Evolution and Pathogenicity of the H1 and H3 Subtypes of Swine Influenza Virus in Mice between 2016 and 2019 in China

Pigs are considered a “mixing vessel” that can produce new influenza strains through genetic reassortments, which pose a threat to public health and cause economic losses worldwide. The timely surveillance of the epidemiology of the swine influenza virus is of importance for prophylactic action. In this study, 15 H1N1, one H1N2, and four H3N2 strains were isolated from a total of 4080 nasal swabs which were collected from 20 pig farms in three provinces in China between 2016 and 2019. All the isolates were clustered into four genotypes. A new genotype represented by the H1N2 strain was found, whose fragments came from the triple reassortant H1N2 lineage, classical swine influenza virus (cs-H1N1) lineage, and 2009 H1N1 pandemic virus lineage. A/Sw/HB/HG394/2018(H1N1), which was clustered into the cs-H1N1 lineage, showed a close relationship with the 1918 pandemic virus. Mutations determining the host range specificity were found in the hemagglutinin of all isolates, which indicated that all the isolates had the potential for interspecies transmission. To examine pathogenicity, eight isolates were inoculated into 6-week-old female BALB/c mice. The isolates replicated differently, producing different viral loadings in the mice; A/Swine/HB/HG394/2018(H1N1) replicated the most efficiently. This suggested that the cs-H1N1 reappeared, and more attention should be given to the new pandemic to pigs. These results indicated that new reassortments between the different strains occurred, which may increase potential risks to human health. Continuing surveillance is imperative to monitor swine influenza A virus evolution.


Introduction
The swine influenza, caused by the swine influenza virus (IAV-S), is an acute respiratory disease in pigs, which is characterized by fever, labored breathing, and coughing [1]. Swine are considered as a "mixing vessel" for human, avian, and swine influenza viruses, in which new IAV can be produced through genetic reassortments because of the presence of a mammalian-like receptor, α2-6 linked sialic acids (SIA), and avian-like receptor α2-3 linked SIA in porcine respiratory epithelial cells [2]. The new reassorted strains can then be transmitted to other species, expanding the lineage infection and endangering animal and human health [3].

Virus Isolation and DNA Sequencing
The nasal swabs were thawed and centrifuged at 5000× g for 3 min at 4 • C. The supernatant was then collected (0.2 mL) and inoculated into two 10-day-old SPF chicken embryos through the allantoic cavity. The inoculated chicken embryos were incubated at 37 • C for 72 h. After three blind passages, embryo allantoic fluids were collected and used for the hemagglutination (HA) assay. The samples with chicken blood agglutination activity were saved and the total RNA was extracted according to the instructions of the Viral RNA Mini Kit (Tiangen, China). The RNA was reverse-transcribed into cDNA by Uni12 universal primers (5 -AGCAAAAGCAGG-3 ) using the ReverTra Ace qPCR RT Kit (TOYOBO, Japan). Complete viral genomes were amplified according to Hoffmann's method [26]. The PCR products were purified by 0.8% agarose gel electrophoresis and sequenced at Shanghai Sangon Biotech Co., Ltd. (Shanghai, China).

Virus Purification and Titration
The isolated strains were purified using 10-day-old SPF chicken embryos by inoculation with 0.1 mL of supernatant diluted with 10 2 -10 4 -fold. The viral titrations were measured by an egg-infectious dose 50% (EID50) using three 10-day-old SPF chicken embryos with 0.1 mL of each supernatant, diluted by 10 1 −10 9 -fold. After incubating at 37 • C for 48 h, the EID 50 titer in the allantoic fluid was determined according to the method of Reed-Muench. For viral titration in organs, tissues were homogenized by 1:10 dilution with sterile PBS (containing penicillin and streptomycin, 2000 U/mL) and centrifuged at 8000× g for 5 min at 4 • C. The supernatant was collected and inoculated into chicken embryos as described above.

Phylogenetic Analysis
The fragments of the IAV-S were aligned and analyzed using the MegAlign program in the DNAStar package (DNASTAR, Madison, USA). Complete genomes of 256 A/H1 and 67 A/H3 swine influenza viruses isolated in China were downloaded from the Influenza Virus Resource at the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html) and the Viruses 2020, 12, 298 4 of 14 GISAID database on 10 February 2019. Repetitive sequences in the two databases were removed by matching strain names in Bioedit v7.1.3.0 [27]. The remaining sequences were combined with reference sequences, as described previously [28][29][30], and those generated in the present study. The eight datasets corresponding to the eight gene segments of the influenza A virus were first aligned using MAFFT v7.450 [31] and then adjusted manually in Bioedit v7.1.3.0 [27]. Phylogenetic analysis was conducted using RAxML v8.2.9 with the GTRGAMMA applied as a nucleotide substitution model [32] and 1000 bootstrap replicates. Trees were visualized using FigTree (http://tree.bio.ed.ac.uk/software/figtree/).

Replication and Pathogenicity of IAV-S in Mice
To evaluate the replication and pathogenicity, eight isolates , and A/Swine/SD/LY396/2016(H1N2) represented by different genotypes were selected to learn the pathogenicity in mice. Fifty-four 6-week-old female BALB/c mice were randomly divided into nine groups (including eight virus-infected groups and one mock-infected group). The mice were inoculated intranasally with 10 6 EID 50 of purified IAV-S after anesthetizing with CO 2 , which was approved by the Animal Care and Use Committee of Shandong Agricultural University. The project identification code is 2017-041, which was approved on 12 April 2017 (12/04/2017). Three mice in each group were sacrificed at 3 days post-infection (DPI), and tissues, including the brain, nasal turbinate, lung, spleen, and kidney, were collected and maintained at −70 • C for virus titration, or fixed in 4% neutral formaldehyde for pathological observations. The remaining mice were monitored for 14 days to assess body weight loss and mortality. A weight-loss rate of more than 30% was reported as a death. All surviving mice were euthanized by CO 2 at 14 DPI.

Virus Isolation and Phylogenetic Analysis
The supernatant of nasal swab extracts were inoculated into 10-day-old, specific-pathogen-free (SPF) chicken embryos and passaged blindly three times. Twenty strains, including 15 H1N1, one H1N2, and four H3N2 IAV-S subtypes were successfully isolated ( Table 1). The results showed that different subtypes of IAV-S were circulating in China and H1N1 was the dominant subtype. The complete genomes of all isolates were sequenced, and then phylogenetic analysis was performed. Compared with 256 reference H1 viruses isolated during 2002-2018, distinct clusters were formed within the phylogenetic trees corresponded to ea-H1N1, H1N1pdm09, cs-H1N1, and hl-H1N1 ( Figure 1). In this study, Of the 16 H1 influenza viruses isolated from 2016 to 2018, 14 were in the ea-H1N1 lineage, one (A/Swine/HB/HG394/2018(H1N1)) in the cs-H1N1 lineage, and one (A/Swine/SD/LY396/2016(H1N2)) in the H1N1pdm09 lineage. The nucleotide similarity between the 16 H1 fragments was 72.9-100%. Compared with 67 H3 viruses isolated during 2002-2019, the HA fragment of our four H3N2 viruses were in the lineage of hl-H3N2 and shared 99.8-100% identity at the nucleotide level. Similarly to the HA fragments, 14 N1 NA fragments were clustered in the ea-H1N1 lineage and one (A/Swine/HB/HG394/2018(H1N1)) N1 NA fragment was clustered in the cs-H1N1 lineage ( Figure 1) with nucleotide similarities of 81.8-100% among the 15 strains. N2 fragments of the four H3N2 strains were from the hl-H3N2 lineage, while the N2 fragment of the H1N2 strain was clustered into the tr-H1N2 lineage. The nucleotide similarity of the four N2 fragments from H3N2 was 99.6-100% and shared 72.9-100% identity at the nucleotide level between all five N2 fragments. The results also showed that the PB2, PB1, PA, MP, and NP fragments of the 20 strains showed the same clustering pattern and were clustered into the H1N1pdm09 and cs-H1N1 lineages. The PB2, PB1, PA, MP, and NP fragments of all isolates were clustered into the H1N1pdm09 lineage, except those from A/Swine/HB/HG394/2018(H1N1), which were clustered into the cs-H1N1 lineage. The nucleotide similarities of PB2, PB1, PA, MP, and NP of 20 isolates were 96-100%, 96.2-100%,

Genotyping Analysis
According to the phylogenetic analysis of the gene segments, the 20 isolates were divided into four genotypes, 1, 2, 3, and 4 ( Table 2). Fourteen out of 15 H1N1 strains carried TRIG, with fragments from the ea-H1N1 lineage (HA and NA fragments), the cs-H1N1 lineage (NS fragment), and the H1N1pdm09 lineage (PB2, PB1, PA, NP, and M fragments). All fragments of

Genotyping Analysis
According to the phylogenetic analysis of the gene segments, the 20 isolates were divided into four genotypes, 1, 2, 3, and 4 ( Table 2). Fourteen out of 15 H1N1 strains carried TRIG, with fragments from the ea-H1N1 lineage (HA and NA fragments), the cs-H1N1 lineage (NS fragment), and the H1N1pdm09 lineage (PB2, PB1, PA, NP, and M fragments). All fragments of A/Swine/HB/HG394/2018(H1N1) belonged to the cs-H1N1 lineage. The H3N2 strains were double-reassortant, with fragments from the hl-H3N2 lineage (HA and NA fragments), and H1N1pdm09 lineage (PB2, PB1, PA, NP, M, and NS fragments). The H1N2 strain was a TRIG strain, with fragments from the tr-H1N2 lineage (NA fragment), cs-H1N1 lineage (NS fragment), and H1N1pdm09 lineage (PB2, PB1, PA, HA, NP, and M fragments). It is worth noting that the 19 strains contained five H1N1pdm09-originating internal genes (PB2, PB1, PA, NP, and M fragments), indicating the high prevalence of the H1N1pdm09 subtype. The H1 subtype also had an NS fragment derived from cs-H1N1. In the H3 subtype, six internal genes were from H1N1pdm09.  A/Swine/HB/HG394/2018(H1N1) belonged to the cs-H1N1 lineage. The H3N2 strains were doublereassortant, with fragments from the hl-H3N2 lineage (HA and NA fragments), and H1N1pdm09 lineage (PB2, PB1, PA, NP, M, and NS fragments). The H1N2 strain was a TRIG strain, with fragments from the tr-H1N2 lineage (NA fragment), cs-H1N1 lineage (NS fragment), and H1N1pdm09 lineage (PB2, PB1, PA, HA, NP, and M fragments). It is worth noting that the 19 strains contained five H1N1pdm09-originating internal genes (PB2, PB1, PA, NP, and M fragments), indicating the high prevalence of the H1N1pdm09 subtype. The H1 subtype also had an NS fragment derived from cs-H1N1. In the H3 subtype, six internal genes were from H1N1pdm09.

Molecular Characteristics Analysis of the Isolates
To understand the molecular characteristics of the isolated IAV-S strains, key amino acid sites were analyzed. HA was initially translated into a single molecule, HA0, which was then cleaved by host proteases into HA1 and HA2, where HA1 and HA2 are covalently linked by a disulfide bond [33]. The cleavage sites of all isolated H1 subtype IAV-S were PSIQSR/G, while those of the H3 subtype IAV-S were PEKQTR/G. Such cleavage sites are characteristic of low pathogenicity of avian IAV ( Table 3). The receptor-binding site of the HA protein determines the receptor-binding characteristics of IAV-S and is directly related to host tropism [34]. The receptor binding sites of the HA protein from our isolated H1 subtype IAV-S were at the V155, N159, H183, D/N/V190, D/E/G225, Q/R226, A/E227, and G228 positions, respectively ( A/Swine/HB/HG394/2018(H1N1) belonged to the cs-H1N1 lineage. The H3N2 strains were doublereassortant, with fragments from the hl-H3N2 lineage (HA and NA fragments), and H1N1pdm09 lineage (PB2, PB1, PA, NP, M, and NS fragments). The H1N2 strain was a TRIG strain, with fragments from the tr-H1N2 lineage (NA fragment), cs-H1N1 lineage (NS fragment), and H1N1pdm09 lineage (PB2, PB1, PA, HA, NP, and M fragments). It is worth noting that the 19 strains contained five H1N1pdm09-originating internal genes (PB2, PB1, PA, NP, and M fragments), indicating the high prevalence of the H1N1pdm09 subtype. The H1 subtype also had an NS fragment derived from cs-H1N1. In the H3 subtype, six internal genes were from H1N1pdm09.

Molecular Characteristics Analysis of the Isolates
To understand the molecular characteristics of the isolated IAV-S strains, key amino acid sites were analyzed. HA was initially translated into a single molecule, HA0, which was then cleaved by host proteases into HA1 and HA2, where HA1 and HA2 are covalently linked by a disulfide bond [33]. The cleavage sites of all isolated H1 subtype IAV-S were PSIQSR/G, while those of the H3 subtype IAV-S were PEKQTR/G. Such cleavage sites are characteristic of low pathogenicity of avian IAV ( Table 3). The receptor-binding site of the HA protein determines the receptor-binding characteristics of IAV-S and is directly related to host tropism [34]. The receptor binding sites of the HA protein from our isolated H1 subtype IAV-S were at the V155, N159, H183, D/N/V190, D/E/G225, Q/R226, A/E227, and G228 positions, respectively ( A/Swine/HB/HG394/2018(H1N1) belonged to the cs-H1N1 lineage. The H3N2 strains were doublereassortant, with fragments from the hl-H3N2 lineage (HA and NA fragments), and H1N1pdm09 lineage (PB2, PB1, PA, NP, M, and NS fragments). The H1N2 strain was a TRIG strain, with fragments from the tr-H1N2 lineage (NA fragment), cs-H1N1 lineage (NS fragment), and H1N1pdm09 lineage (PB2, PB1, PA, HA, NP, and M fragments). It is worth noting that the 19 strains contained five H1N1pdm09-originating internal genes (PB2, PB1, PA, NP, and M fragments), indicating the high prevalence of the H1N1pdm09 subtype. The H1 subtype also had an NS fragment derived from cs-H1N1. In the H3 subtype, six internal genes were from H1N1pdm09.

Molecular Characteristics Analysis of the Isolates
To understand the molecular characteristics of the isolated IAV-S strains, key amino acid sites were analyzed. HA was initially translated into a single molecule, HA0, which was then cleaved by host proteases into HA1 and HA2, where HA1 and HA2 are covalently linked by a disulfide bond [33]. The cleavage sites of all isolated H1 subtype IAV-S were PSIQSR/G, while those of the H3 subtype IAV-S were PEKQTR/G. Such cleavage sites are characteristic of low pathogenicity of avian IAV ( Table 3). The receptor-binding site of the HA protein determines the receptor-binding characteristics of IAV-S and is directly related to host tropism [34]. The receptor binding sites of the HA protein from our isolated H1 subtype IAV-S were at the V155, N159, H183, D/N/V190, D/E/G225, Q/R226, A/E227, and G228 positions, respectively ( A/Swine/HB/HG394/2018(H1N1) belonged to the cs-H1N1 lineage. The H3N2 strains were doublereassortant, with fragments from the hl-H3N2 lineage (HA and NA fragments), and H1N1pdm09 lineage (PB2, PB1, PA, NP, M, and NS fragments). The H1N2 strain was a TRIG strain, with fragments from the tr-H1N2 lineage (NA fragment), cs-H1N1 lineage (NS fragment), and H1N1pdm09 lineage (PB2, PB1, PA, HA, NP, and M fragments). It is worth noting that the 19 strains contained five H1N1pdm09-originating internal genes (PB2, PB1, PA, NP, and M fragments), indicating the high prevalence of the H1N1pdm09 subtype. The H1 subtype also had an NS fragment derived from cs-H1N1. In the H3 subtype, six internal genes were from H1N1pdm09.

Molecular Characteristics Analysis of the Isolates
To understand the molecular characteristics of the isolated IAV-S strains, key amino acid sites were analyzed. HA was initially translated into a single molecule, HA0, which was then cleaved by host proteases into HA1 and HA2, where HA1 and HA2 are covalently linked by a disulfide bond [33]. The cleavage sites of all isolated H1 subtype IAV-S were PSIQSR/G, while those of the H3 subtype IAV-S were PEKQTR/G. Such cleavage sites are characteristic of low pathogenicity of avian IAV ( Table 3). The receptor-binding site of the HA protein determines the receptor-binding characteristics of IAV-S and is directly related to host tropism [34]. The receptor binding sites of the HA protein from our isolated H1 subtype IAV-S were at the V155, N159, H183, D/N/V190, D/E/G225, Q/R226, A/E227, and G228 positions, respectively ( A/Swine/HB/HG394/2018(H1N1) belonged to the cs-H1N1 lineage. The H3N2 strains were doublereassortant, with fragments from the hl-H3N2 lineage (HA and NA fragments), and H1N1pdm09 lineage (PB2, PB1, PA, NP, M, and NS fragments). The H1N2 strain was a TRIG strain, with fragments from the tr-H1N2 lineage (NA fragment), cs-H1N1 lineage (NS fragment), and H1N1pdm09 lineage (PB2, PB1, PA, HA, NP, and M fragments). It is worth noting that the 19 strains contained five H1N1pdm09-originating internal genes (PB2, PB1, PA, NP, and M fragments), indicating the high prevalence of the H1N1pdm09 subtype. The H1 subtype also had an NS fragment derived from cs-H1N1. In the H3 subtype, six internal genes were from H1N1pdm09.

Molecular Characteristics Analysis of the Isolates
To understand the molecular characteristics of the isolated IAV-S strains, key amino acid sites were analyzed. HA was initially translated into a single molecule, HA0, which was then cleaved by host proteases into HA1 and HA2, where HA1 and HA2 are covalently linked by a disulfide bond [33]. The cleavage sites of all isolated H1 subtype IAV-S were PSIQSR/G, while those of the H3 subtype IAV-S were PEKQTR/G. Such cleavage sites are characteristic of low pathogenicity of avian IAV ( Table 3). The receptor-binding site of the HA protein determines the receptor-binding characteristics of IAV-S and is directly related to host tropism [34]. The receptor binding sites of the HA protein from our isolated H1 subtype IAV-S were at the V155, N159, H183, D/N/V190, D/E/G225, Q/R226, A/E227, and G228 positions, respectively (

Molecular Characteristics Analysis of the Isolates
To understand the molecular characteristics of the isolated IAV-S strains, key amino acid sites were analyzed. HA was initially translated into a single molecule, HA0, which was then cleaved by host proteases into HA1 and HA2, where HA1 and HA2 are covalently linked by a disulfide bond [33]. The cleavage sites of all isolated H1 subtype IAV-S were PSIQSR/G, while those of the H3 subtype IAV-S were PEKQTR/G. Such cleavage sites are characteristic of low pathogenicity of avian IAV ( Table 3). The receptor-binding site of the HA protein determines the receptor-binding characteristics of IAV-S and is directly related to host tropism [34]. The receptor binding sites of the HA protein from our isolated H1 subtype IAV-S were at the V155, N159, H183, D/N/V190, D/E/G225, Q/R226, A/E227, and G228 positions, respectively ( Table 3). All of the H1 subtype IAV-S that we isolated exhibited HA T155V, T159N, and E190D mutations, except for A/Swine/SD/LY236/2016(H1N1), A/Swine/HB/HG332/2018(H1N1), and A/Swine/HB/HG354/2018(H1N1). Those results indicated that most of the HA genes from the isolated H1N1 strains may have the potential for transmission between avian and pig species.  (Table 3). Our hl-H3N2 virus has characteristic residues found in human-adapted seasonal H3N2 viruses, including 226I and 228S [35]. H1 HAs had potential N-glycosylation sites, including 14NST, 26NVT, 57NCS, 90NGT, 277NCT, 290NTS, 484NGT, and 543NGS. Only A/Swine/SD/LY236/2016(H1N1) had glycosylation sites at 57NCS, and A/Swine/SD/LY396/2016 had glycosylation sites at 90NGT and 290NTS. Four H3 HAs contained six potential N-glycosylation sites, including 22NGT, 63NCT, 126NWT, 246NST, 285NGS, and 483NGT. Viruses with lysine at position 627 were pathogenic in mice, whereas those with glutamic acid were nonpathogenic in these animals [36]. Only A/Swine/HB/HG394/2018(H1N1) possessed a K at position 627, an A at position 271, and an R at position 591 of PB2. Twenty strains of the influenza virus contained D at position 701 with no mutations ( Table 2). H274Y in the N1 gene, and E119V and R292K in the N2 gene served as reference sites for neuraminidase inhibitor (NAI) resistance [37,38]. No isolates had NAI-resistant amino acids (Table 3), indicating that these viruses are sensitive to NAIs, such as oseltamivir. In the M2 genes which contained the key amino acid target of amantadine drugs [39], the key amino acid sites, L26, V/I27, A30, N/S31, and G34 were found in our isolated H1 subtype, while L26, I27, A30, N31, and G34 were found in the H3 subtype (Table 3).

Pathogenicity of the Isolates in Mice
To evaluate the replication and pathogenicity, eight isolates, including three H1N1  (Figure 2A). The mice infected with A/Swine/SD/LY396/2016(H1N2) lost up to 10.42% body weight by 9 DPI, which then increased slowly until it was restored at 11 DPI (Figure 2A). At 3 DPI, three mice in each group were sacrificed and tissues were collected for detection of viral loads. The results showed that different viral loads in the same organs were caused by different strains. Viral loads in the lung and nasal turbinate of mice infected with A/Swine/HB/HG394/2018 were higher ( Figure 2B) than that of other H1N1 subtypes.
In mice infected with the four H3N2 subtype viruses of A/Swine/HN/NY428/2016, A/Swine/HN/NY426/2016, A/Swine/HN/NY430/2016, and A/Swine/HN/NY125/2016, a higher titer was found in the nasal turbinate than in the lungs, while in mice infected with A/Swine/SD/LY236/2016(H1N1), A/Swine/SD/LY396/2016(H1N2), A/Swine/HN/NY127/2017(H1N1), and A/Swine/HB/HG394/2018(H1N1) a higher virus load was detected in the lungs than in the nasal turbinate ( Figure 2B). No virus was detected in the kidney, brain, and spleen. The histopathological changes in the lungs showed that similar changes of alveolar wall thickening, inflammatory cell infiltration, capillary congestion, and epithelial cell shedding were observed in all infected groups ( Figure 2C). The lungs of A/Swine/HB/HG394/2018(H1N1)-infected mice showed more severe changes, with larger amounts of epithelial cell shedding and a larger volume of infiltrating inflammatory cells.

Pathogenicity of the Isolates in Mice
To evaluate the replication and pathogenicity, eight isolates, including three H1N1

Discussion
IAV-S, as a zoonotic disease, has an important impact on public health [39]. Since 2009, the reassortant viruses derived from H1N1pdm09 and other swine influenza viruses have been isolated

Discussion
IAV-S, as a zoonotic disease, has an important impact on public health [39]. Since 2009, the reassortant viruses derived from H1N1pdm09 and other swine influenza viruses have been isolated in pigs in some countries [40,41]. Sporadic cases of human infection with such swine-origin novel reassortant viruses have been reported [42]. All these indicated that IAV-S poses a serious threat to the health of human beings. In this study, 20 IAV-S strains, including 15 H1N1, one H1N2, and four H3N2 subtypes were identified, which indicated that different subtypes have been co-circulating in pigs in China (Table 1). Based on the genetic and phylogenetic analyses, four genotypes were clustered ( Table 2). Five or six gene fragments of most strains were derived from swine H1N1pdm09, indicating that recently, swine H1N1pdm09 has been the dominant lineage. This provides direct evidence that the H1N1pdm09 internal gene complex has been successfully incorporated into different subtypes of IAV-S. Notably, except for A/swine/Hubei/HG394/2018(H1N1), the fragments of all H1N1 viruses were in the same reassortant pattern-HA and NA from the ea-H1N1 lineage, PB1, PB1, PA, NP, and MP from the H1N1pdm09 lineage, and NS from the cs-H1N1 lineage, which indicated that this may be to the dominant genotypes during their adaption in swine.
Since the isolation of the first IAV-S by Shope from pigs in 1930, these cs-H1N1 viruses likely derived from the 1918 pandemic IAV [43]. Based on the phylogenetic analysis results, A/swine/Hubei/HG394/2018(H1N1) was clustered into the cs-H1N1 lineage, which is closely related with the 1918 pandemic virus ( Figure 1). Interestingly, a sub-lineage within the cs-H1N1 lineage was formed. In this sub-lineage, only A/swine/Guangdong/L3/2009(H1N1) are from the mainland of China [44]. Other strains are from the USA or UK, most of which are related to early human influenza. This indicates that the previous IAV-S appeared again, and more attention should be paid to this virus and also on whether a new pandemic to pigs would be caused by the existence of this subtype virus. Eight isolates represented by four different genotypes were inoculated into mice to compare their pathogenicity. The results showed that mice infected with A/Swine/HB/HG394/2018 (H1N1 subtype) experienced rapid weight loss and resulted in 100% death (Figure 2A). Additionally, high viral loads ( Figure 2B) were detected in the lung and nasal turbinate from mice infected with A/Swine/HB/HG394/2018(H1N1). Severe interstitial pneumonia was also observed in the lungs of mice with A/Swine/HB/HG394/2018(H1N1) ( Figure 2C). Mice infected with other H1 subtypes had high virus titers in their lungs, although such levels caused no clinical signs or obvious weight loss. For H1 subtype strains, the substitutions of E190D are critical for the shift from α-2,3to α-2,6-linked SIA receptor recognition [45]. In this study, an N/V190 was found in A/Swine/SD/LY236/2016(H1N1), A/Swine/HB/HG332/2018(H1N1), and A/Swine/HB/HG354/2018(H1N1) isolates, while 190D was found in all other H1 fragments ( Table 3).
The presence of specific amino acids in PB2 are important for the replication and virulence of influenza viruses in mammals [46]. E627K or D701N mutations in PB2 are markers for enhanced polymerase activity and viral replication in mammalian cells, as well as the pathogenicity of H7N9 viruses in the BALB/c mouse model [47]. The presence of PB2-627K enhances viral replication and pathogenicity in mammalian model systems, and is associated with lethality in humans infected with H5N1 and H7N7 avian viruses [48]. For the H1N1pdm09 virus, it was reported that the amino acid alanine (A) at position 271 of PB2 enhances the transmissibility of influenza A in ferrets, and arginine (R) at position 591 is important for H1N1pdm09 in mammalian adaptation [49]. Nineteen out of 20 strains of the influenza virus contained E and D at positions 627 and 701, respectively, with no mutations. A lysine (K) at amino acid position 627 was found in A/Swine/HB/HG394/2018 (Table 3). It was reported that R at position 591 of PB2 can compensate for the lack of K at position 627 and confers efficient viral replication to H1N1pdm09 viruses in mammals [50]. In this study, only A/swine/Hubei/HG394/2018(H1N1) possessed K627, T271, and Q591 of PB2, which is characteristic of mammalian influenza viruses and which may be a reason for the high rate of death in mice infected with the A/swine/Hubei/HG394/2018(H1N1) virus.
A glutamic acid (E) at amino acid position 225 was found in all H1 genes, except for A/Swine/SD/LY396/2016(H1N2), A/Swine/HN/NY127/2017(H1N1), and A/Swine/HB/HG394/ 2018(H1N1). The substitution of glycine (G) for glutamic acid (E) at position 225 (E225G) plays a critical role in the transmission of the ea-H1N1 virus by increasing the efficiency of viral assembly and budding [51]. Thus, the difference in amino acid mutations of the HA gene may explain why H1 subtypes replicated more efficiently in the lung than in the nasal turbinate. For H3 viruses, the substitutions of Q226L and G228S in HA was accepted as a complete switch from α-2,3to α-2,6-linked receptor-binding specificity [52,53]. In all four H3N2 subtype viruses, only G228S was observed, which indicated that the receptor-binding specificity was partially changed. As a result, all four H3N2 subtype strains replicated less efficiently in the lungs than the nasal turbinate.
The amino acid residue at position 31 of the M2 protein of the 19 out of 20 influenza viruses were all mutated to N, indicating that all such viruses are resistant to amantadine (Table 3). Of the 20 influenza viruses, no sites in the NA protein were linked to NAI resistance, indicating that these viruses are sensitive to NAIs, such as oseltamivir.

Conclusions
Based on this study, different IAV-S subtypes have been co-circulating in pigs in China. Swine H1N1pdm09 has recently been the dominant lineage. A new genotype of the H1N2 subtype was found. Molecular characteristics analysis indicated that the isolates have the potential for interspecies transmission. A cs-H1N1 strain (A/swine/Hubei/HG394/2018(H1N1)) was isolated, which is closely related to the 1918 pandemic virus. All the isolates replicated differently in mice, and A/swine/Hubei/HG394/2018(H1N1) replicated more efficiently than other subtypes. These results indicate that new reassortments between the different strains occurred, and that continued surveillance is required to monitor the evolution of the swine influenza A virus.