TYPE Original Research PUBLISHED 13 March 2023 DOI 10.3389/fcimb.2023.1111143 Vaccination against swine OPEN ACCESS influenza in pigs causes different EDITED BY Rabeh El-Shesheny, National Research Centre, Egypt drift evolutionary patterns upon REVIEWED BY Sook-San Wong, swine influenza virus The University of Hong Kong, Hong Kong SAR, China Philippe Colson, experimental infection and IHU Mediterranee Infection, France *CORRESPONDENCE reduces the likelihood of José I. Núñez joseignacio.nunez@irta.cat genomic reassortments Álvaro López-Valiñas alvaro.lopezv@irta.cat SPECIALTY SECTION Álvaro López-Valiñas 1,2,3*, Marta Valle 1,2,3, Miaomiao Wang 1,2,3, This article was submitted to Ayub Darji 1,2,3, Guillermo Cantero 1,2,3, Chiara Chiapponi 4, Virus and Host, a section of the journal Joaquim Segalés 2,3,5, Llilianne Ganges 1,2,3,6 and Frontiers in Cellular and Infection Microbiology José I. Núñez 1,2,3* RECEIVED 29 November 2022 1 IRTA, Programa de Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Campus de la ACCEPTED 14 February 2023 Universitat Autònoma de Barcelona (UAB), Barcelona, Spain, 2 Unitat mixta d’Investigació IRTA-UAB en PUBLISHED 13 March 2023 Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), Barcelona, Spain, 3 WOAH Collaborating Centre for the Research and Control of CITATION Emerging and Re-Emerging Swine Diseases in Europe (IRTA-CReSA), Barcelona, Spain, 4 WOAH López-Valiñas Á, Valle M, Wang M, Darji A, Reference Laboratory for Swine Influenza, Istituto Zooprofilattico Sperimentale della Lombardia ed Cantero G, Chiapponi C, Segalés J, Emilia-Romagna, Brescia, Italy, 5 Departament de Sanitat i Anatomia Animals, Universitat Autònoma de Ganges L and Núñez JI (2023) Vaccination Barcelona, Barcelona, Spain, 6 WOAH Reference Laboratory for Classical Swine Fever, IRTA-CReSA, against swine influenza in pigs causes Barcelona, Spain different drift evolutionary patterns upon swine influenza virus experimental infection and reduces the likelihood of genomic reassortments. Front. Cell. Infect. Microbiol. 13:1111143. Influenza A viruses (IAVs) can infect a wide variety of bird and mammal species. doi: 10.3389/fcimb.2023.1111143 Their genome is characterized by 8 RNA single stranded segments. The low COPYRIGHT proofreading activity of their polymerases and the genomic reassortment © 2023 López-Valiñas, Valle, Wang, Darji, Cantero, Chiapponi, Segalés, Ganges and between different IAVs subtypes allow them to continuously evolve, Núñez. This is an open-access article constituting a constant threat to human and animal health. In 2009, a distributed under the terms of the Creative pandemic of an IAV highlighted the importance of the swine host in IAVs Commons Attribution License (CC BY). The use, distribution or reproduction in other adaptation between humans and birds. The swine population and the forums is permitted, provided the original incidence of swine IAV is constantly growing. In previous studies, despite author(s) and the copyright owner(s) are credited and that the original publication in vaccination, swine IAV growth and evolution were proven in vaccinated and this journal is cited, in accordance with challenged animals. However, how vaccination can drive the evolutionary accepted academic practice. No use, dynamics of swine IAV after coinfection with two subtypes is poorly studied. In distribution or reproduction is permitted which does not comply with these terms. the present study, vaccinated and nonvaccinated pigs were challenged by direct contact with H1N1 and H3N2 independent swine IAVs seeder pigs. Nasal swab samples were daily recovered and broncho-alveolar lavage fluid (BALF) was also collected at necropsy day from each pig for swine IAV detection and whole genome sequencing. In total, 39 swine IAV whole genome sequences were obtained by next generation sequencing from samples collected from both experimental groups. Subsequently, genomic, and evolutionary analyses were carried out to detect both, genomic reassortments and single nucleotide variants (SNV). Regarding the segments found per sample, the simultaneous presence of segments from both subtypes was much lower in vaccinated animals, indicating Frontiers in Cellular and Infection Microbiology 01 frontiersin.org López-Valiñas et al. 10.3389/fcimb.2023.1111143 that the vaccine reduced the likelihood of genomic reassortment events. In relation to swine IAV intra-host diversity, a total of 239 and 74 SNV were detected within H1N1 and H3N2 subtypes, respectively. Different proportions of synonymous and nonsynonymous substitutions were found, indicating that vaccine may be influencing the main mechanism that shape swine IAV evolution, detecting natural, neutral, and purifying selection in the different analyzed scenarios. SNV were detected along the whole swine IAV genome with important nonsynonymous substitutions on polymerases, surface glycoproteins and nonstructural proteins, which may have an impact on virus replication, immune system escaping and virulence of virus, respectively. The present study further emphasized the vast evolutionary capacity of swine IAV, under natural infection and vaccination pressure scenarios. KEYWORDS swine influenza virus, vaccination, coinfection, virus evolution, reassortant 1 Introduction from the reassortment of two circulating swine IAVs, the H1N1 Eurasian “avian-like” (EA) swine strain and a previous circulating Influenza A viruses (IAVs) are worldwide distributed pathogens human-like H1N2 virus harboring avian, human and swine IAVs whose natural hosts are many bird and mammal species including segments (Smith et al., 2009; Trifonov et al., 2009). This H1N2 humans and pigs. IAVs genome is composed of 8 RNA negative strain arose in 1994 a consequence of reassortment between two sense strand segments which code for: two polymerases basic (PB1 strains circulating in pigs, H1N1 and H3N2 (Brown et al., 1998). and PB2) and one polymerase acidic (PA), the hemagglutinin (HA), Lately, in China, a new viral strain H1N1 named as G4 EA, with the nucleoprotein (NP), the neuraminidase (NA), the matrix pandemic and triple reassortant segments, became predominant in proteins (M) and the nonstructural proteins (NS1) (Breen et al., the swine population. This strain is capable of infecting humans 2016). The evolution of IAVs, like other RNA viruses, is described and, therefore, could pose an imminent pandemic risk (Sun et al., under the quasispecies theory (Domingo et al., 2012; Domingo and 2020). Hence, pigs play an important role in the generation of new Perales, 2019). On one hand, IAVs genome can rapidly accumulate IAVs strains with pandemic potential as they are considered point mutations along the whole genome, because of the low “mixing vessels”, being susceptible to avian, human, and swine proofreading activity of their polymerases (Chen and Holmes, IAVs (Ito et al., 1998; Brown, 2000; Ma et al., 2009). Accordingly, 2006; Shao et al., 2017), which could confer adaptative advantages pigs could serve as intermediaries in the adaptation of avian IAVs to to progeny. Also, the evolution of the surface glycoproteins, HA and humans and vice versa (Medina, 2018). NA, are constantly under immune pressure if the host has pre- In the last decade, the most circulating subtypes among pig existing immunity against swine IAV. Therefore, both segments, population on European farms have been EA swine H1N1, “human- could achieve a new antigenic pattern after genetic diversification, like” swine H1N2, “human-like” reassortment swine H3N2 and A avoiding pre-existing immunity (Carrat and Flahault, 2007). On the (H1N1) pdm09 viruses (Simon-Grifé et al., 2011; Torremorell et al., other hand, genomic segment reassortments between different IAVs 2012; Kyriakis et al., 2013; Simon et al., 2014; Li and Robertson, subtypes generate higher levels of variability. This genetic 2021). The mortality rate caused by swine IAV in swine is usually low mechanism can occur when a host is simultaneously coinfected if no major co-infections do occur, but it is a highly contagious by different IAVs and, as a result, new viruses could arise with disease whose morbidity rate could reach 100% in exposed animals different host range, antigenicity, and virulence properties. (Er et al., 2014; Li and Robertson, 2021). Both the high seroprevalence Historically, these reassortment events were responsible for the and the ever-increasing pig population make reassortment events emergence of important flu pandemics such as the Asian flu in 1957 possibilities being more feasible. Nowadays, biosecurity measures and (H2N2) and the Hong Kong flu of 1968 (H3N2) (Scholtissek et al., trivalent vaccine application are the main strategies to avoid disease 1978; Kawaoka et al., 1989; Belshe, 2005). and reduce swine IAV prevalence (Thacker and Janke, 2008; Salvesen In 2009, a new pandemic influenza strain A(H1N1)pdm09 of and Whitelaw, 2021), although vaccination is limitedly performed in swine origin quickly spread by human to human contact through Europe reaching only 10-20% of the pig population (Reeth et al., more than 30 countries and it has been calculated that caused 2016). The most used trivalent vaccine against swine IAV in the EU between 151,700 and 574,400 human deaths associated with the includes H1N1, H3N2, and H1N2 subtypes in its formulation. It infection (Swine influenza - WOAH - World Organisation for reduces virus spread and disease, although it does not generate Animal Health, n.d.; Smith et al., 2009). This new strain arose sterilizing immunity in the host, allowing viral replication (Thacker Frontiers in Cellular and Infection Microbiology 02 frontiersin.org López-Valiñas et al. 10.3389/fcimb.2023.1111143 and Janke, 2008; Ma and Richt, 2010). It is known that swine IAV In the present study, the commercial inactivated swine IAV breakthrough infections are common in pigs because virus evolution vaccine (RESPIPORC FLU3, IDT®, Dessau-Rosslau, Germany) was favors the escape to the host immunity (Murcia et al., 2012), this used. Vaccine formulation includes the H1N1 (Haselünne/ situation can promote the circulation of the virus in vaccinated pigs IDT2617/2003), H3N2 (Bakum/IDT1769/2003), and H1N2 (Chamba Pardo et al., 2018). Previous studies suggest that the (Bakum/1832/2000) strains. application of the trivalent vaccine can influence on the evolutionary dynamics of swine IAV viral populations, being different in vaccinated and nonvaccinated animals (Ló pez-Valiñas 2.2 Experiment design et al., 2021; Ló pez-Valiñas et al., 2022).Thus, the aim of the present study was to evaluate the evolutionary dynamics of swine IAV during Twenty 5-week-old domestic pigs free from swine IAV and its a coinfection trial in pigs with EA swine H1N1 and “human-like” antibodies were selected. Animals were equally distributed in two swine H3N2 strains in vaccinated and nonvaccinated pigs. Overall, different boxes of the animal biosafety level 3 (aBSL3) facilities at our study retrieved by NGS 39 complete viral quasispecies collected IRTA-CReSA (Figure 1). In one box, seeder animals H1N1 (No. 1 from vaccinated and nonvaccinated pigs, finding different and 2), seeder animals H3N2 (No. 5 and 6) and animals from group evolutionary patterns, and nonsynonymous substitutions that may A (No. 9 to 14) were allocated. Seeders H1N1 (No. 3 and 4), seeders play an important role in virulence and the evasion of the swine H3N2 (No. 7 and 8), and animals from group B (No. 15 to 20) were immune system. housed in the second box. After an acclimation period of one week, animals from group A received the first dose of the commercial trivalent vaccine according 2 Material and methodologies to manufacturer’s instructions, administering 2 mL intramuscularly in the neck muscle. In parallel, animals from group B received 2 mL 2.1 Cells, viruses, and vaccine of phosphate buffered saline (PBS) in the same manner. Twenty- one days post-vaccination (21 dpv), animals from group A and B, Viral titration and production were performed using Madin- received the second dose of the vaccine and PBS, respectively. Darby Canine Kidney (MDCK, ATCC CCL-34). For cell culture, Three weeks after the second vaccination dose (42 dpv), animals Dulbecco’s Modified Eagle Medium (DMEM) was used 1 to 4 and 5 to 8 were challenged with A/swine/Spain/SF11131/2007 supplemented with fetal bovine serum (FBS) (10%), L-glutamine (H1N1) and A/Swine/Spain/SF32071/2007 (H3N2), respectively. (1%) and penicillin/streptomycin (1%). The cell culture conditions For the inoculation, two administration routes, intranasal and were 37°C with 5% CO2 atmosphere in an incubator. endotracheal, were used with a viral concentration of 107 TCID50 The A/Swine/Spain/01/2010 H1N1 and the A/Swine/Spain/ in a final volume of 2 and 5 mL, respectively. For the intranasal SF32071/2007 H3N2 viruses were propagated in MDCK administration, 1 mL of inoculum per nostril was administered with monolayer cell cultures at a multiplicity of infection (MOI) of a diffuser device (MAD Nasal, Teleflex, Morrisville, NC, USA). On 0.01 and harvested 48 hours later. Virus cell entry was facilitated by the other hand, for the endotracheal administration, animals were 10 µg/mL of porcine trypsin (Sigma-Aldrich, Madrid, Spain) intubated. For both, a nose snare was used to restrain animals addition. For both virus titrations, serial dilutions in MDCK cells (Ló pez-Valiñas et al., 2021). Experimentally inoculated animals were carried out to calculate the 50% tissue culture infection dose were separated from the rest by a physical barrier. One day post (TCID50) (Reed and Muench, 1938). inoculation (1 dpi), barriers were lifted to allow direct contact. FIGURE 1 Experimental design and animal distribution. Vaccinated and nonvaccinated pigs are represented in blue and yellow figures, respectively. Seeders animals are represented in grey until the day of their challenge when pigs challenged with H1N1 are represented in red meanwhile those challenged with H3N2 are in green. Frontiers in Cellular and Infection Microbiology 03 frontiersin.org López-Valiñas et al. 10.3389/fcimb.2023.1111143 After the challenge, animals were daily monitored for rectal A sample from each lung section collected in formalin was temperature, clinical signs, and animal behavior in a blind manner subsequently dehydrated and embedded in paraffin wax. Later, two by trained veterinarians. Clinical signs were scored as previously 3–5 mm thick sections were cut for both, hematoxylin-eosin (H&E) described (Galindo-Cardiel et al., 2013). Moreover, after the first staining and immunohistochemistry (IHC), for light microscopy day post-contact (1 dpc), the swine IAV load in nasal swab samples examination (Busquets et al., 2010; Sisteré -Oró et al., 2019). For was daily measured by RTq-PCR (explained below). When viral IHC, the two-step polymer method (Envision TM) was conducted loads started to drop, animals were euthanized. using a monoclonal one against influenza A virus (IAV) (Hb65 Nasal swab samples were collected before first and second from the ATCC) and system + HRP-labeled polymer Anti-Mouse vaccinations, at day of challenge and daily from 1 dpc to animal (K4001, Dako) as primary and secondary antibodies, respectively euthanasia. Blood samples were collected at vaccinations, challenge, (Sabattini et al., 1998). The degree of lung lesion according to the and euthanasia days. From each euthanized animal, lung, nasal number of airways affected and the amount of immunoreactivity turbinate (NT) and broncho-alveolar lavage fluid (BALF) were were evaluated, on H&E and IHC examination, respectively, using a collected and stored at -80°C. BALF from each animal was previously described semiquantitative scoring system (Detmer collected after filling the right lung with 150 mL of PBS. By last, et al., 2013). lung samples (apical, middle and cranial part of diaphragmatic lobes) was fixed by immersion in 10% buffered formalin. Procedures were approved by the animal ethics committee from 2.5 Swine IAV genome detection the Generalitat de Catalunya, under the project number 10856, following the Spanish and European regulations. Lung and NT were homogenized in brain heart infusion medium (10% weight/volume) with TissueLyser II (Qiagen, Düsseldorf, Germany). RNA from nasal swab, BALF, lung and 2.3 Evaluation of the humoral response NT samples were extracted using the MagAttract 96 Cador against swine IAV Pathogen kit ® (Qiagen, Düsseldorf, Germany) according to manufacturer’s instructions. To detect and quantify swine IAV Antibody levels against influenza NP was performed by ID RNA on each sample a RT-qPCR based on M segment Screen®—influenza A Antibody Competition ELISA kit (ID VET, amplification was performed in the Fast7500 equipment (Applied Grabels, France). The inhibition percentages were calculated Biosystem) (Spackman et al., 2002). Samples with a cycle threshold according to the manufacturer’s instructions where values below (Ct) value under 40 were considered positive whereas no 45% were considered positive, greater than 50% negative, and fluorescence detection were considered negative (Spackman et al., between both values doubtful. 2002; Ló pez-Valiñas et al., 2021). Furthermore, the hemagglutination inhibition (HI) assay was performed in samples collected at day of seeders challenge and each pig euthanasia day as previously described (Ló pez-Serrano et al., 2.6 Whole swine IAV genome sequencing 2021). All tested serum samples were first treated with RDE II Seiken (Denka Chemicals, Tokyo, Japan) at 37°C during 18 h and later RNA from A/Swine/Spain/01/2010 H1N1 and A/Swine/Spain/ inactivated at 56°C for 1 h. Unspecific hemadsorption inhibitors were SF32071/2007 H3N2 inocula, and RNA extracted from nasal swab removed by adding a volume of 50% chicken red blood cells (RBCs) and BALF samples with a Ct value lower than 35 were proposed for and later diluted in PBS (1:10). Treated pig sera were two-fold diluted, whole genome sequencing (Zhou et al., 2009; Ló pez-Valiñas et al., in v-bottomed 96 well plates, with PBS up to the 1:1024 dilution. 2021). First, a whole swine IAV genome amplification was Subsequently, 25 µL of each viral isolates used on challenge diluted up performed using 0.5 mL of each forward MBTuni-12 and reverse to 4 Hemagglutination Units (HAU), were dispensed in each well in MBTuni-13 primers, both at 0.2 mM. Moreover, 0.5 mL of parallel and incubated during 1 h at room temperature. Afterward, 25 SuperScript® III One-Step RT-PCR System with Platinum™ Taq µL of 0.5% of chicken RBCs were dispensed and again incubated for High Fidelity DNA Polymerase (Thermo Fisher Scientific, 1 h at room temperature. The hemagglutination titer of each serum Waltham, MA, USA), 12.5 mL of reaction mix included in the kit, was established as the reciprocal dilution at which inhibition was 8.5 mL of RNase free water and 2.5 mL of swine IAV RNA. In complete. Titers ≥1/40 were considered protective. parallel, a second amplification was performed to enhance biggest swine IAV segments amplification replacing the forward primer by MBTuni12G, keeping the remaining conditions (Lycett et al., 2012). 2.4 Pathological analyses and Only samples with whole swine IAV amplification were further immunohistochemistry in lung selected (Zhou et al., 2009). For sequencing, a multiplexed sequencing library was prepared per sample following the The lung parenchyma was macroscopically examined during Nextera-XT DNA Library Prep protocol (Illumina®, San Diego, each animal necropsy. The percentage of lung-affected area was also CA, USA). Subsequently, the library sequencing was performed determined through an image analysis as previously described using Miseq Reagent Kit v2 in a 150-cycle paired-end run on Miseq (Sibila et al., 2014), using ImageJ® software (ImageJ, n.d.). Instrument (Illumina®, San Diego, CA, USA). Frontiers in Cellular and Infection Microbiology 04 frontiersin.org López-Valiñas et al. 10.3389/fcimb.2023.1111143 2.7 Bioinformatic workflow protein domain delimitation was inferred as previously reported for quasispecies genomic and (Sriwilaijaroen and Suzuki, 2012; Hu et al., 2014; McAuley et al., evolutionary analysis 2019). Besides, HA and NA 3D protein representations were also performed with the PyMOL Molecular Graphics System (v.4.6). In the present study, a bioinformatic workflow was developed to Illumina reads alignment and variant calling, with tools widely used for the study of viral quasispecies (Sobel Leonard et al., 2016; Borges 2.9 Statistical analysis et al., 2018; Cacciabue et al., 2020). First, Illumina adapters were automatically removed. Read quality was checked with FastQC (v The T-test was used to compare the distribution of the antibody levels 0.11.8) (Andrews, 2010) and low-quality reads (Phread < 30) were against influenza NP on the day of the challenge, and Ct values from 4 to 9 trimmed by Trimmomatic (v0.39) (Bolger et al., 2014). To dpi, between vaccinated and nonvaccinated animals. The Chi-squared test determine swine IAV H1N1 and H3N2 inoculum consensus was used to study differences in proportions between the numbers of sequences, reads from both were aligned against the H1N1 genomic segments detected and synonymous and nonsynonymous (JX908038-45) (Martı́n-Valls et al., 2014) and H3N2 (HE774666- percentages in vaccinated and nonvaccinated pigs. Subsequently, the 73) (Mussá et al., 2013) genome sequences using the Burrows- Bonferroni correction was applied. Finally, an analysis of variance Wheeler alignment (BWA) tool mem function (v0.7.17) (Li and (ANOVA) and subsequent post-hoc Kruskal-Wallis test by rank, were Durbin, 2009). After mapping, unmapped and low quality mapped applied to compare p means between vaccinated and nonvaccinated (< 30) reads were removed from the analysis using Samtools animals on different dpi and in different segments. (v.0.39) (Danecek et al., 2021). Moreover, PCR duplicates and r e a d s r e c a l i b r a t i o n w e r e p e r f o r m e d w i t h t h e Pi c a r d “MArkDuplicatesSpark” and “BaseRecalibrator” options included 3 Results in GATK4 (v4.1). Indeed, the sequence depth on each genome position was calculated with “-depth” function included on 3.1 Humoral response against swine IAV Samtools (v1.9) and plotted using ggplot2 library in Rstudio (RStudio | Open source & professional software for data science Competition percentage means of antibodies against NP were greater teams - RStudio, n.d.; Wickham, 2016). Subsequently, consensus at all sampling days in vaccinated animals. From day of challenge until the sequences were generated using consensus option from Bcftools end of the experiment, difference between groups were significant (p= (v.1.9) (Danecek et al., 2021). 0.001881; t-test). At day of challenge, 2 out of 6 serum samples collected All samples were simultaneously aligned, against both inocula from vaccinated animals were considered positive, but in the whole group consensus, following the previous described workflow. Finally, each an increase in the antibody titers has been observed, indicating that an sample was again aligned, using only as a reference the segments immune response has taken place (Figure 2). that were present on each specific sample. In all samples, SNV Regarding HI titers against strains used for the inoculation at the reported because of misalignment reads due to the high percentage day of challenge showed no positive titers against H1N1 subtype in of similarity between H1N1 and H3N2 sequences, were parsed and vaccinated pigs, while all sera had positive titers against H3N2 subtype eliminated using R (v.4.1.2) (RStudio | Open source & professional (Table 1), even though the percentage of amino acid identity is similar software for data science teams - RStudio, n.d.). between the vaccine viruses and the challenge viruses (96,47% for H3 All variants found along the whole genome were noted using and 95,76% for H1). Additionally, from the 50 positions involved in LoFreq software with default parameters (Wilm et al., 2012). antigenic sites, only position 204 (antigenic site Ca1) shows different Moreover, the effect of each substitution noted on variants was amino acids between vaccine and virus challenge (D and V, predicted with SnpEff software (v.4.3) (Cingolani et al., 2012). respectively). On the day of euthanasia, titers against H1N1 subtype Previously, a database for both subtypes was created with “build– were only detected in vaccinated animals, specifically in 3 out of 6 gtf22” function, using the previously annotated genomes (Mussá animals. On the other hand, positive titers against H3N2 were observed et al., 2013; Martı́n-Valls et al., 2014). The following requirements in all samples collected from vaccinated animals, whereas were only were considered to call a single nucleotide variant (SNV); p value detected in nonvaccinated pig sera at 8 and 9 dpi with low titers. <0.01 and at least 50 and 10 reads of depth and alternative base count respectively (Ló pez-Valiñas et al., 2021). Finally, the nucleotide diversity (p) per genomic segment per sample were 3.2 Swine IAV genome was detected calculated with SNPGenie software (Nelson et al., 2015). in both experimental groups after seeder contact 2.8 Protein structure representation Swine IAV RNA was detected in nasal swab samples collected from seeders, vaccinated and nonvaccinated animals, from 2 dpi Allocations of nonsynonymous substitutions with an allele until the end of the experiment (Figure 3A; Supplementary Table frequency greater than 5% were pointed in each swine IAV S1). swine IAV was not detected in any nasal swab sample collected protein. Lolliplot proteins representation was done with the before the challenge (42 dpv) (Figure 3A). As average, lower Ct trackViewer package from Bioconductor (Ou and Zhu, 2019). All values were detected in samples collected from nonvaccinated Frontiers in Cellular and Infection Microbiology 05 frontiersin.org López-Valiñas et al. 10.3389/fcimb.2023.1111143 3.3 Pathological findings On average, the lung affected area was greater in vaccinated animals, although statistical differences were not found. Vaccinated animals No. 9 and No. 10 had higher percentage of pneumonic lesions, 17.89 and 31.89% respectively. The remaining pigs did not exceed 6% of lung affected area (Table 2). All pigs, irrespectively from their vaccination status, developed a mild-to-severe broncho-interstitial pneumonia, with scores ranging from 1.5 to 3. In vaccinated pigs, three animals had the highest FIGURE 2 histopathological scoring, whereas the remaining ones obtained the Swine IAV NP antibody kinetics in serum samples represented in second highest score value, with 2.5. By contrast, an average lower boxplots. The percentage of competition is expressed in the ordinate axis whereas the sampling day is indicated on the abscissas microscopic lesion scores were detected in nonvaccinated pigs. one. Blue boxplots show samples collected from vaccinated Regarding immunohistochemistry, all lungs had scattered amount of animals, whereas the yellow ones represent nonvaccinated ones, and the whiskers show quartile variability. Different shapes illustrate immunoreactivity except nonvaccinated animals No. 16 and 19, with the ID number of each pig. Values above the red line (<45%), below low amounts, and vaccinated pig No. 11, with no immunoreactivity. the grey line (>50%) and between both lines are considered positive, negative, and doubtful respectively. *P < 0.05. 3.4 Whole genome sequencing of inocula, animals in comparison with vaccinated ones, being this difference nasal swab, and BALF collected from significant at 6 dpi (t-test; p=0.0186) with a mean Ct value of 24,23 vaccinated and nonvaccinated animals ± 1,33 and 27,68 ± 2,51, respectively. In total, 32 out of 39 and 31 out of 40 nasal swab samples analyzed from vaccinated and The whole swine IAV genome from inocula A/swine/Spain/ nonvaccinated animals, respectively, were RT-qPCR swine IAV SF11131/2007 (H1N1) and A/Swine/Spain/SF32071/2007 (H3N2) positive. Besides, 23 vaccinated and 26 nonvaccinated samples was sequenced (Figure 3A). From samples collected after contact obtained Ct values lower than 35. challenge, 39 swine IAV genomes were obtained, 20 from vaccinated Regarding BALF, lung, and nasal turbinate samples, swine IAV animals and 19 from nonvaccinated ones (Supplementary Table S1). was detected in all samples except in BALF from vaccinated animal After sequenced samples alignment, a total of 2.467.438 reads of No. 11 and lung from animal No. 2. No significative differences swine IAV were obtained, 1.347.544 from vaccinated samples and were observed between groups, showing similar mean Ct values 1.119.899 from nonvaccinated ones; 67% of these reads matched (Figure 3B; Supplementary Table S1). All positive BALF samples against the H1N1 genome whereas the remaining 33% did it against had a Ct value lower than 35. H3N2 subtype (Supplementary Table S2). TABLE 1 HI titers of sera samples collected from vaccinated and nonvaccinated animals against H1N1 (left) and H3N2 (right) strains used on the challenge. HI titers against H1N1 HI titers against H3N2 Euthanasia Day Euthanasia Day Group Pig ID 42 dpv 6 dpi 7 dpi 8 dpi 9 dpi 42 dpv 6 dpi 7 dpi 8 dpi 9 dpi 9 0 0 160 80 10 0 20 80 80 11 0 320 40 80 Vaccinated Animals 12 0 0 80 40 13 0 0 80 160 14 0 160 160 320 15 0 0 0 10 16 0 0 0 40 17 0 0 0 0 Nonvaccinated Animals 18 0 0 40 40 19 0 0 0 80 20 0 0 0 0 Sampling days are specified on the top of each table. Empty cells indicate no sampling. Titers greater than 40 are considered positive. Frontiers in Cellular and Infection Microbiology 06 frontiersin.org López-Valiñas et al. 10.3389/fcimb.2023.1111143 A B FIGURE 3 Swine IAV genome detection in samples collected from H1N1 and H3N2 seeders, vaccinated and nonvaccinated pigs (A) Daily swine IAV RNA detection in samples collected from nasal swabs. (B) Swine IAV genome detection in BALF, lung, and nasal turbinates collected on the euthanasia day. The Ct values are represented as a heat map where the maximum intense red colors represent higher viral loads and vice versa. RT-qPCR CT values greater than 40 were considered negative, represented in white. No sampling collected was represented as grey. The ID of each animal and the day at which each sample was collected are represented in ordinate and abscissa axes, respectively. Regarding the depth per position and the coverage (Figure 4), between H1N1 and H3N2 sequences (Supplementary Table S3). 74.8% of H1N1 swine IAV whole genome positions sequenced were Hence, 597 SNVs were further analyzed. represented with a depth of more than 50 reads. On the other hand, the 6% of the H3N2 position sequenced were also represented by more than 50 reads. Those positions were selected for further 3.5 Determination of H1N1 and H3N2 variant analysis. In general, NS and M segments obtained the genomic segment per sample greatest median depth values (Figure 4), meanwhile, the biggest segments PA, PB1 and PB2 got the lowest values. In relation to the subtype segments profile per sample, in A total of 1947 out of 2544 called variants were deleted because vaccinated animals we obtained 95 H1N1 segments, 18 H3N2 of misalignment reads due to the high percentage of similarity segments, and 41 segments where both subtypes were simultaneously TABLE 2 Lung pathological results according to the percentage of total lung-affected area, and the semi-quantitative scoring for the degree of airways affectation and amount of immunoreactivity. Group Pig ID Euthanasia day Lung affected area (%) Histopathological score Immunohistochemical score 9 8 dpi 17.96 3 ++ 10 8 dpi 31.89 3 ++ 11 8 dpi 5.52 2.5 – Vaccinated pigs 12 6 dpi 3.19 3 ++ 13 6 dpi 0.19 2.5 ++ 14 9 dpi 6.09 2.5 ++ mean 10.81 2.75 15 8 dpi NO DATA 1.5 ++ 16 9 dpi 4.86 3 + 17 6 dpi 0.48 2 ++ Nonvaccinated pigs 18 8 dpi 5.43 2.5 ++ 19 9 dpi 5.65 3 + 20 6 dpi 0.62 1.5 ++ mean 3.41 2.25 Histopathological score: absence (0), few inflammatory cells isolated (0.5), a localized cluster of inflammatory cells (1), several clusters of inflammatory cells (1.5–2), severely several (2.5), and many airways affected (3). Moreover, minimal (1.5), mild (2) interstitial infiltrate, and plus moderate interstitial and alveolar infiltrate (2.5–3). Immunohistochemical score: absence (-), low (+), scattered (++), moderate (+++), and abundant (++++) amount of immunoreactivity. Frontiers in Cellular and Infection Microbiology 07 frontiersin.org López-Valiñas et al. 10.3389/fcimb.2023.1111143 A B FIGURE 4 Illumina sequencing reads aligned against each subtype genomic profile. (A) Illumina sequencing profile of swine IAV H1N1 (plotted in red) and H3N2 (plotted in green) used as inocula. In the x axis each position per genomic segment is indicated whereas the depth of read is indicated in y axis in the logarithm scale. (B) Heat map of the median depth value obtained in all sequenced samples from vaccinated and nonvaccinated animals per H1N1 (left) and H3N2 (right) genomic segments. Genomic segments per subtype and samples are indicated in the x and y axes, respectively. The name of each sequenced sample indicates the origin and the day of its collection. BALF samples were collected at each pig necropsy. found (Figure 5). Meanwhile, in nonvaccinated pigs, 42 H1N1, 50 of allele frequency whereas none of them did it for the H3N2 H3N2, and 60 segments with both subtypes were found (Figure 5). The inoculum (Table 3). distribution of H1N1 and H3N2 segments was statistically different These SNV were later detected in samples collected from vaccinated between groups (P = 0.0002, P = 0.000018, respectively; chi-squared), and nonvaccinated animals in the HN1 inoculum (Figure 6). In most of being greater the number of H1N1 segments in vaccinated animals and the sequenced samples, the SNVs G211R (NS1), V80V (M1), X70E greater the number of H3N2 segments in nonvaccinated ones. (M2), L395L, and V418 (PB1) increased their allele frequency over time. Regarding the number of segments in which sequences from both On the other hand, allele frequencies of NS1 K108T, M1 N87N and subtypes were found, in vaccinated animals this number was lower Y132Y, NA N50K, PA E325G, PB1 N382N, and PB2 substitutions than that of nonvaccinated ones, although this difference was not tended to decrease until they were no longer detected, with some significant (P = 0.0587; chi-squared). exceptions (Figure 6). Notably, all SNV detected in H3N2 inoculum were no later detected in sequenced samples. 3.6 Variants detected in inocula and their evolution in samples sequenced from 3.7 De novo SNVs identification; infected pigs synonymous and nonsynonymous proportion and allocation along swine From both inocula, 14 and 9 SNV were detected from H1N1 IAV genome segments and H3N2 sequences, respectively (Table 3). For H1N1 sequences, 8 out of 14 substitutions found were synonymous, whereas for H3N2 A total of 313 SNVs were found in all analyzed samples, 187 in ones all SNV detected were nonsynonymous. Furthermore, most of vaccinated animals and 126 in nonvaccinated ones (Figures 7 and the SNV detected in H1N1 sequences (12 out of 14) exceed the 5% 8). In vaccinated pigs, 172 were found in H1N1 and 15 in H3N2 Frontiers in Cellular and Infection Microbiology 08 frontiersin.org López-Valiñas et al. 10.3389/fcimb.2023.1111143 nonsynonymous variants were found in polymerase segments in samples from vaccinated animals, while only 14 were reported in nonvaccinated pigs. In the HA and NA segments, 12 and 13 nonsynonymous substitutions were reported from vaccinated animals while only 3 and 5 were found in the nonvaccinated ones, respectively. Regarding the NS, a total of 17 nonsynonymous variants were identified in vaccinated animals and 9 in nonvaccinated pigs. Lastly, segments in which the least nonsynonymous substitutions appeared were in the M and the NP proteins, with 6 and 7 in vaccinated and 4 and 2 in nonvaccinated pigs, respectively (Figure 8A). Regarding SNV found in the H3N2 subtype, only 8 nonsynonymous substitutions were found in vaccinated animals, 2 in PB2, 2 in PB1, 2 in M, and only 1 in NA and NS. Nevertheless, no nonsynonymous substitutions were reported in PA, HA, and NP (Figure 8B). On the other hand, in the nonvaccinated group, the largest number of nonsynonymous variants were also found in the polymerase segments, 11 in PA, 11 in PB1 and 8 in PB2. In the remaining segments, 4 nonsynonymous variants were detected in NS, 2 in M, and only one was reported in HA, NA, and NP (Figure 8B). 3.8 De novo nonsynonymous SNV with an allele frequency greater than 5% are allocated on protein domains FIGURE 5 For the H1N1 subtype, the following nonsynonymous SNV Profile subtype identification per genomic segment in each sequenced sample heatmap. H1N1 subtype segments are indicated exceeding the 5% of allele frequency were reported in vaccinated in red cells, H3N2 ones are indicated in green cells and yellow ones animals: NS1 (T91I and L146P), ion channel (M2) (I51N and indicate that both subtypes were simultaneously found. Genomic G58D), NA (V394I), NP(A286T, T360A, S407N, and V456L), HA segments and samples collected from vaccinated and nonvaccinated animals are represented on the x and y axes (E111K, N275S, S278P, T408A, V466I, and K467R), PA (T528I, respectively. The number of each sequenced sample indicate the V645A, and V645I), PA-X (E114K, R208K, and V218A), PB1 animal from which each sample was collected. Sample collection (R187G, A370V, F512C, C625G, S642N, W666L, and S720F), day are also indicated as dpi in nasal swab samples; BALF samples were collected on the day of necropsy. PB1-F2 (K29R and P33L), and PB2 (L77V, D195N, T364K, I394T, Q566L, and D671G). Meanwhile, in nonvaccinated animals, substitutions were allocated in NS1 (K66E, L79I, and subtypes, meanwhile, in nonvaccinated animals 67 and 59 were F137Y), M2 (G58D), matrix protein (M1) (Q158L and E204V), identified per subtype, respectively. Regarding SNVs per subtype, NA (S90P and N209K), HA (V216I and V466I), PA (I407V and 239 were found in H1N1 and 74 in H3N2 (Figure 7). V505I), PA-X (R208K), PB1 (A370V and K388R), PB1-F2 (F57Y), The proportions of the total synonymous and nonsynonymous and PB2 (D195N) (Figures 9 and 10). de novo SNV were studied from 1 to 10% allele frequency in Regarding substitutions found in the H3N2 subtype in sequenced samples from vaccinated and nonvaccinated animals vaccinated pigs, SNVs were noted in PB2 (N9D and P14S), PB1- (Figure 7). Regarding the H1N1 subtype, the proportion of F2 (H15L), and NA (S333G). On the other hand, in nonvaccinated nonsynonymous variants was greater than synonymous ones for animals substitutions found were allocated in NS1 (W102L and all allele frequencies analyzed in both experimental groups. This R211G), nuclear export protein (NS2) (T33I), HA (I351K), PA difference was greater in vaccinated animals although no statistical (I120L, G316E, W317L, S388G, Y393C, I407V and M561V), PB1 differences between groups were found (Figure 7A). In contrast, (T57S, Q202K. C625G, A652D, and F699L), PB1-F2 (H15L), and these proportions were very close to 50% in nonvaccinated animals, PB2 (N71T, T215P, N659I, L665R, and K702E) (Figures 9 and 10). being this trend consolidated throughout the allele frequencies All the substitutions found in the present study, including those analyzed. For H3N2, the proportion of nonsynonymous SNVs with lower allele frequency, were noted in Supplementary Table 4. was greater in both scenarios, specifically in samples from nonvaccinated animals (Figure 7B). Considering those variants whose allele frequency was greater 3.9 Nucleotide diversity than 1%, SNV were allocated along the whole genome of both studied subtypes (Figure 8; Supplementary Table S4). In general, more Within the H1N1 subtype, the nucleotide diversity (p) values substitutions were found in samples from vaccinated animals for fluctuated over time in both experimental groups (Figure 11A). The the H1N1 subtype (Figure 8A). Specifically, a total of 54 de novo p significantly increased in vaccinated animals from 5 to 6 dpi, and Frontiers in Cellular and Infection Microbiology 09 frontiersin.org López-Valiñas et al. 10.3389/fcimb.2023.1111143 TABLE 3 SNV detected in H1N1 and H3N2 inoculum samples. Aminoacidic Nucleotide change change Depth of ref. ! Alt. Base Allele Effect on ref. ! Gene read position alt. count frequency variant position alt. 860 323 A!C 107 12.44 nsyn 108 K!T NS1 731 631 G!A 228 31.19 nsyn 211 G!R 681 240 C!T 435 63.88 syn 80 V!V M1 702 261 T!C 23 3.28 syn 87 N!N 961 396 C!T 14 1.46 syn 132 Y!Y 603 896 R!A 312 51.74 nsyn 70 X!K M2 603 896 R!G 291 48.26 nsyn 70 X!E Inoculum H1N1 NA 274 150 T!A 56 20.44 nsyn 50 N!K PA 189 1055 A!G 10 5.29 nsyn 352 E!G 244 1146 T!C 52 21.31 syn 382 N!N PB1 256 1185 C!T 48 18.75 syn 395 L!L 322 1254 Y!C 132 40.99 syn 418 V!V 405 1054 T!C 98 24.2 syn 352 L!L PB2 990 168 G!A 224 22.63 syn 56 P!P HA 589 1111 C!T 15 2.55 nsyn 371 H!Y 369 1046 A!G 15 4.06 nsyn 349 E!G 574 290 C!T 13 2.26 nsyn 97 T!I PA 4042 2047 C!A 161 3.98 nsyn 683 L!I Inoculum 760 1852 G!A 10 1.32 nsyn 618 V!I H3N2 PB1 827 1308 C!T 12 1.45 nsyn 436 Y!Y 3026 153 G!A 33 1.09 nsyn 51 M!I PB2 440 395 C!T 15 3.41 nsyn 132 P!L 1044 2033 A!C 11 1.05 nsyn 678 D!A ref. (reference), alt. (alternative), nsyn (Nonsynonymous) and syn (Synonymous). from 7 to 8 dpi (P = 0.03134 and 0.003948, respectively; Kruskal- 4 Discussion wallis). However, p decreased at 5 and 7 dpi in comparison with each previous day, although this decrease is not significant. Within In this work, a commercial trivalent vaccine was applied to the H3N2 subtype, the p only significantly increased from 5 to 6 dpi immunize animals from the vaccinated group. On the day of the (P = 0.000221; Kruskal-wallis). In general, p means were greater in seeders contact, higher levels of antibodies against NP swine IAV vaccinated animals. However, the p mean in nonvaccinated animals protein were detected in vaccinated animals, with the highest was slightly higher at 7 dpi, and significantly higher at 9 dpi (P = percentage of competition in pig No. 9. Regarding HI activity, 0.04804; Kruskal-wallis). On the other hand, within H3N2 subtype, before the challenge, only sera from vaccinated animals had higher levels of p were observed in samples from nonvaccinated hemagglutinating titers against H3N2 subtype. On the contrary, pigs at 5, 6, and 8 dpi, whereas in vaccinated animals the highest no sera had HI activity against H1N1 at the time of contact with levels of p were observed at 9 dpi. seeders, although it was subsequently detected in vaccinated pigs Regarding p per genomic segment per group, the diversity was No. 10, 11, and 12 at necropsy day. This is a common effect that the higher within the H1N1 subtype in comparison with the H3N2 one vaccine provides a priming effect, and a boost is observed when the (Figure 11B). A greater p mean was observed in all segments of the virus challenge is inoculated. This has been widely described for H1N1 subtype in vaccinated animals, except in M and NS segments. influenza and other viruses (Ganges et al., 2008; Dı́az et al., 2013; The highest p was found in the HA. No significant differences per Tarradas et al., 2014; Kim et al., 2021; Pliasas et al., 2022). Thus, we genomic segment and group were observed. consider that the vaccine against H1N1 has a limited specific Frontiers in Cellular and Infection Microbiology 10 frontiersin.org López-Valiñas et al. 10.3389/fcimb.2023.1111143 FIGURE 6 Evolution of the allele frequencies of nonsynonymous substitutions detected in H1N1 inoculum and later in samples collected from both experimental groups. The day of variant detection and the allele frequency percentage is represented in ordinate and abscissa axes, respectively. The “In.” in x axis indicates the percentage obtained in the inoculum, whereas 4 to 9 numbers indicate the days post-inoculation. In each column, the different animals are represented while in each row NS1, M1, M2, NA, PA, PB1, and PB2 proteins are indicated. Each mutation is plotted in different colors. immune effect. According to swine IAV detection, the virus was affected area and histopathological score means were greater in detected in seeders, vaccinated, and nonvaccinated animals. Hence, vaccinated animals, meanwhile similar results between groups were the reproduction of a direct contact infection with swine IAV was observed in relation to the immunohistochemical score. In achieved. Besides, the Ct value means indicated that, from 4 to 9 vaccinated animals, animals No. 9 and 10 had the highest dpi, the viral loads were greater in nonvaccinated animals, especially percentage of lung lesions with 17.96 and 31.89%, respectively. at 6 dpi when mean Ct value is 3,45 times lower than vaccinated The remaining animals had similar values to pigs from the ones. Therefore, the vaccine had an effect in terms of modestly nonvaccinated group. Hence, pigs No. 9 and 10 seemed to be reducing viral seeding, so selective pressure on virus genome is considered outliers. It cannot be ruled out that those animals may feasible to study due to humoral immunity caused by vaccination be suffering from vaccine-associated enhanced respiratory disease (Murcia et al., 2012; Ló pez-Valiñas et al., 2021; Ló pez-Valiñas (VAERD), that has been previously described for influenza et al., 2022). immunization (Vincent et al., 2008; Gauger et al., 2011; Kimble On the contrary, the effect of the vaccine was not as effective et al., 2022) This adverse effect is produced when the inactivated regarding the pathological findings, as the percentage of lung vaccine and the infection strains are of the same subtypes but with A B FIGURE 7 De novo synonymous and nonsynonymous SNV proportion bars in samples from vaccinated and nonvaccinated animals at different allele frequencies. (A) SNV proportions found in the H1N1 subtype are represented in red bars (B) SNV proportions found in the H3N2 subtype are represented in green bars. The total number of SNV and its percentages are indicated in each bar. Dark and soft colors represent nonsynonymous and synonymous SNV, respectively. Frontiers in Cellular and Infection Microbiology 11 frontiersin.org López-Valiñas et al. 10.3389/fcimb.2023.1111143 A B FIGURE 8 De novo synonymous and nonsynonymous SNV with an allele frequency greater than 1% allocation along swine IAV genome. (A) SNVs found in the H1N1 subtype are represented in red circles. (B) SNV found in the H3N2 subtype are indicated in green circles. Nonsynonymous and synonymous substitutions are represented in dark and light colors, respectively. Circle size indicates the total number of SNV found per genomic segment and samples, in abscissa and ordinate axes respectively. heterologous antigenicity. In consequence, and overall, the vaccine et al., 2022). The amino acid identity percentage matrix among HAs worked in terms of viral shedding reduction, but it did not avoid the and NAs from both, challenge, and vaccine strains, are available in swine IAV clinical and pathological manifestations. the Supplementary Table S5. Herein, 41 swine IAV quasispecies were analyzed by NGS, Regarding the SNV and the nucleotide diversity found in both including 2 inocula, 20 samples from vaccinated animals, and 19 inocula, the H1N1 strain used resulted more genetically diverse from nonvaccinated ones. In samples from vaccinated animals, than the H3N2 one, since more SNVs were reported, and they were H1N1 segments alone were more widely detected in each genomic also represented with a higher allele frequency. Substitutions found profile (95 times), meanwhile, H3N2 segments alone were barely in the H1N1 inoculum were also detected in samples collected from detected (18 times). On the contrary, in the nonvaccinated group, both experimental groups, although SNVs allele frequencies were the presence of both segments simultaneously found was the most later decreasing over time, or they were no longer detected. frequent profile detected, followed by H3N2 alone. These results However, there were also some substitutions such as, PB2 P56P, indicate that vaccination modified the proportion of viral PB1 V418V, M1 V80V, M2 X70K/E, and NS1 G211R, whose allele populations, favoring the dominance of the H1N1 subtype, frequencies increased in some samples over time. These variants probably because of the absence of strong detectable specific continue belonging to the viral population despite having suffered a immunity against this subtype. Such effect seemed to decrease the bottleneck effect, as showed in Figure 11 where p decreased in viral probability of two subtypes simultaneously coexistence, therefore samples recovered from infected pigs in comparison with the resulting in a decreased probability of genomic reassortment. inoculum. As opposed to that observed for H3N2, the lack of a Contrary, because of the dominant presence of the H1N1 subtype strong detectable neutralizing activity against H1N1 in vaccinated in vaccinated pigs, a greater increase in swine IAV nucleotide animals may support the higher genomic diversification of H1N1 diversity, and the greatest number of de novo SNVs (total of 170) subtype. Considering the quasispecies theory, these mutants could were found. The reduction in the presence of H3N2 segments is also display a beneficial effect on virus fitness, since they were present in reflected in the number of de novo SNV in this subtype (total of 15). the initial viral population mutant spectrum, and they remained Thus, in this scenario, the application of the vaccine caused the after a selection event, meanwhile the remaining disappeared. On H1N1 swine IAV prevailing over H3N2, which has an impact on its the other hand, all SNVs detected in the inoculum H3N2 viral evolution. On one hand, the probability of reassortments was quasispecies spectrum were no longer detected in virus collected reduced in vaccinated pigs, as has been previously reported (Li from any pig samples. All variants detected in both inocula were et al., 2022). On the other hand, swine IAV H1N1 genetic variability probably generated, and maintained over time, during virus increased in this group of animals. In the long term, this could allow passages in MDCK cells, because of virus adaptation to cells. viral genomic diversification and, in consequence, increasing the Thus, the loss of these variants is probably due to the re- likelihood of immune escape mutants generation, as previously adaptation of the virus to its natural host. reported in IAV (Zharikova et al., 2005; Sitaras et al., 2014; The proportion of nonsynonymous and synonymous de novo Yasuhara et al., 2017; Park et al., 2020; Sitaras et al., 2020; Xu SNV found may indicate that the viral quasispecies evolution is Frontiers in Cellular and Infection Microbiology 12 frontiersin.org López-Valiñas et al. 10.3389/fcimb.2023.1111143 A B C D E F G H I J FIGURE 9 Lolliplot representation of nonsynonymous SNV allocation on swine IAV proteins. Substitutions found in NS1 (A), NS2 (B), M1 (C), M2 (D), NP (E), PA (F), PA-X (G), PB2-F2 (H), PB1 (I) and PB2 (J) proteins with an allele frequency greater than 5%. In the ordinate axis, the allele frequency of each substitution is indicated. Substitutions noted in blue and yellow show substitutions found in vaccinated and nonvaccinated animals. Besides, the circle shape indicates that substitution was reported in H1N1 subtype, whereas substitutions found in H3N2 are represented by square shapes. The number inside each shape indicates the pig ID of each reported substitution. Figure legends indicate the most important domains of each protein. under selective pressure (Sobel Leonard et al., 2016). In the H1N1 nonsynonymous substitutions were generated in vaccinated subtype, in vaccinated animals the proportion is greater, indicating animals, which could be indicating that, additionally, the humoral that viral evolution may be under positive selection in this scenario immune pressure is acting as an evolutionary selection force. By (Fitch et al., 1991; Li et al., 2011). However, neutral selection may be contrast, in the H3N2 subtype, in nonvaccinated animals, the acting in nonvaccinated animals, as the same proportion of SNVs highest proportion of nonsynonymous mutations may be was detected (Frost et al., 2018). Therefore, in both scenarios, indicating that the virus is also under positive selection. Similar genetic diversification is occurring. Many of the new mutations results have been previously found with the same strain; therefore, generated in H1N1 subtype during the experimental trial probably virions may be poorly adapted to swine hosts, probably because of play a neutral role in viral fitness, such as the synonymous variants, the readaptation from cell culture (Sobel Leonard et al., 2016; while others are generated because of the viral readaptation from Ló pez-Valiñas et al., 2022). In vaccinated animals, as previously cell culture to its natural host. However, a higher proportion of mentioned, the viral shedding was greatly reduced and, Frontiers in Cellular and Infection Microbiology 13 frontiersin.org López-Valiñas et al. 10.3389/fcimb.2023.1111143 A B FIGURE 10 Localization of nonsynonymous substitutions, with an allele frequency greater than 5%, found in HA and NA surface glycoproteins on 3D and lolliplot protein representation. (A) HA trimer structure (PDB accession no. 3LZG (Xu et al., 2010)) and domains representation by lolliplot. HA1 and HA2 domains are represented in different tones of blues and reds respectively. (B) NA tetramer structure (PDB accession no. 4B7Q (van der Vries et al., 2012)) and domains representation by lolliplot. In both 3D representations, substitutions are highlighted in blue, yellow and orange if it were found in samples from vaccinated, nonvaccinated and both, respectively. In the lolliplot representation, substitutions are plotted in blue and yellow (vaccinated or nonvaccinated groups, respectively), circle and squared shapes (H1N1 and H3N2 subtypes, respectively) and number on shapes indicate the animal in which each substitution was found. In the ordinate axis, the substitution allele frequency is indicated. The different surface glycoprotein domains are indicated in lolliplot legends. consequently, not enough variants have been detected. Therefore, Diaz et al., 2015; Ló pez-Valiñas et al., 2021; Ló pez-Valiñas et al., the main force that drives viral evolution in this scenario could not 2022), since 313 de novo synonymous and nonsynonymous SNV be inferred. were reported from all the quasispecies herein analyzed. During In this study, the rapid evolutionary capacity and plasticity of virus replication, many generated mutations are not able to generate swine IAV are again evidenced (Murcia et al., 2012; Diaz et al., 2013; virus progeny, so they are lost. However, those mutations that are A B FIGURE 11 Evolution of the nucleotide diversity (p) in H1N1 and H3N2 viral quasispecies in vaccinated and nonvaccinated animals over time (A) and per genomic segment (B). Dots in the boxplot indicates p in each genomic segment. Boxplots indicate means, lower and upper quartile, and standard deviation. The nucleotide diversity of H1N1 and H3N2 inocula, and samples collected from vaccinated and nonvaccinated animals are represented in red, green, blue, and yellow respectively. *P < 0.05 and **P < 0.001.. Frontiers in Cellular and Infection Microbiology 14 frontiersin.org López-Valiñas et al. 10.3389/fcimb.2023.1111143 beneficial for viral fitness are naturally selected and will increase Schulze, 1991; Murcia et al., 2012; Thompson and Paulson, 2021). their allelic representation in the viral quasispecies (Domingo and Notably, the N275S substitution was reported with a 97.58% of Perales, 2019). With this regard, in the present study, most of the allele frequency in the vaccinated animal No. 9 at 4 dpi, although its substitutions were not detected with an allele frequency greater than allele frequency decreased to 5.58% in BALF at necropsy of this pig. 5%, so they do not pose an adaptive evolutionary advantage for the Finally, the remaining 3 de novo substitutions, T408A, V466I, and virus. For instance, in the H1N1 subtype in vaccinated animals, only K467R, were detected on the stalk domain of the protein. Two of 63% of all nonsynonymous SNVs found did not exceed the 5% of these substitutions were also reported from animal No. 4, reaching allele frequency. By contrast, 41 (H1N1) and 4 (H3N2) 36.64% of allele frequency for K467R. In a previous similar nonsynonymous substitutions with an allele frequency greater experiment, substitutions in this domain were also found in the than 5% were found in viral samples from vaccinated animals. H3N2 subtype in vaccinated animals, finding exactly one These substitutions were allocated in all swine IAV proteins, except substitution at position 467 (T467I) (Ló pez-Valiñas et al., 2022). M1 and NS2 ones. Regarding the polymerase segments (PB2, PB1, Notably, relevant nonsynonymous substitutions in vaccinated PA), 25 nonsynonymous substitutions were found in the H1N1 animals were not found on the H3N2 HA protein. subtype of vaccinated animals. Single substitutions within these NA is a surface glycoprotein that plays an important role in proteins have been related to an increase in IAV polymerase virion progeny realizing (McAuley et al., 2019). In vaccinated activity, increasing its virulence and facilitating host interspecies animals, only two nonsynonymous substitutions, whose allele jump (Subbarao et al., 1993; Song et al., 2015; Yu et al., 2015; Cai frequency exceeded 5%, were reported, both in the head domain et al., 2020). In the H1N1 NS1 protein, two substitutions were found of the protein. These substitutions, S333G in NA of the H3N2 in the effector binding domain, and both were detected in animal subtype and V394I in NA of the H1N1 one, were located on the No. 9. Substitution T91I is allocated in the domain that recruits the exposed part of the protein. The first one is allocated in the epitopic host eukaryotic translation initiation factor 4G1 (eIF4G), favoring region F’329–339 (Ge et al., 2022), while V394I is nearby to viral mRNA translation instead of host mRNA (de la Luna et al., previously described epitopes(Wan et al., 2015; Jiang et al., 2016). 1995; Aragó n et al., 2000). In addition, substitution L146P is Interestingly, V394I was also detected with 73.7% allelic frequency allocated in the nuclear export signal; according to a previous in viral samples from animal No. 9. Therefore, in this animal, 4 study, leucine at this position is required for NS1 cytoplasmatic important substitutions have been detected in both surface localization. Although such substitution was detected in the glycoproteins. This pig, together with animal No. 10, had the mentioned study, it was only found on day 5 dpi with an allele highest gross pathological score. Therefore, in this animal, apart frequency of 13% (Li et al., 1998). In relation to the NP, 4 from the potential VAERD effect, these mutations may be affecting nonsynonymous substitutions were found in the H1N1 subtype, viral fitness, increasing its replicative capacity, and eventually its while none were identified in the H3N2 one. These results were virulence. Altogether, all substitutions found in both HA and NA, detected in previous studies carried out in our group, where the may have an impact on evading the immune system by the virus, evolutionary capacity of H1N1 and H3N2 subtypes was separately although further analyses with reverse genetic technology would be evaluated, and 6 nonsynonymous substitutions were detected in the required (Li et al., 2021). H1N1 subtype and none in the H3N2 one (Ló pez-Valiñas et al., In conclusion, our findings support the wide capacity of evolution 2021; Ló pez-Valiñas et al., 2022). and adaptation to changing environments of swine IAV. In the Surface glycoproteins HA and NA proteins are highly present study, a lower probability of viral reassortment was observed immunogenic, constituting the main target to generate in vaccinated animals, as has been recently reported (Li et al., 2022). neutralizing antibodies by the host after IAV infection or However, the vaccine did not avoid the genetic diversification of vaccination (Li et al., 2011; Eichelberger and Wan, 2014; Ma, swine IAV. Herein, nonsynonymous substitutions were found in viral 2020). Thus, both proteins are under high immune pressure when quasispecies collected in vaccinated animals, and the role that they a previously immunized host becomes infected with the virus, could play in the evasion of the immune system was hypothesized which triggers a greater genetic drift (Li et al., 2011; Krammer, according to previous literature. Finally, the present study underlines 2019). The HA is a viral surface glycoprotein that attaches to sialic the importance of performing surveillance and genomic studies of acid allocated in the swine host respiratory tract, allowing virus cell swine IAV, as well as increasing the vaccination rate in pigs, to reduce entry (Russell, 2021). In the present study, 6 de novo substitutions the circulation of the virus in the field as much as possible, reducing (E111K, N275S, S278P, T408A, V466I, and K467R) with an allele the reassortment events and minimizing the risk of pandemics in frequency greater than 5% were detected in vaccinated animals. The both, humans, and pigs. E111K in the vestigial esterase subdomain is allocated in a previously described epitope region of the protein(Eisenlohr et al., 1988; Yewdell, 2010). Substitutions N275S and S278P were found in the receptor binding domain (RBD) of the protein, where the main Data availability statement antigenic sites of the virus are allocated (Xu et al., 2010; Guo et al., 2018). It has been already described that single substitutions in this The datasets presented in this study can be found in domain are related to the virus interspecies switch (Aytay and online repositories. The names of the repository/repositories Frontiers in Cellular and Infection Microbiology 15 frontiersin.org López-Valiñas et al. 10.3389/fcimb.2023.1111143 and accession number(s) can be found below: https:// Acknowledgments www.ncbi.nlm.nih.gov/, PRJNA902942. The authors would like to thank Laura Baioni for the sequencing libraries preparation. The authors also want to thank Ethics statement the IRTA-CReSA animal facilities staff for their work during the in vivo experiment. Lastly, we would like to thank Marta Pé rez and all The animal study was reviewed and approved by Animal ethics the people from CReSA who were involved in the experiment committee from the Generalitat de Catalunya. during the COVID-19 lockdowns. Conflict of interest Author contributions The authors declare that the research was conducted in the Conceptualization: JIN. Methodology: Á L-V, MV, MW, JIN, absence of any commercial or financial relationships that could be LG, and JS. HTS libraries preparation: CC. Bioinformatic analysis: construed as a potential conflict of interest. Á L-V. Animal experiment: GC, JS, and JIN. Formal analysis: Á L-V and JIN. Resources: AD and JIN. Writing—original draft Publisher’s note preparation: Á L-V. Writing—review and editing: Á L-V, JS, LG, and JIN. Supervision: JIN. project administration: JIN. Funding All claims expressed in this article are solely those of the authors acquisition: AD and JIN. All authors contributed to the article and and do not necessarily represent those of their affiliated approved the submitted version. organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Funding This research was funded by grants AGL2016–75280-R from Supplementary material Ministerio de Ciencia, Innovació n y Universidades from the Spanish government. Á L-V has a pre-doctoral fellowship FPI The Supplementary Material for this article can be found online 2017, Ministerio de Ciencia, Innovació n y Universidades from the at: https://www.frontiersin.org/articles/10.3389/fcimb.2023.1111143/ Spanish government. full#supplementary-material References Andrews, S. (2010) FastQC: A quality control tool for high throughput sequence data. Busquets, N., Segalé s, J., Có rdoba, L., Mussá , T., Crisci, E., Martı́n-Valls, G. E., et al. Available at: https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ (Accessed (2010). 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