Phylogenetic Analysis of a Swine Influenza A(H3N2) Virus Isolated in Korea in 2012

Influenza A viruses (IAVs) in swine can cause sporadic infections and pandemic outbreaks among humans, but how avian IAV emerges in swine is still unclear. Unlike domestic swine, feral swine are free ranging and have many opportunities for IAV exposure through contacts with various habitats and animals, including migratory waterfowl, a natural reservoir for IAVs. During the period from 2010 to 2013, 8,239 serum samples were collected from feral swine across 35 U.S. states and tested against 45 contemporary antigenic variants of avian, swine, and human IAVs; of these, 406 (4.9%) samples were IAV antibody positive. Among 294 serum samples selected for antigenic characterization, 271 cross-reacted with ≥1 tested virus, whereas the other 23 did not cross-react with any tested virus. Of the 271 IAV-positive samples, 236 cross-reacted with swine IAVs, 1 with avian IAVs, and 16 with avian and swine IAVs, indicating that feral swine had been exposed to both swine and avian IAVs but predominantly to swine IAVs. Our findings suggest that feral swine could potentially be infected with both avian and swine IAVs, generating novel IAVs by hosting and reassorting IAVs from wild birds and domestic swine and facilitating adaptation of avian IAVs to other hosts, including humans, before their spillover. Continued surveillance to monitor the distribution and antigenic diversities of IAVs in feral swine is necessary to increase our understanding of the natural history of IAVs.IMPORTANCE There are more than 5 million feral swine distributed across at least 35 states in the United States. In contrast to domestic swine, feral swine are free ranging and have unique opportunities for contact with wildlife, livestock, and their habitats. Our serological results indicate that feral swine in the United States have been exposed to influenza A viruses (IAVs) consistent with those found in both domestic swine and wild birds, with the predominant infections consisting of swine-adapted IAVs. Our findings suggest that feral swine have been infected with IAVs at low levels and could serve as hosts for the generation of novel IAVs at the interface of feral swine, wild birds, domestic swine, and humans.

Influenza A viruses (IAVs) pose a major public health threat due to their wide host range and pandemic potential. Pigs have been proposed as “mixing vessels” for avian, swine, and human IAVs, significantly contributing to influenza ecology. In the United States, IAVs are enzootic in commercial swine farming operations, with numerous genetic and antigenic IAV variants having emerged in the past two decades. However, the dynamics of intensive swine farming systems and their interactions with ecological factors influencing IAV evolution have not been systematically analysed. This review examines the evolution of swine IAVs in commercial farms, highlighting the role of multilevel ecological factors. A total of 61 articles published after 2000 were reviewed, with most studies conducted after 2009 in Midwestern US, followed by Southeast and South-central US. The findings reveal that ecological factors at multiple spatial scales, such as regional transportation networks, interconnectedness of swine operations, farm environments, and presence of high-density, low-genetic diversity herds, can facilitate virus transmission and enhance virus evolution. Additionally, interactions at various interfaces, such as between commercial swine and feral swine, humans, or wild birds contribute to the increase in genetic diversity of swine IAVs. The review underscores the need for comprehensive studies and improved data collection to better understand the ecological dynamics influencing swine IAV evolution. This understanding is crucial for mitigating disease burden in swine production and reducing the risk of zoonotic influenza outbreaks.

Genetic correlation between current circulating H1N1 swine and human influenza viruses

Unexpected outbreaks caused by the H5N1 highly pathogenic avian influenza virus (HPAIV) in dairy cows in the United States (US) have raised significant veterinary and public health concerns. When and how the H5N1 HPAIV was introduced into dairy cows and the broader epidemiology of influenza A virus (IAV) infections in cattle in the US remain unclear. Herein, we performed a retrospective study to screen more than 1,700 cattle serum samples collected from different bovine breeds in the US from January 2023 to May 2024 using an enzyme-linked immunosorbent assay (ELISA) targeting the nucleoprotein (NP) to detect IAV infections, and the positive samples were further tested by hemagglutination inhibition (HI) assay. Results showed that 586 of 1,724 samples (33.99%) from 15 US states were seropositive by the NP ELISA assay, including 78 samples collected in 2024 and 508 samples collected in 2023. Moreover, the HI assay revealed that 45 of these ELISA-positive samples were positive to human seasonal H1N1 and H3N2 and swine H3N2 and H1N2 viruses, and some were positive to two or three tested IAVs. Surprisingly, none of these ELISA-positive samples were HI positive for the circulating bovine H5N1 strain. Our results demonstrate that IAVs other than H5N1 can infect cattle, infections are not limited to dairy cows, and that bovine infections with swine and human IAVs have occurred prior to the H5N1 outbreaks. All results highlight the value in monitoring IAV epidemiology in cattle, as the viruses might adapt to cattle and/or reassort with the currently circulating H5N1 HPAIV, increasing risk to humans.IMPORTANCEInfluenza A virus (IAV) is an important zoonotic pathogen that can infect different species. Although cattle were not historically considered vulnerable to IAV infections, an unexpected outbreak caused by H5N1 highly pathogenic avian influenza virus in dairy cows in the United States (US) in early 2024 has raised significant concerns. When and how the virus was introduced into dairy cows and the wider impact of IAV infections in cattle in the US remain unclear. Our retrospective serological screen provided evidence of human and swine H1 and H3 IAV infections in different cattle breeds in addition to dairy cows, although no H5N1 infection was detected. Our results underline the necessity to monitor IAV epidemiology in cattle, as reassortment of IAVs from different species may occur in cattle, generating novel viruses that pose threats to public and animal health.

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. 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. 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.

Replication of swine and human influenza viruses in juvenile and layer turkey hens

Novel triple-reassortant H1N1 swine influenza viruses in pigs in Tianjin, Northern China

Pigs and humans have shared influenza A viruses (IAV) since at least 1918, and many interspecies transmission events have been documented since that time. However, despite this interplay, relatively little is known regarding IAV circulating in swine around the world compared with the avian and human knowledge base. This gap in knowledge impedes our understanding of how viruses adapted to swine or man impacts the ecology and evolution of IAV as a whole and the true impact of swine IAV on human health. The pandemic H1N1 that emerged in 2009 underscored the need for greater surveillance and sharing of data on IAV in swine. In this paper, we review the current state of IAV in swine around the world, highlight the collaboration between international organizations and a network of laboratories engaged in human and animal IAV surveillance and research, and emphasize the need to increase information in high-priority regions. The need for global integration and rapid sharing of data and resources to fight IAV in swine and other animal species is apparent, but this effort requires grassroots support from governments, practicing veterinarians and the swine industry and, ultimately, requires significant increases in funding and infrastructure.

Southern China has long been considered to be an epicenter of pandemic influenza viruses. The special environment, breeding mode, and lifestyle in southern China provides more chances for wild aquatic birds, domestic poultry, pigs, and humans to be in contact. This creates the opportunity for interspecies transmission and generation of new influenza viruses. In this study, we reported a novel reassortant H1N2 influenza virus from pigs in southern China. According to the phylogenetic trees and homology of the nucleotide sequence, the virus was confirmed to be a novel triple-reassortant H1N2 virus containing genes from classical swine (PB2, PB1, HA, NP, and NS genes), triple-reassortant swine (PA and M genes), and recent human (NA gene) lineages. It indicated that the novel reassortment virus among human and swine influenza viruses occurred in pigs in southern China. The isolation of the novel reassortant H1N2 influenza viruses provides further evidence that pigs are “mixing vessels,” and swine influenza virus surveillance in southern China will provide important information about genetic evaluation and antigenic variation of swine influenza virus to formulate the prevention and control measures for the viruses.

Influenza A virus (IAV), a deadly zoonotic pathogen, poses a tremendous threat and burden to global health systems. Pigs act as "mixing vessel" hosts to support and generate new pandemic viruses. Preventing the spread of IAV in pigs effectively can delay or even block cross-species transmission. Universal vaccines based on the highly conserved ectodomain of influenza matrix protein 2 (M2e) have been widely reported, but have not been applied due to inadequate protection. Porcine circovirus type 2 (PCV2) causes immunosuppression and promotes swine influenza virus (SIV) infection. Here, M2e is inserted into capsid protein of PCV2 without burying the neutralizing epitopes and self-assembles to form a bivalent nanovaccine. Inoculation with the nanovaccine induces robust M2e- and PCV2-specific immune responses. The nanovaccine confers protection against lethal challenges of IAV from different species in mice, and significantly reduces SIV titers in pigs' respiratory tract and blocks SIV transmission. These results indicate that the nanovaccine is an economical and promising PCV2 and universal IAV bivalent vaccine, and it will synergistically and powerfully offer potential ability to block IAV cross-species reassortment and transmission.

Influenza A viruses (IAVs) can infect a wide variety of bird and mammal species. Their genome is characterized by 8 RNA single stranded segments. The low proofreading activity of their polymerases and the genomic reassortment between different IAVs subtypes allow them to continuously evolve, constituting a constant threat to human and animal health. In 2009, a pandemic of an IAV highlighted the importance of the swine host in IAVs adaptation between humans and birds. The swine population and the incidence of swine IAV is constantly growing. In previous studies, despite vaccination, swine IAV growth and evolution were proven in vaccinated and challenged animals. However, how vaccination can drive the evolutionary dynamics of swine IAV after coinfection with two subtypes is poorly studied. In the present study, vaccinated and nonvaccinated pigs were challenged by direct contact with H1N1 and H3N2 independent swine IAVs seeder pigs. Nasal swab samples were daily recovered and broncho-alveolar lavage fluid (BALF) was also collected at necropsy day from each pig for swine IAV detection and whole genome sequencing. In total, 39 swine IAV whole genome sequences were obtained by next generation sequencing from samples collected from both experimental groups. Subsequently, genomic, and evolutionary analyses were carried out to detect both, genomic reassortments and single nucleotide variants (SNV). Regarding the segments found per sample, the simultaneous presence of segments from both subtypes was much lower in vaccinated animals, indicating that the vaccine reduced the likelihood of genomic reassortment events. In relation to swine IAV intra-host diversity, a total of 239 and 74 SNV were detected within H1N1 and H3N2 subtypes, respectively. Different proportions of synonymous and nonsynonymous substitutions were found, indicating that vaccine may be influencing the main mechanism that shape swine IAV evolution, detecting natural, neutral, and purifying selection in the different analyzed scenarios. SNV were detected along the whole swine IAV genome with important nonsynonymous substitutions on polymerases, surface glycoproteins and nonstructural proteins, which may have an impact on virus replication, immune system escaping and virulence of virus, respectively. The present study further emphasized the vast evolutionary capacity of swine IAV, under natural infection and vaccination pressure scenarios.

PB1-F2 protein, the 11th influenza A virus (IAV) protein, is considered to play an important role in primary influenza virus infection and postinfluenza secondary bacterial pneumonia in mice. The functional role of PB1-F2 has been reported to be a strain-specific and host-specific phenomenon. Its precise contribution to the pathogenicity and transmission of influenza virus in mammalian host, such as swine, and avian hosts, such as turkeys, remain largely unknown. In this study, we explored the role of PB1-F2 protein of triple-reassortant (TR) H3N2 swine influenza virus (SIV) in pigs and turkeys. Using the eight-plasmid reverse genetics system, we rescued wild-type SIV A/swine/Minnesota/1145/2007 (H3N2) (SIV 1145-WT), a PB1-F2 knockout mutant (SIV 1145-KO), and its N66S variant (SIV 1145-N66S). The ablation of PB1-F2 in SIV 1145 modulated early-stage apoptosis but did not affect the viral replication in swine alveolar macrophage cells. In pigs, PB1-F2 expression did not affect nasal shedding, lung viral load, immunophenotypes, and lung pathology. On the other hand, in turkeys, SIV 1145-KO infected poults, and its in-contacts developed clinical signs earlier than SIV 1145-WT groups and also displayed more extensive histopathological changes in intestine. Further, turkeys infected with SIV 1145-N66S displayed poor infectivity and transmissibility. The more extensive histopathologic changes in intestine and relative transmission advantage observed in turkeys infected with SIV 1145-KO need to be further explored. Taken together, these results emphasize the host-specific roles of PB1-F2 in the pathogenicity and transmission of IAV. Novel triple-reassortant H3N2 swine influenza virus emerged in 1998 and spread rapidly among the North American swine population. Subsequently, it showed an increased propensity to reassort, generating a range of reassortants. Unlike classical swine influenza virus, TR SIV produces a full-length PB1-F2 protein, which is considered an important virulence marker of IAV pathogenicity. Our study demonstrated that the expression of PB1-F2 does not impact the pathogenicity of TR H3N2 SIV in pigs. On the other hand, deletion of PB1-F2 caused TR H3N2 SIV to induce clinical disease early and resulted in effective transmission among the turkey poults. Our study emphasizes the continuing need to better understand the virulence determinants for IAV in intermediate hosts, such as swine and turkeys, and highlights the host-specific role of PB1-F2 protein.

Influenza A viruses (IAVs) have both zoonotic and anthroponotic potential and are of public and veterinary importance. Swine are intermediate hosts and 'mixing vessels' for generating reassortants, progenies of which may harbour pandemic propensity. Swine handlers are at the highest risk of becoming infected with IAVs from swine but there is little information on the ecology of IAVs at the human-animal interface in Africa. We analysed and characterized nasal and throat swabs from swine and farmers respectively, for IAVs using RT-qPCR, from swine farms in the Ashanti region, Ghana. Sera were also analysed for IAVs antibodies and serotyped using ELISA and HI assays. IAV was detected in 1.4% (n=17/1,200) and 2.0% (n=2/99) of swine and farmers samples, respectively. Viral subtypes H3N2 and H1N1pdm09 were found in human samples. All virus-positive swine samples were subtyped as H1N1pdm09 phylogenetically clustering closely with H1N1pdm09 that circulated among humans during the study period. Phenotypic markers that confer sensitivity to Oseltamivir were found. Serological prevalence of IAVs in swine and farmers by ELISA was 3.2% (n=38/1,200) and 18.2% (n=18/99), respectively. Human H1N1pdm09 and H3N2 antibodies were found in both swine and farmers sera. Indigenous swine influenza A viruses and/or antibodies were not detected in swine or farmers samples. Majority (98%, n=147/150) of farmers reported of not wearing surgical mask and few (4%, n=6) reported to wear gloves when working. Most (n=74, 87.7%) farmers reported of working on the farm when experiencing influenza-like illness. Poor husbandry and biosafety practices of farmers could facilitate virus transmission across the human-swine interface. Farmers should be educated on the importance of good farm practices to mitigate influenza transmission at the human-animal interface.

During the last decade, endemic swine H1 influenza A viruses (IAV) from six different genetic clades of the hemagglutinin gene caused zoonotic infections in humans. The majority of zoonotic events with swine IAV were restricted to a single case with no subsequent transmission. However, repeated introduction of human-seasonal H1N1, continual reassortment between endemic swine IAV, and subsequent drift in the swine host resulted in highly diverse swine IAV with human-origin genes that may become a risk to the human population. To prepare for the potential of a future swine-origin IAV pandemic in humans, public health laboratories selected candidate vaccine viruses (CVV) for use as vaccine seed strains. To assess the pandemic risk of contemporary US swine H1N1 or H1N2 strains, we quantified the genetic diversity of swine H1 HA genes, and identified representative strains from each circulating clade. We then characterized the representative swine IAV against human seasonal vaccine and CVV strains using ferret antisera in hemagglutination inhibition assays (HI). HI assays revealed that 1A.3.3.2 (pdm09) and 1B.2.1 (delta-2) demonstrated strong cross reactivity to human seasonal vaccines or CVVs. However, swine IAV from three clades that represent more than 50% of the detected swine IAVs in the USA showed significant reduction in cross-reactivity compared to the closest CVV virus: 1A.1.1.3 (alpha-deletion), 1A.3.3.3-clade 3 (gamma), and 1B.2.2.1 (delta-1a). Representative viruses from these three clades were further characterized in a pig-to-ferret transmission model and shown to exhibit variable transmission efficiency. Our data prioritize specific genotypes of swine H1N1 and H1N2 to further investigate in the risk they pose to the human population.

In this comprehensive large-scale study, conducted from 2015 to 2019, 7,209 wild boars across South Korea were sampled to assess their exposure to influenza A viruses (IAVs). Of these, 250 (3.5%) were found to be IAV-positive by ELISA, and 150 (2.1%) by the hemagglutination inhibition test. Detected subtypes included 23 cases of pandemic 2009 H1N1, six of human seasonal H3N2, three of classical swine H1N1, 13 of triple-reassortant swine H1N2, seven of triple-reassortant swine H3N2, and seven of swine-origin H3N2 variant. Notably, none of the serum samples tested positive for avian IAV subtypes H3N8, H5N3, H7N7, and H9N2 or canine IAV subtype H3N2. This serologic analysis confirmed the exposure of Korean wild boars to various subtypes of swine and human influenza viruses, with some serum samples cross-reacting between swine and human strains, indicating potential infections with multiple IAVs. The results highlight the potential of wild boar as a novel mixing vessel, facilitating the adaptation of IAVs and their spillover to other hosts, including humans. In light of these findings, we recommend regular and frequent surveillance of circulating influenza viruses in the wild boar population as a proactive measure to prevent potential human influenza pandemics and wild boar influenza epizootics.