INTRODUCTION
Influenza A virus (IAV) is an economically important pathogen of swine that has the ability to evolve and evade the host immune response, which presents a challenge to current control strategies. The negative-sense, single-stranded RNA genome consists of eight noncontiguous gene segments that are known to encode between 10 and 17 proteins (
1–3). The segmented genome structure creates the opportunity for reassortment when two or more IAV strains concurrently infect the same host, resulting in novel gene combinations and increased diversity at the genomic level (
4,
5). IAV can also rapidly accumulate mutations due to errors introduced by viral polymerase (
6). Swine have both α2,6- and α2,3-Gal-linked sialic acid on the surface of their respiratory epithelial cells; therefore, in addition to swine-to-swine transmission, they have the potential for infection from human- and avian-origin IAVs (
7,
8). Consequently, observed IAV diversity in swine is increased by the transmission, occasional establishment, and evolution of avian and human IAVs in swine populations.
The genetic diversity of IAV translates to a similarly large breadth of antigenic diversity in the hemagglutinin (HA) and neuraminidase (NA) proteins. The accumulation of amino acid substitutions from polymerase mutation in the surface glycoproteins often results in changes in the antigenic phenotype of IAV (
9,
10). For the H3 subtype, a small number of amino acid residues have a disproportionate effect on antigenic phenotype in both humans and swine (
11–13). Six of these amino acid positions (145, 155, 156, 158, 159, and 189 [H3 mature peptide numbering {
14}]) are referred to as the H3 antigenic motif in swine (
15). These 6 residues are located on the globular head of the HA protein and are adjacent to the receptor binding site. There may be constraints on substitution flexibility at these positions due to the necessary conservation of receptor binding functionality, as shown for position 145 (
13). Minimal genetic change may result in significant antigenic change, reducing the efficacy of current IAV vaccines against clinical disease and transmission in swine (
16). Vaccination with whole inactivated virus (WIV) with an oil-in-water adjuvant is common in swine in the United States. However, WIVs are most efficacious when the vaccine and challenge strains are closely related (
17,
18). Thus, an important utility of IAV surveillance and sequence data in swine populations is to inform vaccine strain selection.
In 1998, investigations into severe respiratory disease in swine in the United States revealed the introduction of the H3N2 subtype of IAV into swine. The H3N2 viruses that persisted had triple-reassortant internal genes (TRIGs) with HA, NA, and PB1 gene segments derived from human seasonal H3N2; PB2 and PA gene segments from avian IAV; and nucleoprotein (NP), M, and NS gene segments from classical swine H1N1 (
19–21). The HA gene from this introduction established the colloquially named H3 cluster IV (C-IV) in the United States (named the 1990.4 lineage in global H3 nomenclature [
21]). The C-IV HA continued to circulate in the United States and evolved into genetically distinct clades A through F (
22). C-IV clade A (C-IVA) began to increase in detection frequency beginning in 2010 and was the predominant H3 clade in U.S. swine until 2016 when it was surpassed by H3 2010.1, a more recent human seasonal incursion that subsequently became established as a swine lineage (
21,
23,
24). In recognition of the animal and public health relevance of IAV in swine, the U.S. Department of Agriculture (USDA) implemented a surveillance system beginning in 2010 (
25). The system allows producers to voluntarily and anonymously submit samples collected from swine exhibiting influenza-like illness to regional veterinary diagnostic laboratories for diagnostics and sequencing of the HA and NA genes. The surveillance system has resulted in the collection of 9,391 IAV isolates and the publication of 9,040 HA sequences as of 30 September 2021 (
26). The data collected within the system have been applied to monitor spatial and temporal trends in the genetic and antigenic diversity of IAV in swine across the sampled areas of the United States (
11,
27).
In this study, we used the USDA IAV-in-swine surveillance data set, supplemented with other publicly available sequence data, to describe a resurgence in C-IVA sequence detections in 2019 as well as a relative decrease in the detection of H3 2010.1. We quantified genetic and antigenic characteristics associated with the increased detection frequency of the C-IVA clade. Concurrently, we adapted the Nextstrain platform (
28) to IAV in swine to provide near-real-time phylogenetic visualization of surveillance data for the H3 subtype. Collectively, these analyses provide insight into the factors contributing to the expansion of swine IAV clades and improve our ability to predict mechanisms that allow IAV to evade current control measures in swine populations.
DISCUSSION
This study investigated the recent increase in the detection of H3 C-IVA viruses in U.S. swine and how circulating H3 diversity within swine populations could help explain genetic clade turnover and dominance dynamics. The H3 2010.1 clade that emerged in 2012 began to supplant the C-IVA clade in 2016 (
Fig. 1A). There was limited serological cross-reactivity between 2010.1 and C-IVA swine H3N2 viruses (
24). Animals in the population in 2016 were more likely to have natural immunity against 2010.1 viruses, while animals with natural immunity against C-IVA viruses gradually decreased.
Vaccine immunity likely plays a significant role in the dynamics of HA clades detected in the United States. There is only one fully licensed commercial swine IAV vaccine, and this does not contain the 2010.1 antigen; instead, it has an H3 C-IVA antigen, an H3 C-IVB antigen, and two H1 antigens (H1N1 gamma and H1N2 delta1) (
32). Consequently, instead of a commercial product, many swine in production systems were likely vaccinated against the 2010.1 clade of viruses with custom or autogenous vaccines following its emergence and dominance over C-IVA (
16,
33). In 2012, 60% of producers of large breeding herds reported the use of an influenza vaccine, and 47% of those were using autogenous vaccines rather than a fully licensed product (
34). Thus, waning immunity against C-IVA viruses due to decreased natural immunity and the increased use of vaccines containing 2010.1 viruses could have provided an advantage to the C-IVA viruses. This is supported by the balanced shape of the HA phylogeny of the major clade after 2019, which suggests an absence of immune- or vaccine-driven selection within the C-IVA major clade (
Fig. 2 and
3).
Although the number of C-IVA detections increased since 2018, the relative genetic diversity within the clade did not increase until mid-2020. The 2018–2019 clonal expansion of C-IVA with low diversity suggests that a selective sweep occurred in the population. Sweep-related changes have been identified in human seasonal H3N2 IAV and are most often detected at amino acid sites located on the HA (
35,
36). In 2019, the relative genetic diversity began to rise, likely as the result of the success, spread, and subsequent diversification of the virus. It is important to note that detection frequencies of this clade and other swine IAV clades in USDA IAV-in-swine data are not a measure of prevalence: these data are derived from the passive sampling of clinically sick pigs (
37). Despite this limitation, the surveillance provides a 10-year time series for each genetic clade circulating in U.S. swine, allowing the meaningful identification of changes in detection frequency relative to historical counts. We suggest that a pattern of increasing detection frequency paired with low relative genetic diversity can act as an early warning signal that can be used to flag genetic clades of swine IAV that require characterization and risk assessment for swine agriculture and pandemic preparedness.
To determine whether a selective sweep occurred due to antigenic drift, we identified amino acid substitutions sustained in the major and minor clades that were circulating as the detection frequency increased from 2018 to 2020. We identified an N156H substitution in the HA of the major clade. Amino acid position 156 was previously identified as having an effect on the antigenic phenotype, usually in combination with substitutions in other positions on the HA (
11,
15,
38), but the impact of an amino acid substitution depends on the biological properties of the specific amino acid(s) that changed in the context of the overall HA1 amino acid sequence (
39,
40). This N156H substitution prompted the antigenic characterization of the major and minor clades via HI assays to assess for a potential loss in cross-reactivity. A significant loss in cross-reactivity of the contemporary 156H from previous strains with 156N would suggest a potential lack of population immunity that could explain the increased frequency of the major clade. However, our data did not demonstrate that the substitution caused significant antigenic drift. Antibodies raised against ancestral C-IVA demonstrated HI cross-reactivity against the more recent strains regardless of the substitution. Our results support previous findings that variation at position 156 alone did not cause significant antigenic drift (
15,
38). The limited change in antigenic phenotype suggests that the N156H substitution may not have been the primary cause of the observed clonal expansion of the C-IVA major clade.
With no evidence of significant antigenic drift in the HA between the contemporary C-IVA major and minor clades, the viruses were analyzed for evidence of other genetic signatures associated with expansion. The minor clade recently reassorted to acquire N2-2002A.2 genes, while the major clade remained paired with N2-2002B.2. A phylodynamic analysis suggested that the most likely donor of the N2-2002A.2 gene was an H1 beta virus. This H1 genetic clade is not frequently detected, with only 104 detections from January 2011 to March 2021 (see
Fig. S1 in the supplemental material). Not all regions of the United States are sampled equally in the passive surveillance program; there can be geographical areas and production systems that are underrepresented (
27), and swine and their IAVs may move into the United States from regions with far less surveillance (e.g., see reference
41). Our analysis supports the proposition that infrequently detected clades of IAV can contribute to IAV reassortment and increase genetic diversity, and this may result in novel viruses that have the capacity to expand across the United States (
31).
We further investigated the antigenic effects of this reassortment event with a panel of NI antisera previously used to describe antigenic variation among and between swine N2 lineages (
30). The 2002B.2 of the C-IVA major clade viruses retained close antigenic relationships with the 2002B.2 reference antigen, but antigenic variation existed between clade representatives within the 2002 lineage. Since the early-emerging 2010.1 viruses were paired with 2002A at the time when they began to outnumber the previously circulating C-IVA viruses, population immunity may have been skewed toward both mismatched HA and NA. The 2002B.2 clade of N2 now represents the majority of circulating N2 detected in the swine population, so the importance of NA immunity for the maintenance of the major C-IVA H3 clade paired with 2002B is not clear.
We analyzed whole-genome sequencing (WGS) data for evidence of reassortment of the internal genes. The major C-IVA clade was determined to have reassorted with other endemic swine viruses, likely an H1 delta1 virus, to acquire an NP of the H1N1pdm09 lineage. The H1 delta1 clade was fairly common from 2011 to 2017, making up 17 to 47% of yearly IAV (H1 and H3) detections (
Fig. S1). The phylodynamic analysis also revealed other NP reassortment events in the major clade of C-IVA, suggesting that although the C-IVA NPs all share the same NP pdm evolutionary lineage circulating in swine, they are derived from different donor H1 and H3 swine viruses. The genotype of swine IAV internal genes was summarized as a concatenation of one-letter codes representing the genetic lineage of each gene segment (PB2, PB1, PA, NP, M, and NS) without the HA and NA segments. The C-IVA internal gene constellation in 2010 was
TTTTTT, with all internal genes coming from the TRIG lineage. C-IVA viruses then acquired a matrix gene from the H1N1pdm09 virus, with constellation
TTTTPT, and this was common between 2009 and 2016 (
42). This constellation was also involved in an H3N2v outbreak in humans from 2011 to 2012, causing 340 cases across 13 U.S. states (
43). However, beginning in 2017, the internal gene constellation found in the C-IVA major clade that increased in detection frequency was
TTTPPT with an H1N1pdm09 NP gene. This constellation was observed previously but was uncommon (22 of the 368 isolates between 2009 and 2016) (
42). Influenza NP is characterized as a structural RNA binding protein that forms the ribonucleoprotein (RNP) particle (
44), and its genetic variation may impact functions such as the temporal regulation of apoptosis or the import and export of viral RNPs (vRNPs) from the nucleus (
45,
46). Results from a transmission study in pigs have demonstrated that H3 viruses with the
TTTPPT constellation are more effective in viral transmission than H3 strains with a
TTTTPT constellation (
42). Consequently, our data suggest that the success of this clade of viruses could be explained by differences between the pdm09 and TRIG genetic lineages of the NP acquired following reassortment in 2017.
Since H3 C-IVA viruses continue to make up roughly one-half of H3N2 detections within the national USDA influenza A virus-in-swine surveillance program in 2021, the increased detection frequency of C-IVA suggests that vaccines should include antigens from this clade of IAV. Subsequent surveillance is necessary to determine if vaccination against C-IVA will result in a decrease in detection; however, this would require additional knowledge of farm-specific vaccines and vaccination strategies. Matching vaccine components to circulating diversity and understanding how swine transportation patterns and biosecurity practices affect the transmission of swine IAV H3 clades can help improve animal health. Our analysis also demonstrates how low-frequency or regionally restricted genotypes donated gene segments to a different HA clade that subsequently led to the expansion of newly reassorted gene combinations. This dynamic and the resurgence of the H3 C-IVA clade create concern for public health, with the knowledge that a virus from the same clade was involved in causing a human outbreak in the context of reassortment. Our HI assay included a representative strain (A/swine/New York/A01104005/2011) that was genetically similar to the H3N2v from the 2011–2012 outbreaks and showed that the contemporary C-IVA representatives tested had not undergone significant antigenic drift. However, dominant swine H3N2 clades have caused numerous zoonotic events through human-swine agricultural interfaces (
43,
47,
48), and more recent contemporary C-IVA swine strains may be antigenically drifted from the pandemic preparedness candidate vaccine virus A/Minnesota/11/2010 (
49). Thus, understanding the factors that contribute to IAV in swine clade expansion is necessary to inform and improve prediction methods for more successful control measures and to provide insight into pandemic preparedness efforts.
ACKNOWLEDGMENTS
We gratefully acknowledge pork producers, swine veterinarians, and laboratories for participating in the USDA influenza A virus-in-swine surveillance system and publicly sharing sequences.
This work was supported in part by the Iowa State University Presidential Interdisciplinary Research Initiative; the Iowa State University Veterinary Diagnostic Laboratory; the U.S. Department of Agriculture (USDA) Agricultural Research Service (ARS) (project number 5030-32000-231-000-D); the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services (contract number 75N93021C00015); the USDA Agricultural Research Service Research Participation Program of the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. Department of Energy (DOE) and the USDA Agricultural Research Service (contract number DE-AC05-06OR23100); the Department of Defense, Defense Advanced Research Projects Agency, Preventing Emerging Pathogenic Threats program (contract number HR00112020034); and the SCInet project of the USDA-ARS (project number 0500-00093-001-00-D). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA, DOE, ORISE, DARPA, or ISU. The USDA is an equal opportunity provider and employer.