TYPE Original Research PUBLISHED 15 September 2023 DOI 10.3389/fcimb.2023.1258321 Genetic diversification patterns OPEN ACCESS in swine influenza A virus EDITED BY Lester J. Perez, Abbott, United States (H1N2) in vaccinated and REVIEWED BY Mingbin Liu, nonvaccinated animals Nanchang University, China Velmurugan Balaraman, Kansas State University, United States Álvaro López-Valiñas 1,2,3*, Marta Valle 1,2,3, Marta Pérez 1,2,3, *CORRESPONDENCE Ayub Darji 1,2,3, Chiara Chiapponi 4, Llilianne Ganges 1,2,3,5, José I. Núñez joseignacio.nunez@irta.cat Joaquim. Segalés 2,3,6 and José I. Núñez 1,2,3* ́ lvaro López-Valiñas A 1 IRTA, Programa de Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Bellaterra, alvarolvd@gmail.com Barcelona, Spain, 2 Unitat Mixta d’Investigació IRTA-UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain, 3 WOAH RECEIVED 13 July 2023 Collaborating Centre for the Research and Control of Emerging and Re-Emerging Swine Diseases in ACCEPTED 25 August 2023 Europe (IRTA-CReSA), Barcelona, Spain, 4 WOAH Reference Laboratory for Swine Influenza, Istituto PUBLISHED 15 September 2023 Zooprofilattico Sperimentale della Lombardia ed Emilia-Romagna, Brescia, Italy, 5 WOAH Reference CITATION Laboratory for Classical Swine Fever, IRTA-CReSA, Barcelona, Spain, 6 Departament de Sanitat i López-Valiñas Á, Valle M, Pérez M, Darji A, Anatomia Animals, Universitat Autònoma de Barcelona, Barcelona, Spain Chiapponi C, Ganges L, Segalés J and Núñez JI (2023) Genetic diversification patterns in swine influenza A virus (H1N2) in vaccinated and nonvaccinated animals. Front. Cell. Infect. Microbiol. 13:1258321. Influenza A viruses (IAVs) are characterized by having a segmented genome, low doi: 10.3389/fcimb.2023.1258321 proofreading polymerases, and a wide host range. Consequently, IAVs are COPYRIGHT constantly evolving in nature causing a threat to animal and human health. In © 2023 López-Valiñas, Valle, Pérez, Darji, 2009 a new human pandemic IAV strain arose in Mexico because of a Chiapponi, Ganges, Segalés and Núñez. This is an open-access article distributed under reassortment between two strains previously circulating in pigs; Eurasian the terms of the Creative Commons “avian-like” (EA) swine H1N1 and “human-like” H1N2, highlighting the Attribution License (CC BY). The use, importance of swine as adaptation host of avian to human IAVs. Nowadays, distribution or reproduction in other forums is permitted, provided the original although of limited use, a trivalent vaccine, which include in its formulation H1N1, author(s) and the copyright owner(s) are H3N2, and, H1N2 swine IAV (SIAV) subtypes, is one of the most applied strategies credited and that the original publication in this journal is cited, in accordance with to reduce SIAV circulation in farms. Protection provided by vaccines is not accepted academic practice. No use, complete, allowing virus circulation, potentially favoring viral evolution. The distribution or reproduction is permitted evolutionary dynamics of SIAV quasispecies were studied in samples collected which does not comply with these terms. at different times from 8 vaccinated and 8 nonvaccinated pigs, challenged with H1N2 SIAV. In total, 32 SIAV genomes were sequenced by next-generation sequencing, and subsequent variant-calling genomic analysis was carried out. Herein, a total of 364 de novo single nucleotide variants (SNV) were found along all genetic segments in both experimental groups. The nonsynonymous substitutions proportion found was greater in vaccinated animals suggesting that H1N2 SIAV was under positive selection in this scenario. The impact of each substitution with an allele frequency greater than 5% was hypothesized according to previous literature, particularly in the surface glycoproteins hemagglutinin and neuraminidase. The H1N2 SIAV quasispecies evolution capacity was evidenced, observing different evolutionary trends in vaccinated and nonvaccinated animals. KEYWORDS swine influenza virus, viral evolution, vaccination, NGS, quasispecies Frontiers in Cellular and Infection Microbiology 01 frontiersin.org López-Valiñas et al. 10.3389/fcimb.2023.1258321 1 Introduction reassortment swine H3N2, and A(H1N1) pdm09 virus, are the most circulating subtypes among the pig population (Simon-Grifé et al., Influenza A viruses (IAVs) are worldwide distributed pathogens 2011; Torremorell et al., 2012; Kyriakis et al., 2013; Simon et al., 2014; Li with a very wide host range, being able to infect many birds and and Robertson, 2021). Nowadays, vaccination against SIAV, although mammal species, and with increasing zoonotic potential (Webster limited, is the most effective and used strategy to control SIAV in the et al., 1992). The IAV genome is composed of 8 single-stranded viral swine population, being administered to approximately 10-20% of the RNA segments of negative polarity, with a total length of 13.6 kb European pig population (Reeth et al., 2016). One of the most used (Zhou et al., 2009). The genomic segments have different sizes, vaccines against SIAV is the trivalent one, which includes in its ordered from largest to smallest size as follows: the polymerase basic formulation inactivated strains of the H1N1, H3N2, and H1N2 2 (PB2), polymerase basic 1 (PB1), polymerase acid (PA), surface SIAV subtypes. This vaccine reduces both, disease signs and viral glycoproteins hemagglutinin (HA), nucleoprotein (NP), shedding. However, due to the genomic distance between farm neuraminidase (NA), matrix segment (M), and non-structural circulating and vaccine formulation strains, the protection generated (NS) (Breen et al., 2016). Each segment codes for at least one during vaccination is not complete, allowing virus circulation, protein with the same name, except M and NS segments that also potentially favoring viral evolution (Thacker and Janke, 2008; Ma code for the ion channel protein (M2) and the nuclear export and Richt, 2010). In previous studies, the viral evolution patterns of the protein (NS2/NEP), respectively, by alternative RNA splicing SIAV H1N1 and H3N2 subtypes were analyzed in vaccinated and (Lamb et al., 1980; Lamb et al., 1981). Furthermore, there are nonvaccinated pigs, showing different evolutionary patterns in both mRNAs with open reading frames that code for accessory viral experimental groups (Ló pez-Valiñas et al., 2021; Ló pez-Valiñas et al., proteins recently identified, such as the polymerase basic 1 frame 2 2022; Ló pez-Valiñas et al., 2023). However, the evolutionary pressure (PB1-F2), and the PA-X proteins (Wise et al., 2009; Jagger et al., generated by the trivalent vaccine on SIAV H1N2 subtype is still 2012; Hao et al., 2020). poorly studied. The evolution of IAVs, such as other RNA viruses, is described Considering this background, the present work aimed to study the under the quasispecies theory, where a viral population is composed evolutionary dynamics of SIAV H1N2 quasispecies under immunity of a spectrum or cloud of mutants (Eigen, 1971; Eigen and Schuster, pressure and the identification of de novo nonsynonymous 1977; Bull et al., 2005; Lauring and Andino, 2010; Schuster, 2016; substitutions that may have an impact on viral fitness and host Domingo and Perales, 2019). This implies an adaptive advantage for immune system evasion. Therefore, 32 whole SIAV genomes the virus because of the phenotypic variants reservoir that could be recovered at different time points from challenged vaccinated and beneficial for virus adaptability in pressure events, such as immune nonvaccinated pigs were determined by next-generation sequencing escape mutants and drug resistance (Domingo et al., 2012; Schuster, (NGS), showing different evolutionary patterns between both 2016). There are three molecular mechanisms that provide genetic experimental groups. variability to the IAV genomes, influencing its evolution: point mutations, genomic reassortments, and recombination, although the last phenomenon does not seem to play an important role 2 Materials and methods during IAV evolution (Chare et al., 2003; Krammer et al., 2018). The addition of point mutations in IAVs is due to the low rate of 2.1 Cells, viruses, and vaccine fidelity of their polymerases. Consequently, RNA viruses can add and accumulate a lot of point mutations in their genomes during Madin-Darby Canine Kidney (MDCK, ATCC CCL-34) cells their replication. The IAVs mutation rate is high, on the order were used for viral production and titration. Dulbecco’s Modified between 10-3 and 10 -4 substitution per gen per generation, allowing Eagle Medium (DMEM) supplemented with foetal bovine serum rapid and progresSIAVe evolution of the virus (Ahlquist, 2002). (FBS) (10%), L-glutamine (1%) and penicillin/streptomycin (1%) IAV genome regions such as the epitope regions of the surface was used for cell culture in an incubator at 37°C with 5% glycoproteins HA and NA are constantly under positive CO2 atmosphere. evolutionary pressure since they are the main target for the host The strain (A/Swine/Spain/80598LP4/2007(H1N2)) used in this immune system. Over time, these regions can evolve until they are study was first propagated in MDCK monolayer cell culture at a no longer recognized by the previous immunity of the host multiplicity of infection (MOI) of 0.01. Virus cell infection was population, in case of existing, therefore generating escape promoted applying 10 µg/mL of porcine trypsin (Sigma-Aldrich, variants (Shao et al., 2017). This phenomenon is known as Madrid, Spain). After 48 hours, infected cells were harvested. For antigenic drift, which can generate new viral subtypes (Nelson viral titration, the 50% tissue culture infection dose (TCID50) was and Holmes, 2007; Russell et al., 2008). The accumulation of calculated carrying out a serial dilution in MDCK cells (Reed and point mutations could influence on IAVs host range, virulence, Muench, 1938). and antiviral resistance (Domingo et al., 2012; Shao et al., 2017). In the present study, a commercial vaccine against SIAV Therefore, due to the pandemic risk posed by swine IAVs (SIAVs), (RESPIPORC FLU3, Ceva®, Libourne, France), which includes deeper virus genetic evolution mechanism understanding and the H1N2 (Bakum/1832/2000), H1N1 Haselünne/IDT2617/2003), constant surveillance studies in the field are required. Currently, and H3N2 (Bakum/IDT1769/2003) inactivated strains in its “avian-like” swine H1N1, “human-like” swine H1N2, “human-like” formulation, was used. Frontiers in Cellular and Infection Microbiology 02 frontiersin.org López-Valiñas et al. 10.3389/fcimb.2023.1258321 2.2 Experiment design and A sample of each lung showing gross lesion was fixed by immersion samples collection in 10% buffered formalin. After that, each lung section was dehydrated, and embedded in paraffin wax. For light microscopy examination, Sixteen 5-week-old naïve domestic pigs were used. Pigs were hematoxylin-eosin (HE) staining and immunohistochemistry (IHC) randomly distributed in two groups (A and B) of 8 animals each, were done separately in sections of tissue with a thickness of 3-5 mm located in different boxes at the animal biosafety level 3 (BSL3) facilities (Busquets et al., 2010; Sisteré-Oró et al., 2019). For IHC, the two-step at IRTA-CReSA (Bellaterra, Spain). The animals spent an acclimation polymer method (Envision TM) was performed using a primary and period of 1 week. After that, pigs from group A were vaccinated secondary antibody (Sabattini et al., 1998). For the evaluation of the lung according to manufacturer’s instruction, with administration of 2 mL histopathological findings and the amount of immunoreactivity on HE intramuscularly in the neck. Animals from group B, received in parallel and IHC slides, respectively, previously described semi-quantitative 2 mL of phosphate buffered saline (PBS) administrated as previously scoring methods were used (Detmer et al., 2013). mentioned. A second vaccination dose were carried out 21 days post- vaccination (21 dpv), where animals from group A and B received the 2.5 SIAV RNA detection, whole genome commercial vaccine and PBS, respectively. amplification, and sequencing All animals were challenged with (A/Swine/Spain/80598LP4/2007 (H1N2) strain, three weeks after the second vaccination dose (42 dpv). The MagAttract 96 Cador Pathogen kit ® (Qiagen, Düsseldorf, The virus was inoculated thorough two different administration routes, Germany) was used to extract RNA from nasal swabs, BALF, lung, and intranasal and endotracheal. The final viral concentration inoculated NT samples, following the manufacturer’s instructions. Lung and NT was 107 TCID50 in a final volume of 2 mL for the intranasal and 5 mL samples were previously homogenized with TissueLyser II (Qiagen, for the endotracheal inoculation, as previously described (Ló pez- Düsseldorf, Germany) in brain heart infusion medium (10% Valiñas et al., 2021). After challenged, animals were serially weight/volume). euthanized, 3 from each group 2 days post inoculation (2 dpi), 3 per SIAV RNA detection and quantification were performed on group at 5dpi, and the remaining 4 pigs by the end of the in vivo each sample using an RT-qPCR assay for IAV in the Fast7500 experiment (9 dpi). equipment (Applied Biosystem) (Spackman et al., 2002). Samples Nasal swab samples were collected at each immunization, at that showed fluorescence were considered positive and the cycle challenge, and daily post-challenge until the euthanasia day, for threshold (Ct) value parameter obtained was used for SIAV SIAV detection and genome sequencing. Blood samples were quantification (Spackman et al., 2002; Ló pez-Valiñas et al., 2021). collected at each immunization, challenge, and necropsy day for The inoculum used on the challenge and all BALF and nasal antibody detection against SIAV. At necropsy, lung, nasal turbinate swab samples with a Ct value lower than 35 in RT-qPCR assay were (NT) and broncho-alveolar lavage fluid (BALF) were collected and proposed for whole SIAV genome sequencing (Zhou et al., 2009; stored at -80°C, for SIAV detection and NGS. Furthermore, a lung Ló pez-Valiñas et al., 2021). extra sample was stored in formalin for pathological analysis. First, a whole SIAV genome amplification was performed. The After challenge, rectal temperature, clinical signs (Galindo- PCR reaction mix conditions were as follows: forward MBTuni-12 and Cardiel et al., 2013)and animal behaviour were daily monitored. reverse MBTuni-13 primers both at 0.2 mM (0.5 mL each), Procedures were approved by the animal ethics committee from the SuperScript® III One-Step RT-PCR System with Platinum™ Taq Generalitat de Catalunya, under the project number 10442, High Fidelity DNA Polymerase (Thermo Fisher Scientific, Waltham, following the Spanish and European regulations. MA, USA) (0.5 mL), reaction mix included in the kit (12.5 mL), RNasa free water (8.5 mL) and SIAV RNA from each sample(2.5 mL) (Zhou et al., 2009). Furthermore, to enhance the biggest SIAV segments 2.3 Evaluation of the humoral response amplification another RT-PCR was carried parallelly out, replacing against SIAV MBTuni12 primer with MBTuni12G (Lycett et al., 2012). The rest of the RT-PCR conditions were maintained. All amplified samples In serum samples collected, the antibody levels against influenza showing a whole genome amplification profile by agarose gel NP were calculated using ID Screen®—influenza A Antibody electrophoresis were selected for sequencing (Zhou et al., 2009). Competition ELISA kit (ID VET, Grabels, France), following Illumina technology was used for whole genome SIAV sequencing. Nextera-XT DNA Library Prep protocol (Illumina®, manufacturer instructions. Inhibition percentages lower than 45% indicate positive sera, greater than 50% negative ones, and San Diego, CA, USA) was followed to obtain a multiplexed percentages between both values indicate doubtful results sequencing library per sample. Miseq Reagent Kit v2 in a 150- according to kit instructions. cycle paired-end run on Miseq Instrument was used. 2.4 Pathological analyses and 2.6 Bioinformatic workflow for immunohistochemistry in lung quasispecies genomic and evolutionary analysis The percentage of the lung-affected area of each necropsied animal was measured with ImageJ ® software, as previously Herein, a bioinformatics workflow for aligning Illumina reads described (Sibila et al., 2014). against a known reference and variant calling was developed in the Frontiers in Cellular and Infection Microbiology 03 frontiersin.org López-Valiñas et al. 10.3389/fcimb.2023.1258321 bash programming language. In this pipeline, tools widely used for competition ELISA, pig rectal temperatures, RT-qPCR Ct viral genome analysis were applicated. (Sobel Leonard et al., 2016; values, and % of lung affected area, in vaccinated and Borges et al., 2018; Cacciabue et al., 2020). nonvaccinated animals at different time points. Before that, all First, FastQC (v 0.11.8) (Andrews, 2010) software was used for variable distributions and means variances were studied using illumina reads quality checking. Low-quality reads (Phread < 30) Shapiro and Levene tests, respectively. Paired t-test was also were trimmed by Trimmomatic (v0.39) (Bolger et al., 2014). applied to observe increases in rectal temperatures within the Inoculum reads were first aligned against the reference genome same group on different days. The differences between H1N2 sequence (HF674912-19) (Baratelli et al., 2014), as explained synonymous and nonsynonymous proportions at different allele below. Subsequently, the inoculum consensus sequence was frequencies per group were analyzed by applying the Chi-squared generated using the consensus option from Bcftools (v.1.9) test and the subsequent Bonferroni correction. To compare p (Danecek et al., 2021). For the subsequent variant calling and means, an analysis of variance (ANOVA) and a post-hoc Kruskal- genomic analysis, all sequenced samples were mapped against the Wallis test by rank were applied. All statistical analyses were inoculum consensus sequence. carried out using R (RStudio, 2021). For reads mapping the Burrows-Wheeler alignment (BWA) tool mem function (v0.7.17) was used (Li and Durbin, 2009). After that, unmapped and low-quality mapped (<30) reads were 3 Results removed using Samtools (v.0.39) (Danecek et al., 2021). Moreover, the Picard “MArkDuplicatesSpark” and 3.1 Humoral response, viral replication, and “BaseRecalibrator” algorithms included in GATK4 (v4.1) were pig rectal temperature kinetics used for both; PCR duplicates removal and reads recalibration. The depth of reading of samples on each genomic segment was At challenge day, the NP antibody levels mean detected in sera calculated by “-depth” option included on Samtools (v1.9). The samples from vaccinated animals (58.14 ± 13.54)) were higher than coverage and depth plots were performed with the ggplot2 library those of nonvaccinated ones (88.16 ± 6.89), being this difference in Rstudio (RStudio, 2021; Wickham, 2016). significant (t-test;p = 0.00000663) (Figure 1A). At this time point, All variants found along the SIAV genome from sequenced vaccinated animals No. 3 and 4 were considered positive, No. 1 and samples in comparison with the inoculum consensus sequence 8 doubtful, and the remaining ones negative. On the other hand, all were noted using LoFreq software with default parameters (Wilm sera samples from nonvaccinated animals were considered negative, et al., 2012). After that, the effect on each variant detected was where any antibody levels detected exceeded the lowest value of predicted by SnpEff software (v.4.3) (Cingolani et al., 2012). For vaccinated ones. After the challenge, the SIAV NP ELISA values this purpose, a database has previously been built using as a increased over time, maintaining as average higher values in reference a prior annotated H1N2 genome (Baratelli et al., 2014) vaccinated animals. using “build–gtf22” function included in SnpEff. For single In the present study, SIAV RNA was detected after the nucleotide variant (SNV) calling the following requirement challenge in both experimental groups. On average, viral loads were considered; p value < 0.01, 100 reads of depth, and 10 detected along time in nasal swab samples were always higher in reads of alternative base count (Ló pez-Valiñas et al., 2021). nonvaccinated animals reaching significant differences at 4 and 5 Finally, the nucleotide diversity (p) was calculated by using dpi (t-test; p=0.00628, and p= 0.02927, respectively) (Figure 1B; SNPGenie software (Nelson et al., 2015). Table 1). No SIAV was detected on the day of the challenge. Furthermore, SIAV genome was detected in all BALF, lung, and NT samples from both experimental groups at 2 and 5 dpi with no 2.7 Protein structure representation significant differences in Ct values between groups. At 9 dpi, the detected Ct values were close to the detection limit, being negative For the Lolliplot protein representation, the trackViewer in all samples collected from vaccinated animal No. 7. SIAV RNA package from Bioconductor (Ou and Zhu, 2019) was used. In was not detected in NT samples at 9 dpi. addition, the HA and NA 3D protein was represented by the In total, 42 and 46 out of 51 nasal swabs, BALF and tissue PyMOL Molecular Graphics System (v.4.6). The functional samples analyzed after the challenge tested positive for SIAV in domains of all protein representations were delimited according vaccinated and nonvaccinated animals, respectively. In to previous literature (Sriwilaijaroen and Suzuki, 2012; Hu et al., vaccinated animals only 60% of the samples reached Ct values 2014; McAuley et al., 2019). In the protein structure lower than 35, while in nonvaccinated animals this percentage representations, the nonsynonymous substitutions found with an increased until 84% of the total samples analyzed within the allele frequency greater than 5% were pointed out. group (Table 1). Regarding BALF, lung, and nasal turbinate samples, SIAV RNA was detected in all samples collected at 2 and 5 dpi with ct values 2.8 Statistical analysis lower than 35 (Figure 1B; Table 1). Notably, in euthanized animals at 9 dpi, SIAV was also detected in BALF and lung samples from T-test statistical analysis was herein used to determine animals 8 and 15, and also in BALF from animal 15, all with a ct significant differences between the average values of % value greater than 35. SIAV was not detected in NT samples Frontiers in Cellular and Infection Microbiology 04 frontiersin.org López-Valiñas et al. 10.3389/fcimb.2023.1258321 A B C FIGURE 1 Evaluation of the vaccine effect through humoral response, viral replication, and pig rectal temperature profile. (A) SIAV NP antibody titres boxplot. The percentage of competition is expressed in the ordinate axis. Boxplot whiskers show quartile variability. Values below 50% (grey line) are considered negative, while values above 45% (red line) are considered positive. Values between both lines are considered doubtful. (B) SIAV detection in nasal swab samples heatmap. In the y-axis, each ID animal number from both experimental groups is indicated. The heat map shows cells with different red intensities, from the darkest to the lightest, indicating a range of viral loads from higher to lower, respectively depending on the Ct value obtained per nasal swab sample. White cells represent SIAV negative samples (Ct values > 40) meanwhile, grey ones indicate no sample was collected at that time point. (C) Pig rectal temperature kinetics after SIAV challenge. The temperature measured in °C is represented in the ordinate axis. Horizontal lines represent the temperature profile of each pig over time. Pigs with values above 40°C were considered to have fever. In all figures, the sampling time is indicated in the abscissa axis. Vaccinated, and nonvaccinated animals are represented in blue and green dots, boxplots, and lines, respectively. Besides, the dot shape and number indicate each animal ID. * p < 0.05 and *** p < 0.0001. Paired t-test was used for statistical analysis. collected at 9 dpi. No significant differences were observed between moderate amount of labelling was detected in vaccinated pigs No. groups, showing similar mean values 5, and 6, both necropsied at 5 dpi. By contrast, the highest One day after H1N2 SIAV challenge, 4 vaccinated and 6 immunoreactivity score was detected at 2 dpi in nonvaccinated nonvaccinated animals had fever (Figure 1C). On average, higher animals. No labelling was detected in animals necropsied at 9 dpi. rectal temperatures were found at 1 dpi in nonvaccinated animals Airway histopathological scoring: absence (0), few isolated (40.93 ± 0.33) in comparison with vaccinated ones (39.83 ± 0.296), inflammatory cells (0.5), localized cluster of inflammatory cells being this difference significant (t-test; p = 0.0075) (Figure 1C; (1), several clusters of inflammatory cells in few affected airways Supplementary Table S1). Furthermore, temperatures only (1.5), several clusters of inflammatory cells in several affected increased significantly from challenge day to 1 dpi, and from 1 airways (2), extenSIAVe clusters of inflammatory cells in several dpi to 2 dpi, in nonvaccinated animals (paired t-test; p = 0,044 and affected airways (2.5), and extenSIAVe inflammation in many p=0.0196, respectively.) (Supplementary Table 1). The remaining airways (3). Immunoreactivity scoring: absence (-), low (+), days, no significant differences between groups were found, except scattered (++), moderate (+++), and abundant (++++) amount of at 3 dpi where the temperature was also higher in nonvaccinated SIAV antigen. pigs (t-test; p = 0.01579). 3.3 Whole genome SIAV sequencing of 3.2 Pathological findings inoculum, nasal swab, and BALF samples On average, the percentage of lung affected areas was lower in The SIAV H1N2 strain (A/Swine/Spain/80598LP4/2007 vaccinated animals, although the difference between both groups (H1N2)) whole genome used in the challenge was determined was not significant (Table 2). In vaccinated pigs, the gross (Figure 2A.1). Regarding samples collected after the challenge, a pneumonic lesion percentage was lower than 1 in all animals, total of 32 samples were sequenced, 12 samples from vaccinated except in animal No. 1 which percentage reached the highest animals, and 20 from nonvaccinated ones (Figures 2A.2, value among all necropsied animals at 2 dpi (5.12%). On the 2A.3; Table 1). other hand, in nonvaccinated animals the highest percentage of After quality control and mapping, a total 1.932.820 Illumina lung-affected area were recorded at 5 dpi. SIAV H1N2 reads were obtained; 32.936, 664.005, and 1.232.879 reads Regarding airway histopathological scoring, vaccinated animals in inoculum, vaccinated, and nonvaccinated sequenced samples. The showed as average the lowest score. However, vaccinated animal maximum number of mapped reads obtained per sample were No. 1, together with nonvaccinated animal No. 15 scored the 178.966 in BALF sample collected from nonvaccinated animal No. maximum value. Regarding immunohistochemistry labelling, a 12, and 177.727 reads in nasal swab sample collected at 3 dpi from Frontiers in Cellular and Infection Microbiology 05 frontiersin.org López-Valiñas et al. 10.3389/fcimb.2023.1258321 TABLE 1 SIAV RNA detection in nasal swabs, BALF, lung, and NT samples collected from vaccinated and nonvaccinated pigs. Nasal swab samples Tissue Sample Group Pig ID 0dpi 2dpi 3dpi 4dpi 5dpi 6dpi 9dpi Euthanasia day BALF Lung NT 1 Neg. 32.6 2 dpi 24.22 23.91 25.1 2 Neg. 31.5 29.73 31.5 27.42 3 Neg. 29.15 27.93 19.56 24.58 4 Neg. 34.72 32.44 Neg. 37.49 5 dpi 31.12 31.71 28.41 5 Neg. 25.66 26.41 28.72 28.76 27.67 23.92 28.35 Vaccinated Animals 6 Neg. 35.22 38.55 33.88 36.59 26.13 30.2 32.64 7 Neg. Neg. 33.17 36.37 Neg. Neg. Neg. 9 dpi Neg. Neg. Neg. 8 Neg. 37.38 35.47 32.68 31.56 37.55 37.08 37.86 36.97 Neg. mean 32.32 33.21 32.91 33.60 37.55 37.08 29.24 28.25 27.75 sd 3.683 4.011 2.762 3.594 0.000 0.000 4.091 5.548 2.642 9 Neg. 29.66 2 dpi 29.99 27.44 32.43 10 Neg. 32.22 29.73 25.22 27.42 11 Neg. 28.26 23.62 23.15 23.85 12 Neg. 29.61 27.26 24.74 31.02 5 dpi 26.7 25.3 27.66 13 Neg. 32.1 33.57 28.1 28.45 25.91 22.38 28.35 Nonvaccinated Animals 14 Neg. 30.38 28.09 26.07 27.65 24.33 26.26 26.26 15 Neg. 27.11 25.35 27.21 30.08 33.98 Neg. 9 dpi 39.01 36.48 Neg. 16 Neg. 27.77 29.18 27.55 28.01 Neg. 32.97 35.04 Neg. Neg. mean 29.64 28.69 26.73 29.04 33.98 32.97 29.29 26.60 27.66 sd 1.89 3.07 1.34 1.44 5.39 4.69 2.82 Neg. (negative). Bold numbers indicate sequenced samples. The table shows the RT-qPCR Ct values obtained per sample. The columns below nasal swab samples indicate the experiment day of collection. The euthanasia day of each pig is also indicated. Means and standard deviation per day, per sample, and per group are indicated. animal No. 7. On the other hand, the minimum number of mapped detected in NS1, NA, PA, and PB1 SIAV genes. Most of the reads obtained were 14.269, and 14.169 in samples collected from the genetic substitutions herein reported were found with a low allelic nonvaccinated animal No. 13 at 4 dpi, and the vaccinated pig No. 5 at frequency, although 4 of them (NS1(E187K), NA(E156E), PB2 5 dpi, respectively (Supplementary Table S2). (V327V and E399E)) exceeded 10% of allele frequency Regarding the depth and coverage obtained per whole SIAV representation (Table 3). genome sequenced sample, 88.91% of each genome position Most of these SNVs substitutions were later detected in sequenced was represented by at least 50 Illumina reads of depth. sequenced samples collected from both, vaccinated and SIAV H1N2 sequenced positions with more than 50 reads of nonvaccinated animals (Figure 3). All these substitutions tended representation were selected for further variant analysis. In to reduce their allelic representation over time or were not even general, the smallest SIAV segments NS and M were sequenced detected again in most animal sequenced samples, with a few with greater depth representation. By contrast, segments PA and exceptions. Nonsynonymous substitution in the NS1 gene only PB1 obtained the lowest depth read representation, followed by the increased its allele frequency in pig No. 14, exceeding in both cases HA segment (Figure 2B; Supplementary Table S2). the 25% of allele frequency. Regarding synonymous substitutions, the E74E (NA) allele frequency increased in many of the samples, especially in the vaccinated animal No. 7. In the E593E (PA) 3.4 Inoculum SNVs and their subsequent substitution, the allele frequency only increased in pig No. 14. By detection in pig collected samples last, in the PB2 segment, the E538E substitution increased its allelic frequency in many of the samples later sequenced, especially in In the inoculum sequence, a total of 10 SNVs were detected; 3 animals No. 4, 8, and 14. Likewise, the frequency of the V403V nonsynonymous and 7 synonymous variants. SNVs were only substitution also tends to increase, particularly in animals 4 and 14. Frontiers in Cellular and Infection Microbiology 06 frontiersin.org López-Valiñas et al. 10.3389/fcimb.2023.1258321 TABLE 2 Lung pathology. Pig Euthanasia Lung affected area Airway histopathological Immunoreactivity Group ID day (%) score score 1 5.12 3 ++ 2 2 dpi 0.71 1 + 3 0.50 2 + 4 1.00 2 – Vaccinated Animals 5 5 dpi 0.75 1 +++ 6 0.28 1 +++ 7 0.00 0.5 – 9 dpi 8 0.21 0.5 – mean 1.07 1.38 9 1.78 2.5 ++ 10 2 dpi 1.90 2.5 +++ 11 0.68 2.5 +++ 12 6.78 2.5 + Nonvaccinated 13 5 dpi 5.14 2.5 + Animals 14 3.19 2.5 + 15 0.64 3 – 9 dpi 16 0.00 0.5 – mean 2.51 2.31 Total lung-affected area percentage, airways affected semi-quantitative scoring and amount of immunoreactivity. Immunoreactivity score: absence (-), low (+), scattered (++), moderate (+++), and abundant (++++) amount of immunoreactivity. Finally, the L281L substitution tended to decrease its allelic (Figure 4B; Supplementary Table S3). In samples collected from frequency, except in animals No. 6 and 8 (Figure 3). nonvaccinated animals, most of the de novo nonsynonymous SNVs with an allele frequency greater than 1 were found in the polymerases (PB2, PB1, and PA), M, and NS segments. Regarding these types of 3.5 De novo SNVs identification; SNVs in nonvaccinated pigs, 9, 11, and 10 out of 17, 17, and 20 synonymous and nonsynonymous substitutions exceeded an allelic frequency of 5% in PB2, PB1, and PA, proportion and allocation along respectively. Notably, only 4 out of the 21 nonsynonymous SIAV genome substitutions found in the M segment exceed the 5% of allele frequency. The largest number of nonsynonymous SNVs counted in In the present study, a total of 367 de novo SNV were detected, nonvaccinated animals was found in the NS segment, although only 3 112 in samples collected from vaccinated animals and 255 in out of 39 SNVs were detected with an allelic frequency greater than 5%. samples collected from nonvaccinated ones (Figure 4A). On the contrary, the lowest number of mutations per segment in this Considering the number of sequences obtained per group, on group of animals was found in the NP segment with only 5 average 9.33 substitutions per sample were produced in nonsynonymous SNVs (Figure 4B; Supplementary Table S3). vaccinated animals, while 12.75 were produced in nonvaccinated On the other hand, in vaccinated animals, the highest number of ones, being this difference not significant (t-test). nonsynonymous substitutions (n=17) with an allele frequency greater In relation to the proportion of synonymous and nonsynonymous than 1 was also found in the NS segment, and 5 out of them de novo SNVs counted per group, the nonsynonymous SNVs were exceeded 5%. In this group of animals, most of the nonsynonymous proportionally more abundant. Interestingly, in vaccinated animals, substitutions in polymerase segments were found in PB2. In the PA, the proportion of nonsynonymous substitutions was always higher only 2 substitutions were found, being the lowest number of than in nonvaccinated animals along all allelic frequencies analyzed, nonsynonymous SNVs found. As in nonvaccinated animals, most although significant differences were found (chi-squared substitutions found in polymerase segments did not exceed 5% allelic test) (Figure 4A). representation. In NP and M segments, 7 and 6 nonsynonymous De novo SNVs variants were allocated along all SIAV genome substitutions were found, reaching 5% of allele frequency 4 and 1 of segments in sequenced samples from both experimental groups them, respectively. Frontiers in Cellular and Infection Microbiology 07 frontiersin.org López-Valiñas et al. 10.3389/fcimb.2023.1258321 A.1 A.2 A.3 B FIGURE 2 Inoculum, vaccinated, and nonvaccinated NGS sequencing profile samples per genomic segment in terms of depth and coverage. (A) The Illumina sequencing profile of SIAV H1N2 is plotted in orange (A1). The Illumina profiles of samples collected from vaccinated and nonvaccinated animals are represented in different tones of blues (A2) and greens (A3), respectively. In the abscissa axis, the SIAV genomic segments and each position are represented. In the ordinate axis, the depth of reading is represented in the logarithm scale. (B) Median depth value per sample per genomic segment Heat map. The darker colors in the heat map represent higher median depth values; on the contrary, the lighter ones represent a lower value. Inoculum, vaccinated, and nonvaccinated samples are represented in orange, blue, and green respectively. The sequenced samples are represented in the y-axis, where the name indicates the pig ID and the day of sample collection. BALF samples were collected on the day of each pig necropsy. The SIAV genomic segments are indicated on the x-axis. Regarding nonsynonymous SNVs detected in the surface representation in vaccinated animals while all did so in nonvaccinated glycoproteins HA, and NA, according to the number of sequences ones. In the NA, 10 substitutions were found in nonvaccinated pigs while obtained, proportionally more substitutions were found in vaccinated 7 were found in the vaccinated group. Considering the 5% of allele pigs. In the HA segment, with an allelic frequency of 1%, 7 substitutions frequency, more substitutions were found in vaccinated animals with 5 were found in each group, and 4 of them exceeded 5% allelic substitutions found, meanwhile 4 were found in nonvaccinated group. Frontiers in Cellular and Infection Microbiology 08 frontiersin.org López-Valiñas et al. 10.3389/fcimb.2023.1258321 TABLE 3 Single nucleotide variants detected in the inoculum. Nucleotide Aminoacidic change change ref. → ref. → Depth of Alt. Base Allele fre- Effect on Gene position alt. position alt. read count quency variant 454 G→A 152 E→K 187 20 10.7 Nsyn NS1 363 G→T 121 Q→H 229 11 4.8 Nsyn 1327 A→G 443 I→V 215 18 8.37 Nsyn NA 222 G→A 74 E→E 156 29 18.59 Syn PA 1779 G→A 593 E→E 128 11 8.59 Syn Inoculum H1N2 213 C→T 71 N→N 1085 71 6.54 Syn 843 G→A 281 L→L 392 15 3.83 Syn PB2 1209 G→A 403 V→V 327 51 15.6 Syn 1614 A→G 538 E→E 399 109 27.32 Syn 2265 G→A 755 R→R 880 34 3.86 Syn alt. (alternative), ref. (reference), nsy(Nonsynonymous) and syn (Synonymous). 3.6 De novo nonsynonymous SNV with an nonvaccinated animals nonsynonymous substitutions which allele allele frequency greater than 5% allocation frequency exceed 5% were reported in PB2 (I76M, R80K, D87N, on protein domains H151R, R293K, D309A, M535I, T559I, and V640I), in PB1 (A63V, F94Y, N455D, R572K, S702G, and L753F), in PB1-F2 (D23G, T27I, In the present study, the following de novo SNVs substitutions E31G, S57F, and E70G), PA-X (Y48H, and G66S), PA (A369T, with an allele frequency were detected in vaccinated animals in each E493A, H535Y, and T608I), HA (P100L, S154P, N172D, A278T, SIAV H1N2 protein; in PB2 (T215S, Q257R, and R389G), in PB1 P308A, and V466I), NP(F346I, and N368S), NA (P79S, K296R, and (R727G), PA (C489S, H535Y, and W537R), HA (L335I, V466I, S298Y), matrix protein (M1) (D94N, T169I, and R243W), ion C492Y, and M525I), in NP(E64K, F346I, T373K, and R436K), NA channel (M2) (E75G), NS2 (E81K) and NS1(L166F and D189G). (R60G, K93R, R172K, V398I, and D463Y), M2(T28I) and NS1(S7L, All the substitutions found in this study with an allele frequency E142G, L163S, and R200K) (Figures 5, 6). Meanwhile, in greater than 1% were noted in the Supplementary Table S4. FIGURE 3 Allele frequencies evolution of H1N2 inoculum SNVs over time in pig sequenced samples. The SNV allele frequency percentage is represented in the y-axis. The time is represented in the x-axis, where “In.” is the day of the inoculation, and 2, 3, 4, 5, and 9 are the days post-inoculation. Each animal and genomic segment are represented in columns and rows, respectively. Dots show SNV detection plotted in different colors, while lines join the same mutation detected at different time points in the same animal. nsyn (Nonsynonymous) and syn (Synonymous). Frontiers in Cellular and Infection Microbiology 09 frontiersin.org López-Valiñas et al. 10.3389/fcimb.2023.1258321 A B.1 B.2 FIGURE 4 De novo H1N2 SNVs detected in samples collected from Vaccinated and nonvaccinated animals after challenge. (A) Synonymous and non- synonymous SNVs proportion bars with allele frequencies greater or equal to 2.5, 5, 7.5, and 10 per group. The total number of variants found and the percentage that it represents by type of variant, group, and allele frequency studied are indicated. (B) H1N2 de novo SNV allocation bubble plot representation with an allele frequency greater than 1 (B.1) and 5% (B.2). The bubble size proportionally represents the total number of SNVs found per genome segment (x-axis) and sequenced sample (y-axis). Synonymous and nonsynonymous substitutions are represented in light and dark orange, respectively. 3.7 Nucleotide diversity Nevertheless, the effect of vaccine application on H1N2 SIAV evolution through NGS technology is poorly studied. The study Regarding nucleotide diversity, it was high for NA at day 2 and of the evolution of this subtype could provide an enriching point of 3 dpi, while the trend at days 4 and 5 dpi was decreasing in both view about virus evolution, in addition to the fact that it is one of the experimental groups, although significant differences were not main currently circulating SIAV in swine population (Simon et al., found (Figure 7). 2014; Ma, 2020), and one of the subtypes from which the pandemic A(H1N1)pdm virus arose (Garten et al., 2009). Hence, the present work focuses on the study of the evolutionary dynamics of SIAV 4 Discussion human-like H1N2 subtype under the effect of the most widely used vaccine in the field. The high capacity of mutation and adaptability of IAVs, the Herein, a trivalent SIAV vaccine was applied to one group of wide host range, and their interspecies transmission, make of IAVs animals. At day of the challenge, the antibody titters against SIAV a constant threat to both, human and animal health (Mostafa et al., NP were statistically higher in vaccinated animals. Regarding SIAV 2018). Due to the important role of swine in human-to-avian IAVs detection, it was detected in both experimental groups after the adaptation, the study of SIAVs evolution might be of great value to challenge. The viral loads detected in vaccinated animals were lower understand their genetic diversity and interaction with the host to in nasal swab, BALF, lung, and NT in comparison with avoid new strain emergence. Nowadays, vaccine application is used nonvaccinated ones. Moreover, the number of samples that tested in swine population to reduce the SIAV circulation and mitigate the RT-PCR positive were also lower in vaccinated pigs. After challenge, disease (Reeth et al., 2016; Salvesen and Whitelaw, 2021). However, rectal temperatures were lower in vaccinated animals, increasing the genetic differences between SIAV strains used in vaccine temperature significantly in animals that did not receive the formulation and current circulating strains, make that the vaccine. In relation to pathological findings, the same pattern was immunity generated by vaccine is not complete, allowing virus found, with vaccinated animals showing less severe and extenSIAVe replication and evolution. Previous studies have shown different lesions. Hence, in the present study, although limited, the effect of evolution patterns on H1N1 and H3N2 SIAV subtypes, that may be the vaccine in viral shedding and clinical signs reduction was caused by different host immune pressures (Ló pez-Valiñas et al., proved. However, viral replication was not prevented by 2021; Ló pez-Valiñas et al., 2022; Ló pez-Valiñas et al., 2023). vaccination, facilitating the virus to acquire novel nucleotide Frontiers in Cellular and Infection Microbiology 10 frontiersin.org López-Valiñas et al. 10.3389/fcimb.2023.1258321 A B C D E F G H I J FIGURE 5 Nonsynonymous SNV allocation on SIAV proteins lolliplot representation. 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 ordinate axis, the allele frequency of each substitution is indicated. Substitutions noted in blue circles and green squares show substitutions found in vaccinated and nonvaccinated animals, respectively. Number inside each shape indicates the pig ID of each reported substitution. Figure legends indicate the most important domains of each protein. variants, as previously found (Murcia et al., 2012; Ló pez-Valiñas nonsynonymous ones (E152K and Q229H in NS1, and I443V in et al., 2021; Ló pez-Valiñas et al., 2022; Ló pez-Valiñas et al., 2023). NA). All substitutions were also later detected in samples collected In the present work, 33 SIAV whole genome sequences were from both experimental groups, although their allelic frequencies in analyzed by NGS, including the H1N2 inoculum, 12 samples general decreased in comparison with the initial allelic frequency of collected from vaccinated pigs, and 20 collected from the inoculum, or even they were no longer detected. Therefore, nonvaccinated ones. Regarding the SNVs detected in the these mutations did not imply a benefit on viral fitness, so they were inoculum, a total of 10 SNVs were detected, 3 of them lost over time. Considering that virus was grown on MCDK cells Frontiers in Cellular and Infection Microbiology 11 frontiersin.org López-Valiñas et al. 10.3389/fcimb.2023.1258321 A B FIGURE 6 Location of nonsynonymous SNVs found with an allele frequency greater than 5% on Tri-Dimensional and Lolliplot surface SIAV glycoproteins HA and NA representation. (A) HA trimer (PDB accession no. 3LZG (Xu et al., 2010)). HA1 domains are represented in blue, while HA2 ones are represented in red colors. (B) NA tetramer (PDB accession no. 4B7Q (van der Vries et al., 2012)). In lolliplot representation, substitutions found in vaccinated and nonvaccinated animals are represented in blue circles and green squares, respectively. The numbers within each mutation marked on the lolliplot show the animal in which it was detected. The numbers below each protein representation indicate the amino acid boundaries between the different domains. The allele frequency at which each substitution was reported is indicated in the ordinate axis. before challenge, the nucleotide diversity loss of the inoculum could SIAV subtypes (Ló pez-Valiñas et al., 2021; Ló pez-Valiñas et al., be explained by the virus readaptation of their natural host after a 2022; Ló pez-Valiñas et al., 2023). On the other hand, this bottleneck effect (Sobel Leonard et al., 2016; Cortey et al., 2018). proportion was around 50% for sequences from nonvaccinated In the present study, the rapid evolutionary capacity and animals suggesting that a neutral selection could be acting in this plasticity of SIAV H1N2 under vaccination and nonvaccinated scenario (Frost et al., 2018). Thus, the genome of SIAV is scenarios is again proved, as 367 substitutions were reported from diversifying in both scenarios (vaccination versus non- sequences of all studied pigs, further supporting previous results vaccination), acquiring synonymous substitutions that could play with other SIAV strains (Murcia et al., 2012; Diaz et al., 2013; Diaz a neutral role in the evolution of the virus, and nonsynonymous et al., 2015; Ló pez-Valiñas et al., 2021; Ló pez-Valiñas et al., 2022). substitutions that could have negative or positive role in The nonsynonymous and synonymous de novo SNVs its evolution. proportions, could provide an idea about which is the main force According to the quasispecies evolution theory, those mutations that is driving the virus evolution under different scenarios. In this that poses a beneficial for virus fitness subsequently appear in the work, the proportion of nonsynonymous SNVs was greater than viral progeny, being naturally selected and, consequently, its allele synonymous ones in both experimental groups. Notably, the frequency increases on virus population over time (Domingo and proportion of nonsynonymous substitutions was always higher in Perales, 2019). Herein, 142 out of 367 (38.7%) de novo SNVs were vaccinated animals, maintaining this dynamic throughout all the reported with an allele frequency greater than 5%. Therefore, most variant allelic frequencies analyzed. This could be indicating that of the substitutions generated in the SIAV genome during the trial the immune pressure of the vaccinated pigs was additionally acting could have a negative impact on viral fitness or, alternatively, more as positive selection force in the evolution of the virus (Fitch et al., time is required for one variant to become more dominant. For 1991; Li et al., 2011), as previously reported for H1N1 and H3N2 instance, a total of 135 nonsynonymous SNVs were found in PB2, FIGURE 7 Nucleotide diversity (p) over time and per genomic segment. Boxplots indicate means, lower and upper quartile, and standard deviation. The p represented in blue and green colors correspond to samples collected from vaccinated and nonvaccinated animals, respectively. No significant differences were found. ANOVA test was used for statistical analysis. Frontiers in Cellular and Infection Microbiology 12 frontiersin.org López-Valiñas et al. 10.3389/fcimb.2023.1258321 PB1, PA, and NS segments, although only 45 reached the 5% of proteins are under positive selection forces, acquiring more genetic allele frequency. However, those substitutions with an allele diversification (Li et al., 2011; Krammer, 2019). The percentages of frequency greater than 5% found in vaccinated animals could nucleotide identity between the H1N2 vaccine subtype and the virus play an important role in the adaptation of the viral quasispecies, challenge are 93.19% for HA and 90,14% for NA, which are similar posing an evolutionary advantage against host immune pressure. to other isolates. The HA is the protein responsible for the virus No substitutions were found in NS2, M1, PA-X and PB1-F2 sialic acid cell attachment and subsequent entry (Russell, 2021). proteins in vaccinated animals. Furthermore, in the polymerase Herein, 4 de novo substitutions with an allele frequency greater than segments (PB2, PB1, PA), the number of nonsynonymous 5% were detected in vaccinated animals while 6 were detected in substitutions found were lower in samples from vaccinated nonvaccinated ones. In vaccinated animals, substitutions L335I in animals, being only one detected in PA protein. Substitutions the fusion domain, V466I and C492Y in the stalk domain, and found in polymerase proteins could increase the IAV polymerase M525I in the transmembrane domain, were allocated in the HA activity, the virulence, and interspecific host jump, according to with an allele frequency greater than 5%. In previous studies, previous literature (Subbarao et al., 1993; Song et al., 2015; Yu et al., relevant substitutions in quasispecies collected from vaccinated 2015; Cai et al., 2020). animals were found in the stalk domain of the protein (Ló pez- In the NS1 protein, 6 de novo SNVs were found in vaccinated Valiñas et al., 2021; Ló pez-Valiñas et al., 2022; Ló pez-Valiñas et al., animals meanwhile only 2 were reported from nonvaccinated ones. 2023). According to previous literature, the stalk region of the Substitution S7L in the RNA Binding Domain was simultaneously protein evolves independently from immune pressure (Kirkpatrick reported with a high allele frequency in two vaccinated animals (No. et al., 2018), although our results suggest that this domain could be 4 and 6). The remaining SNVs detected in vaccinated animals, evolving under immune pressure. Furthermore, substitutions in the E142G, L163S, and R200K, were found in the Effector Binding transmembrane domain of the HA were also reported in a similar Domain of the protein. According to previous literature, study with the H1N1 subtype (Ló pez-Valiñas et al., 2021). substitutions on this domain may be involved in the regulation of Substitutions in this domain could affect its interaction with the interferon (IFN) type I and in the viral mRNA translation surrounding bilayer lipids and, as a SIAV result of the protein enhancement, causing as a result the increase of viral replication differently fold, its antigenic exposure can be modified and virulence (Nemeroff et al., 1998; Aragó n et al., 2000; Jia et al., (Kubiszewski-Jakubiak and Worch, 2020). 2010; Thepmalee et al., 2013; Nogales et al., 2017). In a previous Regarding NA, this protein is responsible for releasing the viral similar study with the SIAV H1N1 subtype, substitutions in the NS1 progeny at the end of the lytic viral cycle (McAuley et al., 2019). In protein were only reported from vaccinated animals (Ló pez-Valiñas vaccinated animals, a total of 5 nonsynonymous substitutions were et al., 2021). Furthermore, the substitution L163S (as well as the reported with an allele frequency greater than 5%; the R60G G161E substitution reported in the previous mentioned study) was mutation is in the stalk domain, while the remaining K93R, allocated in a region equivalent to the human leukocyte antigen B7 R172K, V398I, and D463Y are allocated in the head domain of (HLA-B7) binding motif (Gianfrani et al., 2000) that could be the protein, in the most exposed part. Deletions in stalk domine related with swine leukocyte antigen B7 (SLA-B7). Hence, herein it have been related with a major viral shedding and a major could be again hypothesized that the plasticity of the NS1 could help proportion of animals remaining potentially infectious (Deblanc virus adaptability under immune pressure. In relation with the NP, et al., 2020), thus, R60G can be involved in vaccine escape. 7 substitutions were found, 4 of which in samples from vaccinated Substitution D463Y is allocated in a previous described epitope animals. In previous studies, substitutions were also found in N2 region L’463–468 (Ge et al., 2022). Nonsynonymous vaccinated animals previously challenged with the H1N1 subtype substitutions were also found in nonvaccinated animals, K296R (Ló pez-Valiñas et al., 2021). However, substitutions were not and S298Y, both in the head domain. Proportionally to the number reported in the H3N2 subtype (Ló pez-Valiñas et al., 2022; Ló pez- of sequences obtained per group, more substitutions were found in Valiñas et al., 2023). Hence, it could be inferred that the plasticity of the vaccinated animals. Further analyses with reverse genetic NP is different depending on the subtype, being greater in H1N1 technology would be required (Li et al., 2021) to determine the and H1N2 ones, while H3N2 NP seems to be more conserved. effect of each substitution herein found on surface glycoproteins. According to previous studies, the existence of highly conserved NP The nucleotide diversity analysis disclosed a higher value for NA sequences between different subtypes, would open the avenue for a segment in both groups, particularly at 2 and 3dpi, although a high good candidate for IAV universal vaccine development (Zheng value was presented in the inoculum. This is in contrast with the et al., 2014; Hu et al., 2017; Withanage et al., 2022). Therefore, these values found for H1N2 subtype where HA presented a higher substitutions generated in H1N1, and H1N2 NP are probably not nucleotide diversity (Ló pez-Valiñas et al., 2022). This opposite able to establish themselves in the SIAV genome over time, behaviour of the HA from H1N2 subtype and the HA from ultimately resulting in its loss. H1N2 reveals how reassortments can modify the dynamic of the The nonsynonymous SNVs found in the surface glycoproteins, evolution of SIAV. HA, and NA, may be changing the antigenicity of the virus, as these Although the function of de novo SNVs in HA should be further proteins are the main target for the host to generate neutralizing determined to shed light on viral pathogenesis and protection antibodies after SIAV exposure by natural infection or vaccination against this virus, the obtained results highlight the wide (Li et al., 2011; Eichelberger and Wan, 2014; Ma, 2020). Thus, both evolution and plasticity capacity of IAVs, assuming an Frontiers in Cellular and Infection Microbiology 13 frontiersin.org López-Valiñas et al. 10.3389/fcimb.2023.1258321 evolutionary success due to the quick adaptation of viruses to Methodology, Writing – review & editing. JS: Methodology, different environments. Such results further support a continuous Writing – review & editing. JIN: Methodology, Writing – review IAV circulation through ecosystems and evolution, posing a threat & editing, Conceptualization, Funding acquisition. to global health. Hence, the improvement of biosecurity conditions on farms, sustainable meat consumption to contribute to a more rational livestock production, the increase in genetic and Funding surveillance studies, the expansion of current vaccines use against IAV and the development of universal vaccines are necessary This research was funded by grants AGL2016–75280-R from measures to minimize virus circulation and, therefore, reduce its Ministerio de Ciencia, Innovació n y Universidades from the endemic and pandemic risks. Spanish government. Á L-V has a pre-doctoral fellowship FPI 2017, Ministerio de Ciencia, Innovació n y Universidades from the Spanish government. 4.1 Permission to reuse and copyright Permission must be obtained for use of copyrighted material Acknowledgments from other sources (including the web). Please note that it is compulsory to follow figure instructions. The authors thank the IRTA-CReSA animal facilities staff for their work and help during the in vivo experiment. Data availability statement Conflict of interest The datasets presented in this study can be found in online repositories. The names of the repository and accession number can The authors declare that the research was conducted in the be found below: https://www.ncbi.nlm.nih.gov/genbank/, absence of any commercial or financial relationships that could be PRJNA994299. construed as a potential conflict of interest. Ethics statement Publisher’s note The animal study was approved by Animal ethics committee All claims expressed in this article are solely those of the authors from the Generalitat de Catalunya (project number 10442). The and do not necessarily represent those of their affiliated organizations, study was conducted in accordance with the local legislation and or those of the publisher, the editors and the reviewers. Any product institutional requirements. that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Author contributions Supplementary material ÁL-V: Methodology, Software, Writing – original draft. MV: Methodology, Writing – review & editing. MP: Methodology, The Supplementary Material for this article can be found online Writing – review & editing. AD: Funding acquisition, Writing – at: https://www.frontiersin.org/articles/10.3389/fcimb.2023.1258321/ review & editing. CC: Methodology, Writing – review & editing. LG: full#supplementary-material References Ahlquist, P. (2002). RNA-dependent RNA polymerases, viruses, and RNA silencing. Bolger, A. M., Lohse, M., and Usadel, B. (2014). Trimmomatic: a flexible trimmer for Science 296, 1270–1273. doi: 10.1126/SCIENCE.1069132 Illumina sequence data. Bioinformatics 30, 2114–2120. doi: 10.1093/bioinformatics/ Andrews, S. (2010) FastQC: A Quality Control Tool for High Throughput Sequence btu170 Data. Available at: https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ Borges, V., Pinheiro, M., Pechirra, P., Guiomar, R., and Gomes, J. P. (2018). (Accessed September 1, 2021). INSaFLU: an automated open web-based bioinformatics suite “from-reads” for Aragó n, T., de la Luna, S., Novoa, I., Carrasco, L., Ortı́n, J., and Nieto, A. (2000). influenza whole-genome-sequencing-based surveillance. Genome Med. 10. Eukaryotic translation initiation factor 4GI is a cellular target for NS1 protein, a doi: 10.1186/S13073-018-0555-0 translational activator of influenza virus. Mol. Cell Biol. 20, 6259–6268. doi: 10.1128/ Breen, M., Nogales, A., Baker, S. F., and Martı́nez-Sobrido, L. (2016). Replication- MCB.20.17.6259-6268.2000 competent influenza A viruses expressing reporter genes. Viruses 2016 Vol. 8 Page 179 Baratelli, M., Có rdoba, L., Pérez, L. J., Maldonado, J., Fraile, L., Nú ñez, J. I., et al. (2014). Genetic 8, 179. doi: 10.3390/V8070179 characterization of influenza A viruses circulating in pigs and isolated in north-east Spain during Bull, J. J., Meyers, L. A., and Lachmann, M. (2005). Quasispecies made simple. PloS the period 2006-2007. Res. Vet. Sci. 96, 380–388. doi: 10.1016/J.RVSC.2013.12.006 Comput. Biol. 1, 0450–0460. doi: 10.1371/JOURNAL.PCBI.0010061 Frontiers in Cellular and Infection Microbiology 14 frontiersin.org López-Valiñas et al. 10.3389/fcimb.2023.1258321 Busquets, N., Segalé s, J., Có rdoba, L., Mussá , T., Crisci, E., Martı́n-Valls, G. E., et al. Jagger, B. W., Wise, H. M., Kash, J. C., Walters, K. A., Wills, N. M., Xiao, Y. L., et al. (2010). Experimental infection with H1N1 European swine influenza virus protects (2012). An overlapping protein-coding region in influenza A virus segment 3 pigs from an infection with the 2009 pandemic H1N1 human influenza virus. Vet. Res. modulates the host response. Science 337, 199–204. doi: 10.1126/SCIENCE.1222213 41, 74. doi: 10.1051/vetres/2010046 Jia, D., Rahbar, R., Chan, R. W. Y., Lee, S. M. Y., Chan, M. C. W., Wang, B. X., et al. Cacciabue, M., Currá , A., Carrillo, E., König, G., and Gismondi, M. I. (2020). A (2010). Influenza virus non-structural protein 1 (NS1) disrupts interferon signaling. beginner’s guide for FMDV quasispecies analysis: sub-consensus variant detection and PloS One 5, 13927. doi: 10.1371/JOURNAL.PONE.0013927 haplotype reconstruction using next-generation sequencing. Brief Bioinform. 21, 1766– Kirkpatrick, E., Qiu, X., Wilson, P. C., Bahl, J., and Krammer, F. (2018). The 1775. doi: 10.1093/bib/bbz086 influenza virus hemagglutinin head evolves faster than the stalk domain. Sci. Rep. 8. Cai, M., Zhong, R., Qin, C., Yu, Z., Wen, X., Xian, J., et al. (2020). The R251K doi: 10.1038/S41598-018-28706-1 substitution in viral protein PB2 increases viral replication and pathogenicity of Krammer, F. (2019). The human antibody response to influenza A virus infection eurasian avian-like H1N1 swine influenza viruses. Viruses 12, 52. doi: 10.3390/ and vaccination. Nat. Rev. Immunol. 19, 383–397. doi: 10.1038/s41577-019-0143-6 V12010052 Krammer, F., Smith, G. J. D., Fouchier, R. A. M., Peiris, M., Kedzierska, K., Doherty, Chare, E. R., Gould, E. A., and Holmes, E. C. (2003). Phylogenetic analysis reveals a P. C., et al. (2018). Influenza. Nat. Rev. Dis. Primers 4, 1–21. doi: 10.1038/s41572-018- low rate of homologous recombination in negative-sense RNA viruses. J. Gen. Virol. 84, 0002-y 2691–2703. doi: 10.1099/VIR.0.19277-0 Kubiszewski-Jakubiak, S., and Worch, R. (2020). Influenza A H1 and H3 Cingolani, P., Platts, A., Wang, L. L., Coon, M., Nguyen, T., Wang, L., et al. (2012). A transmembrane domains interact differently with each other and with surrounding program for annotating and predicting the effects of single nucleotide polymorphisms, membrane lipids. Viruses 12. doi: 10.3390/V12121461 SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly (Austin) 6, 80–92. doi: 10.4161/fly.19695 Kyriakis, C. S., Rose, N., Foni, E., Maldonado, J., Loeffen, W. L. A. , Madec, F., et al. (2013). Influenza A virus infection dynamics in swine farms in Belgium, France, Italy Cortey, M., Arocena, G., Pileri, E., Martı́n-Valls, G., and Mateu, E. (2018). an d S p a i n 2 00 6 - 2 00 8 . V e t . Mi c ro b i o l . 1 6 2 , 5 4 3 – 55 0 . d o i : 1 0 .1 0 1 6 / Bottlenecks in the transmission of porcine reproductive and respiratory syndrome J.VETMIC.2012.11.014 virus (PRRSV1) to naïve pigs and the quasi-species variation of the virus during infection in vaccinated pigs. Vet. Res. 49. doi: 10.1186/s13567-018-0603-1 Lamb, R. A., Choppin, P. W., Chanock, R. M., and Lai, C. J. (1980). Mapping of the two overlapping genes for polypeptides NS1 and NS2 on RNA segment 8 of influenza Danecek, P., Bonfield, J. K., Liddle, J., Marshall, J., Ohan, V., Pollard, M. O., et al. virus genome. Proc. Natl. Acad. Sci. U.S.A. 77, 1857–1861. doi: 10.1073/pnas.77.4.1857 (2021). Twelve years of SAMtools and BCFtools. Gigascience 10, 1–4. doi: 10.1093/ gigascience/giab008 Lamb, R. A., Lai, C. J., and Choppin, P. W. (1981). Sequences of mRNAs derived from genome RNA segment 7 of influenza virus: Colinear and interrupted mRNAs Deblanc, C., Qué guiner, S., Gorin, S., Chastagner, A., Hervé , S., Paboeuf, F., et al. code for overlapping proteins. Proc. Natl. Acad. Sci. U.S.A. 78, 4170–4174. doi: 10.1073/ (2020). Evaluation of the pathogenicity and the escape from vaccine protection of a new pnas.78.7.4170 antigenic variant derived from the European human-like reassortant swine H1N2 influenza virus. Viruses 12. doi: 10.3390/V12101155 Lauring, A. S., and Andino, R. (2010). Quasispecies theory and the behavior of RNA viruses. PloS Pathog. 6, 1–8. doi: 10.1371/JOURNAL.PPAT.1001005 Detmer, S. E., Gunvaldsen, R. E., and Harding, J. C. (2013). Comparison of influenza a virus infection in high- and low-birth-weight pigs using morphometric analysis. Li, H., and Durbin, R. (2009). Fast and accurate short read alignment with Burrows- Influenza Other Respir. Viruses 7, 2–9. doi: 10.1111/irv.12199 Wheeler transform. Bioinformatics 25, 1754–1760. doi: 10.1093/BIOINFORMATICS/ BTP324 Diaz, A., Allerson, M., Culhane, M., Sreevatsan, S., and Torremorell, M. (2013). Antigenic drift of H1N1 influenza A virus in pigs with and without pasSIAVe Li, Y., and Robertson, I. (2021). The epidemiology of swine influenza. Anim. Dis. 1, immunity. Influenza Other Respir. Viruses 7 Suppl 4, 52–60. doi: 10.1111/IRV.12190 1–14. doi: 10.1186/S44149-021-00024-6 Diaz, A., Enomoto, S., Romagosa, A., Sreevatsan, S., Nelson, M., Culhane, M., et al. Li, W., Shi, W., Qiao, H., Ho, S. Y. W., Luo, A., Zhang, Y., et al. (2011). Positive (2015). Genome plasticity of triple-reassortant H1N1 influenza A virus during infection selection on hemagglutinin and neuraminidase genes of H1N1 influenza viruses. Virol. of vaccinated pigs. J. Gen. Virol. 96, 2982. doi: 10.1099/JGV.0.000258 J. 8. doi: 10.1186/1743-422X-8-183 Domingo, E., and Perales, C. (2019). Viral quasispecies. PloS Genet. 15. doi: 10.1371/ Li, Z., Zhong, L., He, J., Huang, Y., and Zhao, Y. (2021). Development and JOURNAL.PGEN.1008271 application of reverse genetic technology for the influenza virus. Virus Genes 57, 151. doi: 10.1007/S11262-020-01822-9 Domingo, E., Sheldon, J., and Perales, C. (2012). Viral quasispecies evolution. Microbiol. Mol. Biol. Rev. 76, 159–216. doi: 10.1128/mmbr.05023-11 Ló pez-Valiñas, Á ., Baioni, L., Có rdoba, L., Darji, A., Chiapponi, C., Segalé s, J., et al. (2022). Evolution of swine influenza virus H3N2 in vaccinated and nonvaccinated pigs Eichelberger, M. C., and Wan, H. (2014). Influenza neuraminidase as a vaccine antigen. after previous natural H1N1 infection. Viruses 14. doi: 10.3390/V14092008/S1 Curr. Top. Microbiol. Immunol. 386, 275–299. doi: 10.1007/82_2014_398/COVER Ló pez-Valiñas, Á ., Sisteré -Oró , M., Ló pez-Serrano, S., Baioni, L., Darji, A., Eigen, M. (1971). Selforganization of matter and the evolution of biological Chiapponi, C., et al. (2021). Identification and characterization of swine influenza macromolecules. Naturwissenschaften 58, 465–523. doi: 10.1007/BF00623322 virus h1n1 variants generated in vaccinated and nonvaccinated, challenged pigs. Eigen, M., and Schuster, P. (1977). A principle of natural self-organization. Viruses 13, 2087. doi: 10.3390/V13102087/S1 Naturwissenschaften 64, 541–565. doi: 10.1007/BF00450633 Ló pez-Valiñas, Á ., Valle, M., Wang, M., Darji, A., Cantero, G., Chiapponi, C., et al. Fitch, W. M., Leiter, J. M., Li, X. Q., and Palese, P. (1991). Positive Darwinian (2023). Vaccination against swine influenza in pigs causes different drift evolutionary evolution in human influenza A viruses. Proc. Natl. Acad. Sci. U.S.A. 88, 4270–4274. patterns upon swine influenza virus experimental infection and reduces the likelihood doi: 10.1073/pnas.88.10.4270 of genomic reassortments. Front. Cell Infect. Microbiol. 13. doi: 10.3389/ Frost, S. D. W., Magalis, B. R., and Kosakovsky Pond, S. L. (2018). Neutral theory and FCIMB.2023.1111143/BIBTEX rapidly evolving viral pathogens. Mol. Biol. Evol. 35, 1348–1354. doi: 10.1093/ Lycett, S. J., Baillie, G., Coulter, E., Bhatt, S., Kellam, P., McCauley, J. W., et al. (2012). MOLBEV/MSY088 Estimating reassortment rates in co-circulating Eurasian swine influenza viruses. J. Gen. Galindo-Cardiel, I., Ballester, M., Solanes, D., Nofrarı́as, M., Ló pez-Soria, S., Virol. 93, 2326–2336. doi: 10.1099/vir.0.044503-0 Argilaguet, J. M., et al. (2013). Standardization of pathological investigations in the Ma, W. (2020). Swine influenza virus: Current status and challenge. Virus Res., 288. framework of experimental ASFV infections. Virus Res. 173, 180–190. doi: 10.1016/ doi: 10.1016/j.virusres.2020.198118 j.virusres.2012.12.018 Ma, W., and Richt, J. A. (2010). Swine influenza vaccines: current status and future Garten, R. J., Davis, C. T., Russell, C. A., Shu, B., Lindstrom, S., Balish, A., et al. perspectives. Anim. Health Res. Rev. 11, 81–96. doi: 10.1017/S146625231000006X (2009). Antigenic and genetic characteristics of swine-origin 2009 A(H1N1) influenza McAuley, J. L., Gilbertson, B. P., Trifkovic, S., Brown, L. E., and McKimm-Breschkin, viruses circulating in humans. Science 325, 197–201. doi: 10.1126/science.1176225 J. L. (2019). Influenza virus neuraminidase structure and functions. Front. Microbiol. 0. Ge, J., Lin, X., Guo, J., Liu, L., Li, Z., Lan, Y., et al. (2022). The antibody response doi: 10.3389/FMICB.2019.00039 against neuraminidase in human influenza A (H3N2) virus infections during 2018/ Mostafa, A., Abdelwhab, E. M., Mettenleiter, T. C., and Pleschka, S. (2018). Zoonotic 2019 flu season: focusing on the epitopes of 329-N-glycosylation and E344 in N2. Front. potential of influenza A viruses: A comprehenSIAVe overview. Viruses 10. doi: 10.3390/ Microbiol. 13. doi: 10.3389/FMICB.2022.845088/BIBTEX v10090497 Gianfrani, C., Oseroff, C., Sidney, J., Chesnut, R. W., and Sette, A. (2000). Human Murcia, P. R., Hughes, J., Battista, P., Lloyd, L., Baillie, G. J., Ramirez-Gonzalez, R. H., memory CTL response specific for influenza A virus is broad and multispecific. Hum. et al. (2012). Evolution of an Eurasian avian-like influenza virus in naïve and vaccinated Immunol. 61, 438–452. doi: 10.1016/S0198-8859(00)00105-1 pigs. PloS Pathog. 8, e1002730. doi: 10.1371/journal.ppat.1002730 Hao, W., Wang, L., and Li, S. (2020). Roles of the non-structural proteins of influenza Nelson, M. I., and Holmes, E. C. (2007). The evolution of epidemic influenza. Nat. A virus. Pathogens 9, 1–19. doi: 10.3390/PATHOGENS9100812 Rev. Genet. 8, 196–205. doi: 10.1038/NRG2053 Hu, Y., Sneyd, H., Dekant, R., and Wang, J. (2017). Influenza A virus nucleoprotein: Nelson, C. W., Moncla, L. H., and Hughes, A. L. (2015). SNPGenie: Estimating A highly conserved multi-functional viral protein as a hot antiviral drug target. Curr. evolutionary parameters to detect natural selection using pooled next-generation Top. Med. Chem. 17. doi: 10.2174/1568026617666170224122508 sequencing data. Bioinformatics 31, 3709–3711. doi: 10.1093/bioinformatics/ btv449 Hu, Y. J., Tu, P. C., Lin, C. S., and Guo, S. T. (2014). Identification and chronological analysis of genomic signatures in influenza a viruses. PloS One 9. doi: 10.1371/ Nemeroff, M. E., Barabino, S. M. L., Li, Y., Keller, W., and Krug, R. M. (1998). journal.pone.0084638 Influenza virus NS1 protein interacts with the cellular 30 kDa subunit of CPSF and Frontiers in Cellular and Infection Microbiology 15 frontiersin.org López-Valiñas et al. 10.3389/fcimb.2023.1258321 inhibits 3′ End formation of cellular pre-mRNAs. Mol. Cell 1, 991–1000. doi: 10.1016/ Spackman, E., Senne, D. A., Myers, T. J., Bulaga, L. L., Garber, L. P., Perdue, M. L., S1097-2765(00)80099-4 et al. (2002). Development of a real-time reverse transcriptase PCR assay for type A Nogales, A., Chauché , C., DeDiego, M. L., Topham, D. J., Parrish, C. R., Murcia, P. R., influenza virus and the avian H5 and H7 hemagglutinin subtypes. J. Clin. Microbiol. 40, et al. (2017). The K186E amino acid substitution in the canine influenza virus H3N8 3256–3260. doi: 10.1128/JCM.40.9.3256-3260.2002 NS1 protein restores its ability to inhibit host gene expression. J. Virol. 91, 877–894. Sriwilaijaroen, N., and Suzuki, Y. (2012). Molecular basis of the structure and doi: 10.1128/JVI.00877-17 function of H1 hemagglutinin of influenza virus. Proc. Jpn Acad. Ser. B Phys. Biol. Sci. Ou, J., and Zhu, L. J. (2019). trackViewer: a Bioconductor package for interactive and 88, 226–249. doi: 10.2183/pjab.88.226 integrative visualization of multi-omics data. Nat. Methods 16, 453–454. doi: 10.1038/ Subbarao, E. K., London, W., and Murphy, B. R. (1993). A single amino acid in the s41592-019-0430-y PB2 gene of influenza A virus is a determinant of host range. J. Virol. 67, 1761–1764. Reed, L. J., and Muench, H. (1938). A simple method of estimating fifty per cent doi: 10.1128/JVI.67.4.1761-1764.1993 endpoints. Antioch Rev. 27, 493–497. doi: 10.7723/antiochreview.72.3.0546 Thacker, E., and Janke, B. (2008). Swine influenza virus: zoonotic potential and Reeth, K., Vincent, A. L., and Lager, K. M. (2016). “Vaccines and vaccination for swine vaccination strategies for the control of avian and swine influenzas. J. Infect. Dis. 197, influenza: differing situations in Europe and the USA,” in Animal Influenza (Hoboken, NJ, S19–S24. doi: 10.1086/524988 USA: John Wiley & Sons, Inc), 480–501. doi: 10.1002/9781118924341.ch19 Thepmalee, C., Sanguansermsri, P., Suwanankhon, N., Chamnanpood, C., RStudio. (2021). Open source & professional software for data science teams - RStudio. Chamnanpood, P., Pongcharoen, S., et al. (2013). Changes in the NS1 gene of avian Available at: https://www.rstudio.com/ (Accessed September 1, 2021). influenza viruses isolated in Thailand affect expression of type I interferon in primary chicken embryonic fibroblast cells. Indian J. Virol. 24, 365. doi: 10.1007/S13337-013- Russell, C. J. (2021). Hemagglutinin stability and its impact on influenza A virus 0158-8 infectivity, pathogenicity, and transmissibility in avians, mice, swine, seals, ferrets, and humans. Viruses 13, 31-34. doi: 10.3390/V13050746 Torremorell, M., Allerson, M., Corzo, C., Diaz, A., and Gramer, M. (2012). Transmission of influenza A virus in pigs. Transbound Emerg. Dis. 59, 68–84. Russell, C. A., Jones, T. C., Barr, I. G., Cox, N. J., Garten, R. J., Gregory, V., et al. doi: 10.1111/J.1865-1682.2011.01300.X (2008). Influenza vaccine strain selection and recent studies on the global migration of seasonal influenza viruses. Vaccine 26 Suppl 4. doi: 10.1016/J.VACCINE.2008.07.078 van der Vries, E., Collins, P. J., Vachieri, S. G., Xiong, X., Liu, J., Walker, P. A., et al. (2012). H1N1 2009 pandemic influenza virus: resistance of the I223R neuraminidase Sabattini, E., Bisgaard, K., Ascani, S., Poggi, S., Piccioli, M., Ceccarelli, C., et al. (1998). mutant explained by kinetic and structural analysis. PloS Pathog. 8. doi: 10.1371/ The EnVision(TM)+ system: A new immunohistochemical method for diagnostics and journal.ppat.1002914 research. Critical comparison with the APAAP, ChemMate(TM), CSA, LABC, and SABC techniques. J. Clin. Pathol. 51, 506–511. doi: 10.1136/jcp.51.7.506 Webster, R. G., Bean, W. J., Gorman, O. T., Chambers, T. M., and Kawaoka, Y. (1992). Evolution and ecology of influenza A viruses. Microbiol. Rev. 56, 152–179. Salvesen, H. A., and Whitelaw, C. B. A. (2021). Current and prospective control strategies doi: 10.1128/MR.56.1.152-179.1992 of influenza A virus in swine. Porcine Health Manag 7. doi: 10.1186/S40813-021-00196-0 Wickham, H. (2016). Ggplot2: Elegant Graphics for Data Analysis (New York: Schuster, P. (2016). Quasispecies on fitness landscapes. Curr. Top. Microbiol. Springer-Verlag). doi: 10.1007/978-0-387-98141-3 Immunol. 392, 61–120. doi: 10.1007/82_2015_469 Wilm, A., Aw, P. P. K., Bertrand, D., Yeo, G. H. T., Ong, S. H., Wong, C. H., et al. Shao, W., Li, X., Goraya, M. U., Wang, S., and Chen, J. L. (2017). Evolution of (2012). LoFreq: a sequence-quality aware, ultra-sensitive variant caller for uncovering influenza a virus by mutation and re-assortment. Int. J. Mol. Sci. 18. doi: 10.3390/ cell-population heterogeneity from high-throughput sequencing datasets. Nucleic Acids ijms18081650 Res. 40, 11189. doi: 10.1093/NAR/GKS918 Sibila, M., Aragó n, V., Fraile, L., and Segalé s, J. (2014). Comparison of four lung Wise, H. M., Foeglein, A., Sun, J., Dalton, R. M., Patel, S., Howard, W., et al. scoring systems for the assessment of the pathological outcomes derived from (2009). A complicated message: identification of a novel PB1-related protein Actinobacillus pleuropneumoniae experimental infections. BMC Vet. Res. 10. translated from influenza A virus segment 2 mRNA. J. Virol. 83, 8021. doi: 10.1186/1746-6148-10-165 doi: 10.1128/JVI.00826-09 Simon, G., Larsen, L. E., Dürrwald, R., Foni, E., Harder, T., van Reeth, K., et al. Withanage, K., de Coster, I., Cools, N., Viviani, S., Tourneur, J., Chevandier, M., et al. (2014). European surveillance network for influenza in pigs: surveillance programs, (2022). Phase 1 randomized, placebo-controlled, dose-escalating study to evaluate diagnostic tools and Swine influenza virus subtypes identified in 14 European countries OVX836, a nucleoprotein-based influenza vaccine: intramuscular results. J. Infect. Dis. from 2010 to 2013. PloS One 9, e115815. doi: 10.1371/journal.pone.0115815 226, 119–127. doi: 10.1093/INFDIS/JIAB532 Simon-Grifé , M., Martı́n-Valls, G. E., Vilar, M. J., Garcı́a-Bocanegra, I., Mora, M., Xu, R., Ekiert, D. C., Krause, J. C., Hai, R., Crowe, J. E., and Wilson, I. A. (2010). Martı́n, M., et al. (2011). Seroprevalence and risk factors of swine influenza in Spain. Structural basis of preexisting immunity to the 2009 H1N1 pandemic influenza virus. Vet. Microbiol. 149, 56–63. doi: 10.1016/J.VETMIC.2010.10.015 Science (1979) 328, 357–360. doi: 10.1126/science.1186430 Sisteré -Oró , M., Ló pez-Serrano, S., Veljkovic, V., Pina-Pedrero, S., Vergara-Alert, J., Yu, Z., Cheng, K., Sun, W., Zhang, X., Li, Y., Wang, T., et al. (2015). A PB1 T296R Có rdoba, L., et al. (2019). DNA vaccine based on conserved HA-peptides induces substitution enhance polymerase activity and confer a virulent phenotype to a 2009 strong immune response and rapidly clears influenza virus infection from vaccinated pandemic H1N1 influenza virus in mice. Virology 486, 180–186. doi: 10.1016/ pigs. PloS One 14. doi: 10.1371/journal.pone.0222201 J.VIROL.2015.09.014 Sobel Leonard, A., McClain, M. T., Smith, G. J. D., Wentworth, D. E., Halpin, R. A., Zheng, M., Luo, J., and Chen, Z. (2014). Development of universal influenza vaccines Lin, X., et al. (2016). Deep sequencing of influenza A virus from a human challenge based on influenza virus M and NP genes. Infection 42, 251–262. doi: 10.1007/S15010- study reveals a selective bottleneck and only limited intrahost genetic diversification. J. 013-0546-4/TABLES/3 Virol. 90, 11247–11258. doi: 10.1128/jvi.01657-16 Zhou, B., Donnelly, M. E., Scholes, D. T., St. George, K., Hatta, M., Kawaoka, Y., et al. Song, J., Xu, J., Shi, J., Li, Y., and Chen, H. (2015). Synergistic effect of S224P and (2009). Single-reaction genomic amplification accelerates sequencing and vaccine N383D substitutions in the PA of H5N1 avian influenza virus contributes to production for classical and swine origin human influenza A viruses. J. Virol. 83, mamMalian adaptation. Sci. Rep. 5, 1–7. doi: 10.1038/srep10510 10309–10313. doi: 10.1128/jvi.01109-09 Frontiers in Cellular and Infection Microbiology 16 frontiersin.org