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Published in final edited form as: Zoonoses Public Health. 2023 Dec 18;71(3):281–293. doi: 10.1111/zph.13104

Evolution of Influenza A Viruses in Exhibition Swine and Transmission to Humans, 2013–2015

Christine M Szablewski 1,*,†, Dillon S McBride 2,*, Susan C Trock 3, Gregory G Habing 4, Armando E Hoet 5, Sarah W Nelson 6, Jacqueline M Nolting 7, Andrew S Bowman 8,‡
PMCID: PMC10994755  NIHMSID: NIHMS1951537  PMID: 38110691

Summary

Aims

Swine are a mixing vessel for the emergence of novel reassortant influenza A viruses (IAV). Interspecies transmission of swine-origin IAV poses a public health and pandemic risk. In the United States, the majority of zoonotic IAV transmission events have occurred in association with swine exposure at agricultural fairs. Accordingly, this human-animal interface necessitates mitigation strategies informed by understanding of interspecies transmission mechanisms in exhibition swine. Likewise, the diversity of IAV in swine can be a source for novel reassortant or mutated viruses that pose a risk to both swine and human health.

Methods and Results

In an effort to better understand those risks, here we investigated the epidemiology of IAV in exhibition swine and subsequent transmission to humans by performing phylogenetic analyses using full genome sequences from 272 IAV isolates collected from exhibition swine and 23 A(H3N2)v viruses from human hosts during 2013–2015. Sixty-seven fairs (24.2%) had at least one pig test positive for IAV with an overall estimated prevalence of 8.9% (95% CI: 8.3–9.6, Clopper-Pearson). Of the 19 genotypes found in swine, 5 were also identified in humans. There was a positive correlation between the number of human cases of a genotype and its prevalence in exhibition swine. Additionally, we demonstrated that A(H3N2)v viruses clustered tightly with exhibition swine viruses that were prevalent in the same year.

Conclusions

These data indicate that multiple genotypes of swine-lineage IAV have infected humans, and highly prevalent IAV genotypes in exhibition swine during a given year are also the strains detected most frequently in human cases of variant IAV. Continued surveillance and rapid characterization of IAVs in exhibition swine can facilitate timely phenotypic evaluation and matching of candidate vaccine strains to those viruses present at the human-animal interface which are most likely to spillover into humans.

Keywords: disease outbreaks, influenza A virus, public health, swine, United States: viral zoonoses, One Health

Introduction

Influenza A virus is an enveloped virus with a negative strand, 8 segment RNA genome (Bouvier & Palese, 2008). In addition to the well-known seasonal peaks of morbidity and mortality in humans (CDC, 2022), IAV also infects a wide range hosts including avian species and other mammals such as swine (Webster et al., 1992). The broad diversity of IAVs circulating and continuing to evolve in animal hosts can provide a source of novel IAV to which humans are generally naïve and can pose public health and pandemic risk should they adapt to human transmission (Webster, 2002). Swine have periodically been infected with human seasonal IAVs via reverse zoonosis, where both H1 and H3 viruses have become established, leading to further evolution and diversification, and leaving swine as host to a diverse population of enzootic IAVs (Anderson et al., 2021). The vast IAV diversity maintained within swine populations can therefore be generally grouped into evolutionary lineages based up the reverse zoonosis event that established the swine-origin IAV as described by (Anderson et al., 2016, 2021). Some of these reverse zoonoses relevant to the present study include H1 1A derived from the human 1918 pandemic virus (Shope, 1931), H1 1B which spilled into US swine from humans in the early 2000s (Vincent et al., 2009), and H3 3.1990.4 which introduced a new human seasonal H3 in into swine in 1998 where it rapidly diversified and maintains in swine populations today with a recent resurgence following acquisition of different NA and NP genes (Neveau et al., 2022; Zhou et al., 1999).

Susceptibility to avian-origin, human-origin, and endemic swine-origin IAVs allows swine to serve as a potential source of reassortant IAVs that are capable of infecting humans (Ito et al., 1998; W. Ma et al., 2008; Rajão et al., 2017). Perhaps the most noteworthy example of this mixing vessel functionality was in the triple reassortment of human, avian, and swine-origin IAV genes leading the emergence of the H3 3.1990.4 lineage in swine and giving rise to the triple reassortment internal gene (TRIG) constellation which has repeatedly reassorted and persisted in swine since (Vincent et al., 2008; Zhou et al., 1999). Swine-origin IAVs that infect humans are referred to as variant IAV, which are designated with a ‘v’ after the subtype. The public health threat posed by variant IAV was highlighted when a swine-lineage IAV gave rise to the 2009 A(H1N1) influenza pandemic (Garten et al., 2009). The 2009 A(H1N1) pandemic IAV (pdm09) resulted in rapid reverse zoonoses back to swine populations, and has had continued interspecies transmission between swine and humans in both directions since then, only complicating the relationship between swine and human-origin IAV (Markin et al., 2023; Vijaykrishna et al., 2010). Subsequently, starting in 2011, there was a surge of A(H3N2) variant [A(H3N2)v] cases associated with swine exposures. From 2011 through 2023, there have been over 490 variant IAV (H1 and H3 subtypes) cases reported in the United States (https://www.cdc.gov/flu/swineflu/variant-cases-us.htm). Of the cases which had exposure data available i2011–2021, 92% (417/453) had confirmed swine exposure prior to onset of illness, and a majority of those exposures were to exhibition swine at agricultural fairs (Bowman et al., 2017; Centers for Disease Control and Prevention (CDC), 2012; Jhung et al., 2013). Additionally, 88% (429/489) of those cases were reported in children under the age of 18, which is the age group of youth exhibitors spending long hours interacting closely with their swine at the fair. Although humans are exposed to larger populations of swine in U.S. commercial swine production compared with an exhibition setting, access to commercial swine is limited to relatively few individuals due to the biosecurity of those operations. In contrast, millions of individuals who typically do not interact with livestock can be exposed to exhibition swine at agricultural fairs. Since more people come into contact with live swine at agricultural exhibitions than any other setting in the United States, there is an increased risk of IAV transmission from infected swine to humans (Bowman, Nolting, et al., 2012). Understanding the patterns of IAV genomic diversity in exhibition swine populations and strategies to control IAV spread within these populations can help to protect human and animal health.

Influenza A virus was first identified in swine in the 1930s and has been found in the U.S. exhibition swine population since active surveillance began in 2009 (Bowman, Nolting, et al., 2012). Exhibition swine make up approximately 1.5% of the United States’ swineherd, and most of those pigs are raised in small-farm settings primarily for education and/or competition purposes. A unique aspect of livestock exhibitions in the USA is that animals from many source farms commingle for extended periods of time, on many occasions, and with frequent interspecies exposure between humans and swine at the human-animal interface. Commingling naïve animals with one or more infected animals creates the opportunity for viral outbreaks and can facilitate the mixing of diverse IAVs (Bowman, Nolting, et al., 2012; Brown, 2000). Furthermore, individual swine can be exhibited at numerous agricultural exhibitions across multiple states each year, providing repeated opportunities for intrastate and interstate mixing and dissemination of IAVs (Bliss et al., 2017; McBride et al., 2021; Nelson et al., 2020; Nelson, Wentworth, et al., 2016).

Genomic analyses of IAVs in exhibition swine from 2009–2013 indicated that new viral diversity in exhibition swine was primarily introduced from commercial swine (Nelson, Wentworth, et al., 2016). During 2009–2016, 44 different genotypes of A(H3N2) virus were detected in the U.S. swine population, 37 of which contained A(H1N1)pdm09 gene segments (Rajão et al., 2017). One of the predominant genotypes indicated in that study, H3-G1, an A(H3N2) reassortant with the A(H1N1)pdm09 matrix (M) gene, had an estimated prevalence of 32% in U.S. commercial swine from 2009–2016. This genotype was also associated with all 320 A(H3N2)v cases from 2011–2012 (Kitikoon et al., 2013; Rajão et al., 2017).

In response to the increased A(H3N2)v cases in 2012, we significantly expanded our swine IAV surveillance efforts at agricultural fairs. Concurrent with our active swine IAV surveillance program during 2013–2015, A(H3N2)v cases were reported in or near states where we sampled swine. Here we use active surveillance data coupled with public health epidemiologic investigations to demonstrate that during 2013–2015 there were multiple genotypes of IAV circulating in the exhibition swine population and multiple IAV genotypes were transmitted from swine to humans at agricultural fairs. To investigate this, we performed phylogenetic analysis using full genome sequences from 272 IAVs isolated from exhibition swine in 5 states as a part of active surveillance from 2013–2015 and whole genome sequences from 23 of the 25 A(H3N2)v cases described in the U.S. during those same years.

Materials and Methods

Nasal swab or snout wipe samples were collected from selected swine at participating agricultural fairs in Ohio, Indiana, Michigan, Iowa, Texas, Colorado, West Virginia, and Kentucky from 2013–2015 as a part of an ongoing active surveillance project in accordance with The Ohio State University Institutional Animal Care and Use Committee (IACUC) protocol number 2009A0134. We attempted to conduct sampling at 100 fairs per year, with permission from each fair’s organizers, but participation varied year to year. Fairs were selected based on their willingness to participate in the ongoing project and were focused primarily in the midwestern USA as this is where the majority of the previous H3N2v cases were reported (https://www.cdc.gov/flu/swineflu/variant-cases-us.htm). Research personnel traveled to participating fairs and collected nasal swab or snout wipe samples from at least 20 individual swine on the last day of each fair, independent of clinical signs of illness (Bowman, Nolting, et al., 2012; Edwards et al., 2014). Samples were collected on the last day to maximize the IAV detection and reflect the timing of the highest IAV risk (McBride et al., 2022). While most fairs house all swine in one barn, pigs are housed across multiple barns at some exhibitions. Multiple barns are typically located adjacent to each other nearby to the show arena where judging occurs. As such, we collect samples proportionately (based on the approximate number of animals in each space) from all barns and areas where swine are housed to capture viral diversity and the full scope of any potential outbreak should we detect any IAV. Because swine can generally move freely throughout all barns, wash racks, show arena, scales, and sometimes even change pens during a fair, we consider an individual fair to be the relevant epidemiological unit. Swabs or snout wipes were placed in viral transport medium and stored at −80 °C until testing (Nolting et al., 2015). Samples were screened for IAV using a commercially available one-step, real-time reverse transcription-polymerase chain reaction (RRT-PCR) assay (VetMAX-Gold SIV Detection Kit; Applied Biosystems, Austin, TX, USA). The VetMAX Gold kit includes multiple (proprietary) molecular targets for the nucleoprotein and matrix IAV genes and was the only commercial USDA licensed PCR assay for swine influenza detection but is not species or subtype specific (Abente et al., 2017; M. J. Ma et al., 2014; Sharma et al., 2022). RRT-PCR positive samples were then inoculated onto Madin-Darby canine kidney (MDCK) cells for viral isolation, as previously described (Bowman et al., 2013). The complete genomes of representative isolates were sequenced using previously described protocols (Bowman, Sreevatsan, et al., 2012).

Whole genome sequences (WGS) of strains collected as part of the exhibition swine surveillance program from 2013–2015 along with WGS of contemporary swine-lineage IAVs from across the United States retrieved from Genbank (Nelson, Wentworth, et al., 2016) were aligned using MAFFT v7.409 (Katoh & Standley, 2013). Maximum-likelihood trees were inferred under a general time reversible plus gamma evolutionary model with 1,000 bootstrap replicates in RAxML v8.2.10 (Stamatakis, 2014). Trees were visualized and edited using FigTree v1.4.3, Adobe Illustrator, MEGA7, and Geneious 9.1.8 (Kearse et al., 2012; Kumar et al., 2016). Internal gene segments (PB2, PB1, PA, NP, M, and NS) were categorized as TRIG or A(H1N1)pdm09 lineage (pdm09). Gene segments encoding the envelope glycoproteins, HA and NA, were all assigned lineages using octoFLU and based on the previously detailed global nomenclature (Anderson et al., 2016, 2021; Chang et al., 2019). The previously described complete virus genotypes were labeled based on published nomenclature. Eight-segment H3 genotypes were all previously and numbered by (Kitikoon et al., 2013; Rajão et al., 2017). H1 genotypes G1-G9 were previously described by (Nelson, Wentworth, et al., 2016), with four additional (H1-G13 through H1-G16) genotypes described here. Spearman’s rank correlation was estimated using JMP Version 13 (JMP Statistical Discovery LLC).

Novel influenza A virus infections in humans, including variant IAV, have been nationally notifiable to CDC in the U.S. since 2007. Nucleotide sequences from all A(H3N2)v cases reported to CDC during 2013–2015 were downloaded from GISAID. One variant IAV sequence was excluded from analyses because of incomplete sequence data. Two segment sequences were missing from one 2015 variant case; therefore, it was included in the phylogenies but not designated as a genotype. Data identifying the county fair of swine exposure for each variant case were obtained from CDC.

Results

Diverse reassortant genotypes were detected in exhibition swine during 2013–2015.

We conducted surveillance in exhibition swine at 277 agricultural fairs/swine shows across eight states during 2013–2015. IAV was detected in swine at 67 (24.2%) of the fairs/exhibition events in 5 of the 8 states in which we conducted surveillance. The proportion of fairs with at least 1 IAV positive pig per fair decreased significantly between 2013 and 2015 (Table 1). While some fairs/exhibitions were sampled only once during one of the study years, others were sampled once annually throughout the three-year period (three total sampling events). We detected IAV in exhibition swine at 14 repeat exhibitions across multiple years, with IAV being detected at 12 fairs twice, and two fairs in all three study years (Table 2). The larger, regional exhibitions tended to have an average of 2.7 genotypes compared to the local fairs that only had an average of 1.5 genotypes. Additionally, estimated prevalence of IAV in exhibition swine significantly decreased every year from 2013 to 2015 (Table 1), and the overall prevalence across all three years was estimated to be 8.9% (95% CI: 8.3–9.6, Clopper-Pearson).

Table 1:

Detection of Influenza A Virus in exhibition swine from 2013–2015

Year Number of Fairs with ≥1 IAV positive pig 95% CI (Clopper-Pearson) Estimated prevalence in exhibition swine 95% CI (Clopper-Pearson)
2013 31/100 (31%) 22.1 – 41.0 346/2327(14.9%) 13.4 – 16.4
2014 22/73 (30.14%) 19.9 – 42.0 209/2368(8.9%) 7.7 – 10.0
2015 14/104 (13.5%) 7.6 – 21.6 140/3077(4.6%) 3.8 – 5.3
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Table 2:

Influenza A virus genotypes isolated from exhibition swine 2013–2015, displayed by exhibition event.

Exhibition ID No. genotypes per fair Exhibition ID No. genotypes per fair
2013 2014 2015 2013 2014 2015
Iowa A 1 Kentucky A 1
Iowa B 1 Kentucky B 2 2 + mixed
Indiana A 1 2 1 + mixed Ohio A 1
Indiana B 1 1 Ohio B 2
Indiana C 1 Ohio C 1
Indiana D 1 1 Ohio D 2 1
Indiana E 1 Ohio E 1
Indiana F 1 Ohio F 1
Indiana G 2 Ohio G 1
Indiana H 1 Ohio H 1
Indiana I 5 2 Ohio I 1
Indiana J 1 1 Ohio J 1 1
Indiana K 4 2 +mixed 1 Ohio K 1
Indiana L 1 Ohio L 2
Indiana M 2 + mixed Ohio M 3 + mixed 1 +mixed
Indiana N 3 1 Ohio N 1
Indiana O 1 Ohio O 1
Indiana P 2 Ohio P 1
Indiana Q 1 Ohio Q 1
Indiana R 2 1 Ohio R 1
Indiana S 1
Indiana T 1 Texas A 4
Indiana U 1 Texas B 1
Indiana V 3
Indiana W 1 1
Indiana X 1
Indiana Y 1
Indiana Z 1
Indiana AA 1 1
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There were 19 different genotypes detected during 2013–2015 (Figure 1). These genotypes included 5 genetically distinct HA segment lineages (1B.2.2, 1B.2.1, 1A.3.3.3, 3.1990.4.1, and 3.1990.4.2), 3 NA lineages (Classical N1, N2–1998, and N2–2002), and 2 internal gene constellations (TRIG and pdm09). In every year, the PB2, PB1, and NS gene segments were TRIG origin and the M segment was pdm09 origin. The PA and NP segments were either pdm09 or TRIG lineage, but both were detected within and between study years. We described a variety of distinct genotypes each year ranging from 13 genotypes in 2013 to 7 in 2015. We documented 4 new genotypes, all of which were A(H1N2) subtype. The number of genotypes per fair varied from 1 to 5 (Table 2). H3-G1 was the most prevalent A(H3N2) virus detected in 2013 (19.2%) and 2015 (57.1%) (Table 3 and Figure 1). Although there were no H3-G1 isolates in 2014, there were 4 other A(H3N2) genotypes detected in exhibition swine throughout the study period.

Figure 1.

Figure 1.

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Nineteen influenza A virus genotypes found in exhibition swine or humans from 2013–2015. The gene segments encoding for internal proteins (PB2, PB1, PA, NP, M, and NS) of H1N1pdm09 origin are indicated in blue and triple-reassortant internal gene (TRIG) origin segments are indicated in red. The H1 segments are classified as 1B.2.2 (H1δ1, yellow), 1B.2.1 (H1δ2, orange), and 1A.3.3.3 (H1γ, green). The H3 segments are classified as 3.1990.4.1 (H3IV-A, cyan) and 3.1990.4.2 (H3IV-B, pink). The neuraminidase (NA) segments are classified as classical N1 (green), N2–1998 (light blue) or N2–2002 (purple). The A(H1N1)/A(H1N2) genotypes are numbered H1-G1 to H1-G16. The A(H3N2) genotypes are numbered H3-G1 to H3-G21, consistent with genotype nomenclature used previously (Kitikoon et al., 2013; Nelson, Wentworth, et al., 2016; Rajão et al., 2017).

Table 3:

Prevalence of influenza A virus genotypes from swine by fair from 2013–2015

No. positive fairs by year (%)
2013 2014 2015
H1-G1 1 (3.2) 1 (4.5) --
H1-G2 1 (3.2) -- --
H1-G3 1 (3.2) -- --
H1-G4 15 (48.4) 2 (9.1) 1 (7.1)
H1-G5 5 (16.1) -- --
H1-G6 1 (3.2) -- --
H1-G7 1 (3.2) 6 (27.3) 1 (7.1)
H1-G8 3 (9.7) 1 (4.5) --
H1-G9 1 (3.2) 1 (4.5) --
H1-G13 -- 1 (4.5) --
H1-G14 1 (3.2) 1 (4.5) 2 (14.3)
H1-G15 -- -- 1 (7.1)
H1-G16 -- -- 1 (7.1)
H3-G1 10 (32.3) -- 8 (57.1)
H3-G10 3 (9.7) -- --
H3-G11 7 (22.6) -- --
H3-G18 -- 13 (59.1) --
H3-G19 -- 1 (4.5) 4 (28.6)
H3-G21 -- -- --
mixed 2 (6.5) 2 (9.1) 2 (14.3)
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Transmission of swine A(H3N2) to humans.

During this same time period, independently from our swine IAV surveillance, human cases of A(H3N2)v virus infections were reported in Illinois, Indiana, Iowa, Michigan, Ohio, New Jersey, and Minnesota. All 5 of the A(H3) genotypes we isolated from swine during our surveillance were also detected in at least one variant case during the 3-year study period. Of the 25 A(H3) variant cases reported during the study period, 10 (40%) were reported at 4 agricultural fairs at which we conducted swine surveillance. Three out of these four exhibitions had at least one positive pig, and the same genotype was described in swine and humans at two out of the three swine-positive exhibitions.

In all 3 years, the A(H3) genotypes that were isolated from swine at the most fairs also caused variant IAV cases in humans (Figure 1). Spearman’s Rank-Order Correlation (Spearman, 1904) demonstrated a positive correlation between the number of fairs with a particular genotype and the number of human cases of the same genotype (spearmen ρ= 0.67; p-value= 0.0023). Phylogenetic analysis of A(H3N2)v isolate sequences and IAV sequences isolated from exhibition swine in the same respective year demonstrated tight clustering. The HA segments of all A(H3N2)v virus isolates fell within the 3.1990.4.1 and 3.1990.4.2 lineages (cluster IV A and B subclusters, Figure 2a). A(H3N2)v virus isolates from each year had very high genetic similarity with exhibition swine IAV isolates from those years (Figure 2b,2c,2d). All A(H3N2)v virus NA segments, except for a New Jersey isolate from December 2015, clustered closely with swine isolates within the N2–2002 lineage.

Figure 2.

Figure 2.

Figure 2.

Figure 2.

Figure 2.

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The first tree (2a) illustrates the expanded view of the phylogenetic relationships and subclustering (boxes with dotted line) of the H3.1990.4 (Cluster IV) sequences of exhibition swine-origin H3N2 and H3N2v isolates from 2013 (green), 2014 (blue), and 2015 (pink). Human cases clustered closely with exhibition swine samples (2b, 2c, 2d). H3N2v cases are highlighted.

In 2013, 72% (13/18, Figure 1) of sequenced human cases were caused by H3-G1 IAVs, which was the most prevalent A(H3) genotype in swine that year (Table 3). In 2014, we did not identify H3-G1 in swine, but instead saw a predominance of H3-G18 which carried a 3.1990.4.2 HA gene segment. The following year, there was a human case associated with another 3.1990.4.2 genotype (H3-G19).

Discussion

The identification of variant IAVs after exposure to exhibition swine has garnered attention from both the scientific community and mainstream press due to its importance for veterinary and public health (Bowman et al., 2017; Jhung et al., 2013; Lindstrom et al., 2012). Although identification of A(H3N2)v cases decreased from 2013–2015, the year-to-year variation highlights the unpredictable nature of these localized outbreaks. Additionally, identified cases of A(H3N2)v infection have predominantly been children under the age of 18 (Jhung et al., 2013). The higher incidence in children could be because of a lack of antigenic immunity to the A(H3N2)v viruses in this age group. Evidence suggests that nearly all children under the age of 5 years and >80% of children under the age of 14 years in a general population lack neutralizing antibodies to swine-origin A(H3N2) viruses (Skowronski et al., 2012), potentially owing to a lack of prior immunity from vaccination or infection. Additionally, healthy juveniles (born between 2004–2013) in the United States have low or no serum antibody titers cross-reactive with several swine-origin IAVs indicating a potential gap in protection in this age group (Lorbach, Fitzgerald, et al., 2021). Further work is required to characterize the protection status specifically for children with frequent exposure to swine at this type of human-animal interface to fully resolve drivers of zoonotic transmission. Children under the age of 18 years also make up most fair swine exhibitors and, therefore, also have increased swine exposure.

There is concern that a swine-origin IAV will infect susceptible human hosts and undergo subsequent sustained transmission and adaptation. Since exhibition swine are a viral mixing vessel and a source of novel viral diversity, studying the genotypes of IAV circulating in exhibition swine populations can help us better understand the potential risks to swine and human health from mutated or reassortant viruses (Nelson, Stucker, et al., 2016). Genomic analyses with data from over a three-year time-period demonstrates that there are multiple genotypes circulating in exhibition swine each year and these genotypes vary from year to year in both composition and prevalence (Figure 1). IAVs with pandemic potential must be unique enough antigenically to evade population level immunity and must be sufficiently adapted to easily spread from person-to-person (Zimmerman et al., 2012). Genotypic diversity and frequent reassortment of IAVs in swine populations can make them a source of emergent IAVs to which the human population lacks immunity and may lead to the emergence of viruses with unpredictable transmissibility and pathogenesis (Lorbach, Fitzgerald, et al., 2021).

Surveillance early in the swine show season at shows upstream in the exhibition swine IAV transmission chain can enable both proactive interventions prior to the primary zoonotic risk associated with later county fairs as well as the rapid genomic analysis and risk analysis of IAVs circulating in the exhibition swine population (Nelson et al., 2020; Rambo-Martin et al., 2020). Fortunately, A(H3N2)v viruses identified to date have not yet achieved sustained human-to-human transmission (Jhung et al., 2013). During this study period, there were 6 different zoonotic genotypes of IAV that were reported to have infected a range of 1 to 13 humans. Although the H3-G1 genotype has infected more humans than any other genotype to date (Kitikoon et al., 2013; Rajão et al., 2017), we provide evidence for interspecies transmission of other H3 genotypes as well. In our active exhibition swine surveillance during 2013–2015, we detected 5 different A(H3N2) genotypes, and all 5 corresponded to at least 1 human case. Although it remains unclear why H3-G1 surged in humans during 2011–2012 and decreased the during the following years of our study, possible explanations include immunity from previous A(H3N2) exposures in humans and swine, implementation of interventions such as improved cleaning and disinfection at fairs resulting from higher public health awareness, or lower exposure levels due to a decrease in prevalence within pig populations (Nelson, Wentworth, et al., 2016).

Although two relatively novel genotypes, H1-G15 and H1-G3, both H1B.2.1, varied from the more established genotypes, they only differed from each other in the NA segment. The increase in genotypic diversity supports previous studies describing continual reassortment of A(H3N2) viruses with A(H1N1)pdm09 viruses in the swine population (Kitikoon et al., 2013; Nelson, Wentworth, et al., 2016; Rajão et al., 2017). Although the A(H1N1)pdm09 virus was introduced to commercial swine, it is not the dominant IAV in that population. Instead, A(H3N2) viruses that have reassorted with A(H1N1)pdm09 strains predominate (Nelson, Stucker, et al., 2016). The H3-G1 genotype acquired the pdm09 M gene and demonstrated increased viral replication and transmission characteristics in animal models (Pearce et al., 2012). All genotypes that we detected contained the pdm09 M gene, which is the dominant matrix protein in commercial swine IAVs. Additionally, as the prevalence of H3-G1 has ebbed and flowed in the exhibition swine population, we have observed the emergence of other genotypes that contain pdm09 internal genes (Figure 1) (Nelson, Stucker, et al., 2016; Nelson, Wentworth, et al., 2016).

The size and scope of the exhibitions sampled could have some effect on how many genotypes were recovered from swine at that exhibition. The extent of exhibitions sampled ranged from large national livestock expositions hosting thousands of swine, to small, local county fairs with 100–800 swine. The observed higher genetic diversity of IAV at larger exhibitions demonstrates that not only are individual animals a source of reassortment, but that large regional exhibitions foster greater opportunity for IAV reassortment. Expositions that commingle multiple animals from various management backgrounds and geographical regions can seed genetic diversity by bringing together IAVs from different regions into one central location (McBride et al., 2021). Surveillance and possible intervention at these larger exhibitions should serve as a strategy for evaluation and mitigation of zoonotic IAV risk in exhibition swine. The commingling of animal host species at live animal markets has provided a human-animal interface for close contact and zoonotic virus emergence in the past (Choi et al., 2015; Wan et al., 2011; Worobey et al., 2022), and a similar public health risk exists at swine exhibitions in the United States.

A multi-pronged one health approach is paramount to prevent interspecies transmission of the broad IAV diversity from pigs to humans. (W. Ma et al., 2008). Decreasing prevalence of IAV infection in swine and mitigating behaviors in humans that promote viral transmission are critical for the reduction of zoonotic IAV infections. Factors that may contribute to the increased prevalence of IAV in exhibition swine populations include participation in multiple shows and allowing pigs to commingle for multiple days (Bliss et al., 2017; Bowman et al., 2014). Since reassortment of IAV happens readily in swine populations, recommendations that can help to decrease the prevalence of IAV in swine entering fairs and decrease the intra- and inter-species spread of influenza during the exhibitions are critical outcomes for past and future research. Some measures to minimize this risk have been studied including vaccination of swine against IAV (Lorbach, Nelson, et al., 2021) and shortening the duration of swine shows at county fairs (McBride et al., 2022), but further work is still needed to better mitigate the public health risk.

Although IAV can infect any person, some individuals such as those under the age of 5, over the age of 65, pregnant, or immunosuppressed are at an increased risk of complications from infection (Gambhir et al., 2013). Limiting swine contact with these individuals can help to prevent severe IAV infections and reduce the public health burden of variant IAV and the risk of interspecies transmission events. Supplying handwashing or hand sanitizer stations along with signage related to washing hands could increase hand hygiene compliance, a recommendation to decrease IAV spread. Discouraging sleeping in the swine barns is another recommended control strategy. Youth exhibitors are at increased risk for variant IAV infection because of close contact with swine and risky behavioral practices (Nolting et al., 2019). Accordingly, educating youth swine exhibitors on zoonotic disease and disease prevention is a crucial component to mitigate risk (Nolting et al., 2018). A more detailed list of measures to minimize influenza transmission at swine exhibitions for both fair organizers as well as exhibitors, including a brief discussion on vaccination recommendations for humans as well as swine, can be found in the document prepared by (National Assembly of State Animal Health Officials (NASAHO) & National Association of State Public Health Veterinarians (NASPHV), 2018).

Findings from this field study are limited by surveillance sampling constraints. Most samples were obtained from exhibitions in the Midwestern United States during the summer show season. Expanding sampling in other regions of the United States and other times of year might identify different genotypic prevalences and other novel genotypes. Additionally, representative samples taken from each fair ranged from 20 to 200 samples. It is possible that there are other genotypes that are circulating at low levels that were not detected by our surveillance program. Finally, future efforts should increase sampling to accommodate more exhibitions within each state to help ensure more precise prevalence estimates. It is also possible but unlikely that variant IAV cases reported during the study period were the result of humans arriving at the fair already infected with influenza. For certain IAV lineages like pdm09 H1 with frequent reverse zoonoses, it might be difficult to parse out the direction of interspecies transmission. However, the H3 lineages analyzed here are sufficiently diverged from a 1990s human seasonal H3 for the swine-origin descendants to be clearly distinguishable from a contemporary human seasonal H3. In addition to this, agricultural fairs occur throughout the summer season, outside of the typical human seasonal flu season, making the prevalence of IAV in humans quite low during our surveillance periods.

Although there has been a decrease in the detection of IAV in exhibition swine populations from 2013 to 2015, there continues to be human cases of swine-origin H3N2 among people attending swine exhibitions. All five H3 genotypes found in swine during those years were also reported in humans. Even though the exhibition level prevalence of IAV significantly decreased from 2013–2015, we described a positive correlation between the number of fairs with a particular genotype and the number of human cases of that genotype. With an ever-increasing percentage of our population unfamiliar with food production, agricultural fairs and exhibitions are an important component of public agricultural education. However, this education should not carry with it an undue risk to public health. Understanding how influenza A viruses evolve and reassort, both in animal reservoirs and as a mechanism that drives zoonosis, can be helpful in the prediction and prevention of pandemic risk interspecies transmission events. Ideally, active surveillance of influenza in exhibition swine populations would inform annual predictions for the threat of zoonotic IAV. Especially in light of the emergence of H3 3.2010.1 and 3.2010.2 in swine since this study period (Powell et al., 2021; Sharma et al., 2022), surveillance data are more critical than ever to inform vaccine strain selection. Robust and timely IAV surveillance allows for rapid identification and risk assessment of the full breadth of viral diversity in swine such that the OFFLU and WHO vaccine composition meetings can effectively evaluate the sufficiency of current candidate vaccine viruses and ensure we are prepared for any pandemic risk IAV that we may face in the future (OFFLU VCM Summary Reports, n.d.; WHO, 2021).

Supplementary Material

Supinfo

Impacts.

  • Multiple H3 genotypes of swine-lineage influenza A viruses have infected humans in the United States since 2013–2015, which pose a risk for pandemic emergence.

  • The public health risk posed by interspecies influenza A virus transmission in the United States was associated with each virus’ prevalence in swine at agricultural fairs.

  • Efforts to reduce influenza prevalence in exhibition swine could be useful to mitigate zoonotic influenza A virus emergence.

Acknowledgements

The authors thank Nola Bliss, Jody Edwards, Alexa Edmunson, Elise Gerken, Keirsten Harris, Amber Kihm, Grant Price, Jeffrey Workman, Michele Zentkovich, Bret Marsh, Kurt Stevenson, and Tony Forshey for technical assistance and support. Thank you to the US Centers for Disease Control and Prevention, especially Todd Davis, Yunho Jang, and John Barnes, for assistance in the data management for the variant IAV sequences studied here. We thank the agricultural fairs for participating. This work has been funded in part with federal funds from the Centers of Excellence for Influenza Research and Surveillance (CEIRS), National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. HHSN266200700007C and Contract No. HHSN272201400006C.

Footnotes

Conflict of interest statement

The authors declare no conflicts of interest.

Ethics approval statement

All swine surveillance samples were collected in accordance with The Ohio State University Institutional Animal Care and Use Committee (IACUC) approved protocol number 2009A0134.

Contributor Information

Christine M. Szablewski, The Ohio State University, Department of Veterinary Preventive Medicine, Columbus, OH, USA.

Dillon S. McBride, The Ohio State University, Department of Veterinary Preventive Medicine, Columbus, OH, USA.

Susan C. Trock, Centers for Disease Control and Prevention, Atlanta, GA, USA

Gregory G. Habing, The Ohio State University, Department of Veterinary Preventive Medicine, Columbus, OH, USA

Armando E. Hoet, The Ohio State University, Department of Veterinary Preventive Medicine, Columbus, OH, USA

Sarah W. Nelson, The Ohio State University, Department of Veterinary Preventive Medicine, Columbus, OH, USA

Jacqueline M. Nolting, The Ohio State University, Department of Veterinary Preventive Medicine, Columbus, OH, USA

Andrew S. Bowman, The Ohio State University, Department of Veterinary Preventive Medicine, Columbus, OH, USA.

Data availability statement

All of the sequences generated and analyzed as a part of this study are publicly available in NCBI GenBank (swine origin viral sequences) or GISAID (human origin viral sequences) and corresponding accession numbers are available in supporting information tables S1 and S2 respectively.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supinfo
NIHMS1951537-supplement-Supinfo.docx (59.3KB, docx)

Data Availability Statement

All of the sequences generated and analyzed as a part of this study are publicly available in NCBI GenBank (swine origin viral sequences) or GISAID (human origin viral sequences) and corresponding accession numbers are available in supporting information tables S1 and S2 respectively.

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