Seroprevalence of Bovine Viral Diarrhea Virus in Wild Pigs (Sus scrofa) in 17 States in the USA.

Wild pigs (Sus scrofa) are among the most detrimental invasive species in the USA. They are damaging to crops and agriculture, pose a public health risk as reservoirs of zoonotic pathogens, and may also spread disease to livestock. One pathogen identified in wild pigs is bovine viral diarrhea virus (BVDV), a virus that causes an economically important disease of cattle (Bos taurus and Bos indicus). We sought to determine the BVDV seroprevalence in wild pigs in 17 states across the US and to determine whether age category, sex, or location were associated with a positive antibody titer. Serum samples from 945 wild pigs were collected from 17 US states. Virus neutralization assays were performed to determine antibody titers against BVDV-1b and BVDV-2a. Total BVDV seroprevalence for the study area was 5.8% (95% confidence interval [CI], 4.11-8.89). Seroprevalence across all evaluated states was determined to be 4.4% (95% CI, 2.48-6.82) for BVDV-1b and 3.6% (95% CI, 1.54-5.60) for BVDV-2a. The seroprevalence for individual states varied from 0% to 16.7%. There was no statistical difference in median antibody titer for BVDV-1b or BVDV-2a by sex or age category. State seroprevalences for both BVDV-1b and BVDV-2a were associated with wild pig population estimates for those states.

Pandemic Risk Assessment for Swine Influenza A Virus in Comparative In Vitro and In Vivo Models.

Swine influenza A viruses pose a public health concern as novel and circulating strains occasionally spill over into human hosts, with the potential to cause disease. Crucial to preempting these events is the use of a threat assessment framework for human populations. However, established guidelines do not specify which animal models or in vitro substrates should be used. We completed an assessment of a contemporary swine influenza isolate, A/swine/GA/A27480/2019 (H1N2), using animal models and human cell substrates. Infection studies in vivo revealed high replicative ability and a pathogenic phenotype in the swine host, with replication corresponding to a complementary study performed in swine primary respiratory epithelial cells. However, replication was limited in human primary cell substrates. This contrasted with our findings in the Calu-3 cell line, which demonstrated a replication profile on par with the 2009 pandemic H1N1 virus. These data suggest that the selection of models is important for meaningful risk assessment.

Establishment of Swine Primary Nasal, Tracheal, and Bronchial Epithelial Cell Culture Models for the Study of Influenza Virus Infection.

We established primary porcine nasal, tracheal, and bronchial epithelial cells that recapitulate the physical and functional properties of the respiratory tract and have the ability to fully differentiate. Trans-well cultures demonstrated increased transepithelial electrical resistance over time the presence of tight junctions as demonstrated by immunohistochemistry. The nasal, tracheal, and bronchial epithelial cells developed cilia, secreted mucus, and expressed sialic acids on surface glycoproteins, the latter which are required for influenza A virus infection. Swine influenza viruses were shown to replicate efficiently in the primary epithelial cell cultures, supporting the use of these culture models to assess swine influenza and other virus infection. Primary porcine nasal, tracheal, and bronchial epithelial cell culture models enable assessment of emerging and novel influenza viruses for pandemic potential as well as mechanistic studies to understand mechanisms of infection, reassortment, and generation of novel virus. As swine are susceptible to infection with multiple viral and bacterial respiratory pathogens, these primary airway cell models may enable study of the cellular response to infection by pathogens associated with Porcine Respiratory Disease Complex.

Swine influenza A virus isolates containing the pandemic H1N1 origin matrix gene elicit greater disease in the murine model.

Since the 1990s, endemic North American swine influenza A viruses (swFLUAVs) contained an internal gene segment constellation, the triple reassortment internal gene (TRIG) cassette. In 2009, the H1N1 pandemic (pdmH1N1) virus spilled back into swine but did not become endemic. However, the pdmH1N1 contributed the matrix gene (pdmM) to the swFLUAVs circulating in the pig population, which replaced the classical swine matrix gene (swM) found in the TRIG cassette, suggesting the pdmM has a fitness benefit. Others have shown that swFLUAVs containing the pdmM have greater transmission efficiency compared to viruses containing the swM gene segment. We hypothesized that the matrix (M) gene could also affect disease and utilized two infection models, resistant BALB/c and susceptible DBA/2 mice, to assess pathogenicity. We infected BALB/c and DBA/2 mice with H1 and H3 swFLUAVs containing the swM or pdmM and measured lung virus titers, morbidity, mortality, and lung histopathology. H1 influenza strains containing the pdmM gene caused greater morbidity and mortality in resistant and susceptible murine strains, while H3 swFLUAVs caused no clinical disease. However, both H1 and H3 swFLUAVs containing the pdmM replicated to higher viral titers in the lungs and pdmM containing H1 viruses induced greater histological changes compared to swM H1 viruses. While the surface glycoproteins and other gene segments may contribute to swFLUAV pathogenicity in mice, these data suggest that the origin of the matrix gene also contributes to pathogenicity of swFLUAV in mice, although we must be cautious in translating these conclusions to their natural host, swine. IMPORTANCE: The 2009 pandemic H1N1 virus rapidly spilled back into North American swine, reassorting with the already genetically diverse swFLUAVs. Notably, the M gene segment quickly replaced the classical M gene segment, suggesting a fitness benefit. Here, using two murine models of infection, we demonstrate that swFLUAV isolates containing the pandemic H1N1 origin M gene caused increased disease compared to isolates containing the classical swine M gene. These results suggest that, in addition to other influenza virus gene segments, the swFLUAV M gene segment contributes to pathogenesis in mammals.

Heterologous prime-boost H1N1 vaccination exacerbates disease following challenge with a mismatched H1N2 influenza virus in the swine model.

Influenza A viruses (IAVs) pose a significant threat to both human and animal health. Developing IAV vaccine strategies able to elicit broad heterologous protection against antigenically diverse IAV strains is pivotal in effectively controlling the disease. The goal of this study was to examine the immunogenicity and protective efficacy of diverse H1N1 influenza vaccine strategies including monovalent, bivalent, and heterologous prime-boost vaccination regimens, against a mismatched H1N2 swine influenza virus. Five groups were homologous prime-boost vaccinated with either an oil-adjuvanted whole-inactivated virus (WIV) monovalent A/swine/Georgia/27480/2019 (GA19) H1N2 vaccine, a WIV monovalent A/sw/Minnesota/A02636116/2021 (MN21) H1N1 vaccine, a WIV monovalent A/California/07/2009 (CA09) H1N1, a WIV bivalent vaccine composed of CA09 and MN21, or adjuvant only (mock-vaccinated group). A sixth group was prime-vaccinated with CA09 WIV and boosted with MN21 WIV (heterologous prime-boost group). Four weeks post-boost pigs were intranasally and intratracheally challenged with A/swine/Georgia/27480/2019, an H1N2 swine IAV field isolate. Vaccine-induced protection was evaluated based on five critical parameters: (i) hemagglutination inhibiting (HAI) antibody responses, (ii) clinical scores, (iii) virus titers in nasal swabs and respiratory tissue homogenates, (iv) BALf cytology, and (v) pulmonary pathology. While all vaccination regimens induced seroprotective titers against homologous viruses, heterologous prime-boost vaccination failed to enhance HAI responses against the homologous vaccine strains compared to monovalent vaccine regimens and did not expand the scope of cross-reactive antibody responses against antigenically distinct swine and human IAVs. Mismatched vaccination regimens not only failed to confer clinical and virological protection post-challenge but also exacerbated disease and pathology. In particular, heterologous-boosted pigs showed prolonged clinical disease and increased pulmonary pathology compared to mock-vaccinated pigs. Our results demonstrated that H1-specific heterologous prime-boost vaccination, rather than enhancing cross-protection, worsened the clinical outcome and pathology after challenge with the antigenically distant A/swine/Georgia/27480/2019 strain.

H1N1 G4 swine influenza T cell epitope analysis in swine and human vaccines and circulating strains uncovers potential risk to swine and humans.

BACKGROUND: Pandemic influenza viruses may emerge from animal reservoirs and spread among humans in the absence of cross-reactive antibodies in the human population. Immune response to highly conserved T cell epitopes in vaccines may still reduce morbidity and limit the spread of the new virus even when cross-protective antibody responses are lacking. METHODS: We used an established epitope content prediction and comparison tool, Epitope Content Comparison (EpiCC), to assess the potential for emergent H1N1 G4 swine influenza A virus (G4) to impact swine and human populations. We identified and computed the total cross-conserved T cell epitope content in HA sequences of human seasonal and experimental influenza vaccines, swine influenza vaccines from Europe and the United States (US) against G4. RESULTS: The overall T cell epitope content of US commercial swine vaccines was poorly conserved with G4, with an average T cell epitope coverage of 35.7%. EpiCC scores for the comparison between current human influenza vaccines and circulating human influenza strains were also very low. In contrast, the T cell epitope coverage of a recent European swine influenza vaccine (HL03) was 65.8% against G4. CONCLUSIONS: Poor T cell epitope cross-conservation between emergent G4 and swine and human influenza vaccines in the US may enable G4 to spread in swine and spillover to human populations in the absence of protective antibody response. One European influenza vaccine, HL03, may protect against emergent G4. This study illustrates the use of the EpiCC tool for prospective assessment of existing vaccine strains against emergent viruses in swine and human populations.

Development and characterization of an immortalized swine respiratory cell line for influenza A virus research.

Introduction: Swine serve as an important intermediate host species for generating novel influenza A viruses (IAVs) with pandemic potential because of the host's susceptibility to IAVs of swine, human and avian origin. Primary respiratory cell lines are used in IAV research to model the host's upper respiratory tract in vitro. However, primary cell lines are limited by their passaging capacity and are time-consuming for use in industry and research pipelines. We were interested in developing and characterizing a biologically relevant immortalized swine respiratory cell line that could be used for efficient propagation and characterization of swine IAV isolates. Methods: Lung tissue for the generation of primary swine respiratory cells were isolated from the bronchi of an 8-week-old Yorkshire/Hampshire pig, which were immortalized by transduction of the SV40 T antigen using a lentivirus vector. The transduction of the SV40 T antigen was confirmed by Real Time RT-PCR in cells passaged greater than twenty times. Results: Immortalized swine respiratory cells expressed primarily α2,6 sialic acid receptors and were susceptible to both swine and human IAVs, with swine viruses exhibiting higher replication rates. Notably, infection with a swine H3N2 isolate prompted increased IL-6 and IL-1α protein secretion compared to a seasonal human H3N2 virus. Even after 20 passages, the immortalized cells maintained the primary respiratory cell phenotype and remained permissive to IAV infection without exogenous trypsin. Discussion: In summary, our developed immortalized swine respiratory cell line offers an alternative in vitro substrate for studying IAV replication and transmission dynamics in pigs, overcoming the limitations of primary respiratory cells in terms of low passage survivability and cost.

A Novel Neuraminidase Virus-Like Particle Vaccine Offers Protection Against Heterologous H3N2 Influenza Virus Infection in the Porcine Model.

Influenza A viruses (IAVs) pose a global health threat, contributing to hundreds of thousands of deaths and millions of hospitalizations annually. The two major surface glycoproteins of IAVs, hemagglutinin (HA) and neuraminidase (NA), are important antigens in eliciting neutralizing antibodies and protection against disease. However, NA is generally ignored in the formulation and development of influenza vaccines. In this study, we evaluate the immunogenicity and efficacy against challenge of a novel NA virus-like particles (VLPs) vaccine in the porcine model. We developed an NA2 VLP vaccine containing the NA protein from A/Perth/16/2009 (H3N2) and the matrix 1 (M1) protein from A/MI/73/2015, formulated with a water-in-oil-in-water adjuvant. Responses to NA2 VLPs were compared to a commercial adjuvanted quadrivalent whole inactivated virus (QWIV) swine IAV vaccine. Animals were prime boost vaccinated 21 days apart and challenged four weeks later with an H3N2 swine IAV field isolate, A/swine/NC/KH1552516/2016. Pigs vaccinated with the commercial QWIV vaccine demonstrated high hemagglutination inhibition (HAI) titers but very weak anti-NA antibody titers and subsequently undetectable NA inhibition (NAI) titers. Conversely, NA2 VLP vaccinated pigs demonstrated undetectable HAI titers but high anti-NA antibody titers and NAI titers. Post-challenge, NA2 VLPs and the commercial QWIV vaccine showed similar reductions in virus replication, pulmonary neutrophilic infiltration, and lung inflammation compared to unvaccinated controls. These data suggest that anti-NA immunity following NA2 VLP vaccination offers comparable protection to QWIV swine IAV vaccines inducing primarily anti-HA responses.

Bivalent vaccination with NA1 and NA2 neuraminidase virus-like particles is protective against challenge with H1N1 and H3N2 influenza A viruses in a murine model.

Neuraminidase (NA) is the second most abundant glycoprotein on the surface of influenza A viruses (IAV). Neuraminidase type 1 (NA1) based virus-like particles (VLPs) have previously been shown to protect against challenge with H1N1 and H3N2 IAV. In this study, we produced neuraminidase type 2 (NA2) VLPs derived from the sequence of the seasonal IAV A/Perth/16/2009. Intramuscular vaccination of mice with NA2 VLPs induced high anti-NA serum IgG levels capable of inhibiting NA activity. NA2 VLP vaccination protected against mortality in a lethal A/Hong Kong/1/1968 (H3N2) virus challenge model, but not against lethal challenge with A/California/04/2009 (H1N1) virus. However, bivalent vaccination with NA1 and NA2 VLPs demonstrated no antigenic competition in anti-NA IgG responses and protected against lethal challenge with H1N1 and H3N2 viruses. Here we demonstrate that vaccination with NA VLPs is protective against influenza challenge and supports focusing on anti-NA responses in the development of future vaccination strategies.

Novel Vaccine Technologies in Veterinary Medicine: A Herald to Human Medicine Vaccines.

The success of inactivated and live-attenuated vaccines has enhanced livestock productivity, promoted food security, and attenuated the morbidity and mortality of several human, animal, and zoonotic diseases. However, these traditional vaccine technologies are not without fault. The efficacy of inactivated vaccines can be suboptimal with particular pathogens and safety concerns arise with live-attenuated vaccines. Additionally, the rate of emerging infectious diseases continues to increase and with that the need to quickly deploy new vaccines. Unfortunately, first generation vaccines are not conducive to such urgencies. Within the last three decades, veterinary medicine has spearheaded the advancement in novel vaccine development to circumvent several of the flaws associated with classical vaccines. These third generation vaccines, including DNA, RNA and recombinant viral-vector vaccines, induce both humoral and cellular immune response, are economically manufactured, safe to use, and can be utilized to differentiate infected from vaccinated animals. The present article offers a review of commercially available novel vaccine technologies currently utilized in companion animal, food animal, and wildlife disease control.

Peptide ELISA and FRET-qPCR Identified a Significantly Higher Prevalence of Chlamydia suis in Domestic Pigs Than in Feral Swine from the State of Alabama, USA.

Chlamydia suis is an important, highly prevalent, and diverse obligate intracellular pathogen infecting pigs. In order to investigate the prevalence and diversity of C. suis in the U.S., 276 whole blood samples from feral swine were collected as well as 109 fecal swabs and 60 whole blood samples from domestic pigs. C. suis-specific peptide ELISA identified anti-C. suis antibodies in 13.0% of the blood of feral swine (26/276) and 80.0% of the domestic pigs (48/60). FRET-qPCR and DNA sequencing found C. suis DNA in 99.1% of the fecal swabs (108/109) and 21.7% of the whole blood (13/60) of the domestic pigs, but not in any of the assayed blood samples (0/267) in feral swine. Phylogenetic comparison of partial C. suis ompA gene sequences and C. suis-specific multilocus sequencing typing (MLST) revealed significant genetic diversity of the C. suis identified in this study. Highly genetically diverse C. suis strains are prevalent in domestic pigs in the USA. As crowding strongly enhances the frequency and intensity of highly prevalent Chlamydia infections in animals, less population density in feral swine than in domestic pigs may explain the significantly lower C. suis prevalence in feral swine. A future study is warranted to obtain C. suis DNA from feral swine to perform genetic diversity of C. suis between commercial and feral pigs.

Molecular epidemiology and glycomics of swine influenza viruses circulating in commercial swine farms in the southeastern and midwest United States.

Tracking the genetic diversity and spread of swine influenza viruses (SIVs) in commercial swine farms is central for control and to reduce the potential emergence of SIV reassortants. We analyzed the diversity of SIVs in nasal washes or oral fluids from commercial swine farms in North Carolina using influenza M qRT-PCR and hemagglutinin (HA) and neuraminidase (NA) subtyping. We found a predominance of H1 HAs and N2 NAs in the samples examined. The majority of the H1 HAs could be further classified into gamma and delta subclusters. We also identified HAs of the H1 alpha cluster, and those of human novel pandemic origin. Glycan binding profiles from a representative subset of these viruses revealed broad α2,6 sialylated glycan recognition, though some strains exhibited the ability to bind to α2,3 sialic acid. These data show that SIV surveillance can aid our understanding of viral transmission dynamics and help uncover the diversity at the human-swine interface.

Evaluation of a Direct Immunofluorescent Assay and/or Conjunctival Cytology for Detection of Canine Distemper Virus Antigen.

Canine distemper is a common and potentially lethal multisystemic disease caused by the Canine distemper virus (CDV). We evaluated the diagnostic performance of direct immunofluorescent assay (FA) and cytology to detect CDV antigen in conjunctival cells compared with an established polymerase chain reaction (PCR) detection assay used as a gold standard for CDV diagnosis. Samples were collected from 57 young dogs presenting with central nervous system signs compatible with distemper disease. Exfoliative epithelial cells were collected from the right and left conjunctiva of each animal using nylon-bristled cytobrushes for cytology and cotton swabs for FA and PCR. For the direct FA, samples were stained with anti-CDV polyclonal antiserum conjugated to fluorescein isothiocyanate and imaged using a fluorescent microscope. Out of 57 dogs tested, 19 were PCR positive (15 positive in direct FA and 4 positive in cytology, including one that was negative by PCR), whereas 37 dogs were negative in all methods. A good agreement was observed between the FA and PCR, with a κ-value of 0.833 (95% CI: 0.678-0.989). Meanwhile, there was poor agreement between cytology and PCR (κ-value of 0.164; 95% CI: -0.045 to 0.373) and a fair agreement between FA and cytology (κ-value of 0.231; 95% CI: -0.026 to 0.487). Our results indicated a poor performance of cytology for the detection of CDV antigen. In contrast, FA is a 100% specific and an adequately sensitive assay (sensitivity: 78.95%, negative likelihood ratio: 0.21, 95% CI: 0.09-0.50) for antemortem diagnosis of canine distemper.

Molecular epidemiology of swine influenza A viruses in the Southeastern United States, highlights regional differences in circulating strains.

Swine influenza A virus (IAV) can cause widespread respiratory disease with high morbidity, low mortality, and have a substantial economic impact to the swine industry. Swine infection may contribute to pandemic IAV given their susceptibility to both avian and human IAVs. Currently, three IAV subtypes (H1N1, H3N2 and H1N2) circulate in swine in North America frequently combining gene segments from avian or human viruses. This study investigated the prevalence of IAV in commercial swine herds. A total of 1878 oral fluid samples were collected from pigs of all ages from 201 commercial farms located in North Carolina and South Carolina. Sixty-eight oral fluid samples from 35 farms were positive by MP gene PCR with an overall IAV-positivity of 3.6%. On the herd level, the percentage of IAV positivity was 17.4%. Fifty-six viruses were subtyped, while 12 were partly subtyped or not subtyped at all. Using de novo assembly, complete sequences were obtained for 59 HA genes. The majority of IAVs subtyped had an H1 HA demonstrating a considerable prevalence over H3 viruses. Furthermore, only six out of eleven HA types were detected which has implications for the selection of vaccines used by swine producers in the region.

Influenza D in Italy: towards a better understanding of an emerging viral infection in swine.

Influenza D virus (IDV), a new member of the Orthomyxoviridae family, was first reported in 2011 in swine in Oklahoma, and consequently found in cattle across North America and Eurasia. To investigate the circulation of IDV among pigs in Italy, in the period between June 2015 and May 2016, biomolecular and virological tests were performed on 845 clinical samples collected from 448 pig farms affected by respiratory distress located in the Po Valley. Serological tests were conducted on 3698 swine sera, including archive sera collected in 2009, as well as samples collected in 2015 from the same region. Viral genome was detected in 21 (2.3%) samples from 9 herds (2%), while virus was successfully isolated from 3 samples. Genetic analysis highlighted that Italian swine IDVs are closely related to the D/swine/Oklahoma/1334/2011 cluster. Sera collected in 2015 showed a high prevalence of IDV antibody titers (11.7%), while archive sera from 2009 showed statistically significant lower positivity rates (0.6%). Our results indicate an increasing epidemiological relevance of the pathogen and the need for in-depth investigations towards understanding its pathogenesis, epidemiology and possible zoonotic potential of this emerging virus.

MicroRNA-555 has potent antiviral properties against poliovirus.

Vaccination with live-attenuated polio vaccine has been the primary reason for the drastic reduction of poliomyelitis worldwide. However, reversion of this attenuated poliovirus vaccine occasionally results in the emergence of vaccine-derived polioviruses that may cause poliomyelitis. Thus, the development of anti-poliovirus agents remains a priority for control and eradication of the disease. MicroRNAs (miRNAs) have been shown to regulate viral infection through targeting the viral genome or reducing host factors required for virus replication. However, the roles of miRNAs in poliovirus (PV) replication have not been fully elucidated. In this study, a library of 1200 miRNA mimics was used to identify miRNAs that govern PV replication. High-throughput screening revealed 29 miRNAs with antiviral properties against Sabin-2, which is one of the oral polio vaccine strains. In particular, miR-555 was found to have the most potent antiviral activity against three different oral polio attenuated vaccine strains tested. The results show that miR-555 reduced the level of heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNP C) required for PV replication in the infected cells, which in turn resulted in reduction of PV positive-strand RNA synthesis and production of infectious progeny. These findings provide the first evidence for the role of miR-555 in PV replication and reveal that miR-555 could contribute to the development of antiviral therapeutic strategies against PV.

Diagnostic performance of a rapid in-clinic test for the detection of Canine Parvovirus under different storage conditions and vaccination status.

Canine parvovirus (CPV) is one of the most common causes of acute haemorrhagic enteritis in young dogs, while clinical diagnosis is often indecisive. The aim of our study was to evaluate the diagnostic accuracy of an in-clinic rapid test in the detection of CPV infection in dogs. To this end, we compared the Rapid Diagnostic Kit of Canine Parvovirus, Coronavirus and Rotavirus antigen (Quicking(®)) to PCR, which is considered as the most reliable diagnostic method. A total of 78 duplicated faecal samples were collected from diarrhoeic dogs. Vaccination history within a month prior to the onset of diarrhoea was reported for 12 of the sampled dogs. The rapid diagnostic test was performed in 23 of the faecal samples directly, while the rest were placed into a sterile cotton tipped swab suitable for collection and transportation of viruses (Sigma Σ-VCM(®)) and stored at -20 °C. The sensitivity of the Quicking rapid diagnostic test compared to PCR in the total number of samples, in samples from non-vaccinated dogs and in samples tested directly after collection were 22.22% (95% CI: 13.27-33.57%), 26.67% (95% CI: 16.08-39.66%) and 76.47% (95% CI: 50.10-93.04%) respectively, while the specificity of the test was 100% in any case. In conclusion, negative results do not exclude parvoenteritis from the differential diagnosis, especially in dogs with early vaccination history, but a positive result almost certainly indicates CPV infection. An improved sensitivity may be expected when the test is performed immediately.

European surveillance network for influenza in pigs: surveillance programs, diagnostic tools and Swine influenza virus subtypes identified in 14 European countries from 2010 to 2013.

Swine influenza causes concern for global veterinary and public health officials. In continuing two previous networks that initiated the surveillance of swine influenza viruses (SIVs) circulating in European pigs between 2001 and 2008, a third European Surveillance Network for Influenza in Pigs (ESNIP3, 2010-2013) aimed to expand widely the knowledge of the epidemiology of European SIVs. ESNIP3 stimulated programs of harmonized SIV surveillance in European countries and supported the coordination of appropriate diagnostic tools and subtyping methods. Thus, an extensive virological monitoring, mainly conducted through passive surveillance programs, resulted in the examination of more than 9 000 herds in 17 countries. Influenza A viruses were detected in 31% of herds examined from which 1887 viruses were preliminary characterized. The dominating subtypes were the three European enzootic SIVs: avian-like swine H1N1 (53.6%), human-like reassortant swine H1N2 (13%) and human-like reassortant swine H3N2 (9.1%), as well as pandemic A/H1N1 2009 (H1N1pdm) virus (10.3%). Viruses from these four lineages co-circulated in several countries but with very different relative levels of incidence. For instance, the H3N2 subtype was not detected at all in some geographic areas whereas it was still prevalent in other parts of Europe. Interestingly, H3N2-free areas were those that exhibited highest frequencies of circulating H1N2 viruses. H1N1pdm viruses were isolated at an increasing incidence in some countries from 2010 to 2013, indicating that this subtype has become established in the European pig population. Finally, 13.9% of the viruses represented reassortants between these four lineages, especially between previous enzootic SIVs and H1N1pdm. These novel viruses were detected at the same time in several countries, with increasing prevalence. Some of them might become established in pig herds, causing implications for zoonotic infections.