in PRRSV viral loads or affect the alveolar macrophage population in the lungs of super-infected pigs as compared to those of pigs infected with PRRSV alone. However, swIAV pre-infection was associated with an increase in the number of conventional dendritic cells type 1 (cDC1), perforin-expressing T cells and NK-related lymphocytes in bronchoalveolar lavage. This coincided with an increase of PRRSV-specific IFN-γ producing CD4 T cells in blood detected seven days post-PRRSV infection. These findings suggest that a swIAV infection could enhance immune responses during subsequent PRRSV infection by recruiting cDC1 and inducing IL-12, promoting a type-1 immune response, highlighting the complex interplay and often unexpected outcomes of viral co-infections occurring in close temporal proximity. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction On farms, pigs are often exposed to multiple respiratory infections, which are frequently associated with complex respiratory diseases of viral and/or bacterial origin. This phenomenon is referred to as the porcine respiratory disease complex (PRDC) [ 1 – 3 ]. Among the pathogens involved in PRDC, swine influenza A viruses (swIAV) and the porcine reproductive and respiratory syndrome virus (PRRSV) play a key role. SwIAV belongs to the Alphainfluenzavirus genus in the Orthomyxoviridae family. These enveloped viruses have a segmented negative-sense single-stranded RNA genome. The main swIAV subtypes circulating in pig populations worldwide for decades include H1N1, H1N2, and H3N2 [ 4 ]. Swine influenza is a common respiratory disease in pig farms, causing clinical signs such as fever, breathing difficulties, coughing, and nasal discharge. PRRSV, a positive-sense single-stranded RNA virus from the Arteriviridae family, is sub-divided into two species: PRRSV-1, mainly found in Europe and PRRSV-2, predominantly present in North America and Asia [ 5 , 6 ]. This virus causes respiratory signs, reproductive disorders, and growth retardation and increased mortality in piglets. Its virulence varies depending on the strain and host characteristics [ 7 ]. Epidemiological studies conducted in France have shown a significant association between PRRSV and swIAV infections [ 8 , 9 ]. Thus, more than half of the 125 farms studied in the Western part of the country displayed a positive correlation between antibodies against PRRSV-1 and the H1N2 subtype of swIAV [ 8 ]. This would indicate that in regions where these viruses are widespread, the risk of co-infection is particularly high. Given the high risk of simultaneous infections in regions where both PRRSV and swIAV are prevalent, it is important to explore how these co-infections may affect animal health. Research has aimed to explore the interactions between swIAV and PRRSV through simultaneous or sequential infections, with PRRSV inoculated first, followed by swIAV a few days later. This was investigated in several studies, with outcomes varying depending on experimental conditions and virus strains. Some studies have reported aggravation of lung lesions [ 10 , 11 ], while others found no differences compared to single virus infections [ 12 – 14 ]. Recent research also highlighted the complexity of immune responses triggered by co-infections. Bougon et al. (2021) observed that a PRRSV-1 infection eight days before exposure to swIAV H1N2 reduced influenza-like illness while increasing anti-swIAV antibodies and decreasing PRRSV-1 replication in lungs. Interferon-α (IFN-α) appeared to play a key role in the bidirectional interference between the two viral infections. Chrun et al. (2023) showed that a co-infection with swIAV H3N2 and PRRSV-2 did not worsen clinical signs, reduced swIAV H3N2 viral load in the lungs, and even enhanced certain immune parameters, including PRRSV-2-specific CD8 T cell count and swIAV H3N2-specific IgG levels. Most studies have focused on the inoculation of PRRSV followed by swIAV. Since influenza is an acute infection lasting no more than a week, whereas PRRS is an infection that can persist for months, it is more likely that animals infected with PRRSV will subsequently be co-infected with swIAV [ 2 ]. However, the alternative scenario, where animals are first infected with swIAV and secondly by PRRSV infection is plausible on pig farms, although it has never been explored and deserves further investigation. In most cases, infection with influenza A virus (IAV) is reported to weaken the host, facilitating infections by opportunistic pathogens such as Streptococcus suis for instance in pigs [ 16 ] or Streptococcus pneumoniae in humans [ 17 ], ultimately worsening the overall illness. One proposed mechanism following investigations in mice was that influenza infection led to depletion of alveolar macrophages (AM), making the host more prone to secondary infections [ 18 – 20 ]. However, this theory is debated, as other studies in mice have shown AM persistence during influenza infection [ 19 , 21 ]. Furthermore, respiratory viral infections may also have protective effects. Indeed, research in mice revealed that some respiratory viruses, including IAV, can induce a beneficial immune imprint in the lungs, offering better protection against subsequent bacterial infections, such as those caused by Streptococcus pneumoniae or Escherichia coli [ 22 , 23 ]. In this context, the present study aimed to evaluate the impact of swIAV infection on the AM population and the early innate and adaptive immune responses during a PRRSV secondary infection. We examined these interactions between a swIAV H1N2 and PRRSV-1, utilizing two strains representative of viruses that were circulating on pig farms at the same period of time in Brittany, France. Material and methods Virus strains The swIAV strain A/swine/Ille et Vilaine/0415/2011 (H1 hu N2, HA-clade 1B.1.2.3) and the so-called “Finistère” PRRSV strain (referenced as PRRS-FR-2005-29-24-1; PRRSV-1 subtype 1), both isolated from pig farms in Brittany, France, are part of the collections of the French Agency for Food, Environmental, and Occupational Health & Safety (ANSES, Ploufragan, France). Swine IAV was propagated and titrated using Madin-Darby canine kidney (MDCK) cells (ATCC reference CCL-34), whereas primary porcine alveolar macrophages (AM) were used for PRRSV, as previously described [ 15 ]. Animal experiment Thirty specific pathogen-free (SPF) Large White piglets (11.5 weeks old) from ANSES-Ploufragan’s protected animal facilities [ 24 ], were randomly assigned to six groups based on parental origin, weight, and sex (Fig. 1 ). The experiment was conducted at ANSES facilities, which are accredited for animal research by the Direction Départementale de la Protection des Populations des Côtes d’Armor. The animal study received approval from the National Committee for Ethics in Animal Experimentation ANSES/ENVA/UPEC (approval no. 23 − 017 #40450) and authorization was granted by the French Ministry for Research (authorization no. APAFIS #40450-2023012311482365 v3). On D0, three groups ("swIAV," "swIAV/PRRSV-D10," and "swIAV/PRRSV-D14") were intratracheally (IT) inoculated with 10 6 TCID 50 (50% tissue culture infectious dose) of swIAV in 5 mL per pig, as previously described (Bougon et al., 2021). The other three groups ("Mock," "PRRSV-D10," and "PRRSV-D14") received Minimum Essential Medium (MEM). On D7, the groups "swIAV/PRRSV-D10," "swIAV/PRRSV-D14," "PRRSV-D10," and "PRRSV-D14" were intranasally (IN) inoculated with 5 x 10 5 TCID 50 of PRRSV in 5 mL per pig. Rectal temperature, and clinical signs such as cough and sneezes were monitored daily. Hyperthermia was defined by a rectal temperature above 40°C. Coughing and sneezing were counted for 15 min in each room. Individual weights were measured weekly before swIAV inoculation, then twice a week after swIAV inoculation, and food consumption at the room level was recorded daily. Nasal swabs were collected in Virocult (MW915, Sigma Virocult®, MWE medical wire) and the supernatants were frozen at -80°C until RT-qPCR analysis. Animals were euthanized and necropsied according to the following schedule: on D7 for the "swIAV" and "Mock" groups, on D10 for the "PRRSV-D10" and "swIAV/PRRSV-D10" groups, and on D14 for the "PRRSV-D14" and "swIAV/PRRSV-D14" groups. After necropsies, bronchoalveolar lavages (BALs) were performed using sterile phosphate-buffered saline (PBS). A first BAL was performed with 40 mL of PBS, and the collected liquid was centrifuged at 500 × g for 10 minutes at 4°C to purify BAL fluid (BALF) from cells (BALC), and frozen at -20°C for antibody and cytokine assays, and at -80°C for RT-qPCR analyses. Two additional BALs with 100 mL of PBS each were carried out to collect and isolate more BALCs, which were stored in liquid nitrogen in fetal calf serum (FCS) with 10% dimethyl sulfoxide (DMSO) (Sigma-Aldrich). Lung tissue was taken from the right apical lobes, which were clamped during the BALs, and tracheobronchial lymph nodes (TBLN) were collected. These samples were frozen at -80°C for RT-qPCR analyses or fixed in 4% formaldehyde (Merck) for histopathological analyses. Serum samples were collected from clotted blood (centrifuged at 3 000 × g for 5 min) and frozen at -20°C for antibody and cytokine assays, and at -80°C for RT-qPCR analyses. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood using Ficoll density gradient centrifugation with LeucoSep tubes (Greiner Bio One) and stored in liquid nitrogen in FCS with 10% DMSO. Histopathological examination of lungs The formalin-fixed tissues were paraffin-embedded and sectioned into 4 µm slices. The sections were then stained with hematoxylin-eosin-saffron. Lesion intensity was evaluated based on parameters such as alveolar septal thickening, material accumulation in respiratory airways, inflammatory cell cuffing around bronchioles or vessels, proliferative bronchiolitis, alveolar emphysema, and bronchus-associated lymphoid tissue (BALT) hyperplasia. Lesions were scored using two different scoring systems: one with three parameters from Jung et al. (2007) and another composite system with three additional parameters from Larcher et al. (2019). For each system, scores were expressed as a percentage of the theoretical maximum. Viral quantifications Total RNA was extracted from nasal swabs, serum, BALF, lung tissues, and lymph nodes using the ID Gene™ Mag Fast 384 Extraction Kit (Innovative Diagnostics) on the Auto-Pure 96 nucleic acid purification system (Allsheng). For swIAV detection, duplex RT-qPCR targeting the M gene and β-actin was conducted using the Go Script RT mix for 1-Step RT-qPCR (Promega), following the method described by Cador et al. (2016). Quantification of the swIAV genome was achieved through serial dilutions of standardized M and β-actin mRNA, with results expressed as copy numbers of the M gene per 10 6 copies of the β-actin gene. PRRSV quantification was performed using duplex RT-qPCR with the SuperScript III Platinum one-step RT-qPCR kit (Life Technologies). Specific primers and probes targeting ORF5 of the “Finistère” strain and the porcine β-actin were used, as previously described [ 28 ]. Virus genome quantification was based on serial dilutions of standardized in vitro transcribed mRNA of the “Finistère” strain ORF5 RT-PCR amplicon (Eurofins genomics), with PRRSV genome levels reported as copy numbers per mL or per mg of sample. Cytokine quantifications Cytokines in BALF, including GM-CSF, IFN-γ, IL-10, TNF-α, IL-1α, IL-1β, IL-6, IL-8, IL-18, and IL-12, were measured using a porcine-specific cytokine array (Milliplex PCYTMAG-23K, Merck Millipore) following the manufacturer’s instructions on the MAGPIX System (DiaSorin). Flow cytometry analysis For the phenotyping of Tregs in BAL, cells were thawed and seeded in round-bottom 96-well microtiter plates (Nunc MicroWell Plates, Thermo Fisher Scientific) with a staining buffer containing PBS and 10% heat-inactivated porcine plasma. Surface staining was performed using primary monoclonal antibodies (mAbs) against CD3, CD4, CD8α, CD8β, CD25, and ICOS (for details on antibodies, refer to Table 1). Secondary staining involved goat anti-mouse-IgG2b-A488 (Jackson Immuno Research) and Streptavidin-BV650 (BioLegend) for labeling of CD4 and CD8α, respectively. Dead cells were marked with Fixable Viability Dye eFluor780 (Thermo Fisher Scientific) following the manufacturer's guidelines. Cells were then fixed and permeabilized using the eBioscience™ Foxp3/ Transcription Factor Staining Buffer Set (Thermo Fisher Scientific). Intracellular staining was performed with mAbs against Foxp3 and Ki-67. Surface staining was carried out at 4°C for 20 minutes and intracellular staining at 4°C for 30 minutes. After intracellular staining, cells were washed twice and resuspended in Perm/Wash Buffer for analysis. A second antibody panel was applied for characterisation of unconventional T cells and NK cells. Here, antibodies against the surface molecules TCR-γδ, CD8α, and CD161 were used. Secondary staining included rat anti-mouse-IgG2b-BUV395 (BD Biosciences), streptavidin-BV650 (BioLegend), and goat anti-mouse-IgG1-BV421 (Jackson Immuno Research) to label TCR-γδ, CD8α, and CD161, respectively. Additional surface staining was performed with mAbs against CD3, CD8β, CD16, NKp46, CD4, and CD2. The eBioscience™ Foxp3/ Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) was used for fixation and permeabilization as described previously. Intracellular staining was performed with mAbs against T-bet, perforin, and PLZF. Samples from both panels were analyzed on a Cytek Aurora spectral cytometer (Cytek Biosciences) with 5 lasers (UV 355 nm, violet 405 nm, blue 488 nm, yellow-green 561 nm, red 640 nm) and 64 fluorescence detection channels. Spectral unmixing was performed using SpectroFlo software (version 3.2.1, Cytek Biosciences) with single-stained reference samples. Autofluorescence signatures were subtracted using unstained controls. Data from 2 × 10 4 to 3 × 10 5 live lymphocytes per sample were recorded and analyzed using FlowJo software for Windows (version 10.9.0, BD Biosciences). For mononuclear phagocyte cell staining in BAL, cells were thawed and plated in round-bottom 96-well microtiter plates with a staining buffer containing PBS, supplemented with 2 mM ethylenediaminetetraacetic acid (EDTA), 5% swine serum, and 5% goat serum. Surface staining was performed using primary mAbs against MHCII, CD172a, and CD11c. For secondary staining, rat anti-mouse-IgG2a-PE-Cy7 (Thermo Fisher Scientific), goat anti-mouse-IgG2b-APC-Cy7 (Abcam), and goat anti-mouse-IgG1-A647 (Thermo Fisher Scientific) were used to label MHCII, CD172a, and CD11c, respectively. Dead cells were identified using the LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit for 405 nm excitation (Thermo Fisher Scientific) after surface staining. Additional surface staining was performed with mAbs against CD1 and CD163. Staining steps were carried out at 4°C for 30 minutes. Labelled cells were fixed with 1% paraformaldehyde (PFA) for 20 min at room temperature, followed by two washes with PBS. Samples were then acquired in PBS using a MACSQuant 10 cytometer (Miltenyi Biotec), equipped with 3 lasers (violet 405 nm, blue 488 nm, red 635 nm) and eight fluorescence detection channels. Data from 6 × 10 4 to 4 × 10 5 live cells per sample were recorded and analyzed using FlowJo software. Alveolar macrophages (AM), monocyte-derived dendritic cells (moDC), and conventional dendritic cells of types 1 and 2 (cDC1 and cDC2) were identified based on staining for MHC II, CD11c, CD163, CD172a, and CD1, as previously described [ 29 , 30 ]. An anti-CD11c antibody was used to identify myeloid cells [ 31 ]. Intracellular cytokine staining (ICS) For ICS, PBMCs were thawed and plated in round-bottom 96-well microtiter plates with 5 × 10 5 cells per well, in a final volume of 200 µL per well. Cells were cultivated in Roswell Park Memorial Institute (RPMI) 1640 (Sigma-Aldrich) supplemented with 1% penicillin (100 IU/mL) and streptomycin (100 µg/mL) (PS) (Gibco, Thermo Fisher Scientific) and 10% fetal calf serum (FCS) (Life Science Production). They were exposed to either PRRSV-1 ("Finistère" strain) at a multiplicity of infection (MOI) of 1 for 18 hours, phorbol 12-myristate 13-acetate (PMA) (50 ng/mL) and ionomycin (500 ng/mL) as a positive control for the final four hours, or cell culture medium alone. Brefeldin A (BD GolgiPlug™, BD Biosciences) was added to all conditions for the final four hours at a concentration of 1 µg/mL. After incubation, cells were harvested, centrifuged, and resuspended in staining buffer containing PBS with 3% FCS. Cells were then surface-stained with mAbs against CD4, CD8α, and CD8β. Subsequently, goat-anti-mouse-IgG2b-A488 (Jackson Immuno Research) and Streptavidin-BV421 (BioLegend) were used to label CD4 and CD8α, respectively. Dead cells were identified using Fixable Viability Dye eFluor780 (Thermo Fisher Scientific) after surface staining. Cells were then fixed and permeabilized using the BD Cytofix/Cytoperm™ Fixation/Permeabilization Kit (BD Biosciences). Intracellular staining was performed with mAbs against IFN-γ, IL-2 and TNF-α. Incubation steps were performed in the same way as described above. After intracellular staining, cells were washed twice and resuspended in Perm/Wash Buffer (BD Biosciences). Samples were acquired on the Cytek Aurora cytometer. Data from a minimum of 4 × 10 5 live lymphocytes per sample were recorded and analyzed using FlowJo software. Data for TNF-α labelling was not analyzed due to a technical issue. Antibody assessment in sera and BALF Anti-swIAV (NP protein) IgG was detected using the ID Screen Influenza A Nucleoprotein Swine Indirect kit (Innovative Diagnostics) in serum at a 1:100 dilution and in BALF at a 1:2 dilution. Anti-swIAV IgA was measured in BALF at a 1:2 dilution using the same kit, with a modified protocol using a goat anti-pig IgA antibody HRP conjugate (Euromedex) at a 1:3000 dilution and in-house controls to calculate sample-to-positive (S/P) ratios. Anti-PRRSV (N protein) immunoglobulin G (IgG) was measured in serum and BALF using the IDEXX PRRS X3 ELISA kit (IDEXX Laboratories). In serum, the kit was used according to the manufacturer's instructions at a 1:40 dilution, while in BALF, an adapted protocol was applied with a 1:2 dilution of the samples. For detecting anti-PRRSV immunoglobulin A (IgA) in 1:2 diluted BALF and anti-PRRSV immunoglobulin M (IgM) in 1:40 diluted serum, the same kit was employed replacing the anti-pig IgG conjugated antibody by a goat anti-pig IgA or a goat anti-pig IgM HRP conjugated antibody (Euromedex) at a 1:3000 or 1:25000 dilution, respectively. For anti-PRRSV IgA or IgM assays, in-house calibrated negative and positive BALF or serum controls were used to calculate sample-to-positive (S/P) ratios. Statistical analyses Non-normal distribution of the data was determined by the Shapiro-Wilk test. The Kruskal-Wallis test was applied for unpaired comparisons among four groups and significances were directly depicted on Figs. 2 A and 2 B (rectal temperature and average daily weight gain data). Unpaired comparisons between two groups (Mann-Whitney test) were applied for Fig. 2 C to Fig. 8 (histopathological, virological, and immunological data). The Mann-Whitney test was used to compare Mock vs . swIAV, PRRSV-D10 vs. swIAV/PRRSV-D10, and PRRSV-D14 vs . swIAV/PRRSV-D14. However, Kruskal-Wallis test was also applied for unpaired comparisons among four groups on these data, and significances are depicted in Supplementary Table S1 . The absolute number of mononuclear phagocyte cells was calculated by determining the percentage of live cells using flow cytometry, then multiplying this percentage by the total number of BAL cells counted, all divided by 100 and multiplied by the volume of BAL collected. The script used for high-dimensional flow cytometry analysis was developed by Adrian Liston's group [ 32 ] (The Babraham Institute, UK) and is available on GitHub at:. t-SNE plots were generated using R (version 4.3.0). t-SNE algorithm was run on live lymphocytes using the parameters CD3, CD4, CD8α, CD8β, CD16, NKp46, TCR-γδ, CD2, CD161, Perforin, PLZF and T-bet. Samples of five pigs per treatment group (Mock, swIAV, PRRSV-D10, PRRSV-D14, swIAV/PRRSV-D10, swIAV/PRRSV-D14) were used with 5000 cells per sample and 5000 iterations per run. Statistical analyses were performed using GraphPad Prism Software (version 10.2.3). Results Pre-infection with swIAV did not affect the clinical progression of PRRS To compare the clinical outcomes of a single PRRSV inoculation to successive inoculations of swIAV and PRRSV seven days apart (Fig. 1 ), rectal temperature and respiratory signs (coughing, sneezing, and breathing rate) were monitored daily. On D1, the animals that received swIAV intratracheally showed hyperthermia (rectal temperature > 40°C) in four out of five animals in the swIAV group and in five out of ten animals in the swIAV/PRRSV group (mean 40.1 ± 0.5°C and 39.9 ± 0.4°C, respectively). These temperatures were significantly higher than those in the Mock and PRRSV groups (p < 0.05) (Fig. 2 A). By D2, the animals' temperatures returned to normal (below 40°C). On D9, two days after PRRSV inoculation, four out of ten animals in the PRRSV group (mean 39.8 ± 0.4°C) and two out of ten in the swIAV/PRRSV group (mean 39.6 ± 0.3°C) showed an increase in temperature above 40°C. There was no significant difference between the PRRSV and swIAV/PRRSV groups. Some animals showed respiratory signs. On the first day after swIAV inoculation, one out of fifteen animals was coughing. By D9 (two days after PRRSV inoculation), one out of ten animals in the swIAV/PRRSV group was sneezing, and the same animal was coughing on D10 (three days after PRRSV infection). By D10, two out of ten sequentially inoculated animals showed respiratory signs (one was coughing and the other sneezing), compared to one out of ten in the PRRSV-only group, which exhibited a cough. Average daily weight gain (ADWG) was significantly decreased in the swIAV group between D0 and D2, compared to the Mock and PRRSV groups (p < 0.05). However, in the swIAV/PRRSV group, no significant difference was observed between this group and the Mock and PRRSV groups. After PRRSV inoculation, there was no significant growth difference between the PRRSV group and the swIAV/PRRSV group (Fig. 2 B). Microscopic examination of the lung tissue revealed that, compared to control animals, the alveolar walls were significantly thickened in all infected animals, regardless of single or super-infections. The respiratory airways were occluded by necrotic debris and inflammatory cells. Perivascular cuffing by inflammatory cells was observed in the most severely affected animals. The severity of lung lesions was assessed using two different scoring systems (Jung score and Composite score), both of which gave similar results (Figs. 2 C, D). By D7 post-swIAV inoculation, significant lung lesions were observed in the swIAV group compared to the Mock group (p < 0.05). After PRRSV inoculation, no differences were observed on D10 and D14 between the PRRSV group and the swIAV/PRRSV group. Overall, these results suggested that the initial swIAV infection did not worsen the clinical signs and lung lesions caused by PRRSV. Pre-infection with swIAV did not affect PRRSV loads Using RT-qPCR, the viral loads of PRRSV and swIAV genomes were monitored in BALF, lung tissue, tracheobronchial lymph nodes, nasal swab supernatants (swIAV) and serum samples (PRRSV) from all groups. Neither PRRSV nor swIAV genome was detected in the Mock-inoculated group. In animals inoculated with swIAV (both swIAV and swIAV/PRRSV groups), the viral genome was detected in nasal secretions at D4 and D7 post-inoculation, with lower detection levels at D7. At the time of PRRSV inoculation on D7, swIAV was detected in BALF from all the animals in the swIAV group, in lung tissue in two out of five pigs, and in the tracheobronchial lymph nodes in five out of five pigs. By D10 and D14, the swIAV genome was no longer detected in the nasal secretions of pigs in the swIAV/PRRSV group (data not shown). However, it was still detected in BALF in three out of five animals at D10 and in two out of five animals at D14 (Figure S1 B). In lung tissue, swIAV genome was detected in one out of five animals at D10 and D14. In TBLN, it was detected in three out of five animals at D10, but no more at D14 (Figure S1 C, D). PRRSV genetic material was detected at D10 (three days post-PRRSV infection) in all animals from the PRRSV and swIAV/PRRSV groups, in BALF, tracheobronchial lymph nodes and sera. In the lung tissue, PRRSV was detected in all the pigs from the swIAV/PRRSV group but in only two out of five animals in the PRRSV group. By D14 (seven days post-PRRSV infection), PRRSV was detected in all animals and all sample types (Fig. 3 A-D). However, no significant differences were observed between the PRRSV and swIAV/PRRSV groups at D10 or D14. Thus, virological monitoring indicated that PRRSV loads in the lungs, lymph nodes and blood were not influenced by swIAV pre-infection. Interleukin-12 increased trend upon swIAV and swIAV/PRRSV infections in BALF The cytokine profile (GM-CSF, IFN-γ, IL-10, TNF-α, IL-1α, IL-1β, IL-6, IL-8, IL-18, and IL-12) in BALF was assessed on D7 for the Mock and swIAV groups, and on D10 (three days post-PRRSV inoculation) and D14 (seven days post-PRRSV inoculation) for the PRRSV and swIAV/PRRSV groups (Fig. 4 ). The levels of GM-CSF, IFN-γ, IL-10, and TNF-α were below detection thresholds. No significant differences were observed for the cytokines IL-1α, IL-1β, IL-8, and IL-18 (Fig. 4 A, B, D, E). In the swIAV group on D7, a significant increase in the pro-inflammatory cytokines IL-6 and IL-12 (a pro-Th1 cytokine) was observed compared to the Mock group (p < 0.01) (Fig. 4 C, F). On D10, a trend suggested higher IL-12 levels in the swIAV/PRRSV group compared to the PRRSV group, with four out of five animals of the PRRSV group showing lower concentrations than those in the swIAV/PRRSV group, and five out of five compared to the Mock group, although this difference was not statistically significant. This trend continued on D14, with all five animals in the swIAV/PRRSV group exhibiting higher IL-12 concentrations than those in the Mock group. The swIAV/PRRSV group presented a significant IL-12 overexpression when compared with the Mock group using Kruskal-Wallis unpaired comparisons test (Supplementary table 1 ). However, two out of five animals in the PRRSV group displayed IL-12 levels similar to those in the swIAV/PRRSV group. Pre-infection with swIAV increased number of conventional type 1 dendritic cells (cDC1) in BAL during PRRSV infection To characterize the cellular innate immune response in more detail, mononuclear phagocyte cell populations were examined in BAL for all groups. Conventional DC1 were defined as MHCII high CD11c + CD163 − CD172a −/low CD1 − , while cDC2 were defined as MHCII high CD11c + CD163 − CD172a + CD1 + . Monocyte-derived DC (moDC) were identified as MHCII high CD11c + CD163 low CD172a + CD1 − , and macrophages from the BAL were characterized as MHCII high CD11c + CD163 high CD172a + (Figure S2 ). In the Mock and swIAV groups at D7, no differences in the number of identified cell populations were observed (Fig. 5 A-D). Similarly, no significant differences were observed between the PRRSV and swIAV/PRRSV groups on D10 and D14, except for a significant increase in cDC1 on D14 in the swIAV/PRRSV group compared to the PRRSV group (p < 0.01) (Fig. 5 C). This increase in cDC1 remains significant when analyzed using the Kruskal-Wallis unpaired comparisons test (Supplementary Table 1). Pre-infection with swIAV increased activated CD4 + regulatory and conventional CD4 T cells in BAL To further explore the immune response, lymphoid cell populations in the BAL were analyzed by flow cytometry for all groups and all necropsy time points. One set of experiments focused on conventional CD4 + Foxp3 − T cells (Tconv) and CD4 + Foxp3 + Treg cells (Figs. 6 A-D, S3), investigating their distribution within total CD4 T cells. No significant differences were observed in the percentages of Tconv and Treg between the swIAV and Mock groups, or between the PRRSV and swIAV/PRRSV groups (Figs. 6 A, B). Within the two subsets of Tconv and Treg, we also analyzed the co-expression of Inducible T-cell Costimulator (ICOS), involved in anti-inflammatory signalling and Ki-67, a molecule expressed in active stages of the cell cycle. A significant increase in ICOS + Ki-67 + Tconv and Treg cells was observed in the swIAV group compared to the Mock group. For the ICOS + Ki-67 + Tconv, this increase remained on D10 in the swIAV/PRRSV compared to the PRRSV group (Figs. 6 C). Interestingly, swIAV/PRRSV group presented a significantly higher frequency of ICOS + Ki-67 + Tconv cells when compared to the Mock group using Kruskal-Wallis unpaired comparisons test (Supplementary table 1 ). Moreover, a significant difference in ICOS + Ki-67 + Tconv cells was observed at D10 when comparing swIAV/PRRSV to the PRRSV group alone. Overall, this suggested that the swIAV infection resulted in an increase in activated Tregs and Tconv in the BAL that was maintained even in the context of a subsequent PRRSV infection. Swine IAV infection resulted in sustained increases in effector lymphocytes in BAL A third flow cytometry panel focused on potential changes in conventional T cells, unconventional T cells and NK cells. Given the high numbers of addressed markers (n = 12, CD2, CD3, CD4, CD8α, CD8β, CD16, CD161, NKp46, Perforin, PLZF, T-bet, TCR-γδ) we performed dimensionality reduction using t-SNE clustering on live lymphocytes (Figs. 7 A-E, S4). This analysis enabled the identification of natural killer (NK) cells, conventional CD4 and CD8 T cells, and γδ T cells. The t-SNE analysis was set to perform a clustering for twelve clusters (Figures S4, S5). A number of clusters showed significant changes related to treatment groups. In the swIAV group, a significant increase compared to the Mock group was detected for clusters 6, 8, 9 and 10. Cluster 6 contained cells reminiscent of conventional CD4 T cells due to a CD3 + CD4 + CD8α +/− phenotype that was negative for all other markers (Fig. 7 A). Cluster 8 consisted of cells with a phenotype of cytotoxic CD8 T cells (CD2 + CD3 + CD8αβ + perforin +/− , Fig. 7 C). Interestingly, cluster 9 contained cells that were negative for all lineage markers (CD3, TCR-γδ, CD4, CD8β), including NK associated markers NKp46 and CD16 but contained high levels of perforin (Fig. 7 D). Additionally, swIAV infection also led to an increase of cells represented by cluster 10 which consisted of CD3 + T cells that were negative for CD4, CD8β and TCR-γδ, but CD2 + CD8α + perforin dim (Fig. 7 E). Clusters 6, 8, 9 and 10 also presented a significant increase in swIAV/PRRSV group on D10 compared with Mock D7 when using Kruskal-Wallis unpaired comparisons test (Supplementary Table 1). Notably, these elevated populations remained consistently higher in the swIAV/PRRSV groups at D14, showing significant differences compared to the PRRSV group (p < 0.05). An increase in a subset of TCR-γδ cells (cluster 7, Fig. 7 B) with a CD8α + CD2 +/− perforin +/− phenotype was also observed in the swIAV/PRRSV group compared to PRRSV group on D14. Pre-infection with swIAV increased IFN-γ-producing CD4 T cells after PRRSV re-stimulation We investigated IFN-γ and IL-2 production in T cells within PBMCs following in vitro re-stimulation with the autologous PRRSV-1 strain by intracellular cytokine staining. Analyses focused on the PRRSV and swIAV/PRRSV groups, isolated on D14 (seven days post-PRRSV infection) and the Mock group. Cells cultured in medium alone served as negative controls. CD4 expressing lymphocytes were pre-gated and cytokine producing cells separated into CD8α + and CD8α − subsets (Figure S6). Irrespective of CD8α expression, the percentage of IFN-γ-producing CD4 T cells was significantly higher in the swIAV/PRRSV group on D14 compared to PRRSV groups (p < 0.05) (Figs. 8 A, B) and the Mock group when using Kruskal-Wallis unpaired comparisons test (Supplementary Table 1). Notably, the percentages in the PRRSV group remained at levels comparable to those observed in the Mock group (Figs. 8 A, B). No significant differences were observed in the production of IL-2 (Figs. 8 C, D). Of note, the IFN-γ response in PBMCs induced by this PRRSV strain typically begins around two weeks after inoculation [ 15 ]. Local swIAV antibodies continued to rise after swIAV clearance Humoral immune responses to swIAV and PRRSV infections were also assessed by detecting specific antibodies directed against each virus. For animals infected with swIAV, IgA in BALF and IgG in both BALF and serum were detected (Figures S7A-C). S/P ratios continued to rise on D10 and D14, although most pigs had cleared swIAV by those time points, which is typical in single swIAV infections (Figure S1 ). No significant differences in anti-PRRSV IgA and IgG antibody levels were observed in the BALF of the PRRSV and swIAV/PRRSV groups throughout the study (Figures S8A, B). At D14, seven days after PRRSV inoculation, IgA and IgG antibodies began to be detectable in some animals. Similarly, no significant differences in anti-PRRSV IgG and IgM levels were observed in serum between the PRRSV and swIAV/PRRSV groups. The S/P ratios remained low but started to increase at D14 (Figures S8C, D). Discussion Co-infections with different pathogens are often the cause of respiratory diseases in pigs [ 1 , 2 , 33 ]. It is established that infection with IAV increases susceptibility to secondary bacterial infections, such as Streptococcus suis in pigs [ 34 , 35 , 16 ] or Streptococcus pneumoniae in humans [ 36 , 17 , 18 ]. One of the proposed mechanisms for this influenza-related susceptibility is the depletion of AM, as suggested from studies in mice between four and ten days post-IAV inoculation [ 18 – 20 ]. In our study, we did not observe differences in the number of AM in animals from the swIAV group compared to the Mock group at 7 days post-IAV inoculation. This suggests that, differently to IAV infection in mice, the swIAV strain used in this study did not lead to AM depletion in pigs. However, it is important to approach the analysis of resident AM during IAV infection with caution. During this infection various chemokines have increased expression in the lungs, leading to a heterogeneous influx of innate immune cells, such as monocytes-derived macrophages (moMΦ) and moDC [ 18 , 29 , 19 , 20 ], whose surface markers might vary based on inflammatory conditions. It would therefore be interesting to consolidate these results in our study on pigs using multiparametric, unbiased approaches, such as single-cell RNA sequencing, to more precisely distinguish resident cells from recruited ones. While various combinations of viral infections are possible, our study focused on a PRRSV-1 super-infection occurring one week after a swIAV H1N2 infection, both commonly observed on pig farms in France. The objective of our research was to evaluate the impact of a primary swIAV infection on the host’s immune responses during a secondary PRRSV infection. We analyzed the effects on clinical parameters, viral loads, and both innate and adaptive immune responses in pigs. One week after the initial infection with swIAV, clinical signs following the secondary infection with PRRSV did not differ significantly from those observed during a single infection with PRRSV. This suggested that the initial swIAV infection did not exacerbate the animals' health status during the subsequent PRRSV infection. It is noteworthy that in a previous study using the same swIAV H1N2 and PRRSV-1 strains, but with a reversed order of infection (PRRSV inoculation followed by swIAV inoculation eight days later), Bougon et al. (2021) observed a reduction in clinical signs in the co-infected group, suggesting an attenuation of the impact of swIAV H1N2 infection in pigs previously infected with PRRSV-1. Correlation analyses revealed an association between IFN-α production and the onset of clinical signs. PRRSV infection led to a reduction in IFN-α production in PRRSV/swIAV super-infected pigs, consistent with previous observations [ 37 ], which likely contributed to the attenuation of clinical signs and the pro-inflammatory response induced by the influenza infection [ 15 ]. They also reported a transient yet significant decrease in PRRSV viral load in the lungs of super-infected pigs, which was correlated with the induction of IFN-α by swIAV, a cytokine to which PRRSV is particularly sensitive [ 38 ]. Another study by Renson et al. [ 39 ] examined the effects of H1N2 swIAV infection on the replication of a PRRSV-1 modified live vaccine (MLV1) in SPF piglets. SwIAV infection six hours before MLV1 administration delayed MLV1 viremia and post-vaccination seroconversion. The early rise in IFN-α levels following H1N2 swIAV infection likely explained the inhibition of MLV1 replication. These results highlighted that the order of infections may play a key role in modulating clinical responses. By contrast, in this study, the animals were super-infected seven days after the swIAV infection, when IFN-α was no longer detectable in BAL [ 15 ], to avoid any interference from this cytokine. The main objective of the present study was to evaluate the effect of a primary infection with swIAV on the early progression of a subsequent PRRSV infection. No significant differences were observed in the PRRSV viral load in the lungs and blood of pigs super-infected with swIAV and PRRSV compared to those infected only with PRRSV. We also aimed to examine whether swIAV could induce an early immune imprint, similar to what has been observed in the murine IAV [ 23 ] and adenovirus [ 22 ] models. Aegerter et al. showed that primary infection with IAV conferred protection against Streptococcus pneumoniae by recruiting AM derived from monocytes, which persisted in the lungs for an extended period following IAV infection. Similarly, adenovirus promoted a lasting innate immune memory of AM, facilitated by T lymphocytes through the production of IFN-γ, enhancing immunity against S. pneumoniae and E. coli . It is important to note that in these studies, the protective imprint was observed several weeks after IAV inoculation (at least four weeks), while in this experiment, PRRSV infection was performed only one week after swIAV inoculation. However, the present results did not show that primary infection with swIAV offered protection against PRRSV or on contrary enhanced its replication in the early stages following swIAV infection. Early in swIAV infection, pro-inflammatory cytokines (IFN-α, TNF-α, and IL-12) are secreted in the lungs, accompanied by the infiltration of immune cells, particularly DC [ 40 , 29 , 41 ]. In the BAL, a higher frequency of cDC1 cells was observed in swIAV-infected pigs, likely attracted by CD8 T cells, which also increased in the lungs following swIAV infection. CD8 T cells express the X-C motif chemokine ligand 1 (XCL1), a chemokine selectively expressed in NK cells and CD8 T cells, which is further elevated upon stimulation, as shown in murine and human cells [ 42 , 43 ]. XCL1 attracts cDC1 via their XC chemokine receptor 1 (XCR1), promoting IL-12 production in cDC1 [ 30 , 44 ]. Indeed, IL-12 was also elevated in the present study following super-infection with PRRSV. The IL-12 produced by cDC1 stimulates the production of IFN-γ by antigen-specific T cells, thus directing the immune response towards a Th1 and ultimately type-1 immune response [ 30 , 45 ]. In line with this, we observed an increase in CD4 T cells as well as perforin expressing CD8 T cells and less well characterized non-T NK-cell like lymphocytes in the lungs of swIAV/PRRSV group. This coincided with a PRRSV-specific Th1 response in the blood of pigs that were initially infected with swIAV. These results suggest that a primary swIAV infection could promote the rapid induction of an anti-PRRSV-1 immunity via the recruitment of cDC1 and the production of IL-12 by these cells. It is well established that PRRSV infection leads to a delayed activation of PRRSV-specific CD4 and CD8 T cells, typically appearing two to three weeks post-inoculation [ 46 , 15 ]. However, the present study indicated that prior infection with swIAV increased the presence of CD4 and perforin expressing lymphocytes in the lungs during PRRSV infection, potentially contributing to a more pronounced immune response against PRRSV. It remains to be determined whether this increase in effector cells was due to active recruitment of new cells to the lungs or the persistence of cells initially activated by the swIAV infection, but their increase in swIAV-only infected pigs suggests the latter. Indeed, CD4, CD8, and γδ T lymphocytes were present in the BAL as early as six days post-infection with swIAV [ 40 ], and swIAV-specific cells could be detected between D4 and D7 in PBMCs [ 47 , 48 ]. However, this proliferation of lymphocytes did not prevent PRRSV replication at the early stages of infection, and their role in controlling PRRSV at later stages remains to be clarified. A longer-term follow-up of PRRSV infection would be valuable to determine if it is better controlled over time through these cellular responses. Additionally, it would be relevant to explore whether enhanced induction of type-1 responses could contribute to protection against a subsequent homologous or heterologous PRRSV challenge. Conclusion In summary, this study demonstrated that the primary infection with swIAV, occurring one week prior, did not interfere with the early infection of AM by PRRSV. However, several parameters of the cellular antiviral response were significantly elevated, indicating that an enhanced immune response could influence the progression of PRRSV infection at later stages. These results raise questions about the potential impact of this response on the progression of PRRS disease. Therefore, further research is needed to better understand the underlying mechanisms of these immune responses. Declarations Author Contributions Conceptualization, G.S., O.B., N.B., F.M. and J.G.; methodology, G.S., O.B., N.B., W.G., S.S, C.D., P.R. and J.G.; software, S.S.; validation, O.B., G.S., N.B., W.G., S.S, C.D., and P.R.; formal analysis, S.S and J.G.; investigation, O.B., N.B., C.H., A.P., S.S., S.G., C.D., P.R, M.L.D., F.P., T.L. and J.G.; data curation, S.S and J.G.; writing-original draft preparation, J.G.; writing-review and editing: G.S., O.B., N.B., W.G., S.S., F.M., C.H., A.P., F.P., C.D., P.R., S.G., S.Q., M.L.D., T.L., and J.G.; visualization, S.S. and J.G.; supervision, G.S., O.B., N.B., F.P. and W.G.; project administration, W.G., N.B., G.S and O.B.; funding acquisition, W.G., N.B., G.S. and O.B. Acknowledgements We thank the Flow Cytometry and Immunological Toolbox Science Technology Platforms at The Pirbright Institute for supporting this research. We also thank the staff at the Biological Research Facility and Immunological Toolbox at The Roslin Institute (https://www.ed.ac.uk/roslin/facilities-resources/immunological-toolbox) for their assistance in producing the monoclonal antibody to porcine CD161. Additionally, we would also like to thank Nicolas Barbier, Séverine Hervé, Roselyne Fonseca, Gaëtan Pinsard, and Sophie Mahé for their help with the necropsies, as well as to Jean-Marie Guionnet and Gérald Le Diguerher for animal care. Funding JG received a PhD grant co-funded by the French National Research Institute for Agriculture, Food and Environment (INRAE) and the French Agency for Food, Environmental and Occupational Health & Safety (ANSES). JG also received funding from the Directorate for Higher Education, Sites and Europe (DESSE), the ERASMUS+ program, and the Doctoral School for Plant, Animal, Food, Sea, and Environment (VAAME) for mobility in the United Kingdom. SS and WG are supported by the Biotechnology and Biological Sciences Research Council (BBSRC) Strategic Programs to The Pirbright Institute, BB/X011038/1 and BB/X011046/1. The Flow Cytometry and Immunological Toolbox facilities at The Pirbright Institute are supported through the BBSRC National Bioscience Research Infrastructure High and Low Containment Services and Science Platforms (BBS/E/PI/23NB0004, BBS/E/PI/23NB0003). The Biological Research Facility and Immunological Toolbox at the Roslin Institute are supported by funding from UKRI BBSRC (BB/CCG2270/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Conflicts of Interest The authors declare no conflict of interest. References Opriessnig T, Giménez-Lirola LG, Halbur PG (2011) Polymicrobial respiratory disease in pigs. Anim Health Res Rev 12:133–148. https://doi.org/10.1017/S1466252311000120 Saade G, Deblanc C, Bougon J et al (2020) Coinfections and their molecular consequences in the porcine respiratory tract. Vet Res 51:80. https://doi.org/10.1186/s13567-020-00807-8 Meurens F, Zhu J, Renois F (2024) [Viral co-infections targeting the porcine respiratory system: Consequences and limits of the experimental systems]. Virologie (Montrouge) 28:9–21. https://doi.org/10.1684/vir.2024.1032 Chauhan RP, Gordon ML (2020) A Systematic Review Analyzing the Prevalence and Circulation of Influenza Viruses in Swine Population Worldwide. Pathogens 9:355. https://doi.org/10.3390/pathogens9050355 Lunney JK, Benfield DA, Rowland RRR (2010) Porcine reproductive and respiratory syndrome virus: An update on an emerging and re-emerging viral disease of swine. Virus Res 154:1–6. https://doi.org/10.1016/j.virusres.2010.10.009 Adams MJ, Lefkowitz EJ, King AMQ et al (2017) Changes to taxonomy and the International Code of Virus Classification and Nomenclature ratified by the International Committee on Taxonomy of Viruses (2017). Arch Virol 162:2505–2538. https://doi.org/10.1007/s00705-017-3358-5 Balasuriya UB, Carossino M (2017) Reproductive effects of arteriviruses: equine arteritis virus and porcine reproductive and respiratory syndrome virus infections. Curr Opin Virol 27:57–70. https://doi.org/10.1016/j.coviro.2017.11.005 Fablet C, Marois-Créhan C, Simon G et al (2012) Infectious agents associated with respiratory diseases in 125 farrow-to-finish pig herds: A cross-sectional study. Vet Microbiol 157:152–163. https://doi.org/10.1016/j.vetmic.2011.12.015 Fablet C, Marois-Créhan C, Grasland B et al (2016) Factors associated with herd-level PRRSV infection and age-time to seroconversion in farrow-to-finish herds. Vet Microbiol 192:10–20. https://doi.org/10.1016/j.vetmic.2016.06.006 Van Reeth K, Nauwynck H, Pensaert M (1996) Dual infections of feeder pigs with porcine reproductive and respiratory syndrome virus followed by porcine respiratory coronavirus or swine influenza virus: a clinical and virological study. Vet Microbiol 48:325–335. https://doi.org/10.1016/0378-1135(95)00145-X Czyżewska-Dors E, Pomorska-Mól M, Dors A et al (2019) Proinflammatory cytokine changes in bronchoalveolar lavage fluid cells isolated from pigs infected solely with porcine reproductive and respiratory syndrome virus or co-infected with swine influenza virus. J Veterinary Res 63:489–495. https://doi.org/10.2478/jvetres-2019-0063 Pol JMA, van Leengoed LAMG, Stockhofe N et al (1997) Dual infections of PRRSV/influenza or PRRSV/ Actinobacillus pleuropneumoniae in the respiratory tract. Vet Microbiol 55:259–264. https://doi.org/10.1016/S0378-1135(96)01323-5 Pomorska-Mól M, Podgórska K, Czyżewska-Dors E et al (2020) Kinetics of single and dual simultaneous infection of pigs with swine influenza A virus and porcine reproductive and respiratory syndrome virus. J Vet Intern Med 34:1903–1913. https://doi.org/10.1111/jvim.15832 Chrun T, Maze EA, Roper KJ et al (2023) Simultaneous co-infection with swine influenza A and porcine reproductive and respiratory syndrome viruses potentiates adaptive immune responses. Front Immunol 14:1192604. https://doi.org/10.3389/fimmu.2023.1192604 Bougon J, Deblanc C, Renson P et al (2021) Successive Inoculations of Pigs with Porcine Reproductive and Respiratory Syndrome Virus 1 (PRRSV-1) and Swine H1N2 Influenza Virus Suggest a Mutual Interference between the Two Viral Infections. Viruses 13:2169. https://doi.org/10.3390/v13112169 Obradovic MR, Segura M, Segalés J, Gottschalk M (2021) Review of the speculative role of co-infections in Streptococcus suis-associated diseases in pigs. Vet Res 52:49. https://doi.org/10.1186/s13567-021-00918-w McCullers JA (2011) Preventing and Treating Secondary Bacterial Infections with Antiviral Agents. Antivir Ther 16:123–135. https://doi.org/10.3851/IMP1730 Ghoneim HE, Thomas PG, McCullers JA (2013) Depletion of Alveolar Macrophages during Influenza Infection Facilitates Bacterial Superinfections. J Immunol 191:1250–1259. https://doi.org/10.4049/jimmunol.1300014 Califano D, Furuya Y, Metzger DW (2018) Effects of Influenza on Alveolar Macrophage Viability Are Dependent on Mouse Genetic Strain. J Immunol 201:134–144. https://doi.org/10.4049/jimmunol.1701406 Li F, Piattini F, Pohlmeier L et al (2022) Monocyte-derived alveolar macrophages autonomously determine severe outcome of respiratory viral infection. Sci Immunol 7:eabj5761. https://doi.org/10.1126/sciimmunol.abj5761 Janssen WJ, Barthel L, Muldrow A et al (2011) Fas Determines Differential Fates of Resident and Recruited Macrophages during Resolution of Acute Lung Injury. Am J Respir Crit Care Med 184:547–560. https://doi.org/10.1164/rccm.201011-1891OC Yao Y, Jeyanathan M, Haddadi S et al (2018) Induction of Autonomous Memory Alveolar Macrophages Requires T Cell Help and Is Critical to Trained Immunity. Cell 175:1634–1650e17. https://doi.org/10.1016/j.cell.2018.09.042 Aegerter H, Kulikauskaite J, Crotta S et al (2020) Influenza-induced monocyte-derived alveolar macrophages confer prolonged antibacterial protection. Nat Immunol 21:145–157. https://doi.org/10.1038/s41590-019-0568-x Grevelinger J, Bourry O, Meurens FJ et al (2024) Impact of swine influenza A virus on porcine reproductive and respiratory syndrome virus infection in alveolar macrophages. Front Vet Sci 11. https://doi.org/10.3389/fvets.2024.1454762 Jung K, Alekseev KP, Zhang X et al (2007) Altered Pathogenesis of Porcine Respiratory Coronavirus in Pigs due to Immunosuppressive Effects of Dexamethasone: Implications for Corticosteroid Use in Treatment of Severe Acute Respiratory Syndrome Coronavirus. J Virol 81:13681–13693. https://doi.org/10.1128/jvi.01702-07 Larcher T, Fablet C, Renson P et al (2019) Assessment of pulmonary tissue responses in pigs challenged with PRRSV Lena strain shows better protection after immunization with field than vaccine strains. Vet Microbiol 230:249–259. https://doi.org/10.1016/j.vetmic.2019.01.022 Cador C, Hervé S, Andraud M et al (2016) Maternally-derived antibodies do not prevent transmission of swine influenza A virus between pigs. Vet Res 47:86. https://doi.org/10.1186/s13567-016-0365-6 Rose N, Renson P, Andraud M et al (2015) Porcine reproductive and respiratory syndrome virus (PRRSv) modified-live vaccine reduces virus transmission in experimental conditions. Vaccine 33:2493–2499. https://doi.org/10.1016/j.vaccine.2015.03.040 Maisonnasse P, Bouguyon E, Piton G et al (2016) The respiratory DC/macrophage network at steady-state and upon influenza infection in the swine biomedical model. Mucosal Immunol 9:835–849. https://doi.org/10.1038/mi.2015.105 Bordet E, Blanc F, Tiret M et al (2018) Porcine Reproductive and Respiratory Syndrome Virus Type 1.3 Lena Triggers Conventional Dendritic Cells 1 Activation and T Helper 1 Immune Response Without Infecting Dendritic Cells. Front Immunol 9. https://doi.org/10.3389/fimmu.2018.02299 Deloizy C, Bouguyon E, Fossum E et al (2016) Expanding the tools for identifying mononuclear phagocyte subsets in swine: Reagents to porcine CD11c and XCR1. Dev Comp Immunol 65:31–40. https://doi.org/10.1016/j.dci.2016.06.015 Roca CP, Burton OT, Neumann J et al (2023) A cross entropy test allows quantitative statistical comparison of t-SNE and UMAP representations. Cell Rep Methods 3. https://doi.org/10.1016/j.crmeth.2022.100390 Meurens F, Zhu J, Renois F (2024) Viral co-infections targeting the porcine respiratory system: Consequences and limits of the experimental systems. Virologie 28:9–21. https://doi.org/10.1684/vir.2024.1032 Williamson SM, Tucker AW, McCrone IS et al (2012) Descriptive clinical and epidemiological characteristics of influenza A H1N1 2009 virus infections in pigs in England. Vet Rec 171:271–271. https://doi.org/10.1136/vr.100673 Lin X, Huang C, Shi J et al (2015) Investigation of Pathogenesis of H1N1 Influenza Virus and Swine Streptococcus suis Serotype 2 Co-Infection in Pigs by Microarray Analysis. PLoS ONE 10:e0124086. https://doi.org/10.1371/journal.pone.0124086 McCullers JA, Rehg JE (2002) Lethal Synergism between Influenza Virus and Streptococcus pneumoniae: Characterization of a Mouse Model and the Role of Platelet-Activating Factor Receptor. J Infect Dis 186:341–350. https://doi.org/10.1086/341462 Butler JE, Lager KM, Golde W et al (2014) Porcine reproductive and respiratory syndrome (PRRS): an immune dysregulatory pandemic. Immunol Res 59:81–108. https://doi.org/10.1007/s12026-014-8549-5 Albina E, Carrat C, Charley B (1998) Short Communication: Interferon-α Response to Swine Arterivirus (PoAV), the Porcine Reproductive and Respiratory Syndrome Virus. J Interferon Cytokine Res 18:485–490. https://doi.org/10.1089/jir.1998.18.485 Renson P, Deblanc C, Bougon J et al (2021) Concomitant Swine Influenza A Virus Infection Alters PRRSV1 MLV Viremia in Piglets but Does Not Interfere with Vaccine Protection in Experimental Conditions. Vaccines 9:356. https://doi.org/10.3390/vaccines9040356 Khatri M, Dwivedi V, Krakowka S et al (2010) Swine Influenza H1N1 Virus Induces Acute Inflammatory Immune Responses in Pig Lungs: a Potential Animal Model for Human H1N1 Influenza Virus. J Virol 84:11210–11218. https://doi.org/10.1128/JVI.01211-10 Crisci E, Fraile L, Montoya M (2019) Cellular Innate Immunity against PRRSV and Swine Influenza Viruses. Vet Sci 6:26. https://doi.org/10.3390/vetsci6010026 Crozat K, Guiton R, Contreras V et al (2010) The XC chemokine receptor 1 is a conserved selective marker of mammalian cells homologous to mouse CD8α + dendritic cells. J Exp Med 207:1283–1292. https://doi.org/10.1084/jem.20100223 Yamazaki C, Miyamoto R, Hoshino K et al (2010) Conservation of a chemokine system, XCR1 and its ligand, XCL1, between human and mice. Biochem Biophys Res Commun 397:756–761. https://doi.org/10.1016/j.bbrc.2010.06.029 Ghilas S, Ambrosini M, Cancel J-C et al (2021) Natural killer cells and dendritic epidermal γδ T cells orchestrate type 1 conventional DC spatiotemporal repositioning toward CD8 + T cells. iScience 24:103059. https://doi.org/10.1016/j.isci.2021.103059 Annunziato F, Romagnani C, Romagnani S (2015) The 3 major types of innate and adaptive cell-mediated effector immunity. J Allergy Clin Immunol 135:626–635. https://doi.org/10.1016/j.jaci.2014.11.001 Xiao Z, Batista L, Dee S et al (2004) The Level of Virus-Specific T-Cell and Macrophage Recruitment in Porcine Reproductive and Respiratory Syndrome Virus Infection in Pigs Is Independent of Virus Load. J Virol 78:5923–5933. https://doi.org/10.1128/jvi.78.11.5923-5933.2004 Talker SC, Stadler M, Koinig HC et al (2016) Influenza A Virus Infection in Pigs Attracts Multifunctional and Cross-Reactive T Cells to the Lung. J Virol 90:9364–9382. https://doi.org/10.1128/jvi.01211-16 Deblanc C, Quéguiner S, Gorin S et al (2020) Evaluation of the Pathogenicity and the Escape from Vaccine Protection of a New Antigenic Variant Derived from the European Human-Like Reassortant Swine H1N2 Influenza Virus. Viruses 12:1155. https://doi.org/10.3390/v12101155 Tables Table 1 is available in the Supplementary Files section. Supplementary Files Table1Grevelinger2025.pdf Table 1: Primary antibodies used in flow cytometry analyses 1 Conjugation with conjugation kit (Abcam) SupplementaryDataGrevelinger2025.pdf Cite Share Download PDF Status: Published Journal Publication published 08 Jun, 2025 Read the published version in Veterinary Research → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5928429","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":410511366,"identity":"9986c5be-0be9-4366-bcc2-d011a5bc46aa","order_by":0,"name":"Janaïna Grevelinger","email":"","orcid":"","institution":"Oniris, INRAE, BIOEPAR, 44300 Nantes, France","correspondingAuthor":false,"prefix":"","firstName":"Janaïna","middleName":"","lastName":"Grevelinger","suffix":""},{"id":410511367,"identity":"4dc51103-3b10-4f93-83ca-67dca04b60e1","order_by":1,"name":"Olivier Bourry","email":"","orcid":"","institution":"ANSES, Ploufragan-Plouzané-Niort Laboratory, French Agnecy for Food, Environmental and Occupational Health and Safety (ANSES), Ploufragan, 22440, France","correspondingAuthor":false,"prefix":"","firstName":"Olivier","middleName":"","lastName":"Bourry","suffix":""},{"id":410511368,"identity":"399ea12b-19e3-45fc-a2cb-e6c8740c58b4","order_by":2,"name":"Selma Schmidt","email":"","orcid":"","institution":"Pirbright Institute","correspondingAuthor":false,"prefix":"","firstName":"Selma","middleName":"","lastName":"Schmidt","suffix":""},{"id":410511369,"identity":"ac5c796a-a3c6-4c57-9a5f-7abefccf8b8b","order_by":3,"name":"François Meurens","email":"","orcid":"","institution":"Université de Montréal Faculté de médecine vétérinaire: Universite de Montreal Faculte de medecine veterinaire","correspondingAuthor":false,"prefix":"","firstName":"François","middleName":"","lastName":"Meurens","suffix":""},{"id":410511370,"identity":"3f59f219-578e-4ca6-af5f-9c0d81cc3211","order_by":4,"name":"Céline Deblanc","email":"","orcid":"","institution":"ANSES, Ploufragan-Plouzané-Niort Laboratory, Swine Virology Immunology Unit, Ploufragan, France","correspondingAuthor":false,"prefix":"","firstName":"Céline","middleName":"","lastName":"Deblanc","suffix":""},{"id":410511371,"identity":"fc6f06d5-850a-4a7d-9bb0-54a84c94709a","order_by":5,"name":"Caroline Hervet","email":"","orcid":"","institution":"Oniris, INRAE, BIOEPAR, Nantes, France","correspondingAuthor":false,"prefix":"","firstName":"Caroline","middleName":"","lastName":"Hervet","suffix":""},{"id":410511372,"identity":"5c718a05-e57f-4f39-83d8-10a16f574d3a","order_by":6,"name":"Aline Perrin","email":"","orcid":"","institution":"Oniris, INRAE, BIOEPAR, Nantes, France","correspondingAuthor":false,"prefix":"","firstName":"Aline","middleName":"","lastName":"Perrin","suffix":""},{"id":410511373,"identity":"13a5e7ed-c4df-476c-95ab-4e7a31913e41","order_by":7,"name":"Stéphane Gorin","email":"","orcid":"","institution":"ANSES, Ploufragan-Plouzané-Niort Laboratory, Swine Virology Immunology Unit, Ploufragan, France","correspondingAuthor":false,"prefix":"","firstName":"Stéphane","middleName":"","lastName":"Gorin","suffix":""},{"id":410511374,"identity":"c5bf1fa7-09fa-4c2d-8d6e-920fb99de00e","order_by":8,"name":"Mireille Le Dimna","email":"","orcid":"","institution":"ANSES, Ploufragan-Plouzané-Niort Laboratory, Swine Virology Immunology Unit, Ploufragan, France","correspondingAuthor":false,"prefix":"","firstName":"Mireille","middleName":"Le","lastName":"Dimna","suffix":""},{"id":410511375,"identity":"18ff8c0e-5846-4e0b-9ad2-759953dc2f99","order_by":9,"name":"Stéphane Quéguiner","email":"","orcid":"","institution":"ANSES, Ploufragan-Plouzané-Niort Laboratory, Swine Virology Immunology Unit, Ploufragan, France","correspondingAuthor":false,"prefix":"","firstName":"Stéphane","middleName":"","lastName":"Quéguiner","suffix":""},{"id":410511376,"identity":"e1875e4d-dfac-40ca-9263-01ace3446031","order_by":10,"name":"Thibaut Larcher","email":"","orcid":"","institution":"Oniris, INRAE, Apex, Nantes, France","correspondingAuthor":false,"prefix":"","firstName":"Thibaut","middleName":"","lastName":"Larcher","suffix":""},{"id":410511377,"identity":"b70fe531-6374-4e8f-9a54-acca5b1b3159","order_by":11,"name":"Patricia Renson","email":"","orcid":"","institution":"ANSES, Ploufragan-Plouzané-Niort Laboratory, Swine Virology Immunology Unit, Ploufragan, France","correspondingAuthor":false,"prefix":"","firstName":"Patricia","middleName":"","lastName":"Renson","suffix":""},{"id":410511378,"identity":"e0cf9de6-06af-4e0b-9dc2-827a90383447","order_by":12,"name":"Frédéric Paboeuf","email":"","orcid":"","institution":"ANSES, Ploufragan-Plouzané-Niort Laboratory, Swine Virology Immunology Unit, Ploufragan, France","correspondingAuthor":false,"prefix":"","firstName":"Frédéric","middleName":"","lastName":"Paboeuf","suffix":""},{"id":410511379,"identity":"55ada66a-1d61-4062-8b74-70426b0f7f43","order_by":13,"name":"Wilhelm Gerner","email":"","orcid":"","institution":"Pirbright Institute","correspondingAuthor":false,"prefix":"","firstName":"Wilhelm","middleName":"","lastName":"Gerner","suffix":""},{"id":410511380,"identity":"d4d186fc-020f-4f33-985e-201a95fab403","order_by":14,"name":"Nicolas Bertho","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYDACZiB+AMQGELYND0TYhoCWBIgWxmYGhjSoljQCNiFpOcxAUIs5O/PDBwk1dUBG7/PHBRXnZcylDz98wJBwD6cWy2Y2Y4OEY4cZLHuOGzbPOHObx7IvzdiAIaEYpxaDwzxsEglsBxgMbqQxNvO23eYxOMNgJsH4I4GAln91DAb3n4G0nANqYf/+gyGBgJbENmagLWwgLQeAWnjMGPBrAfolse8wj2VPGuNsnjPJIC3FEgn4tJwHhs+Hb3Vy5uzHGD7zVNjZAx228cMHPFpggAeVS1jDKBgFo2AUjAJ8AAB9qEwM1IDJ0AAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-6732-3483","institution":"INRAE UMR1300: BIOEPAR","correspondingAuthor":true,"prefix":"","firstName":"Nicolas","middleName":"","lastName":"Bertho","suffix":""},{"id":410511381,"identity":"bfa88635-2fc6-437c-8143-94afd01c9d2e","order_by":15,"name":"Gaëlle Simon","email":"","orcid":"","institution":"ANSES, Ploufragan-Plouzané-Niort Laboratory, Swine Virology Immunology Unit, Ploufragan, France","correspondingAuthor":false,"prefix":"","firstName":"Gaëlle","middleName":"","lastName":"Simon","suffix":""}],"badges":[],"createdAt":"2025-01-30 07:47:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5928429/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5928429/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13567-025-01536-6","type":"published","date":"2025-06-08T15:57:03+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":75499412,"identity":"28ce841b-1389-4c79-8636-7be437eaa031","added_by":"auto","created_at":"2025-02-05 08:48:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":251560,"visible":true,"origin":"","legend":"\u003cp\u003eDesign of animal experiment\u003c/p\u003e","description":"","filename":"Figure1Grevelinger20251.png","url":"https://assets-eu.researchsquare.com/files/rs-5928429/v1/7edf343fe0aba56602dca6cb.png"},{"id":75497636,"identity":"0a21553e-4631-491c-9547-7140ce0afd1b","added_by":"auto","created_at":"2025-02-05 08:32:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":49993,"visible":true,"origin":"","legend":"\u003cp\u003eClinical signs and lung lesions\u003c/p\u003e\n\u003cp\u003e(A) Rectal temperature. On D0, the red arrow indicates swIAV inoculation, and on D7, the blue arrow indicates PRRSV inoculation. Statistical analysis was performed using the Kruskal-Wallis unpaired, non-parametric test. Different letters (a-d) indicate that the considered group (specified by its color) was significantly different from the Mock group (a), from the swIAV group (b), from the PRRSV group (c) or from the swIAV/PRRSV group (d) with p \u0026lt; 0.05 (mean ± SD; n = 5-10).\u003c/p\u003e\n\u003cp\u003e(B) Average daily weight gain (mean; n = 5-10). Statistical analysis was performed using the Kruskal-Wallis unpaired, non-parametric test, (*) p \u0026lt; 0.05.\u003c/p\u003e\n\u003cp\u003e(C, D) Lung sections were evaluated for histopathological lesions. (C) Jung score and (D) Composite score (mean; n = 5). Statistical analysis was performed using the Mann-Whitney unpaired, non-parametric test, (*) p \u0026lt; 0.05 or (**) p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure1Grevelinger20252.png","url":"https://assets-eu.researchsquare.com/files/rs-5928429/v1/20b3bd37bcf1e13eed601f27.png"},{"id":75497639,"identity":"256645f9-b434-49d9-b1ac-1f67bd4b7ba9","added_by":"auto","created_at":"2025-02-05 08:32:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":36627,"visible":true,"origin":"","legend":"\u003cp\u003ePRRSV load in BALF, lung tissue, tracheobronchial lymph node and serum\u003c/p\u003e\n\u003cp\u003e(A-D) Quantification of PRRSV loads by RT-qPCR in (A) BALF, (B) lung tissue, (C) tracheobronchial lymph nodes, and (D) serum. Statistical analysis was performed using the Mann-Whitney unpaired, non-parametric test (mean; n = 5-10).\u003c/p\u003e","description":"","filename":"Figure1Grevelinger20253.png","url":"https://assets-eu.researchsquare.com/files/rs-5928429/v1/97bbc70d588e5ea698873cfc.png"},{"id":75497637,"identity":"eaddaa1d-fdb4-46b4-867f-22349f01e1a7","added_by":"auto","created_at":"2025-02-05 08:32:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":38224,"visible":true,"origin":"","legend":"\u003cp\u003eCytokine profile in BALF\u003c/p\u003e\n\u003cp\u003e(A-F) Cytokine levels were quantified in BALF using multiplex immunoassays. (A) IL-1α, (B) IL-1β, (C) IL-8, (D) IL-18, (E) IL-6 and (F) IL-12. Statistical analysis was performed using the Mann-Whitney unpaired, non-parametric test, (*) p \u0026lt; 0.05 or (**) p \u0026lt; 0.01 (mean; n = 5).\u003c/p\u003e","description":"","filename":"Figure1Grevelinger20254.png","url":"https://assets-eu.researchsquare.com/files/rs-5928429/v1/70fb921f6778ab66463c728a.png"},{"id":75499115,"identity":"94597bdd-930b-4137-879b-f62e1b59e0f6","added_by":"auto","created_at":"2025-02-05 08:40:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":34773,"visible":true,"origin":"","legend":"\u003cp\u003eAbsolute numbers of myeloid cell populations in the BAL\u003c/p\u003e\n\u003cp\u003e(A-D) The absolute numbers of myeloid cells were determined in the BAL using flow cytometry. (A) Alveolar macrophages (AM), (B) monocyte-derived dendritic cells (moDC), (C) conventional type 1 dendritic cells (cDC1), and (D) conventional type 2 dendritic cells (cDC2). Statistical analysis was performed using the Mann-Whitney unpaired, non-parametric test, (*) p \u0026lt; 0.05 or (**) p \u0026lt; 0.01 (mean; n = 5).\u003c/p\u003e","description":"","filename":"Figure1Grevelinger20255.png","url":"https://assets-eu.researchsquare.com/files/rs-5928429/v1/eab719f7cfcebd337688ee89.png"},{"id":75499413,"identity":"d24cf32b-09e0-4a28-9684-1a24f6dd18ed","added_by":"auto","created_at":"2025-02-05 08:48:00","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":36655,"visible":true,"origin":"","legend":"\u003cp\u003eProportions of regulatory and conventional CD4 T cells in BAL\u003c/p\u003e\n\u003cp\u003e(A-D) The percentages of Tconv and Treg CD4 cells in the BAL were determined using flow cytometry. (A, B) presents the percentages within total CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e cells. (A) shows CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e-/low\u003c/sup\u003eFoxp3\u003csup\u003e-\u003c/sup\u003e Tconv cells. (B) shows CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003ehigh\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003e Treg cells. (C, D) shows the percentages of ICOS\u003csup\u003e+\u003c/sup\u003eKi-67\u003csup\u003e+ \u003c/sup\u003ewithin the respective parental population of (C) Tconv and (D) Treg.\u003c/p\u003e\n\u003cp\u003eStatistical analysis was performed using the Mann-Whitney unpaired, non-parametric test, (*) p \u0026lt; 0.05 or (**) p \u0026lt; 0.01 (mean; n = 5).\u003c/p\u003e","description":"","filename":"Figure1Grevelinger20256.png","url":"https://assets-eu.researchsquare.com/files/rs-5928429/v1/b951295d8913b8a0c86c0d9e.png"},{"id":75499114,"identity":"4c328ff5-3432-4e60-a24d-072ff3aad855","added_by":"auto","created_at":"2025-02-05 08:40:00","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":41306,"visible":true,"origin":"","legend":"\u003cp\u003eProportions of lymphoid cell populations in the BAL\u003c/p\u003e\n\u003cp\u003e(A-E) Lymphocyte populations were clustered using the t-SNE algorithm to analyse flow cytometry data, with clusters 6 to 10 being presented. The t-SNE algorithm is based on live lymphocytes. Statistical analysis was performed using the Mann-Whitney unpaired, non-parametric test, (*) p \u0026lt; 0.05 or (**) p \u0026lt; 0.01 (mean; n = 5).\u003c/p\u003e","description":"","filename":"Figure1Grevelinger20257.png","url":"https://assets-eu.researchsquare.com/files/rs-5928429/v1/0bbd89b3ee1ebe9ec743d1c6.png"},{"id":75497643,"identity":"61ebdc1f-c6c0-4b03-9ef6-54000a0c5425","added_by":"auto","created_at":"2025-02-05 08:32:00","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":25159,"visible":true,"origin":"","legend":"\u003cp\u003ePRRSV-specific CD4 T cell responses in PBMC\u003c/p\u003e\n\u003cp\u003e(A-F) The percentages of IFN-γ and IL-2 producing cells within total CD4 T cells were analyzed in PBMC isolated at D14. Cells were restimulated \u003cem\u003ein vitro \u003c/em\u003efor 18 h with PRRSV (MOI 1) or cultured with medium. (A, B) IFN-γ. (C, D) IL-2. The percentages were calculated by subtracting the percentage of cytokine-producing cells cultured with medium only). Values are obtained by subtracting the percentage obtained in the live PRRSV restimulation condition from the percentage obtained in the unstimulated condition. Statistical analysis was performed using the Mann-Whitney unpaired, non-parametric test, (*) p \u0026lt; 0.05 or (**) p \u0026lt; 0.01 (mean; n = 5).\u003c/p\u003e","description":"","filename":"Figure1Grevelinger20258.png","url":"https://assets-eu.researchsquare.com/files/rs-5928429/v1/de98adaf735979fe4ebf7e5b.png"},{"id":84242484,"identity":"e8b5d63c-9f6d-45bf-8f48-231e3580c763","added_by":"auto","created_at":"2025-06-09 16:08:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1412284,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5928429/v1/0c72b9cb-a820-4d7a-9c0d-40531aafe3a4.pdf"},{"id":75499118,"identity":"d1217c93-accd-41fd-a864-de105acd1484","added_by":"auto","created_at":"2025-02-05 08:40:00","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":425934,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 1:\u003c/strong\u003e Primary antibodies used in flow cytometry analyses\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eConjugation with conjugation kit (Abcam)\u003c/p\u003e","description":"","filename":"Table1Grevelinger2025.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5928429/v1/75b5a126ae5700d6f6ee7ed7.pdf"},{"id":75497644,"identity":"29a18a3e-8ddb-4ea8-80cb-b7e67cc59ad7","added_by":"auto","created_at":"2025-02-05 08:32:00","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2258342,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryDataGrevelinger2025.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5928429/v1/c67462b4602d4888d015d75c.pdf"}],"financialInterests":"","formattedTitle":"Swine Influenza A virus infection sets the local immunological landscape in subsequent infection with Porcine Reproductive and Respiratory Syndrome virus","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOn farms, pigs are often exposed to multiple respiratory infections, which are frequently associated with complex respiratory diseases of viral and/or bacterial origin. This phenomenon is referred to as the porcine respiratory disease complex (PRDC) [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Among the pathogens involved in PRDC, swine influenza A viruses (swIAV) and the porcine reproductive and respiratory syndrome virus (PRRSV) play a key role.\u003c/p\u003e \u003cp\u003eSwIAV belongs to the \u003cem\u003eAlphainfluenzavirus\u003c/em\u003e genus in the \u003cem\u003eOrthomyxoviridae\u003c/em\u003e family. These enveloped viruses have a segmented negative-sense single-stranded RNA genome. The main swIAV subtypes circulating in pig populations worldwide for decades include H1N1, H1N2, and H3N2 [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Swine influenza is a common respiratory disease in pig farms, causing clinical signs such as fever, breathing difficulties, coughing, and nasal discharge.\u003c/p\u003e \u003cp\u003ePRRSV, a positive-sense single-stranded RNA virus from the \u003cem\u003eArteriviridae\u003c/em\u003e family, is sub-divided into two species: PRRSV-1, mainly found in Europe and PRRSV-2, predominantly present in North America and Asia [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. This virus causes respiratory signs, reproductive disorders, and growth retardation and increased mortality in piglets. Its virulence varies depending on the strain and host characteristics [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEpidemiological studies conducted in France have shown a significant association between PRRSV and swIAV infections [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Thus, more than half of the 125 farms studied in the Western part of the country displayed a positive correlation between antibodies against PRRSV-1 and the H1N2 subtype of swIAV [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This would indicate that in regions where these viruses are widespread, the risk of co-infection is particularly high.\u003c/p\u003e \u003cp\u003eGiven the high risk of simultaneous infections in regions where both PRRSV and swIAV are prevalent, it is important to explore how these co-infections may affect animal health. Research has aimed to explore the interactions between swIAV and PRRSV through simultaneous or sequential infections, with PRRSV inoculated first, followed by swIAV a few days later. This was investigated in several studies, with outcomes varying depending on experimental conditions and virus strains. Some studies have reported aggravation of lung lesions [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], while others found no differences compared to single virus infections [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Recent research also highlighted the complexity of immune responses triggered by co-infections. Bougon et al. (2021) observed that a PRRSV-1 infection eight days before exposure to swIAV H1N2 reduced influenza-like illness while increasing anti-swIAV antibodies and decreasing PRRSV-1 replication in lungs. Interferon-α (IFN-α) appeared to play a key role in the bidirectional interference between the two viral infections. Chrun et al. (2023) showed that a co-infection with swIAV H3N2 and PRRSV-2 did not worsen clinical signs, reduced swIAV H3N2 viral load in the lungs, and even enhanced certain immune parameters, including PRRSV-2-specific CD8 T cell count and swIAV H3N2-specific IgG levels.\u003c/p\u003e \u003cp\u003eMost studies have focused on the inoculation of PRRSV followed by swIAV. Since influenza is an acute infection lasting no more than a week, whereas PRRS is an infection that can persist for months, it is more likely that animals infected with PRRSV will subsequently be co-infected with swIAV [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, the alternative scenario, where animals are first infected with swIAV and secondly by PRRSV infection is plausible on pig farms, although it has never been explored and deserves further investigation.\u003c/p\u003e \u003cp\u003eIn most cases, infection with influenza A virus (IAV) is reported to weaken the host, facilitating infections by opportunistic pathogens such as \u003cem\u003eStreptococcus suis\u003c/em\u003e for instance in pigs [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] or \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e in humans [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], ultimately worsening the overall illness. One proposed mechanism following investigations in mice was that influenza infection led to depletion of alveolar macrophages (AM), making the host more prone to secondary infections [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, this theory is debated, as other studies in mice have shown AM persistence during influenza infection [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Furthermore, respiratory viral infections may also have protective effects. Indeed, research in mice revealed that some respiratory viruses, including IAV, can induce a beneficial immune imprint in the lungs, offering better protection against subsequent bacterial infections, such as those caused by \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e or \u003cem\u003eEscherichia coli\u003c/em\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this context, the present study aimed to evaluate the impact of swIAV infection on the AM population and the early innate and adaptive immune responses during a PRRSV secondary infection. We examined these interactions between a swIAV H1N2 and PRRSV-1, utilizing two strains representative of viruses that were circulating on pig farms at the same period of time in Brittany, France.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eVirus strains\u003c/h2\u003e \u003cp\u003eThe swIAV strain A/swine/Ille et Vilaine/0415/2011 (H1\u003csub\u003ehu\u003c/sub\u003eN2, HA-clade 1B.1.2.3) and the so-called \u0026ldquo;Finist\u0026egrave;re\u0026rdquo; PRRSV strain (referenced as PRRS-FR-2005-29-24-1; PRRSV-1 subtype 1), both isolated from pig farms in Brittany, France, are part of the collections of the French Agency for Food, Environmental, and Occupational Health \u0026amp; Safety (ANSES, Ploufragan, France).\u003c/p\u003e \u003cp\u003eSwine IAV was propagated and titrated using Madin-Darby canine kidney (MDCK) cells (ATCC reference CCL-34), whereas primary porcine alveolar macrophages (AM) were used for PRRSV, as previously described [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnimal experiment\u003c/h3\u003e\n\u003cp\u003eThirty specific pathogen-free (SPF) Large White piglets (11.5 weeks old) from ANSES-Ploufragan\u0026rsquo;s protected animal facilities [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], were randomly assigned to six groups based on parental origin, weight, and sex (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe experiment was conducted at ANSES facilities, which are accredited for animal research by the Direction D\u0026eacute;partementale de la Protection des Populations des C\u0026ocirc;tes d\u0026rsquo;Armor. The animal study received approval from the National Committee for Ethics in Animal Experimentation ANSES/ENVA/UPEC (approval no. 23\u0026thinsp;\u0026minus;\u0026thinsp;017 #40450) and authorization was granted by the French Ministry for Research (authorization no. APAFIS #40450-2023012311482365 v3).\u003c/p\u003e \u003cp\u003eOn D0, three groups (\"swIAV,\" \"swIAV/PRRSV-D10,\" and \"swIAV/PRRSV-D14\") were intratracheally (IT) inoculated with 10\u003csup\u003e6\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e (50% tissue culture infectious dose) of swIAV in 5 mL per pig, as previously described (Bougon et al., 2021). The other three groups (\"Mock,\" \"PRRSV-D10,\" and \"PRRSV-D14\") received Minimum Essential Medium (MEM). On D7, the groups \"swIAV/PRRSV-D10,\" \"swIAV/PRRSV-D14,\" \"PRRSV-D10,\" and \"PRRSV-D14\" were intranasally (IN) inoculated with 5 x 10\u003csup\u003e5\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e of PRRSV in 5 mL per pig.\u003c/p\u003e \u003cp\u003eRectal temperature, and clinical signs such as cough and sneezes were monitored daily. Hyperthermia was defined by a rectal temperature above 40\u0026deg;C. Coughing and sneezing were counted for 15 min in each room. Individual weights were measured weekly before swIAV inoculation, then twice a week after swIAV inoculation, and food consumption at the room level was recorded daily.\u003c/p\u003e \u003cp\u003eNasal swabs were collected in Virocult (MW915, Sigma Virocult\u0026reg;, MWE medical wire) and the supernatants were frozen at -80\u0026deg;C until RT-qPCR analysis. Animals were euthanized and necropsied according to the following schedule: on D7 for the \"swIAV\" and \"Mock\" groups, on D10 for the \"PRRSV-D10\" and \"swIAV/PRRSV-D10\" groups, and on D14 for the \"PRRSV-D14\" and \"swIAV/PRRSV-D14\" groups. After necropsies, bronchoalveolar lavages (BALs) were performed using sterile phosphate-buffered saline (PBS). A first BAL was performed with 40 mL of PBS, and the collected liquid was centrifuged at 500 \u0026times; g for 10 minutes at 4\u0026deg;C to purify BAL fluid (BALF) from cells (BALC), and frozen at -20\u0026deg;C for antibody and cytokine assays, and at -80\u0026deg;C for RT-qPCR analyses. Two additional BALs with 100 mL of PBS each were carried out to collect and isolate more BALCs, which were stored in liquid nitrogen in fetal calf serum (FCS) with 10% dimethyl sulfoxide (DMSO) (Sigma-Aldrich). Lung tissue was taken from the right apical lobes, which were clamped during the BALs, and tracheobronchial lymph nodes (TBLN) were collected. These samples were frozen at -80\u0026deg;C for RT-qPCR analyses or fixed in 4% formaldehyde (Merck) for histopathological analyses.\u003c/p\u003e \u003cp\u003eSerum samples were collected from clotted blood (centrifuged at 3 000 \u0026times; g for 5 min) and frozen at -20\u0026deg;C for antibody and cytokine assays, and at -80\u0026deg;C for RT-qPCR analyses. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood using Ficoll density gradient centrifugation with LeucoSep tubes (Greiner Bio One) and stored in liquid nitrogen in FCS with 10% DMSO.\u003c/p\u003e\n\u003ch3\u003eHistopathological examination of lungs\u003c/h3\u003e\n\u003cp\u003eThe formalin-fixed tissues were paraffin-embedded and sectioned into 4 \u0026micro;m slices. The sections were then stained with hematoxylin-eosin-saffron. Lesion intensity was evaluated based on parameters such as alveolar septal thickening, material accumulation in respiratory airways, inflammatory cell cuffing around bronchioles or vessels, proliferative bronchiolitis, alveolar emphysema, and bronchus-associated lymphoid tissue (BALT) hyperplasia. Lesions were scored using two different scoring systems: one with three parameters from Jung et al. (2007) and another composite system with three additional parameters from Larcher et al. (2019). For each system, scores were expressed as a percentage of the theoretical maximum.\u003c/p\u003e\n\u003ch3\u003eViral quantifications\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from nasal swabs, serum, BALF, lung tissues, and lymph nodes using the ID Gene\u0026trade; Mag Fast 384 Extraction Kit (Innovative Diagnostics) on the Auto-Pure 96 nucleic acid purification system (Allsheng).\u003c/p\u003e \u003cp\u003eFor swIAV detection, duplex RT-qPCR targeting the M gene and β-actin was conducted using the Go Script RT mix for 1-Step RT-qPCR (Promega), following the method described by Cador et al. (2016). Quantification of the swIAV genome was achieved through serial dilutions of standardized M and β-actin mRNA, with results expressed as copy numbers of the M gene per 10\u003csup\u003e6\u003c/sup\u003e copies of the β-actin gene.\u003c/p\u003e \u003cp\u003ePRRSV quantification was performed using duplex RT-qPCR with the SuperScript III Platinum one-step RT-qPCR kit (Life Technologies). Specific primers and probes targeting ORF5 of the \u0026ldquo;Finist\u0026egrave;re\u0026rdquo; strain and the porcine β-actin were used, as previously described [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Virus genome quantification was based on serial dilutions of standardized \u003cem\u003ein vitro\u003c/em\u003e transcribed mRNA of the \u0026ldquo;Finist\u0026egrave;re\u0026rdquo; strain ORF5 RT-PCR amplicon (Eurofins genomics), with PRRSV genome levels reported as copy numbers per mL or per mg of sample.\u003c/p\u003e\n\u003ch3\u003eCytokine quantifications\u003c/h3\u003e\n\u003cp\u003eCytokines in BALF, including GM-CSF, IFN-γ, IL-10, TNF-α, IL-1α, IL-1β, IL-6, IL-8, IL-18, and IL-12, were measured using a porcine-specific cytokine array (Milliplex PCYTMAG-23K, Merck Millipore) following the manufacturer\u0026rsquo;s instructions on the MAGPIX System (DiaSorin).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry analysis\u003c/h2\u003e \u003cp\u003eFor the phenotyping of Tregs in BAL, cells were thawed and seeded in round-bottom 96-well microtiter plates (Nunc MicroWell Plates, Thermo Fisher Scientific) with a staining buffer containing PBS and 10% heat-inactivated porcine plasma. Surface staining was performed using primary monoclonal antibodies (mAbs) against CD3, CD4, CD8α, CD8β, CD25, and ICOS (for details on antibodies, refer to Table\u0026nbsp;1). Secondary staining involved goat anti-mouse-IgG2b-A488 (Jackson Immuno Research) and Streptavidin-BV650 (BioLegend) for labeling of CD4 and CD8α, respectively. Dead cells were marked with Fixable Viability Dye eFluor780 (Thermo Fisher Scientific) following the manufacturer's guidelines. Cells were then fixed and permeabilized using the eBioscience\u0026trade; Foxp3/ Transcription Factor Staining Buffer Set (Thermo Fisher Scientific). Intracellular staining was performed with mAbs against Foxp3 and Ki-67. Surface staining was carried out at 4\u0026deg;C for 20 minutes and intracellular staining at 4\u0026deg;C for 30 minutes. After intracellular staining, cells were washed twice and resuspended in Perm/Wash Buffer for analysis.\u003c/p\u003e \u003cp\u003eA second antibody panel was applied for characterisation of unconventional T cells and NK cells. Here, antibodies against the surface molecules TCR-γδ, CD8α, and CD161 were used. Secondary staining included rat anti-mouse-IgG2b-BUV395 (BD Biosciences), streptavidin-BV650 (BioLegend), and goat anti-mouse-IgG1-BV421 (Jackson Immuno Research) to label TCR-γδ, CD8α, and CD161, respectively. Additional surface staining was performed with mAbs against CD3, CD8β, CD16, NKp46, CD4, and CD2. The eBioscience\u0026trade; Foxp3/ Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) was used for fixation and permeabilization as described previously. Intracellular staining was performed with mAbs against T-bet, perforin, and PLZF. Samples from both panels were analyzed on a Cytek Aurora spectral cytometer (Cytek Biosciences) with 5 lasers (UV 355 nm, violet 405 nm, blue 488 nm, yellow-green 561 nm, red 640 nm) and 64 fluorescence detection channels. Spectral unmixing was performed using SpectroFlo software (version 3.2.1, Cytek Biosciences) with single-stained reference samples. Autofluorescence signatures were subtracted using unstained controls. Data from 2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e to 3 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e live lymphocytes per sample were recorded and analyzed using FlowJo software for Windows (version 10.9.0, BD Biosciences).\u003c/p\u003e \u003cp\u003eFor mononuclear phagocyte cell staining in BAL, cells were thawed and plated in round-bottom 96-well microtiter plates with a staining buffer containing PBS, supplemented with 2 mM ethylenediaminetetraacetic acid (EDTA), 5% swine serum, and 5% goat serum. Surface staining was performed using primary mAbs against MHCII, CD172a, and CD11c. For secondary staining, rat anti-mouse-IgG2a-PE-Cy7 (Thermo Fisher Scientific), goat anti-mouse-IgG2b-APC-Cy7 (Abcam), and goat anti-mouse-IgG1-A647 (Thermo Fisher Scientific) were used to label MHCII, CD172a, and CD11c, respectively. Dead cells were identified using the LIVE/DEAD\u0026trade; Fixable Aqua Dead Cell Stain Kit for 405 nm excitation (Thermo Fisher Scientific) after surface staining. Additional surface staining was performed with mAbs against CD1 and CD163. Staining steps were carried out at 4\u0026deg;C for 30 minutes. Labelled cells were fixed with 1% paraformaldehyde (PFA) for 20 min at room temperature, followed by two washes with PBS. Samples were then acquired in PBS using a MACSQuant 10 cytometer (Miltenyi Biotec), equipped with 3 lasers (violet 405 nm, blue 488 nm, red 635 nm) and eight fluorescence detection channels. Data from 6 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e to 4 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e live cells per sample were recorded and analyzed using FlowJo software. Alveolar macrophages (AM), monocyte-derived dendritic cells (moDC), and conventional dendritic cells of types 1 and 2 (cDC1 and cDC2) were identified based on staining for MHC II, CD11c, CD163, CD172a, and CD1, as previously described [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. An anti-CD11c antibody was used to identify myeloid cells [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIntracellular cytokine staining (ICS)\u003c/h3\u003e\n\u003cp\u003eFor ICS, PBMCs were thawed and plated in round-bottom 96-well microtiter plates with 5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well, in a final volume of 200 \u0026micro;L per well. Cells were cultivated in Roswell Park Memorial Institute (RPMI) 1640 (Sigma-Aldrich) supplemented with 1% penicillin (100 IU/mL) and streptomycin (100 \u0026micro;g/mL) (PS) (Gibco, Thermo Fisher Scientific) and 10% fetal calf serum (FCS) (Life Science Production). They were exposed to either PRRSV-1 (\"Finist\u0026egrave;re\" strain) at a multiplicity of infection (MOI) of 1 for 18 hours, phorbol 12-myristate 13-acetate (PMA) (50 ng/mL) and ionomycin (500 ng/mL) as a positive control for the final four hours, or cell culture medium alone. Brefeldin A (BD GolgiPlug\u0026trade;, BD Biosciences) was added to all conditions for the final four hours at a concentration of 1 \u0026micro;g/mL. After incubation, cells were harvested, centrifuged, and resuspended in staining buffer containing PBS with 3% FCS. Cells were then surface-stained with mAbs against CD4, CD8α, and CD8β. Subsequently, goat-anti-mouse-IgG2b-A488 (Jackson Immuno Research) and Streptavidin-BV421 (BioLegend) were used to label CD4 and CD8α, respectively. Dead cells were identified using Fixable Viability Dye eFluor780 (Thermo Fisher Scientific) after surface staining. Cells were then fixed and permeabilized using the BD Cytofix/Cytoperm\u0026trade; Fixation/Permeabilization Kit (BD Biosciences). Intracellular staining was performed with mAbs against IFN-γ, IL-2 and TNF-α. Incubation steps were performed in the same way as described above. After intracellular staining, cells were washed twice and resuspended in Perm/Wash Buffer (BD Biosciences). Samples were acquired on the Cytek Aurora cytometer. Data from a minimum of 4 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e live lymphocytes per sample were recorded and analyzed using FlowJo software. Data for TNF-α labelling was not analyzed due to a technical issue.\u003c/p\u003e\n\u003ch3\u003eAntibody assessment in sera and BALF\u003c/h3\u003e\n\u003cp\u003eAnti-swIAV (NP protein) IgG was detected using the ID Screen Influenza A Nucleoprotein Swine Indirect kit (Innovative Diagnostics) in serum at a 1:100 dilution and in BALF at a 1:2 dilution. Anti-swIAV IgA was measured in BALF at a 1:2 dilution using the same kit, with a modified protocol using a goat anti-pig IgA antibody HRP conjugate (Euromedex) at a 1:3000 dilution and in-house controls to calculate sample-to-positive (S/P) ratios.\u003c/p\u003e \u003cp\u003eAnti-PRRSV (N protein) immunoglobulin G (IgG) was measured in serum and BALF using the IDEXX PRRS X3 ELISA kit (IDEXX Laboratories). In serum, the kit was used according to the manufacturer's instructions at a 1:40 dilution, while in BALF, an adapted protocol was applied with a 1:2 dilution of the samples. For detecting anti-PRRSV immunoglobulin A (IgA) in 1:2 diluted BALF and anti-PRRSV immunoglobulin M (IgM) in 1:40 diluted serum, the same kit was employed replacing the anti-pig IgG conjugated antibody by a goat anti-pig IgA or a goat anti-pig IgM HRP conjugated antibody (Euromedex) at a 1:3000 or 1:25000 dilution, respectively. For anti-PRRSV IgA or IgM assays, in-house calibrated negative and positive BALF or serum controls were used to calculate sample-to-positive (S/P) ratios.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eNon-normal distribution of the data was determined by the Shapiro-Wilk test. The Kruskal-Wallis test was applied for unpaired comparisons among four groups and significances were directly depicted on Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB (rectal temperature and average daily weight gain data). Unpaired comparisons between two groups (Mann-Whitney test) were applied for Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC to Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e8\u003c/span\u003e (histopathological, virological, and immunological data). The Mann-Whitney test was used to compare Mock \u003cem\u003evs\u003c/em\u003e. swIAV, PRRSV-D10 \u003cem\u003evs.\u003c/em\u003e swIAV/PRRSV-D10, and PRRSV-D14 \u003cem\u003evs\u003c/em\u003e. swIAV/PRRSV-D14. However, Kruskal-Wallis test was also applied for unpaired comparisons among four groups on these data, and significances are depicted in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eThe absolute number of mononuclear phagocyte cells was calculated by determining the percentage of live cells using flow cytometry, then multiplying this percentage by the total number of BAL cells counted, all divided by 100 and multiplied by the volume of BAL collected.\u003c/p\u003e \u003cp\u003eThe script used for high-dimensional flow cytometry analysis was developed by Adrian Liston's group [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] (The Babraham Institute, UK) and is available on GitHub at:. t-SNE plots were generated using R (version 4.3.0). t-SNE algorithm was run on live lymphocytes using the parameters CD3, CD4, CD8α, CD8β, CD16, NKp46, TCR-γδ, CD2, CD161, Perforin, PLZF and T-bet. Samples of five pigs per treatment group (Mock, swIAV, PRRSV-D10, PRRSV-D14, swIAV/PRRSV-D10, swIAV/PRRSV-D14) were used with 5000 cells per sample and 5000 iterations per run. Statistical analyses were performed using GraphPad Prism Software (version 10.2.3).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePre-infection with swIAV did not affect the clinical progression of PRRS\u003c/h2\u003e \u003cp\u003eTo compare the clinical outcomes of a single PRRSV inoculation to successive inoculations of swIAV and PRRSV seven days apart (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), rectal temperature and respiratory signs (coughing, sneezing, and breathing rate) were monitored daily. On D1, the animals that received swIAV intratracheally showed hyperthermia (rectal temperature\u0026thinsp;\u0026gt;\u0026thinsp;40\u0026deg;C) in four out of five animals in the swIAV group and in five out of ten animals in the swIAV/PRRSV group (mean 40.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C and 39.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u0026deg;C, respectively). These temperatures were significantly higher than those in the Mock and PRRSV groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). By D2, the animals' temperatures returned to normal (below 40\u0026deg;C). On D9, two days after PRRSV inoculation, four out of ten animals in the PRRSV group (mean 39.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u0026deg;C) and two out of ten in the swIAV/PRRSV group (mean 39.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u0026deg;C) showed an increase in temperature above 40\u0026deg;C. There was no significant difference between the PRRSV and swIAV/PRRSV groups.\u003c/p\u003e \u003cp\u003eSome animals showed respiratory signs. On the first day after swIAV inoculation, one out of fifteen animals was coughing. By D9 (two days after PRRSV inoculation), one out of ten animals in the swIAV/PRRSV group was sneezing, and the same animal was coughing on D10 (three days after PRRSV infection). By D10, two out of ten sequentially inoculated animals showed respiratory signs (one was coughing and the other sneezing), compared to one out of ten in the PRRSV-only group, which exhibited a cough.\u003c/p\u003e \u003cp\u003eAverage daily weight gain (ADWG) was significantly decreased in the swIAV group between D0 and D2, compared to the Mock and PRRSV groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, in the swIAV/PRRSV group, no significant difference was observed between this group and the Mock and PRRSV groups. After PRRSV inoculation, there was no significant growth difference between the PRRSV group and the swIAV/PRRSV group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eMicroscopic examination of the lung tissue revealed that, compared to control animals, the alveolar walls were significantly thickened in all infected animals, regardless of single or super-infections. The respiratory airways were occluded by necrotic debris and inflammatory cells. Perivascular cuffing by inflammatory cells was observed in the most severely affected animals. The severity of lung lesions was assessed using two different scoring systems (Jung score and Composite score), both of which gave similar results (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D). By D7 post-swIAV inoculation, significant lung lesions were observed in the swIAV group compared to the Mock group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). After PRRSV inoculation, no differences were observed on D10 and D14 between the PRRSV group and the swIAV/PRRSV group.\u003c/p\u003e \u003cp\u003eOverall, these results suggested that the initial swIAV infection did not worsen the clinical signs and lung lesions caused by PRRSV.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePre-infection with swIAV did not affect PRRSV loads\u003c/h2\u003e \u003cp\u003eUsing RT-qPCR, the viral loads of PRRSV and swIAV genomes were monitored in BALF, lung tissue, tracheobronchial lymph nodes, nasal swab supernatants (swIAV) and serum samples (PRRSV) from all groups. Neither PRRSV nor swIAV genome was detected in the Mock-inoculated group.\u003c/p\u003e \u003cp\u003eIn animals inoculated with swIAV (both swIAV and swIAV/PRRSV groups), the viral genome was detected in nasal secretions at D4 and D7 post-inoculation, with lower detection levels at D7. At the time of PRRSV inoculation on D7, swIAV was detected in BALF from all the animals in the swIAV group, in lung tissue in two out of five pigs, and in the tracheobronchial lymph nodes in five out of five pigs. By D10 and D14, the swIAV genome was no longer detected in the nasal secretions of pigs in the swIAV/PRRSV group (data not shown). However, it was still detected in BALF in three out of five animals at D10 and in two out of five animals at D14 (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). In lung tissue, swIAV genome was detected in one out of five animals at D10 and D14. In TBLN, it was detected in three out of five animals at D10, but no more at D14 (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC, D).\u003c/p\u003e \u003cp\u003ePRRSV genetic material was detected at D10 (three days post-PRRSV infection) in all animals from the PRRSV and swIAV/PRRSV groups, in BALF, tracheobronchial lymph nodes and sera. In the lung tissue, PRRSV was detected in all the pigs from the swIAV/PRRSV group but in only two out of five animals in the PRRSV group. By D14 (seven days post-PRRSV infection), PRRSV was detected in all animals and all sample types (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-D). However, no significant differences were observed between the PRRSV and swIAV/PRRSV groups at D10 or D14.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThus, virological monitoring indicated that PRRSV loads in the lungs, lymph nodes and blood were not influenced by swIAV pre-infection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eInterleukin-12 increased trend upon swIAV and swIAV/PRRSV infections in BALF\u003c/h2\u003e \u003cp\u003eThe cytokine profile (GM-CSF, IFN-γ, IL-10, TNF-α, IL-1α, IL-1β, IL-6, IL-8, IL-18, and IL-12) in BALF was assessed on D7 for the Mock and swIAV groups, and on D10 (three days post-PRRSV inoculation) and D14 (seven days post-PRRSV inoculation) for the PRRSV and swIAV/PRRSV groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe levels of GM-CSF, IFN-γ, IL-10, and TNF-α were below detection thresholds. No significant differences were observed for the cytokines IL-1α, IL-1β, IL-8, and IL-18 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B, D, E). In the swIAV group on D7, a significant increase in the pro-inflammatory cytokines IL-6 and IL-12 (a pro-Th1 cytokine) was observed compared to the Mock group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, F). On D10, a trend suggested higher IL-12 levels in the swIAV/PRRSV group compared to the PRRSV group, with four out of five animals of the PRRSV group showing lower concentrations than those in the swIAV/PRRSV group, and five out of five compared to the Mock group, although this difference was not statistically significant. This trend continued on D14, with all five animals in the swIAV/PRRSV group exhibiting higher IL-12 concentrations than those in the Mock group. The swIAV/PRRSV group presented a significant IL-12 overexpression when compared with the Mock group using Kruskal-Wallis unpaired comparisons test (Supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, two out of five animals in the PRRSV group displayed IL-12 levels similar to those in the swIAV/PRRSV group.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ePre-infection with swIAV increased number of conventional type 1 dendritic cells (cDC1) in BAL during PRRSV infection\u003c/span\u003e \u003c/p\u003e \u003cp\u003eTo characterize the cellular innate immune response in more detail, mononuclear phagocyte cell populations were examined in BAL for all groups.\u003c/p\u003e \u003cp\u003eConventional DC1 were defined as MHCII\u003csup\u003ehigh\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003eCD163\u003csup\u003e\u0026minus;\u003c/sup\u003eCD172a\u003csup\u003e\u0026minus;/low\u003c/sup\u003eCD1\u003csup\u003e\u0026minus;\u003c/sup\u003e, while cDC2 were defined as MHCII\u003csup\u003ehigh\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003eCD163\u003csup\u003e\u0026minus;\u003c/sup\u003eCD172a\u003csup\u003e+\u003c/sup\u003eCD1\u003csup\u003e+\u003c/sup\u003e. Monocyte-derived DC (moDC) were identified as MHCII\u003csup\u003ehigh\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003eCD163\u003csup\u003elow\u003c/sup\u003eCD172a\u003csup\u003e+\u003c/sup\u003eCD1\u003csup\u003e\u0026minus;\u003c/sup\u003e, and macrophages from the BAL were characterized as MHCII\u003csup\u003ehigh\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003eCD163\u003csup\u003ehigh\u003c/sup\u003eCD172a\u003csup\u003e+\u003c/sup\u003e (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the Mock and swIAV groups at D7, no differences in the number of identified cell populations were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-D). Similarly, no significant differences were observed between the PRRSV and swIAV/PRRSV groups on D10 and D14, except for a significant increase in cDC1 on D14 in the swIAV/PRRSV group compared to the PRRSV group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). This increase in cDC1 remains significant when analyzed using the Kruskal-Wallis unpaired comparisons test (Supplementary Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePre-infection with swIAV increased activated CD4\u003csup\u003e+\u003c/sup\u003e regulatory and conventional CD4 T cells in BAL\u003c/h2\u003e \u003cp\u003eTo further explore the immune response, lymphoid cell populations in the BAL were analyzed by flow cytometry for all groups and all necropsy time points.\u003c/p\u003e \u003cp\u003eOne set of experiments focused on conventional CD4\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e\u0026minus;\u003c/sup\u003e T cells (Tconv) and CD4\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003e Treg cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-D, S3), investigating their distribution within total CD4 T cells. No significant differences were observed in the percentages of Tconv and Treg between the swIAV and Mock groups, or between the PRRSV and swIAV/PRRSV groups (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). Within the two subsets of Tconv and Treg, we also analyzed the co-expression of Inducible T-cell Costimulator (ICOS), involved in anti-inflammatory signalling and Ki-67, a molecule expressed in active stages of the cell cycle. A significant increase in ICOS\u003csup\u003e+\u003c/sup\u003eKi-67\u003csup\u003e+\u003c/sup\u003e Tconv and Treg cells was observed in the swIAV group compared to the Mock group. For the ICOS\u003csup\u003e+\u003c/sup\u003eKi-67\u003csup\u003e+\u003c/sup\u003e Tconv, this increase remained on D10 in the swIAV/PRRSV compared to the PRRSV group (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Interestingly, swIAV/PRRSV group presented a significantly higher frequency of ICOS\u003csup\u003e+\u003c/sup\u003eKi-67\u003csup\u003e+\u003c/sup\u003e Tconv cells when compared to the Mock group using Kruskal-Wallis unpaired comparisons test (Supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Moreover, a significant difference in ICOS\u003csup\u003e+\u003c/sup\u003eKi-67\u003csup\u003e+\u003c/sup\u003e Tconv cells was observed at D10 when comparing swIAV/PRRSV to the PRRSV group alone. Overall, this suggested that the swIAV infection resulted in an increase in activated Tregs and Tconv in the BAL that was maintained even in the context of a subsequent PRRSV infection.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eSwine IAV infection resulted in sustained increases in effector lymphocytes in BAL\u003c/h2\u003e \u003cp\u003eA third flow cytometry panel focused on potential changes in conventional T cells, unconventional T cells and NK cells. Given the high numbers of addressed markers (n\u0026thinsp;=\u0026thinsp;12, CD2, CD3, CD4, CD8α, CD8β, CD16, CD161, NKp46, Perforin, PLZF, T-bet, TCR-γδ) we performed dimensionality reduction using t-SNE clustering on live lymphocytes (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-E, S4). This analysis enabled the identification of natural killer (NK) cells, conventional CD4 and CD8 T cells, and γδ T cells. The t-SNE analysis was set to perform a clustering for twelve clusters (Figures S4, S5).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA number of clusters showed significant changes related to treatment groups. In the swIAV group, a significant increase compared to the Mock group was detected for clusters 6, 8, 9 and 10. Cluster 6 contained cells reminiscent of conventional CD4 T cells due to a CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eCD8α\u003csup\u003e+/\u0026minus;\u003c/sup\u003e phenotype that was negative for all other markers (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Cluster 8 consisted of cells with a phenotype of cytotoxic CD8 T cells (CD2\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003eCD8αβ\u003csup\u003e+\u003c/sup\u003eperforin\u003csup\u003e+/\u0026minus;\u003c/sup\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Interestingly, cluster 9 contained cells that were negative for all lineage markers (CD3, TCR-γδ, CD4, CD8β), including NK associated markers NKp46 and CD16 but contained high levels of perforin (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Additionally, swIAV infection also led to an increase of cells represented by cluster 10 which consisted of CD3\u003csup\u003e+\u003c/sup\u003e T cells that were negative for CD4, CD8β and TCR-γδ, but CD2\u003csup\u003e+\u003c/sup\u003eCD8α\u003csup\u003e+\u003c/sup\u003eperforin\u003csup\u003edim\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Clusters 6, 8, 9 and 10 also presented a significant increase in swIAV/PRRSV group on D10 compared with Mock D7 when using Kruskal-Wallis unpaired comparisons test (Supplementary Table\u0026nbsp;1). Notably, these elevated populations remained consistently higher in the swIAV/PRRSV groups at D14, showing significant differences compared to the PRRSV group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). An increase in a subset of TCR-γδ cells (cluster 7, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eB) with a CD8α\u003csup\u003e+\u003c/sup\u003eCD2\u003csup\u003e+/\u0026minus;\u003c/sup\u003eperforin\u003csup\u003e+/\u0026minus;\u003c/sup\u003e phenotype was also observed in the swIAV/PRRSV group compared to PRRSV group on D14.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003ePre-infection with swIAV increased IFN-γ-producing CD4 T cells after PRRSV re-stimulation\u003c/h2\u003e \u003cp\u003eWe investigated IFN-γ and IL-2 production in T cells within PBMCs following \u003cem\u003ein vitro\u003c/em\u003e re-stimulation with the autologous PRRSV-1 strain by intracellular cytokine staining. Analyses focused on the PRRSV and swIAV/PRRSV groups, isolated on D14 (seven days post-PRRSV infection) and the Mock group. Cells cultured in medium alone served as negative controls. CD4 expressing lymphocytes were pre-gated and cytokine producing cells separated into CD8α\u003csup\u003e+\u003c/sup\u003e and CD8α\u003csup\u003e\u0026minus;\u003c/sup\u003e subsets (Figure S6).\u003c/p\u003e \u003cp\u003eIrrespective of CD8α expression, the percentage of IFN-γ-producing CD4 T cells was significantly higher in the swIAV/PRRSV group on D14 compared to PRRSV groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, B) and the Mock group when using Kruskal-Wallis unpaired comparisons test (Supplementary Table\u0026nbsp;1). Notably, the percentages in the PRRSV group remained at levels comparable to those observed in the Mock group (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, B). No significant differences were observed in the production of IL-2 (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e8\u003c/span\u003eC, D). Of note, the IFN-γ response in PBMCs induced by this PRRSV strain typically begins around two weeks after inoculation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eLocal swIAV antibodies continued to rise after swIAV clearance\u003c/h2\u003e \u003cp\u003eHumoral immune responses to swIAV and PRRSV infections were also assessed by detecting specific antibodies directed against each virus. For animals infected with swIAV, IgA in BALF and IgG in both BALF and serum were detected (Figures S7A-C). S/P ratios continued to rise on D10 and D14, although most pigs had cleared swIAV by those time points, which is typical in single swIAV infections (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNo significant differences in anti-PRRSV IgA and IgG antibody levels were observed in the BALF of the PRRSV and swIAV/PRRSV groups throughout the study (Figures S8A, B). At D14, seven days after PRRSV inoculation, IgA and IgG antibodies began to be detectable in some animals. Similarly, no significant differences in anti-PRRSV IgG and IgM levels were observed in serum between the PRRSV and swIAV/PRRSV groups. The S/P ratios remained low but started to increase at D14 (Figures S8C, D).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCo-infections with different pathogens are often the cause of respiratory diseases in pigs [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. It is established that infection with IAV increases susceptibility to secondary bacterial infections, such as \u003cem\u003eStreptococcus suis\u003c/em\u003e in pigs [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] or \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e in humans [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. One of the proposed mechanisms for this influenza-related susceptibility is the depletion of AM, as suggested from studies in mice between four and ten days post-IAV inoculation [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In our study, we did not observe differences in the number of AM in animals from the swIAV group compared to the Mock group at 7 days post-IAV inoculation. This suggests that, differently to IAV infection in mice, the swIAV strain used in this study did not lead to AM depletion in pigs. However, it is important to approach the analysis of resident AM during IAV infection with caution. During this infection various chemokines have increased expression in the lungs, leading to a heterogeneous influx of innate immune cells, such as monocytes-derived macrophages (moMΦ) and moDC [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], whose surface markers might vary based on inflammatory conditions. It would therefore be interesting to consolidate these results in our study on pigs using multiparametric, unbiased approaches, such as single-cell RNA sequencing, to more precisely distinguish resident cells from recruited ones.\u003c/p\u003e \u003cp\u003eWhile various combinations of viral infections are possible, our study focused on a PRRSV-1 super-infection occurring one week after a swIAV H1N2 infection, both commonly observed on pig farms in France. The objective of our research was to evaluate the impact of a primary swIAV infection on the host\u0026rsquo;s immune responses during a secondary PRRSV infection. We analyzed the effects on clinical parameters, viral loads, and both innate and adaptive immune responses in pigs.\u003c/p\u003e \u003cp\u003eOne week after the initial infection with swIAV, clinical signs following the secondary infection with PRRSV did not differ significantly from those observed during a single infection with PRRSV. This suggested that the initial swIAV infection did not exacerbate the animals' health status during the subsequent PRRSV infection.\u003c/p\u003e \u003cp\u003eIt is noteworthy that in a previous study using the same swIAV H1N2 and PRRSV-1 strains, but with a reversed order of infection (PRRSV inoculation followed by swIAV inoculation eight days later), Bougon et al. (2021) observed a reduction in clinical signs in the co-infected group, suggesting an attenuation of the impact of swIAV H1N2 infection in pigs previously infected with PRRSV-1. Correlation analyses revealed an association between IFN-α production and the onset of clinical signs. PRRSV infection led to a reduction in IFN-α production in PRRSV/swIAV super-infected pigs, consistent with previous observations [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], which likely contributed to the attenuation of clinical signs and the pro-inflammatory response induced by the influenza infection [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. They also reported a transient yet significant decrease in PRRSV viral load in the lungs of super-infected pigs, which was correlated with the induction of IFN-α by swIAV, a cytokine to which PRRSV is particularly sensitive [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Another study by Renson et al. [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] examined the effects of H1N2 swIAV infection on the replication of a PRRSV-1 modified live vaccine (MLV1) in SPF piglets. SwIAV infection six hours before MLV1 administration delayed MLV1 viremia and post-vaccination seroconversion. The early rise in IFN-α levels following H1N2 swIAV infection likely explained the inhibition of MLV1 replication. These results highlighted that the order of infections may play a key role in modulating clinical responses. By contrast, in this study, the animals were super-infected seven days after the swIAV infection, when IFN-α was no longer detectable in BAL [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], to avoid any interference from this cytokine.\u003c/p\u003e \u003cp\u003eThe main objective of the present study was to evaluate the effect of a primary infection with swIAV on the early progression of a subsequent PRRSV infection. No significant differences were observed in the PRRSV viral load in the lungs and blood of pigs super-infected with swIAV and PRRSV compared to those infected only with PRRSV.\u003c/p\u003e \u003cp\u003eWe also aimed to examine whether swIAV could induce an early immune imprint, similar to what has been observed in the murine IAV [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and adenovirus [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] models. Aegerter et al. showed that primary infection with IAV conferred protection against \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e by recruiting AM derived from monocytes, which persisted in the lungs for an extended period following IAV infection. Similarly, adenovirus promoted a lasting innate immune memory of AM, facilitated by T lymphocytes through the production of IFN-γ, enhancing immunity against \u003cem\u003eS. pneumoniae\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e. It is important to note that in these studies, the protective imprint was observed several weeks after IAV inoculation (at least four weeks), while in this experiment, PRRSV infection was performed only one week after swIAV inoculation. However, the present results did not show that primary infection with swIAV offered protection against PRRSV or on contrary enhanced its replication in the early stages following swIAV infection.\u003c/p\u003e \u003cp\u003eEarly in swIAV infection, pro-inflammatory cytokines (IFN-α, TNF-α, and IL-12) are secreted in the lungs, accompanied by the infiltration of immune cells, particularly DC [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In the BAL, a higher frequency of cDC1 cells was observed in swIAV-infected pigs, likely attracted by CD8 T cells, which also increased in the lungs following swIAV infection. CD8 T cells express the X-C motif chemokine ligand 1 (XCL1), a chemokine selectively expressed in NK cells and CD8 T cells, which is further elevated upon stimulation, as shown in murine and human cells [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. XCL1 attracts cDC1 \u003cem\u003evia\u003c/em\u003e their XC chemokine receptor 1 (XCR1), promoting IL-12 production in cDC1 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Indeed, IL-12 was also elevated in the present study following super-infection with PRRSV. The IL-12 produced by cDC1 stimulates the production of IFN-γ by antigen-specific T cells, thus directing the immune response towards a Th1 and ultimately type-1 immune response [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In line with this, we observed an increase in CD4 T cells as well as perforin expressing CD8 T cells and less well characterized non-T NK-cell like lymphocytes in the lungs of swIAV/PRRSV group. This coincided with a PRRSV-specific Th1 response in the blood of pigs that were initially infected with swIAV. These results suggest that a primary swIAV infection could promote the rapid induction of an anti-PRRSV-1 immunity \u003cem\u003evia\u003c/em\u003e the recruitment of cDC1 and the production of IL-12 by these cells.\u003c/p\u003e \u003cp\u003eIt is well established that PRRSV infection leads to a delayed activation of PRRSV-specific CD4 and CD8 T cells, typically appearing two to three weeks post-inoculation [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, the present study indicated that prior infection with swIAV increased the presence of CD4 and perforin expressing lymphocytes in the lungs during PRRSV infection, potentially contributing to a more pronounced immune response against PRRSV. It remains to be determined whether this increase in effector cells was due to active recruitment of new cells to the lungs or the persistence of cells initially activated by the swIAV infection, but their increase in swIAV-only infected pigs suggests the latter. Indeed, CD4, CD8, and γδ T lymphocytes were present in the BAL as early as six days post-infection with swIAV [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], and swIAV-specific cells could be detected between D4 and D7 in PBMCs [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. However, this proliferation of lymphocytes did not prevent PRRSV replication at the early stages of infection, and their role in controlling PRRSV at later stages remains to be clarified.\u003c/p\u003e \u003cp\u003eA longer-term follow-up of PRRSV infection would be valuable to determine if it is better controlled over time through these cellular responses. Additionally, it would be relevant to explore whether enhanced induction of type-1 responses could contribute to protection against a subsequent homologous or heterologous PRRSV challenge.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, this study demonstrated that the primary infection with swIAV, occurring one week prior, did not interfere with the early infection of AM by PRRSV. However, several parameters of the cellular antiviral response were significantly elevated, indicating that an enhanced immune response could influence the progression of PRRSV infection at later stages. These results raise questions about the potential impact of this response on the progression of PRRS disease. Therefore, further research is needed to better understand the underlying mechanisms of these immune responses.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cu\u003eAuthor Contributions\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, G.S., O.B., N.B., F.M. and J.G.; methodology, G.S., O.B., N.B., W.G., S.S, C.D., P.R. and J.G.; software, S.S.; validation, O.B., G.S., N.B., W.G., S.S, C.D., and P.R.; formal analysis, S.S and J.G.; investigation, O.B., N.B., C.H., A.P., S.S., S.G., C.D., P.R, M.L.D., F.P., T.L. and J.G.; data curation, S.S and J.G.; writing-original draft preparation, J.G.; writing-review and editing: G.S., O.B., N.B., W.G., S.S., F.M., C.H., A.P., F.P., C.D., P.R., S.G., S.Q., M.L.D., T.L., and J.G.; visualization, S.S. and J.G.; supervision, G.S., O.B., N.B., F.P. and W.G.; project administration, W.G., N.B., G.S and O.B.; funding acquisition, W.G., N.B., G.S. and O.B.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eAcknowledgements\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Flow Cytometry and Immunological Toolbox Science Technology Platforms at The Pirbright Institute for supporting this research. We also thank the staff at the Biological Research Facility and Immunological Toolbox at The Roslin Institute (https://www.ed.ac.uk/roslin/facilities-resources/immunological-toolbox) for their assistance in producing the monoclonal antibody to porcine CD161.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAdditionally, we would also like to thank Nicolas Barbier, S\u0026eacute;verine Herv\u0026eacute;, Roselyne Fonseca, Ga\u0026euml;tan Pinsard, and Sophie Mah\u0026eacute; for their help with the necropsies, as well as to Jean-Marie Guionnet and G\u0026eacute;rald Le Diguerher for animal care.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eFunding\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJG received a PhD grant co-funded by the French National Research Institute for Agriculture, Food and Environment (INRAE) and the French Agency for Food, Environmental and Occupational Health \u0026amp; Safety (ANSES). JG also received funding from the Directorate for Higher Education, Sites and Europe (DESSE), the ERASMUS+ program, and the Doctoral School for Plant, Animal, Food, Sea, and Environment (VAAME) for mobility in the United Kingdom.\u003c/p\u003e\n\u003cp\u003eSS and WG are supported by the Biotechnology and Biological Sciences Research Council (BBSRC) Strategic Programs to The Pirbright Institute, BB/X011038/1 and BB/X011046/1. The Flow Cytometry and Immunological Toolbox facilities at The Pirbright Institute are supported through the BBSRC National Bioscience Research Infrastructure High and Low Containment Services and Science Platforms (BBS/E/PI/23NB0004, BBS/E/PI/23NB0003).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe Biological Research Facility and Immunological Toolbox at the Roslin Institute are supported by funding from UKRI BBSRC (BB/CCG2270/1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eConflicts of Interest\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eOpriessnig T, Gim\u0026eacute;nez-Lirola LG, Halbur PG (2011) Polymicrobial respiratory disease in pigs. Anim Health Res Rev 12:133\u0026ndash;148. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1017/S1466252311000120\u003c/span\u003e\u003cspan address=\"10.1017/S1466252311000120\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaade G, Deblanc C, Bougon J et al (2020) Coinfections and their molecular consequences in the porcine respiratory tract. Vet Res 51:80. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13567-020-00807-8\u003c/span\u003e\u003cspan address=\"10.1186/s13567-020-00807-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeurens F, Zhu J, Renois F (2024) [Viral co-infections targeting the porcine respiratory system: Consequences and limits of the experimental systems]. Virologie (Montrouge) 28:9\u0026ndash;21. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1684/vir.2024.1032\u003c/span\u003e\u003cspan address=\"10.1684/vir.2024.1032\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChauhan RP, Gordon ML (2020) A Systematic Review Analyzing the Prevalence and Circulation of Influenza Viruses in Swine Population Worldwide. Pathogens 9:355. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/pathogens9050355\u003c/span\u003e\u003cspan address=\"10.3390/pathogens9050355\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLunney JK, Benfield DA, Rowland RRR (2010) Porcine reproductive and respiratory syndrome virus: An update on an emerging and re-emerging viral disease of swine. Virus Res 154:1\u0026ndash;6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.virusres.2010.10.009\u003c/span\u003e\u003cspan address=\"10.1016/j.virusres.2010.10.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAdams MJ, Lefkowitz EJ, King AMQ et al (2017) Changes to taxonomy and the International Code of Virus Classification and Nomenclature ratified by the International Committee on Taxonomy of Viruses (2017). Arch Virol 162:2505\u0026ndash;2538. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00705-017-3358-5\u003c/span\u003e\u003cspan address=\"10.1007/s00705-017-3358-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBalasuriya UB, Carossino M (2017) Reproductive effects of arteriviruses: equine arteritis virus and porcine reproductive and respiratory syndrome virus infections. Curr Opin Virol 27:57\u0026ndash;70. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.coviro.2017.11.005\u003c/span\u003e\u003cspan address=\"10.1016/j.coviro.2017.11.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFablet C, Marois-Cr\u0026eacute;han C, Simon G et al (2012) Infectious agents associated with respiratory diseases in 125 farrow-to-finish pig herds: A cross-sectional study. Vet Microbiol 157:152\u0026ndash;163. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.vetmic.2011.12.015\u003c/span\u003e\u003cspan address=\"10.1016/j.vetmic.2011.12.015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFablet C, Marois-Cr\u0026eacute;han C, Grasland B et al (2016) Factors associated with herd-level PRRSV infection and age-time to seroconversion in farrow-to-finish herds. Vet Microbiol 192:10\u0026ndash;20. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.vetmic.2016.06.006\u003c/span\u003e\u003cspan address=\"10.1016/j.vetmic.2016.06.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVan Reeth K, Nauwynck H, Pensaert M (1996) Dual infections of feeder pigs with porcine reproductive and respiratory syndrome virus followed by porcine respiratory coronavirus or swine influenza virus: a clinical and virological study. Vet Microbiol 48:325\u0026ndash;335. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0378-1135(95)00145-X\u003c/span\u003e\u003cspan address=\"10.1016/0378-1135(95)00145-X\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCzyżewska-Dors E, Pomorska-M\u0026oacute;l M, Dors A et al (2019) Proinflammatory cytokine changes in bronchoalveolar lavage fluid cells isolated from pigs infected solely with porcine reproductive and respiratory syndrome virus or co-infected with swine influenza virus. J Veterinary Res 63:489\u0026ndash;495. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2478/jvetres-2019-0063\u003c/span\u003e\u003cspan address=\"10.2478/jvetres-2019-0063\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePol JMA, van Leengoed LAMG, Stockhofe N et al (1997) Dual infections of PRRSV/influenza or PRRSV/\u003cem\u003eActinobacillus pleuropneumoniae\u003c/em\u003e in the respiratory tract. Vet Microbiol 55:259\u0026ndash;264. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0378-1135(96)01323-5\u003c/span\u003e\u003cspan address=\"10.1016/S0378-1135(96)01323-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePomorska-M\u0026oacute;l M, Podg\u0026oacute;rska K, Czyżewska-Dors E et al (2020) Kinetics of single and dual simultaneous infection of pigs with swine influenza A virus and porcine reproductive and respiratory syndrome virus. J Vet Intern Med 34:1903\u0026ndash;1913. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/jvim.15832\u003c/span\u003e\u003cspan address=\"10.1111/jvim.15832\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChrun T, Maze EA, Roper KJ et al (2023) Simultaneous co-infection with swine influenza A and porcine reproductive and respiratory syndrome viruses potentiates adaptive immune responses. Front Immunol 14:1192604. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fimmu.2023.1192604\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2023.1192604\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBougon J, Deblanc C, Renson P et al (2021) Successive Inoculations of Pigs with Porcine Reproductive and Respiratory Syndrome Virus 1 (PRRSV-1) and Swine H1N2 Influenza Virus Suggest a Mutual Interference between the Two Viral Infections. Viruses 13:2169. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/v13112169\u003c/span\u003e\u003cspan address=\"10.3390/v13112169\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eObradovic MR, Segura M, Segal\u0026eacute;s J, Gottschalk M (2021) Review of the speculative role of co-infections in Streptococcus suis-associated diseases in pigs. Vet Res 52:49. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13567-021-00918-w\u003c/span\u003e\u003cspan address=\"10.1186/s13567-021-00918-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcCullers JA (2011) Preventing and Treating Secondary Bacterial Infections with Antiviral Agents. Antivir Ther 16:123\u0026ndash;135. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3851/IMP1730\u003c/span\u003e\u003cspan address=\"10.3851/IMP1730\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhoneim HE, Thomas PG, McCullers JA (2013) Depletion of Alveolar Macrophages during Influenza Infection Facilitates Bacterial Superinfections. J Immunol 191:1250\u0026ndash;1259. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4049/jimmunol.1300014\u003c/span\u003e\u003cspan address=\"10.4049/jimmunol.1300014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCalifano D, Furuya Y, Metzger DW (2018) Effects of Influenza on Alveolar Macrophage Viability Are Dependent on Mouse Genetic Strain. J Immunol 201:134\u0026ndash;144. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4049/jimmunol.1701406\u003c/span\u003e\u003cspan address=\"10.4049/jimmunol.1701406\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi F, Piattini F, Pohlmeier L et al (2022) Monocyte-derived alveolar macrophages autonomously determine severe outcome of respiratory viral infection. Sci Immunol 7:eabj5761. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/sciimmunol.abj5761\u003c/span\u003e\u003cspan address=\"10.1126/sciimmunol.abj5761\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJanssen WJ, Barthel L, Muldrow A et al (2011) Fas Determines Differential Fates of Resident and Recruited Macrophages during Resolution of Acute Lung Injury. Am J Respir Crit Care Med 184:547\u0026ndash;560. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1164/rccm.201011-1891OC\u003c/span\u003e\u003cspan address=\"10.1164/rccm.201011-1891OC\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYao Y, Jeyanathan M, Haddadi S et al (2018) Induction of Autonomous Memory Alveolar Macrophages Requires T Cell Help and Is Critical to Trained Immunity. Cell 175:1634\u0026ndash;1650e17. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cell.2018.09.042\u003c/span\u003e\u003cspan address=\"10.1016/j.cell.2018.09.042\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAegerter H, Kulikauskaite J, Crotta S et al (2020) Influenza-induced monocyte-derived alveolar macrophages confer prolonged antibacterial protection. Nat Immunol 21:145\u0026ndash;157. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41590-019-0568-x\u003c/span\u003e\u003cspan address=\"10.1038/s41590-019-0568-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrevelinger J, Bourry O, Meurens FJ et al (2024) Impact of swine influenza A virus on porcine reproductive and respiratory syndrome virus infection in alveolar macrophages. Front Vet Sci 11. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fvets.2024.1454762\u003c/span\u003e\u003cspan address=\"10.3389/fvets.2024.1454762\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJung K, Alekseev KP, Zhang X et al (2007) Altered Pathogenesis of Porcine Respiratory Coronavirus in Pigs due to Immunosuppressive Effects of Dexamethasone: Implications for Corticosteroid Use in Treatment of Severe Acute Respiratory Syndrome Coronavirus. J Virol 81:13681\u0026ndash;13693. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/jvi.01702-07\u003c/span\u003e\u003cspan address=\"10.1128/jvi.01702-07\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLarcher T, Fablet C, Renson P et al (2019) Assessment of pulmonary tissue responses in pigs challenged with PRRSV Lena strain shows better protection after immunization with field than vaccine strains. Vet Microbiol 230:249\u0026ndash;259. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.vetmic.2019.01.022\u003c/span\u003e\u003cspan address=\"10.1016/j.vetmic.2019.01.022\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCador C, Herv\u0026eacute; S, Andraud M et al (2016) Maternally-derived antibodies do not prevent transmission of swine influenza A virus between pigs. Vet Res 47:86. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13567-016-0365-6\u003c/span\u003e\u003cspan address=\"10.1186/s13567-016-0365-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRose N, Renson P, Andraud M et al (2015) Porcine reproductive and respiratory syndrome virus (PRRSv) modified-live vaccine reduces virus transmission in experimental conditions. Vaccine 33:2493\u0026ndash;2499. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.vaccine.2015.03.040\u003c/span\u003e\u003cspan address=\"10.1016/j.vaccine.2015.03.040\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaisonnasse P, Bouguyon E, Piton G et al (2016) The respiratory DC/macrophage network at steady-state and upon influenza infection in the swine biomedical model. Mucosal Immunol 9:835\u0026ndash;849. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/mi.2015.105\u003c/span\u003e\u003cspan address=\"10.1038/mi.2015.105\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBordet E, Blanc F, Tiret M et al (2018) Porcine Reproductive and Respiratory Syndrome Virus Type 1.3 Lena Triggers Conventional Dendritic Cells 1 Activation and T Helper 1 Immune Response Without Infecting Dendritic Cells. Front Immunol 9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fimmu.2018.02299\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2018.02299\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeloizy C, Bouguyon E, Fossum E et al (2016) Expanding the tools for identifying mononuclear phagocyte subsets in swine: Reagents to porcine CD11c and XCR1. Dev Comp Immunol 65:31\u0026ndash;40. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.dci.2016.06.015\u003c/span\u003e\u003cspan address=\"10.1016/j.dci.2016.06.015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoca CP, Burton OT, Neumann J et al (2023) A cross entropy test allows quantitative statistical comparison of t-SNE and UMAP representations. Cell Rep Methods 3. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.crmeth.2022.100390\u003c/span\u003e\u003cspan address=\"10.1016/j.crmeth.2022.100390\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeurens F, Zhu J, Renois F (2024) Viral co-infections targeting the porcine respiratory system: Consequences and limits of the experimental systems. Virologie 28:9\u0026ndash;21. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1684/vir.2024.1032\u003c/span\u003e\u003cspan address=\"10.1684/vir.2024.1032\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilliamson SM, Tucker AW, McCrone IS et al (2012) Descriptive clinical and epidemiological characteristics of influenza A H1N1 2009 virus infections in pigs in England. Vet Rec 171:271\u0026ndash;271. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1136/vr.100673\u003c/span\u003e\u003cspan address=\"10.1136/vr.100673\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin X, Huang C, Shi J et al (2015) Investigation of Pathogenesis of H1N1 Influenza Virus and Swine Streptococcus suis Serotype 2 Co-Infection in Pigs by Microarray Analysis. PLoS ONE 10:e0124086. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0124086\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0124086\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcCullers JA, Rehg JE (2002) Lethal Synergism between Influenza Virus and Streptococcus pneumoniae: Characterization of a Mouse Model and the Role of Platelet-Activating Factor Receptor. J Infect Dis 186:341\u0026ndash;350. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1086/341462\u003c/span\u003e\u003cspan address=\"10.1086/341462\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eButler JE, Lager KM, Golde W et al (2014) Porcine reproductive and respiratory syndrome (PRRS): an immune dysregulatory pandemic. Immunol Res 59:81\u0026ndash;108. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12026-014-8549-5\u003c/span\u003e\u003cspan address=\"10.1007/s12026-014-8549-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlbina E, Carrat C, Charley B (1998) Short Communication: Interferon-α Response to Swine Arterivirus (PoAV), the Porcine Reproductive and Respiratory Syndrome Virus. J Interferon Cytokine Res 18:485\u0026ndash;490. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1089/jir.1998.18.485\u003c/span\u003e\u003cspan address=\"10.1089/jir.1998.18.485\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRenson P, Deblanc C, Bougon J et al (2021) Concomitant Swine Influenza A Virus Infection Alters PRRSV1 MLV Viremia in Piglets but Does Not Interfere with Vaccine Protection in Experimental Conditions. Vaccines 9:356. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/vaccines9040356\u003c/span\u003e\u003cspan address=\"10.3390/vaccines9040356\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhatri M, Dwivedi V, Krakowka S et al (2010) Swine Influenza H1N1 Virus Induces Acute Inflammatory Immune Responses in Pig Lungs: a Potential Animal Model for Human H1N1 Influenza Virus. J Virol 84:11210\u0026ndash;11218. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/JVI.01211-10\u003c/span\u003e\u003cspan address=\"10.1128/JVI.01211-10\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCrisci E, Fraile L, Montoya M (2019) Cellular Innate Immunity against PRRSV and Swine Influenza Viruses. Vet Sci 6:26. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/vetsci6010026\u003c/span\u003e\u003cspan address=\"10.3390/vetsci6010026\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCrozat K, Guiton R, Contreras V et al (2010) The XC chemokine receptor 1 is a conserved selective marker of mammalian cells homologous to mouse CD8α\u0026thinsp;+\u0026thinsp;dendritic cells. J Exp Med 207:1283\u0026ndash;1292. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1084/jem.20100223\u003c/span\u003e\u003cspan address=\"10.1084/jem.20100223\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamazaki C, Miyamoto R, Hoshino K et al (2010) Conservation of a chemokine system, XCR1 and its ligand, XCL1, between human and mice. Biochem Biophys Res Commun 397:756\u0026ndash;761. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bbrc.2010.06.029\u003c/span\u003e\u003cspan address=\"10.1016/j.bbrc.2010.06.029\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhilas S, Ambrosini M, Cancel J-C et al (2021) Natural killer cells and dendritic epidermal γδ T cells orchestrate type 1 conventional DC spatiotemporal repositioning toward CD8\u0026thinsp;+\u0026thinsp;T cells. iScience 24:103059. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.isci.2021.103059\u003c/span\u003e\u003cspan address=\"10.1016/j.isci.2021.103059\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnnunziato F, Romagnani C, Romagnani S (2015) The 3 major types of innate and adaptive cell-mediated effector immunity. J Allergy Clin Immunol 135:626\u0026ndash;635. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jaci.2014.11.001\u003c/span\u003e\u003cspan address=\"10.1016/j.jaci.2014.11.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiao Z, Batista L, Dee S et al (2004) The Level of Virus-Specific T-Cell and Macrophage Recruitment in Porcine Reproductive and Respiratory Syndrome Virus Infection in Pigs Is Independent of Virus Load. J Virol 78:5923\u0026ndash;5933. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/jvi.78.11.5923-5933.2004\u003c/span\u003e\u003cspan address=\"10.1128/jvi.78.11.5923-5933.2004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTalker SC, Stadler M, Koinig HC et al (2016) Influenza A Virus Infection in Pigs Attracts Multifunctional and Cross-Reactive T Cells to the Lung. J Virol 90:9364\u0026ndash;9382. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/jvi.01211-16\u003c/span\u003e\u003cspan address=\"10.1128/jvi.01211-16\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeblanc C, Qu\u0026eacute;guiner S, Gorin S et al (2020) Evaluation of the Pathogenicity and the Escape from Vaccine Protection of a New Antigenic Variant Derived from the European Human-Like Reassortant Swine H1N2 Influenza Virus. Viruses 12:1155. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/v12101155\u003c/span\u003e\u003cspan address=\"10.3390/v12101155\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5928429/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5928429/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFarmed pigs are frequently exposed to respiratory infections, with swine influenza A virus (swIAV) and porcine reproductive and respiratory syndrome virus (PRRSV) being key drivers. Most co-infection studies with these viruses have focused on PRRSV infection followed by swIAV. However, the reverse scenario, where swIAV is given first and then PRRSV, has not been explored. This infection sequence is plausible under natural conditions and warrants further study, especially given that influenza A virus has been shown in mice to impair alveolar macrophages, which are the target cells for PRRSV.\u003c/p\u003e \u003cp\u003eThis study aimed to evaluate the impact of swIAV infection on the alveolar macrophage population, clinical signs, immune responses, and viral loads during a secondary infection with PRRSV initiated seven days after the initial swIAV exposure. Results demonstrated that primary swIAV infection did not exacerbate the clinical progression of PRRSV infection, nor did it result in significant differences in PRRSV viral loads or affect the alveolar macrophage population in the lungs of super-infected pigs as compared to those of pigs infected with PRRSV alone. However, swIAV pre-infection was associated with an increase in the number of conventional dendritic cells type 1 (cDC1), perforin-expressing T cells and NK-related lymphocytes in bronchoalveolar lavage. This coincided with an increase of PRRSV-specific IFN-γ producing CD4 T cells in blood detected seven days post-PRRSV infection. These findings suggest that a swIAV infection could enhance immune responses during subsequent PRRSV infection by recruiting cDC1 and inducing IL-12, promoting a type-1 immune response, highlighting the complex interplay and often unexpected outcomes of viral co-infections occurring in close temporal proximity.\u003c/p\u003e","manuscriptTitle":"Swine Influenza A virus infection sets the local immunological landscape in subsequent infection with Porcine Reproductive and Respiratory Syndrome virus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-05 08:31:55","doi":"10.21203/rs.3.rs-5928429/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"35e8a01e-871f-4953-88c3-8b15812ef828","owner":[],"postedDate":"February 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-09T15:59:32+00:00","versionOfRecord":{"articleIdentity":"rs-5928429","link":"https://doi.org/10.1186/s13567-025-01536-6","journal":{"identity":"veterinary-research","isVorOnly":false,"title":"Veterinary Research"},"publishedOn":"2025-06-08 15:57:03","publishedOnDateReadable":"June 8th, 2025"},"versionCreatedAt":"2025-02-05 08:31:55","video":"","vorDoi":"10.1186/s13567-025-01536-6","vorDoiUrl":"https://doi.org/10.1186/s13567-025-01536-6","workflowStages":[]},"version":"v1","identity":"rs-5928429","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5928429","identity":"rs-5928429","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}