This study was conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Animal, Plant, and Fisheries Quarantine and Inspection Agency of Korea. Based on agreement between the Office of University-Industry Cooperation of Hallym University and the Gyeongsangnam-do Livestock Promotion Institute, the experimental protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Hallym University (permit number: Hallym 2012-95). To minimize unnecessary animal suffering, the veterinary inspector collected nasopharyngeal samples only from the pigs exhibiting respiratory symptoms, and the samples were used for the virus isolation and phylogenetic characterization.

Based on the Hallym University IACUC permission, Hallym 2012-95, 108 nasopharyngeal samples were collected from pigs between December 2011 and May 2012 in Gyeongsangnam-do, a southeastern province of Korea. Carried in universal viral transport media (BD, Franklin Lakes, NJ), samples were prepared for the virus isolation. The prepared samples were inoculated into the Madin-Darby canine kidney (MDCK) cell monolayer for 1 h at 37°C. The cells were washed and maintained with growth media for 48–72 h. Viral growth was determined by the morphological changes (cytopathic effects) in the MDCK cells and by a hemagglutination assay of cell supernatants using 0.5% turkey red blood cells (Rockland Immunochemicals Inc.; Gilbertville, PA). The double-positive sample was purified by a standard plaque assay in the MDCK cells and prepared after propagation in fertile chicken eggs.

To determine the genomic sequences of the isolated virus, the viral RNAs of egg-grown samples were extracted using a QIAamp Viral RNA Mini Kit (Qiagen, Venlo, Netherlands). After reverse transcriptase PCR (RT-PCR), the DNA products were used for the sequence readouts. More than three clones per genomic segment were used for the sequence analysis. This virus was subtyped as a H3N2 strain and named A/swine/Korea/PL01/2012 (swPL01). Eight genomic sequences of the swPL01 virus were registered in the NCBI database, with the accession numbers KF382726 to KF382733 from the PB2 gene to the NS.

For the phylogenetic study, the NCBI-registered full-length nucleotide sequences of swine H3N2 viruses were used as references (the total number of full-length sequences after collapsing the identical sequences as of release date June 29, 2013 is as follows: PB2, 341; PB1, 358; PA, 354; HA, 556; NP, 352; NA, 550; M, 400; and NS, 331). By adding various lineage defining sequences or removing redundant sequences, we analyzed the phylogenetic characteristics of the swPL01 virus. The nucleotide sequences of lineage defining human H1N1 (huH1N1; A/New York/494/2002), huH3N2 (A/New York/101/2002), pH1N1 (A/California/04/2009, A/Korea/01/2009, A/Netherlands/602/2009, A/swine/Hong Kong/2995/2009, and A/swine/Hong Kong/189/2010) [20], CS H1N1 (A/swine/Hong Kong/5683/1999, A/swine/Hong Kong/NS143/2000, A/swine/Hong Kong/8690/2001, and A/swine/Hong Kong/227/2002) [20], Eurasian avian-like (EA) H1N1 (A/swine/Hong Kong/638/2003, A/swine/Hong Kong/NS129/2003, A/swine/Hong Kong/435/2007, A/swine/Hong Kong/245/2009) [20], TRIG H1N2 (A/swine/Ontario/52156/2003, A/swine/Ontario/48235/2004, A/swine/Oklahoma/010226-16/2008, A/swine/Texas/008648/2008, A/swine/Hong Kong/1435/2009) [20], [21], H3N2v (A/Indiana/10/2011 and A/Maine/06/2011) [16], and avian H1N1 (avH1N1; A/mallard/Maryland/02-181/2002) and avH3N2 (A/blue-winged teal/Ohio/908/2002) viruses were included in the analysis, except for the HA and NA sequences. For HA gene analysis, only the nucleotide sequences of swine H3N2 and H3N2v viruses were included. For NA gene analysis, only the nucleotide sequences of swine H3N2, TRIG H1N2, and H3N2v viruses were included. Using MEGA5 [22], each sequence of the swPL01 virus was aligned with that of the references using MUSCLE by the unweighted pair group method with the arithmetic mean [23]. The phylogenetic tree was inferred using the Maximum Likelihood (ML) method by obtaining the initial tree(s) for the heuristic search based on the neighbor-joining method through a matrix of pairwise distances evaluated by the maximum composite likelihood (MCL) approach. The bootstrap scores were determined from 500 replicates and are shown below the branches [24]. The sequences that met our bootstrap score standards (>95% for PB2, PB1, PA, and NP or >60% for HA, NA, M, and NS among the sequences registered since the late 1990s) and that were located in close proximity were recalculated with the Korean swine H3N2 and lineage defining sequences for the final ML tree evaluation. Some HA sequences published previously in 2008 [25] and 2013 [26] were included in the final HA tree for the differentiation of HA clusters. The horizontal distances are the nucleotide differences, which were proportional for the number of changes per site. The genetic distances were determined with MEGA5 by the MCL method using bootstrap estimation (500 replicates).

We first analyzed the phylogenetic relations of three polymerase gene segments of the swPL01 virus with other NCBI-registered sequences. In the ML trees, the polymerase genes of Korean swine H3N2 viruses appeared to have evolved from those of the swTx/98 virus (Figure 1). Although some divergences were found in the PB1 gene (A/swine/Korea/CAS05/2004 and A/swine/Korea/CY09/2007) (Figures 1B and S2), the polymerase genes of the Korean isolates were clustered in chronological order. A number of the selected TRIG H3N2 strains identified in Southeast Asia, South America, and North America appeared to reassort with pH1N1 viruses and consisted of different sub-lineages from the swTx/98 virus (Figure 1). Especially for the PA gene, the phylogenetic analysis indicated that nine North American TRIG H3N2 viruses were involved in reassortment events with pH1N1 strains (Figures 1C and S3).

All of the polymerase genes of the swPL01 virus appeared to be evolved from the genes of the swTx/98 virus. However, the swPL01 virus was categorized into a different clade from the other contemporary Korean isolates (Figure 1). These relations were easily appreciated in the ML trees drawn with the overall reference sequences and also seen in maximum parsimony (MP) and neighbor joining (NJ) trees of the selected sequences (Figures S1, S2, and S3). In sequence analysis, the PB2 of A/swine/Ontario/33853/2005 showed the greatest sequence similarity (97.5%) with that of the swPL01 virus whereas maximum 95.7% similarity was seen with the Korean isolates (Table 1). For the PB1 and PA genes, North American isolates in 2005 shared maximum similarity (PB1, 97.8% with A/swine/Minnesota/00611/2005; 97.4% with A/swine/Ontario/33853/2005) (Table 1). On the other hand, 95.7% and 95.9% were the highest sequence similarities found with the PB1 and PA genes of the Korean isolates, respectively (Table 1). Considered together, these findings suggest that the swPL01 virus retains polymerase genes that drifted from the swTx/98 virus but it is phylogenetically different from those of other contemporary Korean strains.

We then analyzed the phylogenetic characteristics of the swPL01 HA and NA genes. In the ML tree of the overall sequences, the HA of swPL01 (indicated as c in Figure S4) virus was distant from those of other contemporary Korean viruses (b, d in Figure S4). We confirmed this phylogenetic distance with the selected HA sequences. In the ML (Figure 2A), MP and NJ (Figure S4) trees, each cluster (clusters I, II, III, and IV) of swine H3 HAs diverged from that of the swTx/98 virus [25]–[27]. In these group denotations, three Korean HAs between 2005–2006 were classified into cluster I, with the parental swTx/98 HA. The HAs of the 2007 Korean isolates consisted of subsequent tertiary and quaternary branches from the HA of the swTx/98 virus whereas that of the A/swine/Korea/CY10/2007 virus that was classified into cluster III in the MP and NJ tree (Figure S4). Farther down in the tree, two Korean HAs in 2004 were categorized as clusters II (A/swine/Korea/CAS05/2004) and III (A/swine/Korea/JNS06/2004), respectively. Under the trunk of cluster III, various HAs were diverged into two large clades. These constituted cluster IV [28], and the HA of the swPL01 virus was included in the upper clade group with others from North America whereas Korean HAs isolated from 2011–2012 were in the lower clade (Figures 2A and S4). In a similar manner to that of the phylogenetic relations of polymerase genes unveiled in Figure 1, the swPL01 HA was located in one of the branches of the cluster IV phylogenetic trunk shared with the other contemporary Korean HAs, but it was categorized in a different group (Figures 2A and S4). These differences were also apparent in the ML tree using the full-length HA sequences of Asian avian, human, and swine H3N2 viruses and North American avian and swine H3N2 viruses (Figure S9). Similarly above, the HA of the swPL01 virus was included in the completely different clade from those of other Korean isolates in the same swine H3 HA trunk. Amino acid differences also explained these phylogenetic distances (Table S1). Compared with the HAs of other Korean isolates, we found 35 amino acid mutations (35/567, 6.17%) from that of the swPL01 virus. 27 mutations (27/345, 7.83%) were located in the HA1 region, and 8 (8/222, 3.6%) were in the HA 2. By concurrent E292N and N294S mutations, the HA of swPL01 virus gained one more potential N-linked glycosylation (NLG; N-Xaa-S/T, any amino acid for Xaa except proline) at HA residue 292 (Table S1). The HA of the A/swine/Ohio/11sw347/2011 virus exhibited the highest sequence similarity (96.6%) with that of the swPL01 virus (Table 1). On the other hand, maximum 94.8% similarity was found with the HAs of the Korean swine H3N2 viruses (Table 1).

The NAs of TRIG H3N2 viruses diverged into various clades, which resulted from reassortments with seasonal human viruses (Figures S6 and S9) [21]. The reassortment event in 1998 introduced human HA, NA, and PB1 genes into the swine influenza viruses and was attributed to the establishment of the TRIG H3N2 lineage in pigs [9]. The later reassortment of TRIG H3N2 viruses occurred with the NA of human H3N2 viruses around 2001–2002. This second reassortment established another NA phylogenetic lineage, clade IV, into swine NA evolution [21]. In our phylogenetic tree of the NA genes, a similar pattern was observed (Figures 2B and S6). Evolved from the NA of the swTx/98 virus, various branch lines were inferred in the N2 lineage. Some NAs were included in the clade of A/swine/Ontario/52156/2003, which could be classified into the N2 lineage of human H1N2 viruses. All of the Korean isolates from 2004–2007 were rooted in the early N2 lineage of the swTx/98 virus (Figures 2B and S6). The NAs of Korean swine H3N2 viruses in 2011–2012 were placed at the end of the evolution tree of cluster IV, right below from the human H3N2 lineage (A/swine/Ontario/48235/2004 and A/swine/Alberta/14722/2005) [21], [28]. However, the NA of the swPL01 virus was classified into a different group from that of the other contemporary Korean viruses (Figures 2B and S6) and exhibited the closest genetic similarity to that of the A/swine/Quebec/4001/2005 virus (96.9%) rather than to any other Korean isolates (less than 94.0%) (Table 1). In addition, 29 (29/470, 6.17%) amino acid differences in the NAs between the swPL01 and other Korean isolates also supported their phylogenetic distances (Table S2). These results suggest that the HA and NA genes of the swPL01 virus are phylogenetically distant from those of the other contemporary Korean isolates.

The phylogenetic analysis of polymerase and surface glycoprotein genes indicated the unique evolutionary position of the swPL01 virus compared with other contemporary Korean isolates (Figures 1 and 2). The overall ML trees of the NP, M, and NS genes presented a similar conclusion (Figures S5, S7, and S8). In the ML, MP, and NJ trees of the selected NP and NS sequences, the swPL01 virus appeared to be evolved from the respective swTx/98 genes but was neither classified within the same clades with other contemporary Korean isolates nor traced by interaction clues with the pH1N1 viruses (NP in Figures 3A and S5; NS in Figures 3C and S8). The NP and NS genes of the swPL01 virus had maximum 96.3% and 97.0% genetic similarities to those of Korean isolates, respectively, whereas 98.1% and 97.9% genetic similarities were observed respectively with the NP of A/swine/Minnesota/00611/2005 and A/swine/Ontario/33853/2005 viruses and the NS of A/swine/Manitoba/12707/2005, A/swine/Ontario/33853/2005/, and A/swine/Quebec/4001/2005 viruses (Table 1).

The ML tree of the swPL01 M gene suggested a different evolution pathway of TRIG H3N2 viruses in Korea (Figures S7 and S9). Most Korean TRIG H3N2 viruses in 2011-2012 appeared to retain the M gene of the pH1N1 lineage, which was derived from the EA lineage (Figures 3B and S7) [2] and termed as H3N2v [29]. However, the swPL01 virus retained the M gene signature of TRIG H3N2 viruses. Located in the phylogenetic tree of TRIG viruses, the swPL01 M segment appeared to be one of the evolutionary branch lines of the swTx/98. Some Korean swine H3N2 viruses around 2004–2007 also retained the M gene of the TRIG lineage (Figures 3B and S7). The M genes of six North American TRIG H3N2 viruses in 2005 exhibited 99.1% sequence similarity with that of the swPL01 virus; however, those of Korean swine H3N2 viruses around 2011–2012 exhibited only ∼87.3% similarity, suggesting a different evolution lineage from the swPL01 M segment (Table 1).