Received: 7 February 2020 | Revised: 23 June 2020 | Accepted: 23 June 2020 DOI: 10.1111/tbed.13709 ORIGINAL ARTICLE Diversity of influenza A viruses retrieved from respiratory disease outbreaks and subclinically infected herds in Spain (2017–2019) Silvana Sosa Portugal1 | Martí Cortey1 | Montserrat Tello1 | Carlos Casanovas2 | Susana Mesonero-Escuredo2 | Sergio Barrabés2 | Pilar Pineda1 | Silke Wacheck3 | Gerard Martín-Valls1 | Enric Mateu1,4 1 Department Sanitat i Anatomia Animals, Facultat de Veterinària, Universitat Abstract Autònoma de Barcelona, Travessera dels The present study was aimed to assess the diversity of influenza A viruses (IAV) cir- Turons s/n, campus UAB, Cerdanyola del Vallès, Spain culating in pig farms in the Iberian Peninsula. The study included two different situa- 2 Ceva Salud Animal, S.A. Avenida Diagonal, tions: farms suffering respiratory disease outbreaks compatible with IAV (n = 211) and Barcelona, Spain randomly selected farms without overt respiratory disease (n = 19). Initially, the pres- 3 Ceva Santé Animale, Libourne, France 4 ence of IAV and lineage determination was assessed by qRT-PCR using nasal swabs. IRTA-CReSA-UAB, edifici CReSA, campus UAB, Cerdanyola del Vallès, Spain IAV was confirmed in 145 outbreaks (68.7%), mostly in nurseries (53/145; 36.5%). Subtyping by RT-qPCR was possible in 94 of those cases being H1avN2hu (33.6%), Correspondence Silvana Sosa Portugal, Department Sanitat H1avN1av (24.3%) and H1huN2hu (18.7%), the most common lineages. H3huN2hu i Anatomia Animals, Universitat Autònoma and H1pdmN1pdm represented 7.5% and 6.5% of the cases, respectively. As for the de Barcelona, Facultat de Veterinària, Cerdanyola del Vallès, Spain. randomly selected farms, 15/19 (78.9%) were positive for IAV. Again, the virus was Email: silvananelly.sosa@e-campus.uab.cat mostly found in nurseries and H1avN2hu was the predominant lineage. Virus isola- tion in MDCK cells was attempted from positive cases. Sixty of the isolates were fully sequenced with Illumina MiSeq®. Within those 60 isolates, the most frequent genotypes had internal genes of avian origin, and these were D (19/60; 31.7%) and A (11/60; 18.3%), H1avN2hu and H1avN1av, respectively. In addition, seven previously unreported genotypes were identified. In two samples, more than one H or N were found and it was not possible to precisely establish their genotypes. A great diversity was observed in the phylogenetic analysis. Notably, four H3 sequences clustered with human isolates from 2004–05 (Malaysia and Denmark) that were considered uncommon in pigs. Overall, this study indicates that IAV is a very common agent in respiratory disease outbreaks in Spanish pig farms. The genetic diversity of this virus is continuously expanding with clear changes in the predominant subtypes and lineages in relatively short periods of time. The current genotyping scheme has to be enlarged to include the new genotypes that could be found in the future. KEYWORDS epidemiology, influenza A, lineages, pig, sequencing This is an open access article under the terms of the Creat​ive Commo​ns Attri​butio​n-NonCo​mmerc​ial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2020 The Authors. Transboundary and Emerging Diseases published by Blackwell Verlag GmbH Transbound Emerg Dis. 2021;68:519–530.  wileyonlinelibrary.com/journal/tbed | 519 520 | SOSA PORTUGAL et al. 1 | I NTRO D U C TI O N Europe (Beato et al., 2016; Howard et al., 2011; Lange et al., 2013; Moreno et al., 2009, 2011; Simon-Grifé et al., 2011). Influenza A virus (IAV) is one of the most important respiratory At present, Spain is an ideal scenario for the examination of such pathogens in pigs being a key agent in the porcine respiratory disease IAV diversity in the European context. As it is one of the few coun- complex (PRDC) of weaners and growers. For many years, IAV infec- tries in Europe where pig population increased in the last years. In tion in pigs was mainly associated with epidemic outbreaks of acute 2019, with 2.2 million sows, a total census of 29 million pigs and respiratory disease affecting most animals in the farm. However, imports of about 1 million animals (Ministerio de Agricultura, Pesca y in the last years, increasing evidence indicates that most farms in Alimentación, 2019), Spain – together with Germany – are the lead- Europe are endemically infected by one or more IAV viruses (Kyriakis ing pork-producing countries in Europe and among the top 5 in the et al., 2011, 2013; Van Reeth et al., 2008; Simon-Grifé et al., 2012) world (Eurostat, 2019). and that reassortment events are not uncommon (Martín-Valls Epidemiological information paired with sequencing and detailed et al., 2014). In those farms, the main circulation of the virus takes phylogenetic analysis are tools that can help understand the circu- place in the farrowing units and nurseries (Ferreira et al., 2017; Ryt- lation routes and mutation patterns of these viruses. The aim of the Hansen et al., 2019), with occasional generalised outbreaks when a present study was to gain a better insight into the diversity of IAV new IAV strain enters the farm for the first time. in Spanish pig farms with outbreaks of respiratory disease or farms The IAV genome consists of eight segments, which encode for with subclinical infections. up to 14 proteins, among them five structural ones: haemagglutinin (HA), neuraminidase (NA), two matrix proteins (M1 and M2) and the nucleoprotein (NP). The combination of haemagglutinin (HA) and neur- 2 | M ATE R I A L S A N D M E TH O DS aminidase (NA) determines the subtype. At present, 18 types of HA and 11 types of NA are known in mammals and birds, but only three 2.1 | Data collection subtypes predominate in pigs: H1N1, H1N2 and H3N2. However, the genetic and antigenic diversity within those subtypes is considerable. The study took place between February 2017 and March 2019 and In Europe, this diversity is particularly high for swine H1N1 and H1N2 included two different situations: farms suffering respiratory dis- viruses. Furthermore, these viruses have evolved into distinct genetic ease and samples from subclinical farms that did not report respira- lineages in different geographic locations. Since the 2009 pandemic, tory disease cases. four lineages are predominantly found in pig farms: Eurasian avi- For the farms suffering respiratory disease, a case was defined an-like H1N1 (H1avN1av), human-like H1N2 (H1huN2hu), human-like as a situation where respiratory disease was noticeable in at least H3huN2hu and the pandemic lineage H1N1 (H1pdmN1pdm). one age group of pigs with predominant signs of cough and fever. While the haemagglutination inhibition test (HI) may distinguish Shipping of suspected influenza cases (nasal swabs) was requested isolates of different phylogenetic origin within a given subtype (Van from field veterinarians in Spain and Portugal. In order to accept a Reeth, Labarque, & Pensaert, 2006), cross-reactivity among strains submission, a data sheet indicating clinical signs of the animals had belonging to subtypes and lineages different to H1 have been re- to be fulfilled. Sampling had to include at least 10 nasal swabs from ported for North American H1 and classical swine H1 (Kyriakis feverish animals. Samples were collected in a virus transport me- et al., 2010). Also, some degree of cross-protection may exist be- dium (Virocult®; Sigma-Aldrich), kept at 4ºC during transport and tween H1pdmN1pdm and H1avN1av (Busquets et al., 2010; Kyriakis immediately processed upon arrival. Occasionally, if dead animals et al., 2010). Altogether, this indicates the need for additional classi- were found, lungs were also sent for analysis. fication schemes. To assess the circulation of IAV in subclinical farms (no obvi- Watson et al. (2015) proposed a genotyping system based on the ous respiratory disease), sampling was performed in 19 farms in origin of their two external (HA and NA) and six internal gene seg- Catalonia. The selection of these farms was done randomly from a ments (NP, M, the three polymerase genes and the non-structural group of different pig producing enterprises representing 20% of protein, also known as internal gene cassette) to classify the swine the Catalan pig industry. The included farms fulfilled two criteria: IAV lineages detected in Europe. At the time of that publication, 1) farms that were not suffering from respiratory outbreaks in any they had classified 23 genotypes, but only four of them presented a production phase and 2) farms that were farrow-to-finish. In those pan-European distribution. In that study, most isolates contained an farms, nasal swabs were taken randomly from suckling pigs, wean- internal gene cassette derived from the Eurasian avian-like lineage ers and fatteners (n = 20 each). Samples were collected using the (67%) or the H1pdmN1pdm lineage (27%). aforementioned virus transport medium and under the mentioned IAV diversity in pigs has been related to the intensification of storage conditions. That sampling strategy would allow us to detect the production system (Van Reeth et al., 2008). In this sense, it has viral circulation in at least one positive farm assuming a 15% herd been suggested that factors such as the continuous introduction of prevalence of subclinical herds and considering a 95% confidence animals from different sources can contribute to the introduction interval. Similarly, the sampling method allowed us to detect at least of new viruses into a farm and the subsequent reassortment among one positive assuming a prevalence of 15% of IAV in each sampled strains. These reassortment events seem to be very common in age (95% confidence interval). SOSA PORTUGAL et al. | 521 2.2 | Processing of samples MiSeq platform at Parc de Genòmica i Recerca-UAB. The output reads in fastaq format (doubled paired) were checked for quality Upon arrival to the laboratory, the Virocoult® transport media using Trimmomatic (matching of forward and reverse sequences containing the nasal swabs were vortexed and the medium was and quality index >20). Next, high-quality reads were filtered using transferred to a sterile 1.5-ml tube. Then, tubes were centrifuged IAV sequence references belonging to all known lineages for each at 4,000 g for 5 min and the supernatant was recovered for further of the eight genome segments (Supplementary material l). Genome processing. Viral RNA was extracted from 150 µl of the supernatant consensuses were generated using the Simple Consensus maker tool using a commercial kit (NucleoSpin® RNA virus, Macherey-Nagel) (available at: https://www.hiv.lanl.gov/conte​nt/seque​nce/CONSE​ according to the manufacturer's instructions. Initial detection of the NSUS/SimpC​on.html) when a viral quasi-species was obtained. The virus was done by means of a RT-qPCR targeted to the IAV matrix segments were then manually aligned and trimmed using BioEdit se- gene (Busquets et al., 2010) using the AgPath-ID™ One-Step RT- quence alignment editor for Windows (Hall, 1999). When filtering PCR reagents (Thermo Fisher). Positive samples underwent a sec- did not yield a viral quasi-species, consensus sequence for that gene ond RT-qPCR for identifying subtypes and European lineages of H was forced using QuASAR software (available at: https://github. and N, including H1av, H1hu, H1pdm, H3hu, N1av, N1pdm and N2hu com/pique​lab/QuASAR). The resulting consensus sequence was (Henritzi et al., 2016, 2019). blasted against available sequences in GenBank, and the closest se- quence was used to re-filter the original fastaq file. 2.3 | Virus isolation 2.5 | Phylogenetic analysis and genotyping Madin–Darby canine kidney (MDCK) cells (ATCC CRL-2936™) were used for the isolation of IAV from RT-qPCR-positive (M gene). Viral Phylogenetic analysis was performed using the MEGA X software isolation was attempted from up to three RT-PCR-positive samples (available for download at: https://www.megas​oftwa​re.net/). An per case (from both respiratory outbreaks and subclinical sampled initial database was built using all sequences available in GenBank farms), to obtain a high-quality isolate for further sequencing. For of Euroasian swine IAV for each genome segment. At first, an initial the isolation process, 100 µl of the sample supernatant was mixed removal of identical sequences (100% of identity in identity matrix) with 900 µl of Minimum Essential Medium Eagle (MEM) supple- was done. A preliminary phylogenetic analysis (neighbour-joining) al- mented with L-glutamine (200 mM), penicillin-streptomycin (10,000 lowed the removal of redundant sequences within a given cluster. units and 10 mg/ml, respectively) and trypsin TPCK-treated for a For each cluster, this corresponded to highly similar isolates (>97% final concentration of 10 µg/ml of trypsin. Trypsin-treated samples identity). Finally, in order to improve the resolution of each tree, a 2 were inoculated in 25cm cell culture flasks. After 1.5 hr of incu- BLAST was done using each of the sequences obtained in the pre- bation at 37ºC, the flasks were filled with 9 ml of medium without sent paper, and the output was used for completing the database trypsin (final concentration of trypsin in the culture 1µg/ml) and cul- for each gene with sequences not initially included. Final databases tures were incubated for up to 5 days. for each gene contained about 150–250 sequences representing the Cells were observed every 24 hr to check for the development of significant clusters in the preliminary analysis. Relevant non-Euro- cytopathic effect (CPE). Once, CPE was observed flasks were frozen pean strain sequences were included when needed. Supplementary at −80ºC, thawed and, after centrifugation, the cell culture super- file 2 shows the GenBank accession numbers of the included se- natant was examined by RT-PCR (M gene) to confirm the replica- quences. Sequences obtained in the present study were added to tion of the virus (lower Cq values compared to the initial inoculum). the corresponding databases for each gene. Final phylogenetic trees Samples were discarded when CPE was not observed after a third were built using the maximum likelihood method (1,000 iterations) blind passage. Up to three samples per case were attempted for viral using the general time-reversible model (GTR) and subtree pruning isolation. and regrafting moves (SPR) option. Once the subtype, lineage and phylogenetic clustering for each isolate and gene was identified, the genotyping was done according to the classification of Watson 2.4 | Next-generation sequencing et al. (2015). Briefly, for internal genes (PB2, PB1, PA, NP, M and NS), genes were classified as 'avian-like' or 'pandemic'. For haemaggluti- In order to have a more precise picture of the IAV circulating in nins, there was a differentiation between the three H1s: avian-like, the studied population, the genome of 60 representative isolates human-like and pandemic. In literature, there is only one type of H3. was fully sequenced using next-generation sequencing technol- For neuraminidases, N1 differentiation was also done depending if ogy (enough to detect at least one genotype with an occurrence they clustered with avian-like isolates and pandemic isolates, and for of ≥5% in the cases, 95% confidence). These cases were selected N2, only the human-like isolate was considered. The combination from the MDCK-isolated viruses, and this included one isolate per of the 8 segments defined the genotype, and previously reported farm as maximum. The total RNA extraction from the 60 selected genotypes have been named according to Watson et al., (2015) viruses isolated in MDCK cells was sequenced using an Illumina® proposal, following the alphabetical order. Previously unreported 522 | SOSA PORTUGAL et al. genotypes were assigned as UG – standing for 'unreported geno- present. Supplementary file 3 shows the geographic distribution of type' – followed by a number. the positive cases for which the precise location of the farm was recorded. The Cq values in IAV-positive cases versus the proportion of in- 3 | R E S U LT S fected animals was also examined (Figure 1). The results showed that the higher the proportion of infected animals, the lower the average 3.1 | Cases of respiratory disease Cq in positive animals (r=−0.33, CI95% = −0.49 to −0.15; p < .0001). Table 1 summarises the results for the submitted cases. During the study period, 211 submissions fulfilled the requirements and were 3.1.1 | Assessment of apparently subclinical farms considered respiratory disease cases. Cases were distributed in all regions of Spain with significant pig production, plus four cases Apparently healthy animals of 17 farrow-to-finish farms and 2 farms submitted from Portugal. Most of the submissions corresponded to without fattening unit were examined. Of these, in 15 farms (78.9%) nursery pigs (39.8%), suckling piglets and fatteners had a consider- animals tested positive for IAV by RT-PCR for the M gene (Table 2). able number of submissions (17.5% and 13.3%, respectively), while The distribution of positive animals did not show differences be- only 7.1% were sow samples, accounting this latter group for the tween suckling piglets and weaners, as 12 farms were detected as lowest percentage of positive submissions (p = .02). Unfortunately, positive in each of these two phases. In 7 farms, both phases were in 22.3% of submissions, the age information could not be provided positive at the same time of sampling. There was one farm that to us for different reasons. Of the 211 submissions, in 145 (68.7%, tested positive for the 3 production phases, being the only one hav- CI95%:60.5%–73.8%), IAV-positive animals (M gene RT-qPCR) were ing positive results not only for suckling piglets and weaners, but also for the fattening unit. TA B L E 1 Distribution of case submissions by age group and proportion of IAV-positive cases (M gene RT-qPCR) 3.1.2 | Lineages detected No. of positive No. of submissions/Total Age group submissions (%) submitted (%) Among the 145 cases with positive samples for the M gene, 94 Suckling piglets 37 (17.5) 26/37 (70.3) (85.4%; CI95%:78.1%–90.6%) could be subtyped using the subtyping RT-qPCR (Table 3). All typable samples yielded Cq values < 30 for Nurseries 84 (39.8) 53/84 (63.1) the M gene. Isolation of the virus was successful in 11/51 cases, in Fatteners 28 (13.3) 22/28 (78.8) which RT-qPCR typing was not possible (21.5%; CI95%:11.8%–35.7%). Sows* 15 (7.1) 6/15 (40)* Those 11 samples were sequenced by NGS and belonged to differ- Other 47 (22.3) 38/47 (80.9) ent subtypes and lineages (H1avN1av, H1avN2hu, H3huN1av and Total 211 (100) 145/211 (68.7) H3huN2hu). For the 40 remaining cases, the complete lineage could *p < .05 not be obtained by RT-qPCR and attempts of isolation in cell culture F I G U R E 1 Distribution and regression of positive animals per case (respiratory outbreaks) and the Cq values. The graph shows the Cq for positive samples distributed by the percentage of positive animals per case SOSA PORTUGAL et al. | 523 were unsuccessful. No geographical pattern in the distribution of of these last three, one could be fully sequenced after isolation and the lineages was found, except for the H3huN1av isolates that were corresponded to H1avN2hu. detected in two farms of the same company in the same province. Regarding the assessment of apparently subclinical farms, the lineage of the IAV virus circulating in the farm could be determined 3.1.3 | Genotyping and phylogenetic analysis by RT-qPCR in 12 cases while in three farms only the H or N lin- eage could be determined. The lineages detected were as follows: Full genome sequencing allowed the determination of the geno- H1avN2hu, H1huN2hu, H1avN1av, H1huN1av and for the three type for 60 isolates (Figure 2). The most common were genotypes D partially subtyped, these were HxN1av, HxN2hu and H1avNx. Out (31.7%) and A (18.3%). In the present study, 7 previously unreported genotypes (UG) were identified and designated as UGx(1-7). For two TA B L E 2 Distribution of influenza A positive pools per farm and additional samples, two different H or N were detected in the sample their average Cq value in herds subclinically infected (by PCR subtyping and NGS sequencing), while all other genes were Positive pools consistent with a single origin. Since it was impossible to establish the genotype of the virus or viruses present, they were designated Positive farms Suckling Average as undetermined genotype (UDX ). For simplification purposes, each (n = 15) piglets Weaners Fatteners Cq value segment has been represented following the colour patterns used Farm 1 4/5 0/5 0/5 31.3 by Watson et al. (2015). In the cases where different lineages were Farm 2 5/5 5/5 1/5 27.6 found, new colours have been proposed. Farm 3 1/5 4/5 N/A a 31.6 A great diversity was observed in the phylogenetic analysis. Farm 4 0/5 3/5 0/5 28.1 Regarding H (Figure 3) and N segments/genes (Figure 4), all isolates Farm 5 0/5 4/5 0/5 29.7 belonged to the expected lineages. However, it is worth noting that Farm 6 4/5 2/5 0/5 31.6 four H3 pig sequences clustered with human isolates from 2004–05 Farm 7 3/5 3/5 0/5 28.9 (Malaysia and Denmark) that were considered uncommon in pigs. Farm 8 4/5 2/5 0/5 27.7 This finding corresponds to isolates dating from the last two years and could possibly mean the disappearance of the classic H3 and its Farm 9 3/5 0/5 0/5 32.8 replacement by this new type. Farm 10 3/5 0/5 0/5 30.3 Interestingly, for some genes, particularly M (Figure 5), local Farm 11 0/5 5/5 0/5 17.9 (Spanish) clustering was observed. Thus, of the 60 sequences ob- Farm 12 4/5 4/5 0/5 26.9 tained in the present study, 37 grouped in two clusters mainly com- Farm 13 5/5 0/5 0/5 28.5 posed of Spanish sequences. Similarly, for the NS gene, 39 Spanish Farm 14 2/5 4/5 0/5 27.8 sequences from the present study clustered together in two groups Farm 15 5/5 5/5 N/A a 27.5 (Supplementary file 8). For PB1 and PB2, a local cluster within the a Farms without a fattening unit. pandemic group was observed; this clustering was not maintained TA B L E 3 Distribution of detected Lineages detected No isolates No isolates No isolates lineages in influenza A positive pigs (n = 95) RT-qPCR NGS Total Percentage samples retrieved from cases of a respiratory disease and one case from an H1avN2hu 31 5 36 33.6 apparently subclinical farm H1avN1av 24 2 26 24.3 H1huN2hu 20 0 20 18.7 H3huN2hu 6 2 8 7.5 H1huN1av 6 0 6 5.6 H1pdmN1pdm 5 0 5 4.7 H3huN1av 1 2 3 2.8 H1pdmN1av/N1pdm 1 0 1 0.9 b H1av/H1pdmN1av 0 1 1 0.9 H3huN1av/N2hub 1 0 1 0.9 Total 95 12 107 100% a Includes one case retrieved from an apparent subclinical farm where the initial RT-qPCR assessment was H1avNx, and after successful isolation, the sequence obtained was found to be H1avN2hu. b In two cases, two H or two N were detected in the RT-qPCR. 524 | SOSA PORTUGAL et al. common agent in those outbreaks in Spanish farms. Nevertheless, it is important to distinguish between a positive IAV result by RT-PCR and attributing to IAV the role of primary agent in the outbreak. In the present study, we did not check for other respiratory pathogens that could have contributed to the clinical features observed in the farm. However, when we performed a random sampling in appar- ently subclinical farms, IAV was also commonly found in the nasal swabs examined. A deeper analysis of the data showed that while in subclinically infected herds the proportion of IAV-positive sam- ples rarely exceeded 50%, in most of the outbreaks the percentage of positive samples was above this value. Moreover, the average Cq values and the proportion of infected animals were significantly related. In addition, in subclinically infected animals, Cqs ≥ 30 were more frequent than in the outbreaks (not shown). Furthermore, in the outbreaks, sample size was smaller as it was directed to animals showing clinical signs, on the contrary, for the random sampling, a larger sample size was needed and it proved to have a higher detec- tion rate. Taken together, these facts suggest that the diagnosis of IAV as a primary agent of a respiratory outbreak requires a large targeted sampling of animals with compatible signs. Our results in- dicate that an influenza outbreak can be presumed when > 50% of the samples are positive and average Cqs are below 30, as seen by the distribution of positive samples per case and their respective Cq (Figure 1). Nonetheless, there are other studies were, under sub- clinical circumstances, the percentage of IAV RT-PCR-positive ani- mals were over 70% (Ferreira et al., 2017; Simon-Grifé et al., 2012). Considering this, a better sampling approach should include an equivalent number of animals not showing clinical signs as control F I G U R E 2 Distribution of genotypes in the 60 influenza A virus isolates sequenced. Designation of the genotypes followed in order to confirm the diagnosis and properly evaluate the relative the classification by Watson et al. (2015). For those genotypes risks. previously unreported, we used the designation ‘unreported On the other hand, most positive animals were found in nurs- genotype’ (UG). †Denominations and strains used as reference eries. This fact correlates with other published studies showing according to Watson et al., 2015. ‡Older closest relative strain similar results (Ferreira et al., 2017; Simon-Grifé et al., 2012). This found in GenBank. §In these cases, two H or two N genes were pattern of respiratory outbreaks is compatible with the presence detected but no differences for all other genes. In these cases, we used the designation undetermined genotype (UD) of IAV endemic circulation, where in most cases the circulation of the virus and the increase of clinical outcome can be observed for PA (Supplementary files 4, 5 and 6). Regarding the NP segment, in the nurseries probably due to the declining of maternally de- two main clusters can be observed (Supplementary file 7). rived antibodies (Gillespie, 1999; Rose et al., 2013; Torremorell et al., 2009). IAV-positive cases were spread all over the territory. We were 4 | D I S CU S S I O N unable to detect any particular distribution of cases or lineages, ex- cept for the two H3huN1av isolates that were found in two adja- Influenza A virus is one of the main components of PRDC cent farms in the same province. Although sampling was based on (Brockmeier, Halbur, & Thacker, 2002). In the present study, about voluntary submissions and, therefore, the number and location of two thirds of the cases submitted because of a respiratory disease detected positive cases do not necessarily correlate with the real episode in the herd were positive for IAV by RT-qPCR. Although proportion and distribution of subtypes and lineages, it is clear that some selection bias may exist, this result indicates that IAV is a very IAV is present in most farms. F I G U R E 3 Maximum likelihood tree of the influenza A positive haemagglutinins H1 and H3 (1,000 iterations). Coloured boxes show the main lineages for Eurasian H1 and H3 as indicated in the figure. Sequences highlighted in black and white letters represent a common ancestor for the whole lineage indicated by the box. Green (avian-like H1), dark red (pandemic H1), purple (human-like H1), orange (new seasonal human H3), and blue (classical human-like swine H3) indicate a strain previously isolated in swine that could be used as a more recent reference of the lineage in swine. Sequences in red are those from the present study. Values in the branches show the bootstrap values (only ≥60 are shown) SOSA PORTUGAL et al. | 525 99 A/swine/Spain/150/2018 (H1N2) D 68 A/swine/Spain/153/2018 (H1N2) C 68 A/swine/Spain/BM55/2019 (H1N2) C H1 A/swine/Spain/1372/2015 (H1N2) UG4 H1hu A/swine/Scotland/410440/1994-like 100 A/swine/Spain/456902/2016 (H1N1) 86 A/swine/Spain/2698/2016 (H1N2) C 99 A/swine/Spain/29257/2012 (H1N2) 100 A/swine/Spain/1297/2016 (H1N2) C 100 A/swine/England/142162/2010 (H1N2) 76 A/swine/England/1382/2010 (H1N2) 62 A/swine/England/33766/2003 (H1N2) 99 A/swine/England/6183/2012 (H1N2) A/swine/England/1380/2011 (H1N2) 100 A/swine/England/7856/2012 (H1N2) 65 A/swine/Germany/Genthin167D/2012 (H1N2) 64 A/swine/Spain/79976/2007 (H1N2) A/swine/Granstedt/IDT3475/2004 (H1N2) A/swine/Netherlands/Haaksbergen136/2011 (H1N2) A/swine/Italy/18/2000 (H1N2) A/swine/Bakum/1832/2000 (H1N2) 96 A/swine/France/56120177/2012 (H1N2) 89 A/swine/France/29130285/2013 (H1N2) 100 A/swine/France/56120285/2012 (H1N2) 90 A/swine/France/35120024/2012 (H1N2) A/swine/France/22110619/2011 (H1N1) 94 99 A/swine/France/IIIeetVilaine0404/2011 (H1N2) A/swine/France/22120391/2012 (H1N2) 78 A/swine/Italy/3592/1999 (H1N2) A/swine/Italy/62/1998 (H1N2) 75 95 A/swine/Italy/233139/2005 (H1N2) 65 A/swine/Italy/50568/2005 (H1N2) 82 82 A/swine/Italy/1662772/2014 (H1N2) A/swine/Italy/198260/2008 (H1N2) 86 A/swine/Italy/70757/2009 (H1N2) A/swine/Italy/81226/2009 (H1N2) 100 77 A/swine/Italy/41350/2011 (H1N2) 99 A/swine/Italy/346150/2013 (H1N2) 90 A/swine/Italy/3346941/2014 (H1N2) A/swine/Scotland/034632/2012 (H1N1) 100 A/swine/England/WVL16/1998 (H1N1) A/swine/England/WVL15/1997 (H1N1) A/swine/Scotland/WVL17/1999 (H1N1) 72 A/swine/England/WVL12/1995 (H1N1) A/swine/England/WVL7/1992 (H1N1) 100 A/swine/England/WVL10/1993 (H1N1) A/swine/France/WVL4/1985 (H1N1) A/swine/Spain/WVL6/1991 (H1N1) A/swine/France/WVL3/1984 (H1N1) A/swine/Potsdam/1/1981 (H1N1) 69 A/swine/Arnsberg/6554/1979 (H1N1) H3 74 A/swine/France/59150357/2015 (H3N2) 98 99 A/swine/Arnsberg/1/1979 (H1N1) A/swine/Italy/v147/1981 (H1N1) A/swine/Italy/94921/2013 (H3N2) 92 A/swine/Belgium/WVL2/1983 (H1N1) 91 A/swine/Italy/671/1987 (H1N1) A/swine/Belgium/Belsele66/2013 (H3N2) 86 92 A/swine/Belgium/WVL5/1989 (H1N1) A/swine/Netherlands/YsselsteynCVI8864A/2012 (H3N2) A/swine/France/WVL13/1995 (H1N1) A/swine/Italy/13697/1994 (H1N1) A/swine/Germany/BadWunnenbergHelmernIDT14829/2012 (H3N2) A/swine/Denmark/WVL9/1993 (H1N1) 95 A/swine/Netherlands/Vragender503/2011 (H3N2) 78 A/swine/Spain/102/2018 (H1N2) UG1 A/swine/Denmark/105474/2006 (H1N1) 84 A/swine/Germany/AttelnIDT14453/2011 (H3N2) A/swine/Denmark/1010482/2011 (H1N1) A/swine/Denmark/124791/2005 (H1N1) A/swine/Netherlands/Weert167B/2012 (H3N2) 100 A/swine/Spain/BM105/2018 (H1N2) D A/swine/France/59120031/2012 (H3N2) A/swine/Spain/BM108/2018 (H1N2) D 74 A/swine/Spain/127/2018 (H1N1) A 100 A/swine/Germany/CoesfeldHarleIDT147411/2012 (H3N2) 95 A/swine/Spain/103/2018 (H1N1) A 96 A/swine/Netherlands/Geesteren346/2011 (H3N2) 63 91 A/swine/Spain/099/2018 (H1N1) A 99 A/swine/Spain/283/2016 (H1N2) D A/swine/Italy/3191593/2010 (H3N2) 100 A/swine/Spain/BM54/2019 (H1N1) M A/swine/Spain/1268/2015 (H1N2) D A/swine/Italy/337155/2012 (H3N2) A/swine/France/01130145/2013 (H1N1) 65 79 A/swine/Italy/2481478/2015 (H3N2) A/swine/Spain/072/2017 (H1N2) D 99 93 A/swine/Spain/124/2018 (H1N2) D A/swine/Italy/3584811/2014 (H3N2) 99 A/swine/Spain/042/2017 (H1N2) D 63 100 A/swine/Italy/13602/2013 (H3N2) 88 91 A/swine/Spain/850/2016 (H1N1) A 78 A/swine/Spain/BM43/2019 (H1N2) D A/swine/Italy/1001761/2013 (H3N2) A/swine/Spain/50047/2003 (H1N1) 99 A/swine/Haseluenne/IDT2617/2003 (H1N1) A/swine/Italy/340032/2013 (H3N2) A/swine/Spain/29113/2012 (H1N1) A/swine/Italy/3410313/2014 (H3N2) A/swine/Italy/58769/2010 (H1N2) A/swine/Italy/13163032/2013 (H1N2) A/swine/Italy/352498/2013 (H3N2) 100 A/swine/Sweden/1021/2009 (H1N2) 98 A/swine/Italy/17234/2015 (H3N2) H3hu A/swine/Ghent/1984 like A/swine/Italy/1324012/2014 (H1N1) A/swine/Hong Kong/201/2010 (H1N1) 99 A/swine/Italy/335254/2014 (H3N2) H1av avian-like A/swine/Guangxi/NS2196/2012 (H1N1) 98 A/swine/Guangdong/717/2012 (H1N2) A/swine/Germany/VisbekIDT13347/2011 (H3N2) 100 A/swine/Hong Kong/1124/2012 (H1N2) A/swine/Germany/BorkenIDT11902/2010 (H3N2) 80 A/swine/Guangxi/3618/2011 (H1N1) 99 A/swine/Hong Kong/1197/2010 (H1N1) A/swine/Italy/1168402/2010 (H3N2) 99 A/swine/Denmark/1025261/2011 (H1N1) 96 A/swine/Italy/245982/2011 (H3N2) A/swine/Denmark/1011441/2011 (H1N2) 100 100 A/swine/Netherlands/Nistelrode530/2011 (H1N1) A/swine/Italy/55925/2011 (H3N2) 84 A/swine/Netherlands/Kootwijkerbroek51/2012 (H1N1) A/swine/Germany/SIV05/2007 (H1N1) A/swine/Hungary/M3/2010 (H3N2) A/swine/Greven/IDT2889/2004 (H1N1) A/swine/Belgium/Valdegem581/2010 (H3N2) 70 A/swine/Belgium/Lichtervelde57/2013 (H1N1) 100 A/swine/Spain/51915/2003 (H1N1) 99 A/swine/Belgium/Bree205/2011 (H3N2) 99 A/swine/Denmark/165082/2008 (H1N1) 99 A/swine/Netherlands/Roosendaal571/2011 (H3N2) A/swine/Belgium/Maldegem553/2010 (H1N1) 100 A/swine/Poland/00109/2010 (H1N1) A/swine/Hungary/13509/2007 (H3N2) 99 A/swine/Spain/071/2017 (H1N1) A A/swine/Spain/007/2017 (H1N1) M A/swine/Spain/16677/2011 (H3N2) 100 A/swine/Spain/112/2018 (H1N2) UG2 94 73 A/swine/Gent/172/2008 (H3N2) A/swine/Spain/32738/2012 (H1N1) A/swine/Italy/140863/2015 (H1N2) 99 63 A/swine/Belgium/Glabbeek284/2012 (H3N2) A/swine/Spain/123/2018 (H1N1) U A/swine/Belgium/113/2013 (H3N2) A/swine/Spain/104/2018 (H1avN1) UD2 100 A/swine/Spain/055/2017 (H1N2) UG3 A/swine/Germany/StadtloderIDT11945/2010 (H3N2) 86 A/swine/Spain/052/2017 (H1N1) M A/swine/Spain/117/2018 (H1N1) A A/swine/Spain/12/2010 (H3N2) 95 100 70 A/swine/Spain/402501/2016 (H1N1) A/swine/Spain/28778/2012 (H3N2) A/swine/Spain/144/2018 (H1N2) UG3 99 A/swine/Spain/1279/2015 (H1N1) A 100 A/swine/Spain/001/2017 (H3N2) N 99 A/swine/Portugal/066/2017 (H1N1) U A/swine/Spain/064/2017 (H1N1) U 100 A/swine/Spain/041/2017 (H3N2) N 100 74 A/swine/Portugal/065/2017 (H1N1) U 100 A/swine/Spain/018/2017 (H3N2) N A/swine/Cotes dArmor/0186/2010 (H1N2) A/swine/Italy/244784/2014 (H1N1) A/swine/Germany/OttendorfIDT11910/2010 (H3N2) 97 A/swine/Italy/269814/2013 (H1N1) 98 A/swine/Spain/27127/2011 (H3N2) A/swine/Spain/53207/2004 (H1N1) A/swine/France/50120350/2012 (H1N1) A/swine/Spain/23885/2011 (H3N2) 100 A/swine/France/53130065/2013 (H1N1) 65 A/swine/Belgium/Gent131/2000 (H3N2) A/swine/France/70130144/2013 (H1N1) 89 86 A/swine/Hungary/19774/2006 (H1N1) A/swine/Hungary/4025/2013 (H3N2) 78 A/swine/Netherlands/Groelnlo186/2011 (H1N1) A/swine/Germany/LohneIDT12137/2010 (H1N1) A/swine/Belgium/Izegem538/2010 (H3N2) 69 100 A/swine/Spain/119/2018 (H1N2) D 100 A/swine/Belgium/Moorslede515/2011 (H3N2) 70 A/swine/Spain/027/2017 (H1N1) A 93 A/swine/Spain/23998/2011 (H1N1) A/swine/Spain/33936/2012 (H3N2) 100 A/swine/Spain/BM151/2018 (H1N1) A 100 A/swine/Spain/31768/2012 (H3N2) A/swine/Spain/008/2017 (H1N2) D 98 100 A/swine/Spain/BM119/2018 (H1N2) D 100 60 A/swine/Spain/18512/2011 (H3N2) A/swine/Spain/049/2017 (H1N2) D 100 A/swine/Spain/29403/2012 (H3N2) A/swine/Spain/019/2017 (H1N1) A 100 A/swine/Spain/085/2018 (H1N2) D 100 A/swine/Italy/NIMR1523/1998 (H3N2) 95 A/swine/Spain/BM130/2018 (H1N2) D A/swine/Spain/BM40/2019 (H1N2) D A/swine/Denmark/1015011/2010 (H3N2) A/swine/Spain/1792/2016 (H1N1) A A/swine/Belgium/Gent1/1984 (H3N2) A/swine/Spain/094/2018 (H1N2) D 66 A/swine/Spain/BM32/2019 (H1N2) D 100 A/swine/Hong Kong/126/1982 (H3N2) 99 A/swine/Spain/118/2018 (H1N2) D A/swine/Kazakhstan/106/1985 (H3N6) 100 A/swine/Hong Kong/39/1977 (H1N1) A/swine/Hong Kong/78/1978 (H1N1) 83 A/swine/England/201635/1992 (H3N2) A/swine/Hokkaido/1980 (H1N1) 99 100 A/swine/England/502321/1994 (H3N2) A/swine/Hong Kong/101/1979 (H1N1) A/swine/Hong Kong/NS837/2001 (H1N1) A/swine/Hong Kong/81/1978 (H3N2) 86 A/swine/Hong Kong/294/2009 (H1N2) 69 93 A/swine/Guangdong/NS3178/2012 (H1N2) A/swine/Iowa/MT 12 07 1931/2012 (H3N2) 72 99 A/swine/Hong Kong/NS1892/2009 (H1N2) 100 A/swine/Korea/S2001/2015 (H3N2) H3 A/Malaysia/29930/2004 like H1pdm “pandemic 2009 like” 99 99 A/swine/Guangdong/1/2012 (H1N2) A/swine/Hong Kong/NS157/2004 (H1N2) A/swine/Hanoi/415/2013 (H3N2) H1cs classical swine 89 A/swine/Hong Kong/NS605/2003 (H1N2) A/swine/Korea/2511/2008 (H1N1) 100 A/swine/Tochigi/14/2013 (H3N2) 100 A/swine/Guangxi/2903/2011 (H1N2) A/swine/Japan/KU-MD4/2013 (H3N2) 100 A/swine/Guangxi/2887/2011 (H1N2) 66 A/swine/Guangxi/2823/2011 (H1N2) A/swine/Guangxi/JG1/2014 (H3N2) 100 A/swine/Guangdong/3584/2011 (H1N2) A/swine/Guangdong/3583/2011 (H1N2) A/Malaysia/29930/2004 (H3N2) 100 88 A/swine/Hong Kong/NS623/2002 (H1N2) A/Denmark/32/2005 (H3N2) A/swine/Hong Kong/1435/2009 (H1N2) A/swine/Hong Kong/4194/2011 (H1N2) A/Denmark/172/2005 (H3N2) 74 A/swine/Netherlands/BargerCompascuumCVI6324A/2012 (H1N2) A/swine/Spain/143/2018 (H3N2) UG5 A/swine/Italy/85429/2009 (H1N1) 99 A/swine/Norway/02 11342/2009 (H1N1) A/swine/Spain/BM37/2019 (H3N1) UG6 A/swine/Italy/116114/2010 (H1N2) A/swine/Hong Kong/NS3817/2010 (H1N1) A/swine/Spain/BM38/2019 (H3N1) UG6 A/swine/Italy/278299 3/2012 (H1N1) A/swine/Spain/062/2017 (H3N1/N2) UD1 A/swine/Spain/BM16/2019 (H1N1) UG7 96 A/swine/Spain/104/2018 (H1pdmN1) UD2 64 A/swine/Spain/100/2018 (H3N2) UG5 A/swine/France/72160174/2016 (H1N1) 96 A/swine/Spain/090/2018 (H3N2) UG5 61 99 A/swine/Spain/BM35/2019 (H1N1) P 99 A/swine/Spain/BM36/2019 (H1N1) P 73 A/swine/Spain/BM114/2018 (H1N1) P 0.05 A/swine/1931 (H1N1) 0.05 526 | SOSA PORTUGAL et al. 100 A/swineFrance/22120146/2012 (H1N1) 99 A/swine/Italy/1103/2015 (H3N2) A/swine/France/22120340/2012 (H1N1) 99 A/swine/Italy/348315/2014 (H3N2) A/swine/Spain/456901/2016 (H3N2) A/swine/France/Cotes d Armor0364/2011 (H1N1) A/swineSpain/35944/2012 (H3N2) 100 A/swine/Cotes d Armor/0070/2010 (H1N1) N1 N2 65 A/swineGermany/BorkenIDT11902/2010 (H3N2) A/swine/Cotes d Armor/0231/2006 (H1N1) A/swine/Hungary/13509/2007 (H3N2) A/swine/France/IIIeetVilaine0354/2011 (H1N1) A/swine/Hungary/13509/2007 (H3N2) A/swine/Gent/172/2008 (H3N2) 89 A/swine/France/53130065/2013 (H1N1) A/swine/Belgium/Glabbeek-284/2012 (H3N2) 100 A/swine/France/29120326/2012 (H1N1) A/swineNetherlands/Roosendaal571/2011 (H3N2) A/swine/Belgorod/ZR/2009 (H1N1) A/swine/Italy/333140/2014 (H1N2) 98 A/swine/Italy/171855/2011 (H3N2) A/swine/Belgium/Heist op den Berg363/2012 (H1N1) 82 A/swine/Spain/82108/2007 (H3N2) 85 A/swine/Netherlands/Putten60/2012 (H1N1) A/swineGermany/Genthin167D/2012 (H1N2) 100 A/swine/Italy/50255/2013 (H1N1) 99 A/swineGermany/DulmenIDT14394/2011 (H1N2) A/swine/Germany/OstbevernIDT13211/2011 (H1N1) A/swine/Spain/12/2010 (H3N2) A/swine/Spain/041/2017 (H3N2) N N2hu A/swine/Ghent/1984 like A/swine/Germany/HagstedtIDT11951/2010 (H1N1) 99 83 A/swine/Spain/28778/2012 (H3N2) A/swine/Italy/278299 1/2012 (H1N1) A/swine/Spain/018/2017 (H3N2) N A/swine/Spain/53207/2004 (H1N1) 65 A/swine/Spain/001/2017 (H3N2) N A/swine/Spain/54008/2004 (H3N2) A/swine/Italy/1598702/2005 (H1N1) A/swineBelgium/Moorslede515/2011 (H3N2) A/swine/Italy/65296/2004 (H1N1) A/swineSpain/18512/2011 (H3N2) 62 A/swine/Italy/53949/2004 (H1N1) A/swine/Spain/BM43/2019 (H1N2) D A/swine/Spain/42386/2002 (H3N2) A/swine/Spain/32738/2012 (H1N1) 95 A/swine/Cloppenburg/IDT4777/2005 (H1N2) A/swine/Spain/33903/2012 (H1N1) 87 99 A/swine/Doetlingen/IDT4735/2005 (H1N2) 65 100 A/swine/Spain/117/2018 (H1N1) A 99 A/swine/Bakum/IDT1769/2003 (H3N2) A/swine/Italy/267505/2010 (H1N1) A/swine/Italy/2721211/2002 (H3N2) 91 100 A/swine/Spain/39139/2002 (H3N2) A/swine/Italy/317775/2010 (H1N1) A/swine/Oedenrode/7c/1996 (H3N2) A/swine/Italy/3179061/2011) 98 A/swine/Spain/33601/2001 (H3N2) A/swine/Italy/138746/2014 (H1N1) 98 A/swine/Hong Kong/NS857/2001 (H1N2) 94 A/swine/Denmark/12687/2003 (H1N2) A/swine/Italy/1334573/2014 (H1N1) A/swineDenmark/1017251/2011 (H1N2) 100 A/swine/Italy/10915/2015 (H1N1) 100 A/swine/Freren/IDT138372/2011 (H1N2) 67 A/swine/Italy/327666/2013 (H1N1) A/swine/Italy/124953/2014 (H1N2) A/swine/Denmark/122311/2005 (H1N2) A/swine/Spain/123/2018 (H1N1) U 89 A/swineDenmark/107792/2012 (H1N2) 72 A/swine/Portugal/066/2017 (H1N1) U A/swine/Spain/1372/2017 (H1N2) UG4 97 100 A/swine/Portugal/065/2017 (H1N1) U A/swine/Denmark/1025291/2007 (H1N2) A/swine/Spain/064/2017 (H1N1) U A/swine/Sweden/9706/2010 (H1N2) 97 A/swine/Denmark/13380/2004 (H1N2) A/swine/Spain/1792/2016 (H1N1) A 100 95 A/swine/Netherlands/Gladau409/2012 (H1N2) A/swine/Spain/850/2016 (H1N1) A A/swine/Spain/102/2018 (H1N2) UG1 A/swine/Spain/027/2017 (H1N1) A 92 A/swine/Spain/2698/2016 (H1N2) C 85 88 A/swine/Spain/094/2018 (H1N2) D A/swine/Spain/BM151/2018 (H1N1) A 100 98 A/swine/Spain/049/2017 (H1N2) D 99 A/swine/Spain/456902/2016 (H1N1) 99 A/swine/Spain/008/2017 (H1N2) D 93 A/swine/Spain/019/2017 (H1N1) A A/swine/Spain/457001/2016 (H1N2) A/swine/Spain/1279/2015 (H1N1) A A/swine/Spain/119/2018 (H1N2) D 100 A/swine/Spain/BM40/2019 (H1N2) D 83 99 A/swine/Spain/BM38/2019 (H3N1) UG6 A/swine/Spain/BM119/2018 (H1N2) D A/swine/Spain/40250-2/2016 (H1N1) 99 A/swine/Spain/BM32/2019 (H1N2) D 77 A/swine/Spain/118/2018 (H1N2) D 99 A/swine/Spain/40240-1/2017 (H1N1) 100 87 A/swine/Spain/085/2018 (H1N2) D A/swine/Spain/402401/2017 (H1N1) 100 A/swine/Spain/BM130/2018 (H1N2) D 84 A/swine/Spain/40192-1/2016 (H1N1) A/swine/Brabant/1984 (H3N2) A/swine/Spain/052/2017 (H1N1) M A/swine/Chachoengsao/2003 (H3N2) A/swine/Italy/526/1985 (H3N2) 100 A/swine/Netherlands/Vlierden220/2012 (H1N1) 81 A/swine/Italy/6/1981 (H3N2) 100 A/swine/Italy/50814/2012 (H1N1) A/swine/Italy/1850/1977 (H3N2) 67 A/swine/Belgium/Neerpelt37/2011 (H1N1) A/swine/Hong Kong/13/77 (H3N2) A/swine/Denmark/125271/2005 (H1N1) 97 A/swine/gasaki/1/1990 (H1N2) 99 A/swine/Ehime/1/1980 (H1N2) 100 A/swine/Spain/071/2017 (H1N1) A A/swine/Hong Kong/82/78 (H3N2) A/swine/Spain/007/2017 (H1N1) M 99 A/swine/Guangdong/01/1998 (H3N2) A/swine/Spain/51915/2003 (H1N1) 100 A/swine/Colorado/1/1977 (H3N2) A/swine/Kagawa/2/1978 (H1N2) A/swine/Denmark/1021761/2010 (H1N1) 99 A/swine/Wisconsin/194/1980 (H3N2) A/swine/Netherlands/Dalfsen12/2012 (H1N1) A/swine/Sichuan/01/2006 (H3N2) 88 A/swine/Germany/LohneIDT12137/2010 (H1N1) A/swine/Denmark/103022/2012 (H1N2) 89 A/swine/Hong Kong/69/2006 (H1N2) A/swine/Denmark/126741/2005 (H1N1) 99 A/swine/Hong Kong/NS246/2004 (H1N2) N1av avian-like 96 A/swine/Denmark/107902/2010 (H1N1) 64 99 A/swine/Guangdong/606/2010 (H1N2) A/swine/Norway/02 11342/2009 (H1N1) 99 A/swine/Hong Kong/NS605/2003 (H1N2) 100 A/swine/Jangsu/295/2010 (H1N1) A/swine/Hong Kong/715/2008 (H1N2) 100 A/swine/Hong Kong/8944/2001 (H1N2) N1pdm “pandemic 2009 like” A/swine/Italy/635211/2013 (H1N1) 98 A/swine/Thailand/PB482/2009 (H3N2) A/swine/Kagoshima/65/2012 (H1N1) 99 A/swine/Thailand/CUS3673N/2012 (H3N2) A/swine/Italy/2253494/2012 (H1N1) 79 A/swine/Thailand/CUP43/2012 (H3N2) A/swine/Chonburi/NIAH106952028/2011 (H3N2) A/swine/HauteLoire/0578/2011 (H1N1) 100 100 A/swine/Thailand/CUS14129N/2013 (H3N2) A/swine/France/18120158/2012 (H1N1) 68 A/swine/Italy/3346941/2014 (H1N2) 69 A/swine/France/12150058/2015 (H1N1) A/swine/Italy/114922/2014 (H1N2) 99 A/swine/Spain/BM16/2019 (H1N1) UG7 A/swine/Italy/337085/2012 (H1N2) 100 A/swine/Italy/282964/2010 (H1N2) A/swine/Thailand/CUSPN47/2010 (H1N1) A/swine/Italy/1262161/2012 (H1N2) 100 64 A/swine/France/35140382/2014 (H1N1) A/swine/Italy/56341/2015 (H1N2) A/swine/Spain/104/2018 (H1N1) UD2 A/swine/Italy/4675/2003 (H1N2) 82 A/swine/Italy/1143471/2006 (H1N2) A/swine/Italy/23721/2015 (H1N1) 84 A/swine/Italy/224721/2012 (H1N2) 92 A/swine/Spain/BM114/2018 (H1N1) P A/swine/Spain/29262/2012 (H1N2) 80 A/swine/Spain/BM37/2019 (H3N1) UG6 A/swine/Finistere/127/99 A/sw/Shizuoka/110/97 (H3N2) A/swine/Spain/BM36/2019 (H1N1) P 93 97 A/swine/Hong Kong/NS2439/2011 (H3N2) A/England/7337/2018 (H1N1) 100 A/swine/Zhejiang/SW64/2014 (H1N2) 68 61 A/swine/France/531800282/2018 (H1N1) A/swine/Hong Kong/1071/2012 (H3N2) 91 A/swine/Guangdong/01/2005 (H3N2) A/Kentucky/7230/2018 (H1N1) 63 A/swine/Guangxi/1/2004 (H3N2) A/Italy/7223/2018 (H1N1) A/swine/Hanoi/415/2013 (H3N2) A/swine/Spain/BM35/2019 (H1N1) P 100 A/swine/Korea/S2001/2015 (H3N2) A/swine/Spain/50047/2003 (H1N1) A/swine/Korea/CY10/2007 (H3N2) A/swine/Hong Kong/78/2003 (H1N2) 91 A/swine/Spain/103/2018 (H1N1) A Spanish 99 A/swine/Korea/CAS09/2006 (H3N2) 72 A/swine/Spain/099/2018 (H1N1) A A/swine/Heilongjiang/10/2007 (H3N2) Only 98 94 A/swine/Spain/062/2017 (H3N1/N2) UD1 A/swine/Guangxi/2893/2011 (H1N2) 88 A/swine/Hong Kong/NS3969/2011 (H1N2) 100 A/swine/Spain/127/2018 (H1N1) A 64 99 A/swine/Guangdong/663/2012 (H1N2) A/swine/Spain/BM54/2019 (H1N1) M A/swine/Hong Kong/915/2004 (H1N2) A/swine/Italy/172336/2001 (H1N1) 80 A/swine/Hong Kong/NS623/2002 (H1N2) 75 A/swine/Cotes d Armor/1624/2002 (H1N1) A/swine/Hong Kong/1479/2009 (H1N2) A/swine/Hong Kong/1562/2005 (H1N2) A/swine/Hong Kong/8512/2001 (H1N1) 89 97 A/swine/Hong Kong/2314/2009 (H1N2) 94 A/swine/Hong Kong/1669/2002 (H1N1) A/swine/Hong Kong/562/2005 (H1N2) A/swine/Hong Kong/NS152/2009 (H1N1) A/swine/Guangdong/2693/2010 (H1N2) 86 99 91 A/swine/Hong Kong/1112/2007 (H1N2) 98 A/swine/Guangxi/LZA3/2015 (H1N1) A/swine/Fujian/12/2010 (H1N2) 7977 A/swine/Hong Kong/421/2006 (H1N1) A/swine/Guangdong/1/2012 (H1N2) A/swine/Hong Kong/NS318/2004 (H1N1) 99 A/swine/Hong Kong/294/2009 (H1N2) A/swine/Hong Kong/1559/2008 (H1N1) 93 A/swine/Hong Kong/NS1892/2009 (H1N2) 100 72 A/swine/Guangdong/NS3166/2012 (H1N2) A/swine/Guangdong/2418/2010 (H1N1) A/swine/Guangdong/L21/2011 (H3N2) 100 A/swine/Hong Kong/72/2007 (H1N1) 62 A/swine/Taiwan/NPUST0005/2013 (H1N2) 100 A/swine/Guangdong/30/2013 (H1N1) A/swine/England/285393/1993 (H3N2) A/swine/khon pathom/NIAH5861/2005 (H3N2) 99 A/swine/Denmark/WVL9/1993 (H1N1) A/swine/England/375017/1993 (H3N2) 65 97 A/swine/Denmark/19216B/1993 (H1N1) A/swine/Nordkirchen/IDT1993/2003 (H3N2) A/swine/Italy/13697/1994 (H1N1) 77 A/swine/Spain/40564/2002 (H1N2) 76 A/swine/Granstedt/IDT3475/2004 (H1N2) 100 A/swine/Italy/15131/1998 (H1N1) A/swine/Bakum/1832/2000 (H1N2) A/swine/Spain/WVL6/1991 (H1N1) 98 99 A/swine/Gent/177/2002 (H1N2) A/swine/England/WVL16/1998 (H1N1) 100 A/swine/Belgium/Oostkamp26/2012 (H1N2) 99 A/swine/England/193667/1998 (H1N1) A/swine/Italy/3592/1999 (H1N2) 73 98 A/swine/Italy/62/1998 (H1N2) A/swine/England/WVL7/1992 (H1N1) A/swine/UnitedKingdom/119404/91 (H3N2) N2hu A/swine/Scotland/410440/1994-like 65 A/swine/England/252411/1992 (H1N1) 65 A/swine/Cotes d Armor/43394/2002 (H1N2) 61 A/swine/England/WVL14/1996 (H1N1) 77 A/swine/France/CotesdArmor0105/2011 (H1N2) A/swine/France/Morbihan0599/2011 (H1N2) A/swine/Scotland/WVL17/1999 (H1N1) 98 80 A/swine/France/22110529/2011 (H1N2) A/swine/England/WVL11/1994 (H1N1) 98 A/swine/France/22130212/2013 (H1N2) A/swine/England/WVL15/1997 (H1N1) A/swine/Cotes d Armor/604/99 (H1N2) 97 99 A/swine/England/167655/1997 (H1N1) 100 A/swine/England/1428/2009 (H1N2) 100 A/swine/England/001428/2009 (H1N2) 85 A/swine/Saraburi/NIAH10076123/2009 (H1N1) 93 A/swine/England/142162/2010 (H1N2) 62 A/swine/Thailand/CB001/2009 (H1N1) A/swine/England/6183/2012 (H1N2) A/swine/Belgium/WVL5/1989 (H1N1) A/swine/England/17394/1996 (H1N2) 70 A/swine/England/61605/1998 (H1N2) 99 A/swine/France/WVL13/1995 (H1N1) A/swine/England/523/2010 (H1N2) 100 A/swine/Italy/670/1987 (H1N1) 99 A/swine/England/P185/2008 (H1N2) A/swine/Italy/671/1987 (H1N1) 100 A/swine/England/112340/2011 (H1N2) A/swine/England/236/2009 (H1N2) A/swine/France/WVL4/1985 (H1N1) 99 A/swine/Spain/29257/2012 (H1N2) A/swine/Belgium/WVL1/1979 (H1N1) A/swine/Spain/07/2011 (H1N2) A/swine/Potsdam/1/1981 (H1N1) 75 A/swine/Spain/072/2017 (H1N2) D 61 60 A/swine/Spain/10/2011 (H1N2) A/swine/France/WVL8/1992 (H1N1) A/Swine/Spain/80598LP4/2007 (H1N2) 69 A/swine/Finnistere/2899/1982 (H1N1) 96 A/swine/Spain/BM105/2018 (H1N2) D A/swine/Eire/89/1996 (H1N1) A/swine/Spain/BM108/2018 (H1N2) D A/swine/Spain/BM55/2019 (H1N2) C 99 A/swine/1931 (H1N1) 100 A/swine/Spain/055/2017 (H1N2) UG3 A/swine/Guangdong/L3/2009 (H1N1) 86 A/swine/Spain/150/2018 (H1N2) D N1cs classical swine A/swine/England/79851/1990 (H1N1) 74 A/swine/Spain/153/2018 (H1N2) C 100 A/swine/Hong Kong/101/1979 (H1N1) A/swine/Spain/144/2018 (H1N2) UG3 100 A/swine/Spain/090/2018 (H3N2) UG5 99 A/swine/Hong Kong/78/1978 (H1N1) A/swine/Spain/062/2017 (H3N1/N2) UG7 A/swine/Hong Kong/33/1977 (H1N1) 85 100 A/swine/Spain/143/2018 (H3N2) UG5 A/swine/Hong Kong/59/1977 (H1N1) A/swine/Spain/100/2018 (H3N2) UG5 A/swine/Hokkaido/1980 (H1N1) 82 A/swine/Spain/1268/2015 (H1N2) D A/swine/Spain/112/2018 (H1N2) UG2 A/swine/Hong Kong/9656/2001 (H1N1) A/swine/Spain/24237/2011 (H1N2) A/swine/Guangdong/34/2006 (H1N1) A/swine/Spain/08/2011 (H1N2) 100 A/swine/Hong Kong/NS837/2001 (H1N1) A/swine/Spain/283/2016 (H1N2) D 65 A/swine/Spain/1297/2017 (H1N2) C 66 A/swine/Hong Kong/4089/1999 (H1N1) A/swine/Spain/124/2018 (H1N2) D 95 A/swine/Spain/042/2017 (H1N2) D 0.05 0.02 SOSA PORTUGAL et al. | 527 F I G U R E 4 Maximum likelihood tree for influenza A positive N1 and N2 gene segments (1,000 iterations). Coloured boxes show the main lineages for Eurasian N1 and N2 as indicated in the figure. Sequences highlighted in black and white letters represent a common ancestor for the whole lineage indicated by the box. Green (avian-like N1), dark red (pandemic N1) and blue (human-like swine N2) indicate a strain previously isolated in swine that could be used as a more recent reference of the lineage in swine. Sequences in red are those from the present study. Values in the branches show the bootstrap values (only ≥60 are shown) The distribution of subtypes and lineages observed agrees with a Cq > 30 could not be isolated neither. Primer mismatching could previous reports from Europe in the last years (Kyriakis et al., 2011; reduce the efficiency of the PCR and this could be the explanation Van Reeth et al., 2008; Simon et al., 2014). In our case, just three for some of the samples that were not subtyped. However, 11 sam- lineages, H1avN2hu, H1avN1av and H1huN2hu, represented 75% ples with very low Cq (17–20) could not be subtyped but were suc- of the cases. It is interesting to note that, as in other European cessfully isolated in cell culture. The design of new primers could countries H3N2 is becoming less common in the herds (Harder have helped to mildly increase the efficiency of PCR subtyping in et al., 2013; Watson et al., 2015). In contrast, in a serological study the present study. However, given the degree of diversity observed, conducted in 2008–2009 in Spain, more than 90% of the examined it can be foreseen in the future, that a continuous update of prim- pigs were seropositive to H3N2 (Simon-Grifé et al., 2011). ers will be necessary, including the problems associated with multi- Watson et al. (2015) proposed a genotype classification for swine plexing at a large scale. Isolation and sequencing by NGS could be a IAV. The classification was based on the composition and origin of suitable alternative, particularly, considering the decreasing costs of the different viral genes. In the original description, 23 genotypes NGS technologies. were reported, with an additional 4% of the strains being unde- In this study, we took the approach of isolating the virus and termined. Afterwards, other reports added new genotypes (Beato then deep sequenced it by using Illumina MiSeq. The reason for et al., 2016). In the present study, we found 7 additional genotypes, including the isolation step was the impossibility of performing di- and in our opinion, more genotypes are very likely to be found in the rect sequencing on samples with Cqs lower than 24. By using this future. Accordingly, it is necessary to establish a widened scheme for approach, maybe we were underestimating some of the diversity the designation of the new variants that will most likely appear. It is of Spanish IAV. This problem could be solved by PCR amplification worth noting that we found four isolates harbouring an infrequent instead of viral isolation (Nirmala et al., 2020). However, this step H3 'human-like'. This H3 was previously reported by Krog, Hjulsager, of PCR amplification could add some bias that would be especially Larsen, and Larsen (2017) in Denmark where a H3N2 virus with an difficult to assess when more than one virus is present in the sam- internal pandemic cassette was detected. In our case, the H3 hu- ple. We first tested the fidelity of this approach by determining man-like isolates were harbouring an avian internal cassette either the divergence rate between direct sequencing and sequencing with the N2 like in Denmark or with an N1av. This is an indication of from a single-passage isolate. The difference resulted to be in the reassortment and spreading out of Denmark. range of 10 –3 (namely 1 discrepancy per every 1,000 nucleotides). Moreover, when the phylogenetic analysis of the different viral Obviously, some bias in the isolation can be produced by using genes was performed, it was evident that either strains of differ- MDCK cells and this could be in part responsible for the inability to ent genotypes could cluster together, or that strains from the same sequence a second virus present in those samples where two H or genotype may be included in different clusters. This suggested that N strains were initially found. However, the advantages of NGS for the complexity of the generation of genetic diversity in swine IAV such diverse viruses like IAV balance the potential disadvantages cannot be covered by just the use of subtype, lineage and genotype of our approach. classifications. The phylogenetic analysis indicated the existence of some local clusters, being this particularly noticeable for the M and NS genes. 5 | CO N C LU S I O N Although the development of local variants is expected, the fact that strains from very diverse subtypes, lineages and genotypes shared a IAV is widely present in most pig farms. The most common de- variant of the M gene locally developed suggests some degree of bio- tected lineages were H1avN2hu and H1avN1av, representing the logical fitness or advantage for this variant. Moreover, some genotypes D and A genotypes, respectively. The genetic diversity of this virus conserved their clusters in some of the genes, while other genotypes is continuously expanding with clear changes in the predominant contained strains grouped into different clusters, disrupting any possi- subtypes and lineages in relatively short periods of time. The cur- ble correlation between the genotype that can be assigned to a specific rent genotyping scheme has to be enlarged to include the new strain and its genetic or phylogenetic implications. Also, this result in- genotypes that can be found. As in any other case, for a good di- dicates a high level of intra- and inter-genotype reassortment events. agnosis of IAV, a differential diagnosis with the investigation of the From a methodological point of view, the present study also pro- presence of other pathogens is needed. A large targeted sampling duced some interesting results. For example, subtyping by RT-qPCR of clinically affected and healthy animals, considering the propor- worked out for most samples with low Cq (<30) in the M gene PCR, tion of positive animals and viral loads in each group may help to but failed for higher Cqs. Interestingly, most of the samples yielding discriminate cases where IAV is the primary agent of the outbreak. 528 | SOSA PORTUGAL et al. A/swine/Spain/042/2017 (H1N2) D F I G U R E 5 Maximum likelihood tree Spanish isolates 67 for influenza A positive Matrix gene Only A/swine/Spain/124/2018 (H1N2) D A/swine/Spain/1268/2015 (H1N2) D 100 M A/swine/Spain/1297/2016 (H1N2) C A/swine/Spain/24237/2011 (H1N2) A/swine/Italy/55925/2011 (H3N2) segment (1,000 iterations). Coloured 100 97 A/swine/Italy/278299 1/2012 (H1N1) A/swine/Italy/100176-1/2013 (H3N2) boxes show the main groups for Eurasian A/swine/Italy/7821/2011 (H1N1) A/swine/Spain/09/2010 (H1N2) A/swine/Spain/850/2016 (H1N1) A M genes. Sequences highlighted in black and white letters represent a common 93 A/swine/Spain/23998/2011 (H1N1) 94 A/swine/Spain/BM151/2018 (H1N1) A A/swine/France/CotesdArmor-0113/2006 (H1N2) A/swine/Hungary/19774/2006 (H1N1) 70 A/swine/Italy/186678-2/2005 (H1N1) ancestor for the whole lineage indicated by the box. Green (avian-like N1), dark red A/swine/Italy/233139/2005 (H1N2) A/swine/France/22-130212/2013 (H1N2) A/swine/France/CotesdArmor-0558/2011 (H1N2) 87 A/swine/Italy/317906-1/2011) A/swine/Italy/271152/2014 (H1N2) (pandemic N1) a strain previously isolated in swine that could be used as a more A/swine/Italy/70757/2009 (H1N2) A/swine/Spain/80598LP1/2007 (H3N2) A/swine/Spain/80598LP4/2007 (H1N2) 100 99 A/swine/Spain/BM37/2019 (H3N1) UG6 A/swine/Spain/BM38/2019 (H3N1) UG6 recent reference of the lineage in swine. Sequences in red are those from the A/swine/Spain/1279/2015 (H1N1) A A/swine/Belgium/111/2013 (H1N1) A/swine/Germany/Bakum-IDT12292/2010 (H1N2) 94 A/swine/Netherlands/Dalfsen-12/2012 (H1N1) A/swine/Germany/Genthin-167D/2012 (H1N2) present study. Clusters containing solely 98 Spanish isolates are indicated. Values in 100 A/swine/Spain/22013/2011 (H1N1) A/swine/Netherlands/Rhezerveen-CVI9121A/2012 (H1N1) 99 A/swine/Italy/319159-3/2010 (H3N2) 88 85 99 A/swine/Belgium/Belsele-66/2013 (H3N2) A/swine/Netherlands/Ysselsteyn-CVI8864A/2012 (H3N2) the branches show the bootstrap values (only ≥60 are shown) A/swine/Italy/302749/2010 (H3N2) 88 95 A/swine/Italy/327666/2013 (H1N1) A/swine/Doetlingen/IDT4735/2005 (H1N2) A/swine/Bakum/1832/00 (H1N2) 97 A/swine/Spain/39139/2002 (H3N2) A/swine/Belgium/Flanders-1/1998 (H3N2) 99 A/swine/Spain/BM43/2019 (H1N2) D A/swine/Spain/18512/2011 (H3N2) A/swine/Gent/177/2002 (H1N2) A/swine/France/Finistere-0011/2011 (H1N1) 73 A/swine/France/56-110525/2010 (H1N1) A/swine/Herzlake/IDT5336/06 (H3N2) A/swine/Spain/13/2010 (H3N2) A/swine/Denmark/1085-1/2012 (H1N1) A/swine/Italy/289171/2003 (H1N1) A/swine/Spain/027/2017 (H1N1) A 88 A/swine/Spain/BM55/2019 (H1N2) C 99 A/swine/Spain/150/2018 (H1N2) D Only Spanish 99 A/swine/Spain/153/2018 (H1N2) C 64 A/swine/Spain/117/2018 (H1N1) A isolates A/swine/Spain/283/2016 (H1N2) D 75 A/swine/Spain/BM108/2018 (H1N2) D A/swine/Spain/104/2018 (H1N1) UD2 A/swine/Spain/103/2018 (H1N1) A 100 A/swine/Spain/072/2017 (H1N2) D 88 A/swine/Spain/BM105/2018 (H1N2) D A/swine/Spain/2698/2016 (H1N2) C A/swine/Denmark/12687/2003 (H1N2) 91 A/swine/Denmark/10-1048-2/2011 (H1N1) M avian-like A/swine/Italy/28762-3/2013 (H1N1) 77 A/swine/Denmark/10345-1/2012 (H1N2) 99 A/swine/Denmark/13608/2004 (H1N2) A/swine/Denmark/10-1391-3/2011 (H1N2) A/swine/Denmark/10-1958-1/2011 (H1N2) A/swine/Denmark/14348-9/2003 (H3N2) A/swine/Denmark/10-1144-1/2011 (H1N2) 100 A/swine/Spain/100/2018 (H3N2) UG5 A/swine/Spain/143/2018 (H3N2) UG5 76 A/swine/Spain/099/2018 (H1N1) A 93 A/swine/Spain/062/2017 (H3N1/N2) UD1 98 A/swine/Spain/127/2018 (H1N1) A Only Spanish isolates 95 A/swine/Spain/071/2017 (H1N1) A 99 A/swine/Spain/45690-1/2016 (H3N2) 98 A/swine/Spain/45690-2/2016 (H1N1) A/swine/Spain/090/2018 (H3N2) UG5 100 A/swine/Spain/008/2017 (H1N2) D A/swine/Spain/45700-1/2016 (H1N2) 93 A/swine/Spain/049/2017 (H1N2) D A/swine/Spain/019/2017 (H1N1) A A/swine/Spain/119/2018 (H1N2) D A/swine/Spain/085/2018 (H1N2) D 99 A/swine/Spain/BM130/2018 (H1N2) D A/swine/Spain/BM119/2018 (H1N2) D A/swine/Spain/1792/2016 (H1N1) A A/swine/Spain/BM40/2019 (H1N2) D A/swine/Spain/BM32/2019 (H1N2) D A/swine/Spain/118/2018 (H1N2) D A/swine/Spain/094/2018 (H1N2) D A/swine/Hong Kong/5200/99 (H3N2) A/swine/Saerbeck/IDT13755/2011 (H1N2) 99 A/swine/Spain/055/2017 (H1N2) UG3 100 A/swine/Spain/052/2017 (H1N1) M 78 A/swine/Spain/40240-1/2017 (H1N1) A/swine/Lohne/IDT13821/2011 (H1N2) A/swine/England/041118/2013 (H1N2) 99 88 A/swine/Spain/041/2017 (H3N2) N 89 A/swine/Spain/001/2017 (H3N2) N A/swine/Spain/018/2017 (H3N2) N A/swine/Spain/BM54/2019 (H1N1) M M “pandemic 2009 like” A/swine/Spain/007/2017 (H1N1) M A/swine/Spain/064/2017 (H1N1) U 100 A/swine/Portugal/065/2017 (H1N1) U 72 A/swine/Portugal/066/2017 (H1N1) U A/swine/England/072865/2013 (H1N1) A/swine/Spain/29113/2012 (H1N1) A/swine/Hong Kong/4133/2011 (H1N2) A/swine/Heudorf-Messkirch/IDT14176/2011 (H1N1) 79 A/swine/Spain/1372/2015 (H1N2) UG4 A/swine/Spain/144/2018 (H1N2) UG3 A/swine/Italy/327497-7/2014 (H1N2) A/swine/Hungary/S137/2013 (H1N1) A/swine/Spain/04/2011 (H1N1) 94 62 A/swine/Spain/112/2018 (H1N2) UG2 A/swine/Spain/102/2018 (H1N2) UG1 A/swine/England/063782/2013 (H1N2) A/swine/Spain/123/2018 (H1N1) U A/swine/South Korea/KS61/2016 (H1N1) A/swine/Spain/BM16/2019 (H1N1) UG7 A/swine/France/57-140136/2014 (H1N1) 93 A/swine/France/35-160233/2016 (H1N1) 89 A/swine/Spain/BM35/2019 (H1N1) P 98 99 A/swine/Spain/BM114/2018 (H1N1) P A/swine/Spain/BM36/2019 (H1N1) P A/swine/England/26029/1998 (H1N1) 81 A/swine/Spain/WVL6/1991 (H1N1) A/swine/Scotland/51190/1999 (H1N2) 99 A/swine/Scotland/53307/2000 (H1N2) 99 A/swine/Italy/671/1987 (H1N1) A/swine/Italy/147/1981 (H1N1) 87 A/swine/France/WVL4/1985 (H1N1) A/swine/Arnsberg/6554/1979 (H1N1) 60 A/swine/Netherlands/25/1980 (H1N1) A/swine/Hong Kong/10/1998 (H9N2) A/swine/Hong Kong/168/1993 (H1N1) 72 A/swine/Guangxi/592/2011 (H5N1) 100 A/swine/Guangdong/2/2014 (H5N6) A/swine/Hong Kong/81/1978 (H3N2) A/swine/May/54 (H1N1) 100 A/swine/Okinawa/2/2005 (H1N1) 97 A/swine/Guangdong/106/2002 (H3N2) 93 A/swine/England/117316/1986 (H1N1) A/swine/Hokkaido/1980 (H1N1) A/swine/Guangdong/1/2012 (H1N2) A/swine/Hong Kong/126/1982 (H3N2) 0.02 SOSA PORTUGAL et al. | 529 AC K N OW L E D G E M E N T S Influenza Viruses in Swine Populations in Northwest Germany. 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