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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.

ABSTRACTSince 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.IMPORTANCEThe 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.

IntroductionSwine 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.MethodsLung 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.ResultsImmortalized 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.DiscussionIn 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.

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.

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.

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.

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.

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.

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.