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We investigated the infection dynamics of 2 influenza A(H1N1) virus isolates from the swine 1A.3.3.2 (pandemic 2009) and 1C (Eurasian, avian-like) lineages. The 1C-lineage virus, A/Pavia/65/2016, although phylogenetically related to swine-origin viruses, was isolated from a human clinical case. This strain infected ferrets, a human influenza model species, and could be transmitted by direct contact and, less efficiently, by airborne exposure. Infecting ferrets and pigs (the natural host) resulted in mild or inapparent clinical signs comparable to those observed with 1A.3.3.2-lineage swine-origin viruses. Both H1N1 viruses could infect pigs and were transmitted to cohoused ferrets. Ferrets vaccinated with a human 2016-17 seasonal influenza vaccine were protected against infection with the antigenically matched 1A pandemic 2009 virus but not against the swine-lineage 1C virus. Our results reaffirm the need for continuous influenza A virus surveillance in pigs and identification of candidate human vaccine viruses.

Swine influenza A virus (swIAV) infection causes substantial economic loss and disease burden in humans and animals. The 2009 pandemic H1N1 (pH1N1) influenza A virus is now endemic in both populations. In this study, we evaluated the efficacy of different vaccines in reducing nasal shedding in pigs following pH1N1 virus challenge. We also assessed transmission from immunized and challenged pigs to naive, directly in-contact pigs. Pigs were immunized with either adjuvanted, whole inactivated virus (WIV) vaccines or virus-vectored (ChAdOx1 and MVA) vaccines expressing either the homologous or heterologous influenza A virus hemagglutinin (HA) glycoprotein, as well as an influenza virus pseudotype (S-FLU) vaccine expressing heterologous HA. Only two vaccines containing homologous HA, which also induced high hemagglutination inhibitory antibody titers, significantly reduced virus shedding in challenged animals. Nevertheless, virus transmission from challenged to naive, in-contact animals occurred in all groups, although it was delayed in groups of vaccinated animals with reduced virus shedding. IMPORTANCE This study was designed to determine whether vaccination of pigs with conventional WIV or virus-vectored vaccines reduces pH1N1 swine influenza A virus shedding following challenge and can prevent transmission to naive in-contact animals. Even when viral shedding was significantly reduced following challenge, infection was transmissible to susceptible cohoused recipients. This knowledge is important to inform disease surveillance and control strategies and to determine the vaccine coverage required in a population, thereby defining disease moderation or herd protection. WIV or virus-vectored vaccines homologous to the challenge strain significantly reduced virus shedding from directly infected pigs, but vaccination did not completely prevent transmission to cohoused naive pigs., (© Crown copyright 2021.)

As high-throughput sequencing technologies are becoming more widely adopted for analysing pathogens in disease outbreaks there needs to be assurance that the different sequencing technologies and approaches to data analysis will yield reliable and comparable results. Conversely, understanding where agreement cannot be achieved provides insight into the limitations of these approaches and also allows efforts to be focused on areas of the process that need improvement. This manuscript describes the next-generation sequencing of three closely related viruses, each analysed using different sequencing strategies, sequencing instruments and data processing pipelines. In order to determine the comparability of consensus sequences and minority (sub-consensus) single nucleotide variant (mSNV) identification, the biological samples, the sequence data from 3 sequencing platforms and the *.bam quality-trimmed alignment files of raw data of 3 influenza A/H5N8 viruses were shared. This analysis demonstrated that variation in the final result could be attributed to all stages in the process, but the most critical were the well-known homopolymer errors introduced by 454 sequencing, and the alignment processes in the different data processing pipelines which affected the consistency of mSNV detection. However, homopolymer errors aside, there was generally a good agreement between consensus sequences that were obtained for all combinations of sequencing platforms and data processing pipelines. Nevertheless, minority variant analysis will need a different level of careful standardization and awareness about the possible limitations, as shown in this study., Competing Interests: The authors have declared that no competing interests exist.

Swine influenza A virus (SwIV) infection has considerable economic and animal welfare consequences and, because of the zoonotic potential, can also have public health implications. The 2009 pandemic H1N1 'swine-origin' infection is now endemic in both pigs and humans. In Europe, avian-like H1 av N1, human-like H1 hu N2, human-like swine H3N2 and, since 2009, pandemic H1N1 (pH1N1) lineage viruses and reassortants, constitute the dominant subtypes. In this study, we used a swine pH1N1 challenge virus to investigate the efficacy of whole inactivated virus vaccines homologous or heterologous to the challenge virus as well as a commercial vaccine. We found that vaccine-mediated protection was most effective when vaccine antigen and challenge virus were homologous and correlated with the specific production of neutralising antibodies and a cellular response to the challenge virus. We conclude that a conventional whole inactivated SwIV vaccine must be antigenically matched to the challenge strain to be an effective control measure., (Copyright © 2019. Published by Elsevier Ltd.)

Influenza is a major health threat, and a broadly protective influenza vaccine would be a significant advance. Signal Minus FLU (S-FLU) is a candidate broadly protective influenza vaccine that is limited to a single cycle of replication, which induces a strong cross-reactive T cell response but a minimal Ab response to hemagglutinin after intranasal or aerosol administration. We tested whether an H3N2 S-FLU can protect pigs and ferrets from heterosubtypic H1N1 influenza challenge. Aerosol administration of S-FLU to pigs induced lung tissue-resident memory T cells and reduced lung pathology but not the viral load. In contrast, in ferrets, S-FLU reduced viral replication and aerosol transmission. Our data show that S-FLU has different protective efficacy in pigs and ferrets, and that in the absence of Ab, lung T cell immunity can reduce disease severity without reducing challenge viral replication., (Copyright © 2018 The Authors.)

Influenza virus infection in pigs is a major farming problem, causing considerable economic loss and posing a zoonotic threat. In addition the pig is an excellent model for understanding immunity to influenza viruses as this is a natural host pathogen system. Experimentally, influenza virus is delivered to pigs intra-nasally, by intra-tracheal instillation or by aerosol, but there is little data comparing the outcome of different methods. We evaluated the shedding pattern, cytokine responses in nasal swabs and immune responses following delivery of low or high dose swine influenza pdmH1N1 virus to the respiratory tract of pigs intra-nasally or by aerosol and compared them to those induced in naturally infected contact pigs. Our data shows that natural infection by contact induces remarkably high innate and adaptive immune response, although the animals were exposed to a very low virus dose. In contacts, the kinetics of virus shedding were slow and prolonged and more similar to the low dose directly infected animals. In contrast the cytokine profile in nasal swabs, antibody and cellular immune responses of contacts more closely resemble immune responses in high dose directly inoculated animals. Consideration of these differences is important for studies of disease pathogenesis and assessment of vaccine protective efficacy.

Influenza A viruses are a major health threat to livestock and humans, causing considerable mortality, morbidity, and economic loss. Current inactivated influenza vaccines are strain specific and new vaccines need to be produced at frequent intervals to combat newly arising influenza virus strains, so that a universal vaccine is highly desirable. We show that pandemic H1N1 influenza virus in which the hemagglutinin signal sequence has been suppressed (S-FLU), when administered to pigs by aerosol can induce CD4 and CD8 T cell immune responses in blood, bronchoalveolar lavage (BAL), and tracheobronchial lymph nodes. Neutralizing Ab was not produced. Detection of a BAL response correlated with a reduction in viral titer in nasal swabs and lungs, following challenge with H1N1 pandemic virus. Intratracheal immunization with a higher dose of a heterologous H5N1 S-FLU vaccine induced weaker BAL and stronger tracheobronchial lymph node responses and a lesser reduction in viral titer. We conclude that local cellular immune responses are important for protection against influenza A virus infection, that these can be most efficiently induced by aerosol immunization targeting the lower respiratory tract, and that S-FLU is a promising universal influenza vaccine candidate., (Copyright © 2016 The Authors.)

Swine influenza presents a substantial disease burden for pig populations worldwide and poses a potential pandemic threat to humans. There is considerable diversity in both H1 and H3 influenza viruses circulating in swine due to the frequent introductions of viruses from humans and birds coupled with geographic segregation of global swine populations. Much of this diversity is characterized genetically but the antigenic diversity of these viruses is poorly understood. Critically, the antigenic diversity shapes the risk profile of swine influenza viruses in terms of their epizootic and pandemic potential. Here, using the most comprehensive set of swine influenza virus antigenic data compiled to date, we quantify the antigenic diversity of swine influenza viruses on a multi-continental scale. The substantial antigenic diversity of recently circulating viruses in different parts of the world adds complexity to the risk profiles for the movement of swine and the potential for swine-derived infections in humans.

Natural killer (NK) cells are important players in the innate immune response against influenza A virus and the activating receptor NKp46, which binds hemagglutinin on the surface of infected cells, has been assigned a role in this context. As pigs are natural hosts for influenza A viruses and pigs possess both NKp46- and NKp46+ NK cells, they represent a good animal model for studying the role of the NKp46 receptor during influenza. We explored the role of NK cells in piglets experimentally infected with 2009 pandemic H1N1 influenza virus by flow cytometric analyses of cells isolated from blood and lung tissue and by immunostaining of lung tissue sections. The number of NKp46+ NK cells was reduced while NKp46- NK cells remained unaltered in the blood 1-3 days after infection. In the lungs, the intensity of NKp46 expression on NK cells was increased during the first 3 days, and areas where influenza virus nucleoprotein was detected were associated with increased numbers of NKp46+ NK cells when compared to uninfected areas. NKp46+ NK cells in the lung were neither found to be infected with influenza virus nor to be undergoing apoptosis. The binding of porcine NKp46 to influenza virus infected cells was verified in an in vitro assay. These data support the involvement of porcine NKp46+ NK cells in the local immune response against influenza virus.

Background: Pigs are thought to act as intermediate hosts in the ecology of influenza viruses of both avian and human origin. The recent development of procedures for pig ex vivo respiratory organ explants has provided new tools for the assessment of influenza virus infection in pigs., Objectives: To use pig ex vivo organ explants to assess the susceptibility of pigs to infection with contemporary viruses, for which there is evidence of human infection and that are thought to pose the greatest threat to pig and human populations., Methods: Pig tracheal, bronchi and lung ex vivo organ explants were infected with both highly pathogenic and low pathogenic avian influenza (AI) virus and the pandemic H1N1 [A(H1N1)pdm/09] virus. Successful infection of explants was detected using a positive-sense RNA real-time RT-PCR assay and anti-nucleoprotein immunohistochemistry. The distribution of cell-surface α2-3- and α2-6-linked sialic acid receptors, the avian- and mammalian influenza A virus-preferred host receptors, respectively, was also characterised for the ex vivo organ cultures and uninfected pig material following necropsy., Results: The α2-3 and α2-6 sialic acid receptor staining on tracheal, bronchi and lung organ explant sections showed similar distributions to those seen for pig tissue following necropsy. While the pig ex vivo organ cultures were susceptible to nearly all viruses tested, lower levels of virus were detected in trachea and bronchi after infection., Conclusion: These results confirm that pigs are susceptible to contemporary viruses that may threaten both veterinary and human health and contribute to the ecology of influenza A viruses., (© 2012 Crown copyright.)

To simulate a field situation in which pigs are in close contact with poultry and thus provide a potential mixing vessel for avian, swine and human influenza viruses, uninfected pigs were placed in contact with Pekin ducks or chickens infected with a H5N1 highly pathogenic avian influenza (HPAI) virus. To sustain prolonged exposure, newly inoculated birds were added at regular intervals. Although influenza virus was detected in birds and environmental samples, 14 days exposure to infected birds failed to produce evidence of infection in the pigs. The ability of pigs to generate reassortant viruses with these particular virus variants (H5N1 clade 2.2.1) may therefore be limited., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

Respiratory epithelial cells and macrophages are the key innate immune cells that play an important role in the pathogenesis of influenza A virus infection. We found that these two cell types from both human and pig showed comparable susceptibilities to initial infection with a highly pathogenic avian influenza (HPAI) H5N1 virus (A/turkey/Turkey/1/05) and a moderately pathogenic human influenza H1N1 virus (A/USSR/77), but there were contrasting differences in host innate immune responses. Human cells mounted vigorous cytokine (tumor necrosis factor alpha [TNF-α] and interleukin-6 [IL-6]) and chemokine (CXCL9, CXCL10, and CXCL11) responses to H5N1 virus infection. However, pig epithelial cells and macrophages showed weak or no TNF-α and chemokine induction with the same infections. The apparent lack of a strong proinflammatory response, corroborated by the absence of TNF-α induction in H5N1 virus-challenged pigs, coincided with greater cell death and the reduced release of infectious virus from infected pig epithelial cells. Suppressor of cytokine signaling 3 (SOCS3), a protein suppressor of the JAK-STAT pathway, was constitutively highly expressed and transcriptionally upregulated in H5N1 virus-infected pig epithelial cells and macrophages, in contrast to the corresponding human cells. The overexpression of SOCS3 in infected human macrophages dampened TNF-α induction. In summary, we found that the reported low susceptibility of pigs to contemporary Eurasian HPAI H5N1 virus infections coincides at the level of innate immunity of respiratory epithelial cells and macrophages with a reduced output of viable virus and an attenuated proinflammatory response, possibly mediated in part by SOCS3, which could serve as a target in the treatment or prevention of virus-induced hypercytokinemia, as observed for humans.

Following the emergence and global spread of a novel H1N1 influenza virus in 2009, two A(H1N1)pdm/09 influenza vaccines produced from the A/California/07/09 H1N1 strain were selected and used for the national immunisation programme in the United Kingdom: an adjuvanted split virion vaccine and a non-adjuvanted whole virion vaccine. In this study, we assessed the immune responses generated in inbred large white pigs (Babraham line) following vaccination with these vaccines and after challenge with A(H1N1)pdm/09 virus three months post-vaccination. Both vaccines elicited strong antibody responses, which included high levels of influenza-specific IgG1 and haemagglutination inhibition titres to H1 virus. Immunisation with the adjuvanted split vaccine induced significantly higher interferon gamma production, increased frequency of interferon gamma-producing cells and proliferation of CD4(-)CD8(+) (cytotoxic) and CD4(+)CD8(+) (helper) T cells, after in vitro re-stimulation. Despite significant differences in the magnitude and breadth of immune responses in the two vaccinated and mock treated groups, similar quantities of viral RNA were detected from the nasal cavity in all pigs after live virus challenge. The present study provides support for the use of the pig as a valid experimental model for influenza infections in humans, including the assessment of protective efficacy of therapeutic interventions.

Background: There is a requirement to detect and differentiate pandemic (H1N1) 2009 (H1N1v) and established swine influenza A viruses (SIVs) by real time reverse transcription (RRT) PCR methods., Objectives: First, modify an existing matrix (M) gene RRT PCR for sensitive generic detection of H1N1v and other European SIVs. Second, design an H1 RRT PCR to specifically detect H1N1v infections., Methods: RRT PCR assays were used to test laboratory isolates of SIV (n = 51; 37 European and 14 North American), H1N1v (n = 5) and avian influenza virus (AIV; n = 43). Diagnostic sensitivity and specificity were calculated for swabs (n = 133) and tissues (n = 116) collected from field cases and pigs infected experimentally with SIVs and H1N1v., Results: The "perfect match" M gene RRT PCR was the most sensitive variant of this test for detection of established European SIVs and H1N1v. H1 RRT PCR specifically detected H1N1v but not European SIVs. Validation with clinical specimens included comparison with virus isolation (VI) as a "gold standard", while field infection with H1N1v in swine was independently confirmed by sequencing H1N1v amplified by conventional RT PCR. "Perfect match" M gene RRT PCR had 100% sensitivity and 95.2% specificity for swabs, 93.6% and 98.6% for tissues. H1 RRT PCR demonstrated sensitivity and specificity of 100% and 99.1%, respectively, for the swabs, and 100% and 100% for the tissues., Conclusions: Two RRT PCRs for the purposes of (i) generic detection of SIV and H1N1v infection in European pigs, and for (ii) specific detection of H1N1v (pandemic influenza) infection were validated.

The declaration of the human influenza A pandemic (H1N1) 2009 (H1N1/09) raised important questions, including origin and host range [1], [2]. Two of the three pandemics in the last century resulted in the spread of virus to pigs (H1N1, 1918; H3N2, 1968) with subsequent independent establishment and evolution within swine worldwide [3]. A key public and veterinary health consideration in the context of the evolving pandemic is whether the H1N1/09 virus could become established in pig populations [4]. We performed an infection and transmission study in pigs with A/California/07/09. In combination, clinical, pathological, modified influenza A matrix gene real time RT-PCR and viral genomic analyses have shown that infection results in the induction of clinical signs, viral pathogenesis restricted to the respiratory tract, infection dynamics consistent with endemic strains of influenza A in pigs, virus transmissibility between pigs and virus-host adaptation events. Our results demonstrate that extant H1N1/09 is fully capable of becoming established in global pig populations. We also show the roles of viral receptor specificity in both transmission and tissue tropism. Remarkably, following direct inoculation of pigs with virus quasispecies differing by amino acid substitutions in the haemagglutinin receptor-binding site, only virus with aspartic acid at position 225 (225D) was detected in nasal secretions of contact infected pigs. In contrast, in lower respiratory tract samples from directly inoculated pigs, with clearly demonstrable pulmonary pathology, there was apparent selection of a virus variant with glycine (225G). These findings provide potential clues to the existence and biological significance of viral receptor-binding variants with 225D and 225G during the 1918 pandemic [5].

We tested serum samples from pigs infected or vaccinated with European swine influenza viruses (SIVs) in hemagglutination-inhibition assays against pandemic (H1N1) 2009 virus and related North American SIVs. We found more serologic cross-reaction than expected. Data suggest pigs in Europe may have partial immunity to pandemic (H1N1) 2009 virus.