viruses Article Evolution of Swine Influenza Virus H3N2 in Vaccinated and Nonvaccinated Pigs after Previous Natural H1N1 Infection Álvaro López-Valiñas 1,2,3 , Laura Baioni 4 , Lorena Córdoba 1,2,3 , Ayub Darji 1,2,3 , Chiara Chiapponi 4 , Joaquim Segalés 2,3,5 , Llilianne Ganges 1,2,3,6 and José I. Núñez 1,2,3, * 1 IRTA, Programa de Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, 08193 Barcelona, Spain 2 Unitat Mixta d’Investigació IRTA-UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, 08193 Barcelona, Spain 3 WOAH Collaborating Centre for the Research and Control of Emerging and Re-Emerging Swine Diseases in Europe (IRTA-CReSA), 08193 Barcelona, Spain 4 WOAH Reference Laboratory for Swine Influenza, Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia-Romagna, 25124 Brescia, Italy 5 Departament de Sanitat i Anatomia Animals, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain 6 WOAH Reference Laboratory for Classical Swine Fever, IRTA-CReSA, 08193 Barcelona, Spain * Correspondence: joseignacio.nunez@irta.cat Abstract: Swine influenza viruses (SIV) produce a highly contagious and worldwide distributed disease that can cause important economic losses to the pig industry. Currently, this virus is endemic in farms and, although used limitedly, trivalent vaccine application is the most extended strategy to control SIV. The presence of pre-existing immunity against SIV may modulate the evolutionary dynamic of this virus. To better understand these dynamics, the viral variants generated in vaccinated Citation: López-Valiñas, Á.; Baioni, and nonvaccinated H3N2 challenged pigs after recovery from a natural A(H1N1) pdm09 infection L.; Córdoba, L.; Darji, A.; Chiapponi, were determined and analyzed. In total, seventeen whole SIV genomes were determined, 6 from C.; Segalés, J.; Ganges, L.; Núñez, J.I. vaccinated, and 10 from nonvaccinated animals and their inoculum, by NGS. Herein, 214 de novo Evolution of Swine Influenza Virus substitutions were found along all SIV segments, 44 of them being nonsynonymous ones with an H3N2 in Vaccinated and allele frequency greater than 5%. Nonsynonymous substitutions were not found in NP; meanwhile, Nonvaccinated Pigs after Previous many of these were allocated in PB2, PB1, and NS1 proteins. Regarding HA and NA proteins, higher Natural H1N1 Infection. Viruses 2022, nucleotide diversity, proportionally more nonsynonymous substitutions with an allele frequency 14, 2008. https://doi.org/10.3390/ greater than 5%, and different domain allocations of mutants, were observed in vaccinated animals, v14092008 indicating different evolutionary dynamics. This study highlights the rapid adaptability of SIV in Academic Editor: Feng Li different environments. Received: 28 June 2022 Accepted: 8 September 2022 Keywords: swine influenza virus; viral evolution; next-generation sequencing (NGS); vaccine; hemag- Published: 10 September 2022 glutinin (HA); neuraminidase (NA) Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- 1. Introduction iations. Swine influenza viruses (SIVs) are the etiological agents of a pig respiratory disease that causes important economic loses in the pig industry. Although the mortality rate associated with SIV infection is usually low, it is a highly contagious disease with morbidities that can Copyright: © 2022 by the authors. reach almost 100% of exposed animals [1,2]. SIVs belong to the Orthomyxoviridae family Licensee MDPI, Basel, Switzerland. type A. The SIV genome, with approximately 13.6 kb size, is characterized by having 8 RNA This article is an open access article negative polarity strain segments: the polymerases (polymerase basic 2 and 1 (PB2 and PB1) distributed under the terms and and acidic polymerase (PA)), the hemagglutinin (HA), nucleoprotein (NP), neuraminidase conditions of the Creative Commons (NA), matrix (M), and the nonstructural (NS) [3]. Attribution (CC BY) license (https:// The surface glycoproteins HA and NA are responsible for interacting with host cells creativecommons.org/licenses/by/ through sialic acid, allowing the entry of viral particles and the exit of virions after the 4.0/). Viruses 2022, 14, 2008. https://doi.org/10.3390/v14092008 https://www.mdpi.com/journal/viruses Viruses 2022, 14, 2008 2 of 24 influenza A virus (IAV) infection cycle [4–6]. Both proteins generate different antigenic patterns used to classify IAVs. Nowadays, H1N1, H1N2, and H3N2 are the most prevalent circulating enzootic subtypes in the swine population worldwide [7]. Moreover, those proteins play an important role in generating protective immunity against SIV, more specifically against HA protein [8]. Currently, though its utilization is still limited, the trivalent vaccine, which contains the three previously mentioned SIV inactivated subtypes, is the most widely used to prevent and control the disease [8–10]. Although the vaccine can reduce disease and virus spread, the host immune system does not generate sterilizing immunity, so viral replication can occur despite vaccination [11,12]. Influenza viruses have a high evolutionary capacity to evade pre-existing immu- nity [13,14]. This ability is related to the low proof-reading fidelity of IAVs’ polymerase, which can lead to the accumulation of point mutations in the IAV RNA segments, achieving new antigenic patterns in a short time, a phenomenon known as antigenic drift [15,16]. HA and NA have a great relevance in the humoral response against IAVs. Punctual nonsynony- mous substitutions in both the HA globular and NA head domains can interfere with host capacity to generate an effective antibody response to the virus [17–19]. Previous studies have shown that the lack of effectiveness of pre-existing immunity against IAVs may favor the virus’s evolution due to a strong antigenic selection driving the rapid generation of new epitopes [20,21]. During the 1990s, SIV H3N2 subtype arose from the triple-reassortment of HA, NA, and PB1 segments from seasonal human H3N2 and PB2, and PA from avian IAV, and NP, M, and NS from the classical H1N1 SIV [22]. This subtype was first identified in swine, and it has been circulating since then all over the world [22–24]. In 2009, A(H1N1) pdm09 triple-reassortment virus arose from the Eurasian “avian-like” swine H1N1 and swine triple-reassortments H1N2 and H3N2 [25]. Thereby, pigs play an important role in the IAVs’ adaptation to different hosts, avian to human and vice versa [26]. The current dominating IAV strains in the European swine population are “avian-like” swine H1N1, “human-like” reassortment swine H1N2, “human-like” reassortment swine H3N2, and A(H1N1) pdm09 virus [2]. Considering the enormous genetic variability and evolutionary capacity of IAVs, their endemic character, the continuous increase in the pig population around the world and the close contact among pigs, birds, and humans, IAV poses a constant threat to human and animal health. Thus, SIV evolution studies including determination of new viral variants emergency are fundamental to improve surveillance and disease control. Against this background, the aim of the present study was to determine the evolu- tionary trends of SIV H3N2 subtype in vaccinated and nonvaccinated pigs in previously naturally H1N1 infected herds. To this end, 14 whole SIV genome samples were sequenced by next generation sequencing, and new viral variants were determined. 2. Materials and Methods 2.1. Cell and Viruses Madin-Darby Canine Kidney (MDCK, ATCC CCL-34) cells were used for both viral titration and production. Dulbecco’s Modified Eagle Medium (DMEM), supplemented with fetal bovine serum (FBS) (10%), L-glutamine (1%) and penicillin/streptomycin (1%), was used for cell culture kept at 37 ◦ C with 5% CO2 atmosphere in an incubator. The A/Swine/Spain/SF32071/2007 H3N2 virus was propagated via infection of a MDCK monolayer cell culture, with addition of 10 µg/mL of porcine trypsin (Sigma- Aldrich, Madrid, Spain), at a multiplicity of infection (MOI) of 0.01. Forty-eight hours later, virus was harvested, and titration was performed by serial dilutions in a MDCK cell culture to calculate the 50% tissue culture infection dose (TCID50 ) according to the Reed and Muench method [27]. Viruses 2022, 14, 2008 3 of 24 2.2. Experimental Design Sixteen 5-week-old domestic pigs were selected from a Spanish commercial farm where the flu vaccine was not applied. Animals were distributed into two groups, Group A (animals from 1 to 8) and Group B (animals 9 to 16). After one week of acclimation period, animals from Group A were immunized with the first dose of the commercial inactivated influenza vaccine (RESPIPORC FLU3, IDT® , Dessau-Rosslau, Germany), according to the manufacturer’s instructions. In the vaccine formulation, the following strains are included H3N2 (Bakum/IDT1769/2003), H1N1 (Haselünne/IDT2617/2003), and H1N2 (Bakum/1832/2000). The vaccine was administered by the intramuscular route using a 2 mL dose injected in the neck muscle. Twenty-one days post-vaccination (dpv), Group A received the second vaccine dose, using the same protocol. In parallel, pigs from Group B were intramuscularly inoculated at 0 and 21 dpv with 2 mL of phosphate buffered saline (PBS). Three weeks after the second vaccination dose (42 dpv), pigs from both experimental groups were challenged with A/Swine/Spain/SF32071/2007 (H3N2) by two administra- tion routes, intranasal and endotracheal, with a viral concentration of 107 TCID50 in a final volume of 2 and 5 mL per route, respectively [28]. For the viral challenge, animals were restrained with a pig lasso, and a diffuser device (MAD Nasal, Teleflex, Morrisville, NC, USA) was used to administrate intranasally 1 mL of inoculum per nostril; animals were intubated for the endotracheal administration [29]. Clinical signs, including rectal temperature and animal behavior, were daily measured by a trained veterinarian after challenge. The registration of clinical signs was scored as previously described [30]. The same number of animals per group was serially euthanized, three animals each at 2 and 5 days post-inoculation (dpi), and the remaining two at 9 dpi. Nasal swabs and sera were collected before vaccination, viral challenge, and on day of euthanasia. In addition, nasal swabs were daily collected from 1 dpi until the euthanasia day. Lung, nasal turbinate (NT) and broncho-alveolar lavage fluid (BALF) were collected and stored at −80 ◦ C. The BALF was collected by filling the right lung of each pig with 150 mL of PBS. An extra lung tissue was collected and fixed by immersion in 10% buffered formalin. Animal experiments were performed in AM Animalia facilities (La Vall de Bianya, Girona, Spain). All procedures were approved by the animal ethics committee from the Generalitat de Catalunya, under project number 10442, following the Spanish and European regulations. 2.3. Humoral Response against SIV The detection of antibodies against influenza NP was performed using the ID Screen® Influenza A Antibody Competition ELISA kit (ID VET, Grabels, France). The inhibition percentage value was calculated, with values higher than 50% considered negative, those below 45% positive, and those between both values doubtful, according to manufacturer’s instructions. Hemagglutination inhibition (HI) assay was performed at 42 dpv (challenge) and on euthanasia day as previously described [29]. Briefly, collected sera from pigs were treated 18 h at 37 ◦ C with RDE II Seiken (Denka Chemicals, Tokyo, Japan) and inactivated for one hour at 56 ◦ C. A volume of 50% chicken red blood cells (RBCs) was added to remove unspecific inhibitors by hemadsorption and subsequently diluted in PBS (1:10). Then, pig sera were twofold diluted in PBS until 1:1024 in v-bottomed 96-well plates. Twenty-five microliters of viral antigen was used on challenge (A/Swine/Spain/SF32071/2007 (H3N2)), diluted to 4 hemagglutination units (HAU) was dispensed. After 1 h of incubation at room temperature, 25 µL of 0.5% of chicken RBC was also added. Finally, after another hour of incubation, hemagglutination was evaluated by tilting the plate. The antibody titers were established as the reciprocal dilution where inhibition was complete, considering seroprotective titers ≥1/40. Viruses 2022, 14, 2008 4 of 24 2.4. Pathological Analyses in Lung and Immunohistochemistry to Detect SIV Macroscopic examination of the lung parenchyma was performed during the necrop- sies of animals. Moreover, an image analysis was performed to calculate the percentage of lung-affected area, as previously described [31], with ImageJ® software (v 1.8) [32]. A sample of each collected lung was fixed in 10% buffered formalin, dehydrated and embedded in paraffin wax. Two sections of 3–5 µm thick were cut and stained with hematoxylin-eosin (HE) for the examination under light microscopy [28,33] to detect SIV antigen by immunohistochemistry (IHC). For the latter, a two-step polymer method (En- vision TM) was performed using a monoclonal antibody against influenza A virus (IAV) (Hb65 from the ATCC) and system + HRP-labeled polymer anti-mouse (K4001, Dako, Santa Clara, CA, USA) as primary and secondary antibodies, respectively [34]. To determine the degree of lung lesions, a semiquantitative method was used based on affected air- ways on HE examination; the amount of immunoreactivity on IHC examination was also assessed [35]. 2.5. SIV RNA Detection Lung and NT were homogenized in brain heart infusion medium (10% weight/volume). RNA was extracted from nasal swab, BALF, lung, and NT using the MagAttract 96 Cador Pathogen kit ® (Qiagen, Düsseldorf, Germany) following the manufacturer’s instructions. Subsequently, the SIV RNA of each sample was quantified by RT-qPCR assay based on the amplification of the conserved segment of the matrix (M) gene [36]. The amplification was performed in the Fast7500 equipment (Applied Biosystem, Foster City, CA, USA). Samples where fluorescence was not detected were considered negative whereas threshold cycle (Ct) values under 40 were considered positive [12,36]. 2.6. Whole Influenza Genome Sequence by NGS The RNA extracted from samples of vaccinated and nonvaccinated animals with a RT-qPCR Ct value lower than 37 were used for whole genome amplification [12,37]. The PCR conditions were: 0.2 µM of forward and reverse primers MBTuni-12 and MBTuni-13, respectively, 2.5 µL of extracted RNA and 0.5 µL of SuperScript® III One-Step RT-PCR System with Platinum™ Taq High Fidelity DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA). Moreover, for the biggest segments (PB2, PB1 and PA), amplification was enhanced by a second amplification with the same conditions but adding a modification of forward primer (MBTuni12G) [38]. Those samples with the eight segments amplified were selected for sequencing [37] by Illumina technology. The Nextera-XT DNA Library Prep protocol (Illumina® , San Diego, CA, USA) was followed for multiplexed sequencing libraries and sequencing was performed by Miseq Reagment Kit v2 in a 150-cycle paired-end run, on a Miseq Instrument (Illumina® , San Diego, CA, USA). Sequencing data were deposited at the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/ (accessed on 10 May 2022)) with the accession number (PRJNA853173). 2.7. De Novo Assambly, Mapping, and Variant Calling After the Miseq run, adapters were automatically removed and reads quality were an- alyzed by FastQC (v 0.11.8) [39]. All reads with a low quality (Phread < 30) were trimmed using Trimmomatic (v0.39) [40]. De novo assembly was performed using the SPAdes algorithm with the very sensitive option [41]. The assemblies generated were screened using NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 10 May 2022)) to determine the strain. To obtain inoculum consensus sequence, inoculum sequencing reads were mapped against A/Swine/Spain/SF32071/2007 (H3N2) reference SIV genome sequences (HE774666-73) [42], using the very sensitive function of the read alignment tool Bowtie2 (v2.3.5) [43]. The consensus sequence was generated using the consensus option from Bcftools (v.1.9) [44]. To detect single nucleotide polymorphisms, reads obtained from Viruses 2022, 14, 2008 5 of 24 each sequenced sample were mapped against it. Post-mapping, all reads with a mapping quality lower than 30 were removed from the analysis using Samtools (v.0.39) [44]. The Pi- card “MArkDuplicatesSpark” option and the “BaseRecalibrator” options, both included in GATK4 (v4.1), were used for removing PCR duplicates and reads recalibration, respectively. For each sample, the number of mapped reads against SIV whole genome and its depth were calculated with the “-depth” function included in Samtools (v1.9) and represented with RStudio using ggplot2 library [45,46]. All variants found in each sample were noted using the default parameters of LoFreq software [47]. A single nucleotide variant was considered if at least 50 reads of depth, 10 reads of the alternative base count, and p value < 0.01 requirements were obtained. The effect of each variant was predicted with SnpEff software (v.4.3) [48]. A previous database from the H3N2 subtype was built with “build–gtf22” function, using the previous annotated genome [42]. 2.8. Nucleotide Diversity (π) The genetic diversity of all sequenced samples per genomic segment was calculated with SNPGenie software (v 1.2) [49]. The nucleotide diversity (π) was calculated as the mean number of pairwise difference per site across each genomic segment. Moreover, the nonsynonymous and synonymous nucleotide diversity (πN and πS ) was calculated as the mean number of nonsynonymous and synonymous differences per nonsynonymous and synonymous sites in each genomic segment, respectively. 2.9. Lolliplot and 3D Structural Representation of SIV Proteins The trackViewer package from Bioconductor was used to visualize the lolliplot pro- teins representation of PB2, PB1, PA, HA, NA, matrix (M1), and nonstructural 1 (NS1) [50]. Moreover, the PyMOL Molecular Graphics System Version 4.6 was used to perform the structural representation of HA and NA proteins. Indeed, all nonsynonymous variants with an allele frequency greater than 5% were pointed in all protein representations. 2.10. Satatistical Analysis Statistical differences in rectal temperatures per day between both groups were studied by T-test application. Pearson’s chi-squared and Kruskal–Wallis tests were also applied to study significant differences between groups in the proportion of synonymous and nonsynonymous substitutions and nucleotide diversity, respectively. 3. Results 3.1. Detection of Natural SIV Circulation in Both Experimental Groups at Vaccination Day At first vaccination day (0 dpv), an antibody response against NP was already detected by ELISA test in pigs from both experimental groups (7 in vaccinated and 4 in nonvaccinated groups) (Figure 1a). Due to such unexpected antibody detection, SIV RNA detection was attempted and confirmed by RT-qPCR in 6 and 7 nasal swab samples from vaccinated and nonvaccinated animals, respectively. High Ct values between 36.3 and 39.14 were found (Figure 1b). Three weeks later (21 dpv), all animals were RNA SIV negative except one animal from each experimental group, being all animals RT-qPCR negative on the day of challenge (42 dpv) (Figure 1b). 3.2. Identification of H1N1 Subtype as Natural Circulating Virus before Challenge After detection of the unexpected SIV infection, the characterization of the circulating virus was performed by NGS. The NCBI BLAST screening of de novo assembly indicated that the circulating strain detected was H1N1, more specifically a strain derived from A(H1N1) pdm09. Viruses2022, Viruses 14,x2008 2022,14, FOR PEER REVIEW 6 of2624 6 of Figure 1. Detection of antibodies against influenza NP at day of first vaccination and SIV detection Figure 1. Detection of antibodies against influenza NP at day of first vaccination and SIV detection before challenge. (a) IgG antibody levels against NP detected by ELISA, where levels are shown before challenge. (a) IgG antibody levels against NP detected by ELISA, where levels are shown on on ordinate axis expressed as percentage of competition. Samples with a value greater than 45 (red ordinate axis expressed as percentage of competition. Samples with a value greater than 45 (red line) line) are are considered considered positive, positive, below below 50 (gray50line) (gray line) negative, negative, and between and between both doubtful. both doubtful. Violet Violet and and orange orange boxplots and dots indicate samples from vaccinated and nonvaccinated groups. boxplots and dots indicate samples from vaccinated and nonvaccinated groups. Each animal is Each animal is represented represented by by oneone sotsot shape. shape. (b)(b) SIVSIV detection detection byby RT-qPCR RT‐qPCR heatmap.Animal heatmap. AnimalIDs IDsare areindicated indicatedinin ordinate ordinateaxis. axis. 3.3.Identification 3.2. Humoral Response of H1N1 againts SubtypeSIVasafter Vaccination Natural and H3N2 Circulating Challenge Virus before Challenge At three weeks after the first immunization (21 dpv) as After detection of the unexpected SIV infection, the characterization well as onofdaytheof challenge circulating (42 dpv), high levels of antibodies against NP were developed in virus was performed by NGS. The NCBI BLAST screening of de novo assembly indicated all vaccinated animals, reaching values of 13.97% of competition detected by ELISA test. that the circulating strain detected was H1N1, more specifically a strain derived from After the H3N2 SIV challenge, A(H1N1) pdm09.high antibody titers were detected, being similar at all time points until the end of the study (Figure 2). Meanwhile, 6 out 8 nonvaccinated animals were NP antibody positive with 3.3. lower antibody Humoral Response values againts andSIV heterogeneous after Vaccinationresponse and H3N2 at Challenge 21 and 42 dpv. After challenge, from 5 dpi until the end of the study, the levels of antibodies from the nonvaccinated group At three reached weeks the same after as values thethose first of immunization (21 dpv) vaccinated ones as 2). (Figure well as on day of challenge (42 dpv), high against HI titers levels of antibodies H3N2 subtype against NP wereindeveloped were detected in all all vaccinated vaccinated pigs at 42 dpv,animals, showing reaching values of 13.97% of competition detected by ELISA test. values greater than 40 in 5 out of 8 sera. After challenge, titers rapidly increased After the H3N2 from 2SIV dpi challenge, to the endhigh antibody of the titers trial in all were detected, vaccinated animalsbeing withsimilar valuesatfrom all time 40 topoints 320. Byuntil the endin contrast, ofnonvaccinated the study (Figure 2). HI animals, Meanwhile, titers were6 not outdetected 8 nonvaccinated animals until 5 dpi, with low were NP antibody values. However, positive at 9 dpi with titerslower antibody increased (Tablevalues 1). and heterogeneous response at 21 and 42 dpv. After challenge, from 5 dpi until the end of the study, the levels of antibodies from the nonvaccinated 3.4. Kinetics of group reached theafter Rectal Temperature sameSIVvalues as those of vaccinated ones (Figure 2). Challenge After H3N2 SIV challenge, most nonvaccinated animals had fever at 1 and 2 dpi. Afterwards, all animals displayed normal rectal temperature until the end of the study, except pig 14, which showed a fever peak at 5 dpi (Figure 3). By contrast, none of the vaccinated pigs developed fever during the trial (Figure 3). The rectal temperature recorded in nonvaccinated animals was statistically significant with respect to vaccinated ones at 1, 2, and 3 dpi (t-test; p = 0.002833, p = 0.02164, p = 0.03623, respectively). Viruses 2022, 14, x FOR PEER REVIEW 7 of 26 Viruses 2022, 14, 2008 7 of 24 Figure 2. Kinetics of antibody levels against SIV NP in serum samples collected from 21 dpv to 9 dpi. Figure 2. Kinetics of antibody levels against SIV NP in serum samples collected from 21 dpv to 9 Percentage dpi. of competition Percentage (%)(%) of competition andand the the dayday of sampling of samplingare are represented in ordinate represented andand in ordinate abscissa axes, abscissa axes, respectively. Violet indicates vaccinated animals, whereas the nonvaccinated group isin respectively. Violet indicates vaccinated animals, whereas the nonvaccinated group is indicated orange box indicated plots, where in orange whiskers box plots, where indicate quartiles’ whiskers variability. indicate quartiles’Each animal ID variability. is represented Each animal ID isby different shapes. Values plotted as the upper red line (<45%) are considered positive, whereas represented by different shapes. Values plotted as the upper red line (<45%) are considered positive, values whereas values of the lower gray line (>50%) are considered negative. Values between of the lower gray line (>50%) are considered negative. Values between both lines (45% and 50%) both lines are (45% and 50%) are considered doubtful. considered doubtful. HI1. titers Table against HI activity H3N2 of sera subtype against were challenge detected strain in all vaccinated pigs(H3N2). A/Swine/Spain/SF32071/2007 at 42 dpv, showing values greater than 40 in 5 out of 8 sera. After challenge, titers rapidly increased HIvaccinated from 2 dpi to the end of the trial in all Antibody Titer Against animals withChallenge Strain values from 40intoSera 320. By contrast, in nonvaccinated animals, HI titers were not detected Euthanasia untilDay 5 dpi, with low Group Pig ID 42 dpv values. However, at 9 dpi titers increased (Table 1).2 dpi 5 dpi 9 dpi 1 10 40 Table 1. HI activity of sera2 against challenge 40 strain A/Swine/Spain/SF32071/2007 320 (H3N2). 3 20 160 4 80 HI Antibody Titer Against80Challenge Strain in Vaccinated 5 80 Sera160 animals 6 20 80 Euthanasia Day Group 7 Pig ID40 42 dpv 160 2 dpi 5 dpi 9 dpi 8 40 80 mean 1 41.3 10 173.3 40 106.7 120 9 2 0 40 0 320 10 0 0 11 3 0 20 0 160 12 4 0 80 10 80 Nonvaccinated 13 0 20 animals Vaccinated animals 5 0 80 14 0 160 15 6 0 20 80 640 16 0 320 mean 7 0 40 0 10 160 480 8 40 80 3.5. SIV Genome Detection after Challenge mean 41.3 173.3 106.7 120 9 All vaccinated pigs were negative for SIV0 RNA detection 0 in nasal swabs at 2 dpi. Later, only animal 4 tested positive at 103, 4, and 5 dpi 0 (time of euthanasia). 0 Meanwhile, pigs 5, 7, and 8 were positive at 5, 3, and 9 dpi, respectively. By contrast, all nonvaccinated pigs 11 0 0 tested positive from Nonvaccinated 2 to 4 dpi, most showing Ct values below 33 (Table 2). From 5 to 9 dpi, animals 12 all nasal swabs analyzed were RT-qPCR 0 negative, 10 except for those of animals 13 and 16 at 5 and 6 dpi, respectively. 13 0 20 14 0 0 After H3N2 SIV challenge, most nonvaccinated animals had fever at 1 and 2 dpi. Afterwards, all animals displayed normal rectal temperature until the end of the study, except pig 14, which showed a fever peak at 5 dpi (Figure 3). By contrast, none of the vaccinated pigs developed fever during the trial (Figure 3). The rectal temperature recorded in nonvaccinated animals was statistically significant with respect to vaccinated Viruses 2022, 14, 2008 8 of 24 ones at 1, 2, and 3 dpi (t‐test; p = 0.002833, p = 0.02164, p = 0.03623, respectively). Figure 3. Kinetics of rectal temperature after H3N2 SIV challenge. Pigs are considered to have fever Figure with3.temperatures Kinetics of rectal temperature greater than 40 ◦ Cafter (red H3N2 dashedSIV challenge. line). Pigsanimals Vaccinated are considered to have fever are represented in violet with temperatures greater than 40 °C (red dashed line). Vaccinated animals are represented in violet whereas nonvaccinated ones are represented in orange. Each line represents the rectal temperature whereas nonvaccinated ones are represented in orange. Each line represents the rectal temperature measured in each pig over time; (*) p ≤ 0.05 and (**) p ≤ 0.01. measured in each pig over time; (*) p ≤ 0.05 and (**) p ≤ 0.01. In the case of BALF samples, SIV genome was detected in those collected from 3 vac- 3.5.cinated SIV Genome Detection pigs at after 2, 5, and Challenge 9 dpi. Likewise, RT-qPCR positive BALF samples were found in All vaccinated pigs 4 nonvaccinated were pigs, negative three at 2 dpiforand SIV the RNA detection other in nasal at 5 dpi. swabstoatlung In relation 2 dpi. sam- Later, ples,only SIVanimal 4 tested was found onlypositive at 3, 4, in the three and 5 dpi animals vaccinated (time of euthanasia). euthanized atMeanwhile, pigs all 2 dpi, whereas 5, 7, and 8 were positive nonvaccinated animals at were 5, 3, and 9 dpi,inrespectively. positive all collectedBy contrast, samples all nonvaccinated in the pigs trial. Finally, all nasal tested positive turbinate from 2from samples to 4 vaccinated dpi, most showing animals Ct werevalues belowwhile negative, 33 (Table 2). From from two samples 5 to 9the nonvaccinated dpi, all nasal swabs group were were analyzed positive (Table 2). RT‐qPCR negative, except for those of animals 13 and From 16 at 5 and all the 6 dpi, RT-qPCR positive samples, 16 SIV whole genomes were amplified and respectively. sequenced. From vaccinated animals, complete SIV sequences were obtained from 3 nasal swab samples (pig 5 from 3 to 5 dpi), two lung samples (pigs 2 and 3), and one BALF sample (pig 1). From nonvaccinated pigs, complete SIV genomes were obtained from seven nasal swab samples (pigs 9, 10, and 11 at 2 dpi, pigs 12, 13, and 15 at 3 dpi and pig 16 at 4 dpi), two lung samples (pigs 9 and 10) and one nasal turbinate sample (pig 10) (Table 2). 3.6. Lung Gross Lesions, Histopathological Assessment, and Immunohistochemistry to Detect SIV Antigen On average, more extensive gross lung lesions were detected in nonvaccinated animals compared to vaccinated ones (Table 3). The highest percentages of lung affected area were recorded in animals 9 and 10 from the nonvaccinated group euthanized at 2 dpi, reaching values of 21.4%. Microscopic lesions were detected in both groups, with a lower scoring in vaccinated animals at all sampling days. Remarkably, the highest scoring values were detected in all lung sections from nonvaccinated animals at 2 dpi, still being high at 5 dpi. No viral labeling by immunohistochemistry in lung sections was detected in vaccinated animals. In contrast, low to moderate immunoreactivity was found in three nonvaccinated animals (Table 3). 3.7. Whole SIV Genome Sequences Obtained from Vaccinated and Nonvaccinated Challenged Animals The complete inoculum sequence (A/Swine/Spain/SF32071/2007 H3N2 strain) used in the viral challenge was determined (Figure 4). In addition, 16 SIV whole genomes were retrieved from samples collected from challenged pigs (Table 1 and Figure 4). Notably, six sequences were recovered from vaccinated pigs and 10 from nonvaccinated ones. From all sequenced samples, a total of 1,198,942 reads were obtained after quality control and 84.36% of them (1,011,412) matched the SIV genome. The maximum number of mapped reads was obtained in the nasal swab collected from nonvaccinated animal 11 at 2 dpi (135,062). By contrast, the minimum number of reads was obtained in the nasal swab of the nonvaccinated animal 12 at 3 dpi (20,581) (Table S1). Viruses 2022, 14, 2008 9 of 24 Table 2. SIV detection in nasal swab samples collected at different time points and from BALF, lung, and nasal turbinate. RT-qPCR Ct values results are shown in the table. Neg.: negative. Highlighted cells represent sequenced samples. Violet and orange cells indicate the group of each animal, vaccinated and nonvaccinated, respectively. Nasal Swab Samples Tissues Samples Group Pig ID Euthanized Day 1 dpi 2 dpi 3 dpi 4 dpi 5 dpi 6 dpi 7 dpi 8 dpi 9 dpi LUNG NT BALF 1 Neg. Neg. 32.96 Neg. 34.45 2 Neg. Neg. 2 dpi 34.48 Neg. Neg. 3 Neg. Neg. 39.36 Neg. Neg. Vaccinated 4 Neg. Neg. 33.45 32.56 37 Neg. Neg. 39.58 animals 5 Neg. Neg. Neg. Neg. 37.42 5 dpi Neg. Neg. Neg. 6 Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. 7 Neg. Neg. 38,56 Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. 38.09 9 dpi 8 Neg. Neg. Neg. Neg. Neg, Neg, Neg. Neg. 37.23 Neg. Neg. Neg. 9 Neg. 31.21 26.49 Neg. 35.98 10 Neg. 33.42 2 dpi 25.27 31.68 34.88 11 39.48 33.39 33.34 Neg. 34.14 Nonvaccinated 12 Neg. 38.28 30.98 Neg. Neg. 34.41 Neg. Neg. animals 13 37.28 35 30.02 33.6 39.48 5 dpi 34.36 39.5 37.14 14 Neg. 38.75 36.96 Neg. Neg. 35.38 Neg. Neg. 15 Neg. 37.13 32.45 34.93 Neg. Neg. Neg. Neg. Neg. 39.78 Neg. Neg. 16 Neg. Neg. 32.8 30.17 Neg. 34.64 Neg. Neg. Neg. 9 dpi 39.54 Neg. Neg. Viruses 2022, 14, 2008 10 of 24 Table 3. Pathological results based on gross (percentage of lung-affected area) and histopathological observations, including the semi-quantitative scorings for HE and IHC in lung samples. Lung Affected Histopathological Immunohistochemistry Group Pig ID Euthanasia Day Area (%) Scoring for SIV 1 1.44 2.5 - 2 2 dpi 0.32 2 - 3 0.32 2 - 4 0.18 1.5 - Vaccinated 5 5 dpi 0 0.5 - animals 6 10 2.5 - 7 1.25 0.5 - 9 dpi 8 0 0 - mean 1.69 1.44 9 21.4 3 ++ 10 2 dpi 20.38 3 +++ 11 1.54 3 - 12 7.3 2.5 - Nonvaccinated 13 5 dpi 2.07 2 + animals 14 4.98 3 - 15 1.11 1 - 9 dpi 16 0.69 1.5 - mean 7.43 2.38 Histopathology scoring: absence (0), few inflammatory cells isolated (0.5), localized cluster of inflammatory cells (1), several clusters of inflammatory cells (1.5–2), severely several (2.5), and many (3) airways affected. Moreover, minimal (1.5), mild (2) interstitial infiltrate, and plus moderate interstitial and alveolar infiltrates (2.5–3). SIV immunohistochemical scoring: absence (-), low (+), scattered (++), moderate (+++), and abundant (++++) amount of immunoreactivity. Regarding coverage per position, 93.91% from all determined SIV genomes was rep- resented with more than 50 reads and selected for further analysis (Figure 4(a.1,a.2)). Specifically, most genome segments were obtained from samples of vaccinated and nonvac- cinated pigs, with a depth median greater than 50. Only the segments PB2, PB1, and PA from lung samples collected from vaccinated pigs 1 and 2, and the HA from lung collected also from vaccinated pig 2, had a depth median lower than 50 (Figure 4b). 3.8. Evolution of Variants Found in Inoculum From the viral inoculum, five nonsynonymous variants were detected, all of them in the polymerase segments: D55N (2.98% allele frequency), E617A (19.05%), V618I (17.5%) and L683I (5.09%) in PA, and Q73K (2.32%) in PB2 (Table 4). Table 4. SNV detected in the inoculum. Depth of Nucleotide Change Alt. Base Allele Aminoacidic Change Gene Read Position Ref. → Alt. Count Frequency Ref. → Alt. Position 503 G→A 163 15 2.99 D→N 55 147 A→C 1850 28 19.05 E→A 617 PA Inoculum 160 G→A 1852 28 17.5 V→I 618 1375 C→A 2047 70 5.09 L→I 683 PB2 863 C→A 217 20 2.32 Q→K 73 Abbreviations: Alt. (alternative) and Ref. (reference). Those variants were later detected in sequenced samples from both experimental groups (Figure 5). For most samples collected during the trial, these substitutions were maintained at the same frequency, or they were lost over time, with a few exceptions. In vaccinated pigs, PA substitutions D55N, E617A, and V618I showed a rapid allele frequency increase in the lung sample from animal 1. The allele frequency of detection of the L683I substitution in PA also increased over time in nasal swabs collected from pig 4 (Figure 5a). Viruses 2022, 14, 2008 11 of 24 Viruses 2022, 14, x FOR PEER REVIEW 11 of 26 On the other hand, in nonvaccinated animals, PB2 Q73K substitution considerably increased its frequency in the nasal swab sample from animal 15 at 3 dpi (Figure 5b). Figure 4. Coverage and depth obtained per genomic segment of SIV H3N2 in inoculum and vac- cinated and nonvaccinated pig samples. (a.1) Illumina sequencing profile reads coverage against H3N2 SIV and its depth in inoculum (plotted in green) and samples from vaccinated animals (plotted in different tones of violet). (a.2) Illumina profile of samples collected from nonvaccinated animals plotted in different orange tones. (b) Heat map of the median depth value per genomic segment in all sequenced samples. The origin and the collection day of each sample are specified in the figure. Virus samples from BALF, lung, and NT were collected on indicated euthanasia day. Viruses Viruses 2022, 2022, 14, 14, 2008x FOR PEER REVIEW 1213of of 24 26 Figure Figure 5. 5. Dynamics Dynamics of of inoculum inoculum variant variant frequencies frequencies over over time. time. (a)(a) Samples Samples from from vaccinated vaccinated animals. animals. (b) Samples from nonvaccinated animals. Rows indicate genomic segments PA and (b) Samples from nonvaccinated animals. Rows indicate genomic segments PA and PB2. Columns PB2. Columns correspond to each animal and sample origin. X axes correspond to post‐inoculation time in which correspond to each animal and sample origin. X axes correspond to post-inoculation time in which each sample was recovered, where “In.” indicates the inoculum. Y axes show the allele frequency. each sample was recovered, where “In.” indicates the inoculum. Y axes show the allele frequency. Each substitution is plotted in a different color. Each substitution is plotted in a different color. 3.9. 3.9. Detection Detection of of DeDe Novo Novo SIV SIV Variants Variants in in Sequenced Sequenced Samples Samples Collected Collected from from Vaccinated Vaccinated andand Nonvaccinated Nonvaccinated Animals Animals From From samples samples collected collected during during thethe trial, trial, single single nucleotide nucleotide variants variants (SNV) (SNV) were were noted noted forfor an allele an allele frequency frequency of 5, of 1, 2.5, 1, 7.5, 2.5,and 5, 10% 7.5, (Figure and 10% (Figure 6). The 6). Theof proportion proportion nonsynony-of nonsynonymous mous variantsthan variants was higher wasthat higher than of the that of the ones synonymous synonymous for the 5 ones allelefor the 5 allele frequencies frequencies analyzed analyzed (Figure 6). No(Figure 6). Nodifferences significant significantbetween differences between groups weregroups found were found applying applying Pearson’s chi‐squared test. Pearson’s chi-squared test. The synonymous and nonsynonymous variants were allocated in all genomic seg- ments recovered from both experimental groups (Figure 6). Regarding SNV with an allele frequency greater than 1%, 56, 33, and 49 were detected in polymerase segments PB2, PB1, and PA, respectively. Notably, most of them were nonsynonymous substitutions (Figure 7A). By contrast, the most of these variations did not exceed an allele frequency of 5% (Figure 7B); we detected only 16 in PB2, 6 in PB1, and 14 in PA(Figure 7B). Viruses2022, Viruses 2022,14, 14,2008 x FOR PEER REVIEW 14 13 of 26 of 24 Figure 6. Proportion of synonymous and nonsynonymous de novo SNVs generated in sequences collected from vaccinated and nonvaccinated pigs. Substitutions with an allele frequency greater than 1% (a), 2.5% (b), 5%(c), 7.5% (d), and 10% (e) are represented. The synonymous and nonsynonymous variants were allocated in all genomic segments recovered from both experimental groups (Figure 6). Regarding SNV with an allele frequency greater than 1%, 56, 33, and 49 were detected in polymerase segments PB2, PB1, and PA, respectively. Notably, most of them were nonsynonymous substitutions Figure Figure 6. (Figure 6. Proportion Proportion of7A). of By contrast, synonymous synonymous and the and most of thesede nonsynonymous nonsynonymous variations de novo novo SNVsdid SNVs not exceed generated generated in an allele in sequences sequences frequency collected of 5% collected from from (Figureand vaccinated vaccinated 7B);nonvaccinated and we detected pigs. nonvaccinated only Substitutions pigs. 16 in PB2, 6 with in PB1, and 14 an allele in PA(Figure frequency frequency greater greater 7B). than 1% than 1% (a), (a), 2.5% 2.5% (b), (b), 5%(c), 5%(c), 7.5% 7.5% (d), (d), and and 10% 10% (e) (e) are arerepresented. represented. The synonymous and nonsynonymous variants were allocated in all genomic segments recovered from both experimental groups (Figure 6). Regarding SNV with an allele frequency greater than 1%, 56, 33, and 49 were detected in polymerase segments PB2, PB1, and PA, respectively. Notably, most of them were nonsynonymous substitutions (Figure 7A). By contrast, the most of these variations did not exceed an allele frequency of 5% (Figure 7B); we detected only 16 in PB2, 6 in PB1, and 14 in PA(Figure 7B). Figure Figure 7.7. De Denovo novo synonymous synonymous andand nonsynonymous nonsynonymous SNV allocation. SNV allocation. Nonsynonymous Nonsynonymous and and synony- synonymous mous variantsvariants are represented are represented in blue, in red and red and blue, respectively. respectively. Circle sizeCircle sizethe indicates indicates the total total number of number of substitutions found per genomic segment (abscissa axis) and sample (ordinate axis). (A) substitutions found per genomic segment (abscissa axis) and sample (ordinate axis). (A) SNVs with SNVs with an allele frequency greater than 1%. (B) SNVs with an allele frequency greater than 5%. an allele frequency greater than 1%. (B) SNVs with an allele frequency greater than 5%. In In relation relation toto HA HA and and NA NA segments, segments, 20 20 and and 16 16 SNVs SNVs were were detected, detected, respectively, respectively, of of which nine and six were nonsynonymous variants with an allele frequency greater which nine and six were nonsynonymous variants with an allele frequency greater than 5% than 5% (Figure (Figure 7). In7). theInNPthegene, NP nogene, no nonsynonymous nonsynonymous variantsvariants were were found found with withfrequency an allele an allele greater Figure 7.than De 5%novo(Figure 7B). Notably, synonymous in the M segment, and nonsynonymous SNV16allocation. SNVs were detected withand Nonsynonymous an allele frequency synonymous greater variants are than 1%, nine represented in of redwhich wererespectively. and blue, nonsynonymous substitutions Circle size found indicates the total only numberin samples from vaccinated of substitutions animals.segment found per genomic Only one nonsynonymous (abscissa and one axis) and sample synonymous (ordinate axis). (A) SNVs with anin substitution allele the Mfrequency segmentgreater gene than were1%. (B) SNVs detected with with anan allelefrequency allele frequencygreater greater than than5%.5% (Figure 7). Lastly, in the NS segment, 19 SNVs were detected, with eight variants exceeding 5% ofInallele relation to HA five frequency, and of NA segments, them 20 and 16 SNVs ones being nonsynonymous were (Figure detected, 7).respectively, of which nine and six were nonsynonymous variants with an allele frequency greater than 5% (Figure 7). In the NP gene, no nonsynonymous variants were found with an allele frequency greater than 5% (Figure 7B). Notably, in the M segment, 16 SNVs were detected with an allele frequency greater than 1%, nine of which were nonsynonymous substitutions found only in samples from vaccinated animals. Only one nonsynonymous and one synonymous substitution in the M segment gene were detected with an allele Viruses 2022, 14, 2008 frequency greater than 5% (Figure 7). Lastly, in the NS segment, 19 SNVs were detected, 14 of 24 with eight variants exceeding 5% of allele frequency, five of them being nonsynonymous ones (Figure 7). In vaccinated animals, 16 amino acid substitutions with an allele frequency greater thanIn5%vaccinated animals,in16PB2 were identified amino acid (M51I substitutions and L697R), PB1with an allele (P64S), frequency PA (E56K, greater N331S, and than 5% were identified in PB2 (M51I and L697R), PB1 (P64S), PA (E56K, N331S, E677K), HA (I339V, T467I, and N505S), NA (F167L, V303I, and G313S), M2 (E70K), and and E677K), HA (I339V, T467I, and N505S), NA (F167L, V303I, and G313S), M2 (E70K), and NS1 NS1 (S99P, S135I, and S135C). By contrast, 14 nonsynonymous substitutions were detected (S99P, S135I, and S135C). By contrast, 14 nonsynonymous substitutions were detected in in nonvaccinated animals: PB2 (T81K, R340K, E472K, and Q591K), PB1(A139D), PA nonvaccinated animals: PB2 (T81K, R340K, E472K, and Q591K), PB1(A139D), PA (M211V (M211V and R256K), HA (V239I, V283A, and N364D), NA (P45L, N329S, and T434A), and and R256K), HA (V239I, V283A, and N364D), NA (P45L, N329S, and T434A), and NS1 NS1 (G72R) ( Figure 8; Figure 9). Further information about all substitutions with an (G72R) (Figure 8; Figure 9). Further information about all substitutions with an allele allele frequency greater than 1% is noted in the Supplementary data (Table S3). frequency greater than 1% is noted in the Supplementary data (Table S3). Figure8.8.Lolliplot Figure Lolliplotrepresenting representingthe theallocation allocationofofall allamino aminoacid acidsubstitutions substitutionswith withan anallele allelefrequency frequency greater than 5% found in PB2 (a), PB1 (b), PA (c), M1 (d), and NS1 (e) SIV proteins. Allele frequency greater than 5% found in PB2 (a), PB1 (b), PA (c), M1 (d), and NS1 (e) SIV proteins. Allele frequency of of each each variant variant is indicated is indicated on ordinate on ordinate axis.axis. If substitution If one one substitution was found was found in more in more thansample, than one one sample, only only the highest allele frequency is indicated. Amino acid substitutions from vaccinated, the highest allele frequency is indicated. Amino acid substitutions from vaccinated, nonvaccinated, and both groups are represented in purple circles, orange squares, and yellow diamonds, respectively. In samples sequenced in animal 4 at different time points, nonsynonymous substitu- tions PA L697R and NS1 S135I, and synonymous substitutions PA G1485A, HA A1500G, and NA A405G were detected in several samples. Indeed, in lung and NT samples collected in nonvaccinated animal 10, the nonsynonymous variant L655V was found (Table S2). Viruses 2022, 14, x FOR PEER REVIEW 16 of 26 Viruses 2022, 14, 2008 15 of 24 nonvaccinated, and both groups are represented in purple circles, orange squares, and yellow diamonds, respectively. 9. Location Figure 9. Figure Location ofofsubstitutions substitutionswith withanan allele frequency allele frequencygreater thanthan greater 5% found in surface 5% found glyco- in surface proteins by both glycoproteins by protein structure both protein and lolliplot structure and representation. The allele frequency lolliplot representation. The allele isfrequency indicated onis indicated on ordinate ordinate axis. (a) HA 3Daxis. (a) HA trimer (PDB3Daccession trimer (PDBno. accession 7VDF) [51]no.and7VDF) [51]domain lolliplot and lolliplot domain representation. representation. HA1 domains are HA1 domains in represented are represented different in blues tones of different tonesdomains and HA2 of bluesinand HA2 tones different domains in of reds. different (b) NA 3Dtones of reds.head tetramer (b) NA 3D tetramer domain head domain (PDB accession (PDB accession no. 4GZO) [52] andno. 4GZO)domains lolliplot [52] andrepresen- lolliplot domains representation. tation. Substitution P45LSubstitution P45L from the stalk from the domain stalkincluded is not domain inis not the included in the limits of the limits of the crystalography crystalography structure used. structure used. In samples sequenced Nonsynonymous in animal substitutions PB24T2090G at different (L697R)time andpoints, nonsynonymous NS1 A403T (S135I), and substitutions PA L697R and NS1 S135I, and synonymous substitutions synonymous substitutions PA G1485A, HA A1500G, and NA A405G were found in animal PA G1485A, HA A1500G, and NA A405G were detected in several samples. Indeed, in 4 at different time points. Indeed, nonsynonymous variant PA T1993G (L655V) was noted lung and NT samples collected in nonvaccinated in lung and NT from animal 10 (Table S2). animal 10, the nonsynonymous variant L655V was foundRegarding (Table S2).stop-gained mutations, 11 different mutations were recorded only in lung Nonsynonymous samples from vaccinated substitutions PB2nonvaccinated animal 1 and T2090G (L697R) and NS1 animals 9 andA403T (S135I), and 10. Mutation S86* synonymous in PB2 was foundsubstitutions PA G1485A, in nonvaccinated HA 9A1500G, animals and NA E739* and 10, whereas A405G in were PB1 wasfound foundin animal 4 at 1different in animals time points. and 10 (Table Indeed,allnonsynonymous S3). Notably, these substitutionsvariant werePA T1993G noted with (L655V) an allele was noted in frequency lungthan lower and5%,NT except from animal C289* 10 (Table in HA S2).animal 1, which was represented with from Regarding an allele stop‐gained frequency of 21.43% mutations, (Figure 9,11 different Table S3). mutations were recorded only in lung samples from vaccinated animal 1 and nonvaccinated animals 9 and 10. Mutation S86* in 3.10.was PB2 Genetic Diversity found in Viral Populations in nonvaccinated animals 9 and 10, whereas E739* in PB1 was found in animals 1 and 10diversity Nucleotide (Table S3). (π)Notably, increasedallinthese both substitutions experimental were groupsnoted with an at similar allele levels at frequency 2 dpi withlower than respect 5%, except(Figure to inoculum C289* 10(a.1)). in HA from At 3animal 1, which was dpi, π decreased represented in viral samples with from nonvaccinated an animals allele frequency and then of 21.43% increased (Figure 9, TableatS3). 4 dpi. In contrast, in vaccinated animals, π decreased slightly at 3 dpi and remained constant the rest of the days. Regarding π by genomic segments, it increased in most samples in both experimental groups compared with the inoculum, in which only diversity was found in PA and PB2 segments (Figure 10(a.2), Table 4). The π only decreased in comparison with inoculum in the segment PA in virus collected from nonvaccinated animals. 3.10. Genetic Diversity in Viral Populations Nucleotide diversity (π) increased in both experimental groups at similar levels at 2 dpi with respect to inoculum (Figure 10a.1). At 3 dpi, π decreased in viral samples from nonvaccinated animals and then increased at 4 dpi. In contrast, in vaccinated animals, π Viruses 2022, 14, 2008 decreased slightly at 3 dpi and remained constant the rest of the days. 16 of 24 Figure 10. Genetic diversity in viral populations collected from vaccinated and nonvaccinated Figure 10.(a)Genetic animals. diversity Nucleotide in viral diversity populations (π) box collected of plot representation from totalvaccinated and nonvaccinated viral population collected from animals. inoculum,(a)vaccinated, Nucleotide and diversity (π) box plot nonvaccinated representation animals, plotted inofgreen, total viral population violet, collected and orange, from respectively. inoculum, vaccinated, and nonvaccinated animals, plotted in green, violet, and orange, respectively. (a.1) Whole genome π per collection day (a.2) Total π per genomic segment. (b) Nonsynonymous and (a.1) Whole genome synonymous π per nucleotide collection diversity (πNday (a.2) and πS)Total π per box plot genomic segment. representation (b) Nonsynonymous of all sequenced samples for and synonymous nucleotide diversity (πN and πS) box plot representation of all sequenced samples NA, HA, and PA segments. The πN is represented in blue whereas πS is represented in red. Mean, for NA, HA, and PA segments. The πN is represented in blue whereas πS is represented in red. lower, and upper quartiles are represented in each box plot, where whiskers indicate the variability Mean, lower, and upper quartiles are represented in each box plot, where whiskers indicate the outside quartiles. variability outside Points represent quartiles. Points each analyzed represent eachsample. analyzed sample. According to the nonsynonymous and synonymous nucleotide diversity (πN and πS, Regarding π by genomic segments, it increased in most samples in both experimental respectively), on average, the πS was greater than πN in NA and HA segments in viral groups compared with the inoculum, in which only diversity was found in PA and PB2 samples collected from vaccinated animals, whereas it was lower in NS. Meanwhile, in segments (Figure 10a.2, Table 4). The π only decreased in comparison with inoculum in samples from nonvaccinated pigs, πS was greater than πN in the NS segment but lower in the segment PA in virus collected from nonvaccinated animals. NA and HA segments (Figure 10b). No significant differences between groups were found According to the nonsynonymous and synonymous nucleotide diversity (πN and πS, applying the Kruskal–Wallis test. respectively), on average, the πS was greater than πN in NA and HA segments in viral samples collected from vaccinated animals, whereas it was lower in NS. Meanwhile, in 4. Discussion samples from nonvaccinated pigs, πS was greater than πN in the NS segment but lower Swine influenza viruses are pathogens distributed worldwide in pigs that cause an in NA and economic important HA segments (Figure loss in 10b). No significant the ever-growing differences pork production between industry groups [7,53]. were According found applying the Kruskal–Wallis test. to the Food and Agriculture Organization (FAO), Spain is the fourth country in the world in terms of the largest pig production, behind China, USA, and Brazil [53]. There are several SIV subtypes worldwide co-circulating in the field, namely H1N1, H3N2, and H1N2 [54,55], with high seroprevalence levels in farms, including in Spain [56]. Nowadays, together with biosecurity measures, vaccination, although limited, is one of the most used strategies to avoid the disease although it does not prevent virus replication [8,12,57]. The latter, together with the current increase in pig population and the capacity of SIV evolution, could change viral host range, antigenicity, and virulence. A previous study showed different evolutionary trends according to pig vaccination status, showing positive selection pressure in trivalent vaccinated animals, whereas purifying selection pressure Viruses 2022, 14, 2008 17 of 24 was noticed in nonvaccinated ones [12]. Considering the capability of IAVs to escape pre-existing immunity acquired by natural infections and vaccine application [14], both scenarios were studied herein. Hence, for a better understanding of the mechanisms that shape the evolution trends of SIVs, a differential whole SIV genomic analysis was performed by deep sequencing after an experimental H3N2 inoculation in vaccinated and nonvaccinated pigs that had suffered a previous natural infection by A(H1N1)pdm09. In the present study, antibodies against NP and SIV RNA were unexpectedly detected in pigs the day of the first vaccination (0 dpv). Viral and antibody detection methods evidenced that virtually all animals had a previous contact with an IAV before challenge. After genome detection and sequencing, the SIV A(H1N1)pdm09 genome was determined in one of these positive samples. Consequently, this virus was circulating in the farm, probably unnoticed, which highlights the high prevalence of this virus in the field, as previously reported [56]. Animals showed levels of antibodies against NP until the end of the experiment, these values being more heterogeneous in nonvaccinated animals. On the day of challenge, only sera from vaccinated pigs showed HI activity against the SIV H3N2 strain. Consequently, the viral replication was lower, and the pathological findings were milder in vaccinated animals, proving again the efficacy of the vaccine [12]. Furthermore, the clinical manifestation of the disease in nonvaccinated animals previously naturally infected with the A(H1N1)pdm09 pandemic virus does not provide apparent cross-protection against the H3N2 SIV subtype [58]. However, as the vaccine does not confer sterilizing protection, viral shedding and clinical–pathological features were also observed in vaccinated animals, as previously described [12,59]. The vaccine effect had a direct impact on sequencing results as viral shedding is lower in vaccinated animals, thus NGS coverage and depth were less favored in collected samples from vaccinated animals. Nonetheless, whole SIV genome sequences could be obtained from six samples recovered from vaccinated animals, allowing the possibility to compare the genetic variability in the viral population collected from both experimental groups. The percentage identity among challenge, vaccine, and pandemic strains HA and NA proteins are available in the Supplementary Table S4. The virus used for pig inoculation was generated after three passages on MDCK cells. This virus, A/Swine/Spain/SF32071/2007 (H3N2), was isolated from a Spanish porcine influenza outbreak [42]. Regarding the genetic diversity found in the inoculum, only five nonsynonymous substitutions were found in PA and PB2 segments. According to the literature, PA D55N substitution was identified as a possible adaptation of IAV H3N2 subtype virus to mammals [60]. Furthermore, the substitution V618I was already noted in a previous similar study, where it was found at 5 dpi in one nonvaccinated animal challenged with H1N1 subtype [12]. In relation to the Q73K substitution found in PB2, it was already detected in the human influenza isolate A/Anhui/1/2013 (H7N9) [61]. Most of the substitutions found in the inoculum were later lost in the sequenced samples from animals after challenge; this result could indicate that these substitutions are implied in the adaptation of field virus in MDCK cells, and they are lost with the readaptation to the natural host. However, the allele frequencies of some substitutions tended to increase over time in some analyzed samples; thus, the presence of these substitutions does not seem to affect the virus fitness when it is readapted to pig. Regarding de novo nonsynonymous and synonymous SNV proportions, they were greater in both experimental groups at all analyzed frequencies, although always slightly higher in samples of vaccinated pigs. Globally, the de novo analysis may support that under both scenarios (vaccination or not), the viral evolution of SIV H3N2 was under positive selection pressure, suggesting that virions are poorly adapted to swine hosts [62]. This positive evolutionary pressure could be influenced by the readaptation of virions from cell culture to their natural host or by host immune pressures. From all sequenced samples collected from vaccinated and nonvaccinated animals, a total of 214 de novo SNVs were detected, proving once again the high mutation rate of RNA viruses such as IAVs [12,16,63]. The appearance of new variants with increased fitness could Viruses 2022, 14, 2008 18 of 24 help virus adaptation to a novel host environment, those variants being naturally selected and therefore perpetuated in viral populations. However, many of these mutations may have a negative effect on the fitness of the virus. For instance, in the polymerase segments (PB2, PB1, and PA), 99 nonsynonymous variants were detected but only 21 exceed 5% of the allele frequency, showing that most mutations that are generated in these segments do not represent a benefit in viral fitness, probably affecting their replicative capacity. In relation to the polymerase segments, in the PB2 protein responsible for host pre- mRNA cap binding [64], M51I and L697R, both retrieved in samples from vaccinated animals, were allocated in the PB1 binding domain of the protein [65]. Notably, substitution R340K was found in the nonvaccinated animal 13 with an allele frequency of 39.13%. According to previous studies, this substitution contributes increasing the activity of the polymerase, mammal adaptation, and virulence of H10N8, H7N9, and H9N2 avian IAV [66,67]. Therefore, this substitution may affect viral replication since it was found in the unique lung sample where immunoreactivity was detected at 5 dpi. The Q591K substitution was observed in the nonvaccinated animal 10, which showed one of the highest pathological scores and the highest rectal temperature registered in the study. This substitution was previously related to mammal adaptation and increased virulence of H5N1, H7M9, and H9N2 IAVs [68]. In the protein with polymerase activity, PB1, the substitution P64S was found in the vaccinated animal 4; this mutation has been previously found circulating in the swine population from Guangdong (China) in an SIV H1N1 virus [69]. With reference to the PA protein, whose key role is cleavage host pre-mRNA and transcription initiation [64], the substitution R256K noted in nonvaccinated animal 10 was found predominant in IAVs H1N1, H2N2, H3N2, and H5N1 [70]. Furthermore, the substitution E349G was found in samples collected from the vaccinated animal 4 and nonvaccinated animals 9 and 10. In previous works, this substitution has been shown to increase the virulence of IAV [71,72]. NS1 protein is implied in viral replication and host immunity regulation. In the present study, five nonsynonymous substitutions with an allele frequency greater than 5% were found in this protein: two in the RNA binding domain in samples from nonvaccinated animals and three in the effector binding domain from vaccinated ones. The two substi- tutions found in nonvaccinated animals, E71K and G72R, were located close to the linker region of the protein, which play an important role in its plasticity. Hence, these substi- tutions could be involved in different protein conformations, affecting their interaction with other molecules and therefore functions [73]. Moreover, the E71K substitution was previously studied by reverse genetics in avian IAV, and it reduced IFN-β expression [74]. This substitution was found in the viral population collected from the nasal swab of the nonvaccinated animal 10. Thus, the E71K substitution may limit IFN-β expression by increasing virus replication and virulence [75]. By contrast, in the vaccinated animal 2, substitutions S99P and S135I were simultaneously found. The first one interacts with the cellular polypeptide tripartite motif-containing protein 25 (Trim25), which regulates the host innate immune response to infection by ubiquitin ligases [76,77]. The second one is implied in host p85β binding, which interacts with phosphoinositide 3-kinase (PI3K), activating a cellular pathway resulting in inhibition of apoptosis by phosphorylation of caspases, contributing to virus replication and virulence [78,79]. Another substitution at position 135 (S135C) was found in the nasal swab from animal 4 at 4 and 5 dpi. It should be highlighted that allele frequency of this substitution increased from one day to the other from 6.35 up to 48.85%. Therefore, this substitution may benefit the viral fitness because of the rapid allelic increase and the viral shedding recorded during 3 consecutive days from this animal. In a previous study, nonsynonymous substitutions in this protein were only found in vaccinated animals, showing that this protein could rapidly change, facilitating the adaptability of the virus under immune pressure [12]. In relation to the M segment, nonsynonymous substitutions were only found in viral populations collected from vaccinated animals. From the seven substitutions identified, only M1 E204K found in the animal 2 exceeded 5% of allelic frequency (14.28%). The role of this protein is virion assembly by interaction with proteins and lipids [80]. A previous study Viruses 2022, 14, 2008 19 of 24 revealed that at this position, it induces filamentous changes in infected cells, affecting virus growth [81]. Notably, only one nonsynonymous substitution was found in the NP protein with an allele frequency of 2.9%. This protein interacts with polymerases and has an important role in the synthesis of RNA and its traffic [82,83]. Nonsynonymous substitutions in this protein could impair viral fitness, as it is a highly conserved protein among IAVs [84]. Therefore, our findings could support NP as a good therapeutic target for antiviral and universal vaccine development [85–87]. However, in a previous study from our group, in which the evolutionary capacity of the H1N1 virus was evaluated, six nonsynonymous substitutions were described in this protein [12]. The surface glycoproteins HA and NA are the main targets to generate antibodies against IAV after natural infection and/or vaccination [20]. Therefore, substitutions in these proteins may play an important role in the generation of mutants that may allow viruses to escape pre-existing immunity. The πN and πS proportion was lower in vaccinated animals, whereas the opposite was found in nonvaccinated ones, pointing to different evolutionary patterns depending on immunological pressure. Thus, under both scenarios, these segments showed different evolutionary tendency, acting to purify and enable natural selection in viruses collected from vaccinated and nonvaccinated animals, respectively. Notably, on average the π was greater in vaccinated animals, and this could indicate that HA and NA are evolving faster in these animals, regardless of whether those substitutions were synonymous or not. Regarding substitutions found with an allele frequency greater than 5%, more changes were found, proportional to the number of obtained sequences per group, in samples from vaccinated animals. In nonvaccinated animals, three substitutions were identified in the HA segment. Substitution V239I was the only one found in the receptor binding domain and it is allocated in one epitope region. This substitution was previously identified in epidemic H3N2 SIV subtype isolated in Southern China in 2012 [88]. Furthermore, V283A and N364D substitutions were found in the vestigial esterase domain and in the fusion peptide. In contrast, four substitutions were found in the fusion (C297* and I339V) and the stalk domains (T467I and N505S) in vaccinated animals. The stop codon mutation, originated by C297* mutation, generates a truncated protein with 296 amino acids. In a previous study, stop codon substitutions were found in HA and those substitutions were transmitted among pigs [89]. Furthermore, avian IAV HA truncated proteins have been previously found in environmental reservoirs [90]. The I339V substitution, close to the cleavage domain, has been previously reported in H3N2 canine IAV in China [91]. Regarding the stalk domain, substitutions T467I and N505S were simultaneously found in the lung sample of the vaccinated animal 2, with allele frequencies of 31.25 and 25%, respectively. According to a previous study, evolution of this domain is slower and independent from immune pressure [92]. Interestingly, our findings suggest that pre-existing immunity may have an implication in the positive evolution of substitutions in this domain, as no substitutions were found in the head domain. Finally, the substitution I351K in the fusion peptide was found in three samples from both experimental groups. Regarding the NA segment, substitutions N329S and T434A, located in the head domain, were recovered from nonvaccinated animals 10 and 16, respectively. In a previous study, substitution N329S was associated with losing one glycosylation site, although it did not alter the activity of the protein [93]. According to a previous study, this substitution is allocated in the antigenic epitope F0 329–339 [94]. In vaccinated animals, substitution F167L was recovered in animal 2, whereas V303I and G313S ones were detected in nasal swab samples from animal 4 at 3 and 4 dpi, respectively. Substitution V303I was previously reported circulating in Bulgaria during the 2019–2020 winter season in H3N2 human IAV [95]. This substitution is allocated in the epitope region E0 302-308 of the N2 protein [94]. Considering that this substitution, like the previously mentioned S135C in NS1 protein, was found in nasal swab samples collected in the only vaccinated animal which showed SIV detection during three consecutive days, this substitution may favor at 3 dpi some adaptative advantage to viral replication. Viruses 2022, 14, 2008 20 of 24 In the present study, the high mutability rate of SIV has been further demonstrated [12], showing that IAVs’ genomes is constantly changing. The high population of pigs and the high persistence of the virus make IAVs a continuous threat to both animal and human health, as virus evolution could affect viral host range, antigenicity, and virulence. Hence, rapid virus evolution contributes to the fact that the virus remains in the field continuously, which simultaneously implies that the virus is constantly evolving. Herein, the impact of each de novo substitution found on virus fitness is hypothesized according to previous literature. Therefore, future research will be necessary to deeply understand the role of these substitutions, either alone or together, through reverse genetic technology studies [96]. Altogether, the risk of future pandemic IAVs, as happened in 2009, is not negligible. To avoid or minimize the damage it could cause, the reduction of SIV circulation as much as possible by applying stricter vaccination schedules and a more rational swine production are highly advisable. Furthermore, SIV surveillance studies in farms and more effective or universal vaccine development are also crucial to prevent future flu pandemics. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/v14092008/s1, Supplementary Table S1. Sequenced reads obtained per sample. The total number of reads and reads mapped against reference genome after post-mapping filter application. Supplementary Table S2. SNV and its allele frequency reported in several samples. Supplementary Table S3. SIV H3N2 single nucleotide variants detected in all samples collected from vaccinated and nonvaccinated pigs. Table S4. HA and NA amino acid percentage identity among challenge, vaccine, and pandemic strains. Author Contributions: Conceptualization, J.I.N.; methodology, Á.L.-V., L.C., J.I.N. and J.S.; HTS libraries preparation, L.B. and C.C.; bioinformatic analysis, Á.L.-V.; formal analysis, Á.L.-V., L.G. and J.I.N.; resources, A.D. and J.I.N.; writing—original draft preparation, Á.L.-V., L.G. and J.I.N.; writing—review and editing, Á.L.-V., J.S., L.G. and J.I.N.; supervision, J.I.N.; project administration, J.I.N.; funding acquisition, A.D. and J.I.N. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by grants AGL2016–75280-R from Ministerio de Ciencia, Inno- vación y Universidades from the Spanish government. Á.L.-V. has a pre-doctoral fellowship FPI 2017, Ministerio de Ciencia, Innovación y Universidades from the Spanish government. Institutional Review Board Statement: The experiment was carried out in accordance with the Spanish and European regulations and was approved by the animal ethics committee from the Generalitat de Catalunya, Spain, under approval code 9657. Informed Consent Statement: Not applicable. Data Availability Statement: Sequencing data were deposited at NCBI, with the accession number (PRJNA853173). Acknowledgments: The authors thank Diego Pérez and Gemma Guevara for their contribution in the field studies. Authors also thank Carlos Neila, Miguel Blanco, and Lola Pailler for their support and advice in bioinformatics biostatistics and figure layout. Conflicts of Interest: The authors declare no conflict of interest. References 1. World Organisation for Animal Health. Swine Influenza. Available online: https://www.oie.int/en/disease/swine-influenza/ (accessed on 22 February 2022). 2. Li, Y.; Robertson, I. The Epidemiology of Swine Influenza. Anim. Dis. 2021, 1, 21. [CrossRef] [PubMed] 3. 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