Influenza A virus (IAV) circulates widely in European pig populations and continues to diversify through frequent introductions from humans, followed by reassortment within swine. Spain represents a particularly dynamic ecological setting due to the coexistence of intensive white pig production, extensive Iberian pig systems, and abundant wild boar populations. This study provides an integrated analysis of IAV evolution and genomic diversity in swine in Spain between 2019 and 2022, expanding on previous surveillance from 2016 to 2019. Sampling across 24 provinces yielded 66 new whole genome sequences from Iberian and white pigs. We identified 18 genotypes, including 11 novel reassortants not detected in our previous survey. Several genotypes, such as H1huN2 G21 and G22, H3N2 G23, and the unusual H3N1 G12, were exclusive to the country. Some genotypes were detected across white pigs, Iberian pigs, and wild boar in Toledo and Badajoz, suggesting viral flow among swine populations. Phylogenetic analyses revealed ongoing introductions of H1N1pdm09 from humans into pigs, generating at least five reassortant genotypes (G10, G16-G19). These lineages incorporated pandemic internal cassettes and, in some cases, human seasonal N2 segments, highlighting the continued role of humans as a source of viral incursions. Conversely, four zoonotic infections (H1N1v) detected in Spain between 2022 and 2026 were linked to genotypes circulating in white pigs, underscoring the bidirectional nature of IAV transmission at the human swine interface. Overall, this study demonstrates that Spain provides ecological conditions conducive to IAV diversification, reassortment, and zoonotic risk. The findings reinforce the need for sustained One Health surveillance.
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Zoonotic infections and genomic evolution of novel reassortant swine influenza A viruses in Spain
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Cross-reactivity between antibodies induced by European swine influenza vaccines and Spanish swine influenza strains
BACKGROUND: Vaccination is an essential tool for controlling the severity of influenza in swine and its spillover risk to humans. Due to fast mutation rates, genomic reassortment, and the high antigenic variability of influenza A viruses (IAV), it is necessary to update vaccine strains to better match and neutralize circulating viruses.
METHODS: In this study, we analyzed the immune cross-reactivity of hemagglutination inhibition (HI) antibodies induced by currently approved European swine influenza vaccines against Spanish swine influenza viruses (SIVs) isolated from 2016 to 2021, as well as human IAV strains, using a murine model. Sera from immunized mice with monovalent (MVV) or trivalent (TVV) swine influenza vaccines were tested against Spanish representative SIVs carrying HAs from different subtypes and lineages using a HI serological assay. Amino acids in the antigenic motifs of the receptor binding site (RBS) of the IAV HA were compared and contrasted among SIV strains to detect changes that may affect protein antigenicity.
RESULTS: Sera from mice immunized with MVV or TVV showed no HI antibodies against a 2019 Eurasian avian-like H1 (EAswH1) SIV. TVV did not induce cross-reactive HI antibodies against two human seasonal H3 SIV from the 2000s (2000s-like H3) or against the human seasonal H3 vaccine strain (HuVacH3). Geometric mean (GM) HI titers of sera from TVV-immunized mice were below the protection threshold (GM < 40) against the recent human seasonal-like H1 (HUswH1) SIV, against an EAswH1 SIV, and against the human seasonal H1 vaccine strain (HuVacH1). HI antibodies induced by MVV showed high cross-reactivity with a 2019 EAswH1 SIV isolate.
CONCLUSION: Currently authorized vaccines do not induce HI antibodies against some contemporary SIV circulating in Spain, nor against human seasonal influenza vaccine strains. Updating vaccine strains to better match new SIVs emerging in Spanish swine is warranted.
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Seasonal Influenza Vaccine 2021/2022 Provides Limited Cross Reactivity Against Contemporary Swine Influenza A Virus Strains in Spain
Swine influenza A viruses (SIVs) pose a zoonotic risk, with variants detected in humans in Europe. This study evaluated the efficacy of the 2021/2022 seasonal influenza vaccine against SIVs. Forty-six postvaccination human sera were tested via hemagglutination inhibition (HI) assay against Spanish SIV genotypes. Seroprotection rate (SPR, HI titer ≥ 40) was 76% (95% CI: 64-88) and 91% (95% CI: 83-99) for human vaccine strains H1 and H3, respectively. SPRs were 67% (95% CI: 53-81) for pandemic swine H1, 64% (95% CI: 50-78) for human seasonal-like H1, 17%-46% for Eurasian avian-like H1, 15% (95% CI: 5-25) for human seasonal-like H3 from the 1970s, and 83%-93% for human seasonal-like H3 from the 2000s SIVs.
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SARS-CoV-2 Neutralizing Antibodies in Free-Ranging Fallow Deer (Dama dama) and Red Deer (Cervus elaphus) in Suburban and Rural Areas in Spain
Effective control of zoonotic infectious diseases requires identifying the animal species involved in the infectious cycle as transmitters or reservoirs where the pathogen could persist and evolve, increasing the risk of reintroduction of new variants in humans and animals. Multiple surveillance studies have detected the presence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) or specific antibodies in free-ranging white-tailed deer (Odocoileus virginianus) in North America, being the wild mammal species with the most reported cases of SARS-CoV-2 infection. However, so far, all attempts to detect the infection in European cervids have been unsuccessful. In this study, we demonstrated the presence of specific antibodies against SARS-CoV-2 in European fallow deer (Dama dama) and red deer (Cervus elaphus) in Spain. However, all samples of European roe deer (Capreolus capreolus), taxonomically related to the American white-tailed deer, were seronegative for the virus. We tested 215 serum samples from roe deer (n = 116), red deer (n = 63), and fallow deer (n = 36) collected in eight provinces of central-southern Spain between 2017 and 2022. We first screened sera by a surrogate virus neutralization test (sVNT) ELISA based on the binding of the receptor-binding domain of SARS-CoV-2 to the angiotensin-converting enzyme 2 (ACE2) receptor and then confirmed positive samples by a conventional virus neutralization test (cVNT) against the Alpha, Delta, and Omicron variants. Four fallow deer and two red deer samples were seropositive in both neutralization assays. Four samples of fallow deer and one of red deer, collected in a suburban park near Madrid in February 2022, had antibodies against the Alpha, Delta, and Omicron variants, while a seropositive sample of red deer, collected in a rural area in February 2021, was specific for the Delta variant. All samples collected before the start of the pandemic in Spain were seronegative for sVNT, which also indicates that there are not cross-reactive antibodies potentially elicited by other viruses antigenically related to SARS-CoV-2. The results indicate probable human-to-deer transmission of SARS-CoV-2, but do not clarify whether there was subsequent spread within herds.
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Evolution of influenza A virus in intensive and free-range swine farms in Spain
Swine harbor genetically diverse influenza A viruses (IAVs) with the capacity to host-switch to humans, causing global pandemics. Spain is the largest swine producer in Europe and has a mixed production system that includes ‘white coat’ pigs raised intensively in modern buildings and free-range Iberian pigs that interface differently with humans, wildlife, and other swine. Through active longitudinal IAV surveillance in nine Spanish provinces during 2015-9, we generated forty-seven complete or near-complete genome sequences from IAVs collected from swine in both systems. Genetically diverse IAVs were identified in intensively raised white pigs and free-range Iberian pigs, including new H3N1 reassortants. Both systems are dynamic environments for IAV evolution, but driven by different processes. IAVs in white pigs were genetically related to viruses found in swine raised intensively in other European countries, reflecting high rates of viral introduction following European trade routes. In contrast, IAVs in Iberian pigs have a genetic makeup shaped by frequent introductions of human IAVs, reflecting rearing practices with high rates of human contact. Transmission between white and Iberian pigs also occurred. In conclusion, Iberian swine with high rates of human contact harbor genetically diverse IAVs and potentially serve as intermediary hosts between white pigs and humans, presenting an understudied zoonotic risk that requires further investigation.