Influenza Virus Infection In Pigs Research Articles

Bacillus subtilis-597 induces changes in lung pathology and inflammation during influenza A virus infection in pigs

In recent years, it has become apparent that imbalances in the gastrointestinal system can impact organs beyond the intestine such as the lungs. Given the established ability of probiotics to modulate the immune system by interacting with gastrointestinal cells, our research aimed to investigate whether administering the probiotic strain Bacillus subtilis-597 could mitigate the outcome of influenza virus infection in pigs. Pigs were fed a diet either with or without the probiotic strain B. subtilis-597 for 14 days before being intranasally inoculated with a swine influenza A H1N2 strain (1 C.2 lineage). Throughout the study, we collected fecal samples, blood samples, and nasal swabs to examine viral shedding and immune gene expression. After seven days of infection, the pigs were euthanized, and lung and ileum tissues were collected for gene expression analysis and pathological examination. Our findings indicate that the administration of B. subtilis-597 exhibit potential in reducing lung lesions, possibly attributable to a general suppression of the immune system as indicated by reduced C-reactive protein (CRP) levels in serum, decreased expression of interferon-stimulated genes (ISGs), and localized reduction of the inflammatory marker serum amyloid A (SAA) in ileum tissue. Notably, the immune-modulatory effects of B. subtilis-597 appeared to be unrelated to the gastrointestinal microbiota, as the composition remained unaltered by both the influenza infection and the administration of B. subtilis-597.

Immune Responses to Pandemic H1N1 Influenza Virus Infection in Pigs Vaccinated with a Conserved Hemagglutinin HA1 Peptide Adjuvanted with CAF®01 or CDA/αGalCerMPEG.

This study aimed to evaluate the immune response and protection correlates against influenza virus (IV) infection in pigs vaccinated with the novel NG34 HA1 vaccine candidate adjuvanted with either CAF®01 or CDA/αGalCerMPEG (αGCM). Two groups of six pigs each were vaccinated intramuscularly twice with either NG34 + CAF®01 or NG34 + CDA/αGCM. As controls, groups of animals (n = 6 or 4) either non-vaccinated or vaccinated with human seasonal trivalent influenza vaccine or NG34 + Freund’s adjuvant were included in the study. All animal groups were challenged with the 2009 pandemic (pdm09) strain of H1N1 (total amount of 7 × 106 TCID50/mL) via intranasal and endotracheal routes 21 days after second vaccination. Reduced consolidated lung lesions were observed both on days three and seven post-challenge in the animals vaccinated with NG34 + CAF®01, whereas higher variability with relatively more severe lesions in pigs of the NG34 + CDA/αGCM group on day three post-infection. Among groups, animals vaccinated with NG34 + CDA/αGCM showed higher viral loads in the lung at seven days post infection whereas animals from NG34 + CAF®01 completely abolished virus from the lower respiratory tract. Similarly, higher IFNγ secretion and stronger IgG responses against the NG34 peptide in sera was observed in animals from the NG34 + CAF®01 group as compared to the NG34 + CDA/αGCM. NG34-vaccinated pigs with adjuvanted CAF®01 or CDA/αGCM combinations resulted in different immune responses as well as outcomes in pathology and viral shedding.

A unique feature of swine ANP32A provides susceptibility to avian influenza virus infection in pigs.

Both the replication and transcription of the influenza virus are catalyzed by the viral polymerase complex. The polymerases of most avian influenza A viruses have poor performance in mammalian cells, which is considered to be one of the important species barriers. Pigs have been long considered as important intermediate hosts for interspecies transmission of the avian influenza virus, because of their susceptibility to infection with both avian and mammalian influenza viruses. However, the molecular basis of influenza polymerase adaptation in pigs remains largely unknown. ANP32A and ANP32B proteins have been identified as playing fundamental roles in influenza virus replication and host range determination. In this study, we found that swine ANP32A (swANP32A), unlike swine ANP32B or other mammalian ANP32A or B, shows stronger supporting activity to avian viral polymerase. Knockout of ANP32A in pig cells PK15 dramatically reduced avian influenza polymerase activity and viral infectivity, suggesting a unique feature of swANP32A in supporting avian influenza viral polymerase. This species-specific activity is mapped to two key sites, 106V and 156S, in swANP32A. Interestingly, the amino acid 106V is unique to pigs among all the vertebrate species studied, and when combined with 156S, exhibits positive epistasis in pigs. Mutation of 106V and 156S to the signature found in ANP32As from other mammalian species weakened the interaction between swANP32A and chicken viral polymerase, and reduced polymerase activity. Understanding the molecular basis of ANP32 proteins may help to discover new antiviral targets and design avian influenza resistant genome edited pigs.

Pulmonary inflammatory response to influenza virus infection in pigs is regulated by DAP12 and macrophage M1 and M2 phenotypes

We delineated the expression of DAP12 (DNAX-Activating Protein) and its associated receptors, TREM-1, TREM-2 and MDL-1 in pig alveolar monocyte/macrophages (AMM) that have attained M1 or M2 phenotypes. Pig AMM stimulated in vitro with IFN-γ and IL-4 induced the expression of M1 (TNFα and iNOS) and M2 (ARG1 and no MMR) phenotypic markers, respectively. In influenza virus infected pigs at seven days post-infection, in addition to substantial modulations in the M1 and M2 markers expression, DAP12, TREM-1 and MDL-1 were downregulated in AMM. Thus, DAP12 signaling promoted the anti-inflammatory pathway in AMM of influenza virus infected pigs.

Human infection with a novel reassortant Eurasian-avian lineage swine H1N1 virus in northern China

ABSTRACT Influenza A virus infections occur in different species, causing mild to severe respiratory symptoms that lead to a heavy disease burden. Eurasian avian-like swine influenza A(H1N1) viruses (EAS-H1N1) are predominant in pigs and occasionally infect humans. An influenza A(H1N1) virus was isolated from a boy who was suffering from fever and headache and designated as A/Tianjin-baodi/1606/2018(H1N1). Full-genome sequencing and phylogenetic analysis revealed that A/Tianjin-baodi/1606/2018(H1N1) is a novel reassortant EAS-H1N1 containing gene segments from EAS-H1N1 (HA and NA), classical swine H1N1(NS) and A(H1N1)pdm09(PB2, PB2, PA, NP and M) viruses. The isolation and analysis of A/Tianjin-baodi/1606/2018(H1) provide further evidence that EAS-H1N1 poses a threat to human health and greater attention should be paid to surveillance of influenza virus infection in pigs and humans.

INFLUÊNCIA DAS CONDIÇÕES DE ARMAZENAMENTO DO VÍRUS INFLUENZA NA DETECÇÃO POR RT-qPCR E ISOLAMENTO VIRAL

Resumo A infecção de suínos pelo vírus influenza causa perdas significativas na suinocultura e a doença tem implicações consideráveis para a saúde pública. Dessa forma, a rápida detecção viral em amostras biológicas de suínos é importante para a vigilância da influenza. Para o diagnóstico, as condições de manutenção das amostras biológicas (modo de acondicionamento, temperatura e período de acondicionamento), desde a colheita das amostras de suínos até o envio ao laboratório, podem interferir negativamente na detecção viral. Neste estudo foi analisada a viabilidade de uma amostra do vírus influenza A H1N1/2009 isolada de suínos, mantida em diferentes modos de acondicionamento (meio comercial UTM, meio in house VTM e sem meio de manutenção) e diferentes temperaturas (4, 23 e 37 °C) por um período de até 120 horas. As amostras foram avaliadas por RT-qPCR e isolamento em ovos embrionados. Foram observados efeitos significativos (p<0,05) para o modo e período de acondicionamento e da interação entre esses dois fatores com a carga viral. Dessa forma, as amostras biológicas enviadas para diagnóstico de influenza devem ser armazenadas, preferencialmente, em meio de manutenção viral a 4 °C e o tempo decorrido entre a colheita da amostra e a chegada ao laboratório deve ser de, no máximo, três dias.

NS1 Protein of 2009 Pandemic Influenza A Virus Inhibits Porcine NLRP3 Inflammasome-Mediated Interleukin-1 Beta Production by Suppressing ASC Ubiquitination

The inflammasome represents a molecular platform for innate immune regulation and controls proinflammatory cytokine production. The NLRP3 inflammasome is comprised of NLRP3, ASC, and procaspase-1. When the NLRP3 inflammasome is activated, it causes ASC speck formation and caspase-1 activation, resulting in the maturation of interleukin-1β (IL-1β). The NLRP3 inflammasome is regulated at multiple levels, with one level being posttranslational modification. Interestingly, ubiquitination of ASC has been reported to be indispensable for the activation of the NLRP3 inflammasome. Influenza A virus (IAV) infection induces NLRP3 inflammasome-dependent IL-1β secretion, which contributes to the host antiviral defense. However, IAVs have evolved multiple antagonizing mechanisms, one of which is executed by viral NS1 protein to suppress the NLRP3 inflammasome. In this study, we compared IL-1β production in porcine alveolar macrophages in response to IAV infection and found that the 2009 pandemic H1N1 induced less IL-1β than swine influenza viruses (SIVs). Further study revealed that the NS1 C terminus of pandemic H1N1 but not that of SIV was able to significantly inhibit NLRP3 inflammasome-mediated IL-1β production. This inhibitory function was attributed to impaired ASC speck formation and suppression of ASC ubiquitination. Moreover, we identified two target lysine residues, K110 and K140, which are essential for both porcine ASC ubiquitination and NLRP3 inflammasome-mediated IL-1β production. These results revealed a novel mechanism by which the NS1 protein of the 2009 pandemic H1N1 suppresses NLRP3 inflammasome activation.IMPORTANCE Influenza A virus (IAV) infection activates the NLRP3 inflammasome, resulting in the production of IL-1β, which contributes to the host innate immune response. ASC, an adaptor protein of NLRP3, forms specks that are critical for inflammasome activation. Here, we report that the NS1 C terminus of the 2009 pandemic H1N1 has functions to suppress porcine IL-1β production by inhibiting ASC speck formation and ASC ubiquitination. Furthermore, the ubiquitination sites on porcine ASC were identified. The information gained here may contribute to an in-depth understanding of porcine inflammasome activation and regulation in response to different IAVs, helping to further enhance our knowledge of innate immune responses to influenza virus infection in pigs.

Evidence of infection with avian, human, and swine influenza viruses in pigs in Cairo, Egypt.

The majority of the Egyptian swine population was culled in the aftermath of the 2009 H1N1 pandemic, but small-scale growing remains. We sampled pigs from piggeries and an abattoir in Cairo. We found virological evidence of infection with avian H9N2 and H5N1 viruses as well as human pandemic H1N1 influenza virus. Serological evidence suggested previous exposure to avian H5N1 and H9N2, human pandemic H1N1, and swine avian-like and human-like viruses. This raises concern about potential reassortment of influenza viruses in pigs and highlights the need for better control and prevention of influenza virus infection in pigs.

Distinct immune responses and virus shedding in pigs following aerosol, intra-nasal and contact infection with pandemic swine influenza A virus, A(H1N1)09.

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-016-0390-5) contains supplementary material, which is available to authorized users.

The infectivity of pandemic 2009 H1N1 and avian influenza viruses for pigs: an assessment byex vivorespiratory tract organ culture*

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.

Swine influenza virus infection dynamics in two pig farms; results of a longitudinal assessment

In order to assess the dynamics of influenza virus infection in pigs, serological and virological follow-ups were conducted in two whole batches of pigs from two different farms (F1 and F2), from 3 weeks of age until market age. Anti-swine influenza virus (SIV) antibodies (measured by ELISA and hemagglutination inhibition) and nasal virus shedding (measured by RRT-PCR and isolation in embryonated chicken eggs and MDCK cells) were carried out periodically. SIV isolates were subtyped and hemagglutinin and neuraminidase genes were partially sequenced and analyzed phylogenetically. In F1, four waves of viral circulation were detected, and globally, 62/121 pigs (51.2%) were positive by RRT-PCR at least once. All F1 isolates corresponded to H1N1 subtype although hemagglutination inhibition results also revealed the presence of antibodies against H3N2. The first viral wave took place in the presence of colostral-derived antibodies. Nine pigs were positive in two non-consecutive sampling weeks, with two of the animals being positive with the same isolate. Phylogenetic analyses showed that different H1N1 variants circulated in that farm. In F2, only one isolate, H1N2, was detected and all infections were concentrated in a very short period of time, as assumed for a classic influenza outbreak. These findings led us to propose that influenza virus infection in pigs might present different patterns, from an epidemic outbreak to an endemic form with different waves of infections with a lower incidence.

Transcription analysis on response of swine lung to H1N1 swine influenza virus

BackgroundAs a mild, highly contagious, respiratory disease, swine influenza always damages the innate immune systems, and increases susceptibility to secondary infections which results in considerable morbidity and mortality in pigs. Nevertheless, the systematical host response of pigs to swine influenza virus infection remains largely unknown. To explore it, a time-course gene expression profiling was performed for comprehensive analysis of the global host response induced by H1N1 swine influenza virus in pigs.ResultsAt the early stage of H1N1 swine virus infection, pigs were suffering mild respiratory symptoms and pathological changes. A total of 268 porcine genes showing differential expression (DE) after inoculation were identified to compare with the controls on day 3 post infection (PID) (Fold change ≥ 2, p < 0.05). The DE genes were involved in many vital functional classes, mainly including signal transduction, immune response, inflammatory response, cell adhesion and cell-cell signalling. Noticeably, the genes associated with immune and inflammatory response showed highly overexpressed. Through the pathway analysis, the significant pathways mainly concerned with Cell adhesion molecules, Cytokine-cytokine receptor interaction, Toll-like receptor signaling pathway and MAPK signaling pathway, suggesting that the host took different strategies to activate these pathways so as to prevent virus infections at the early stage. However, on PID 7, the predominant function classes of DE genes included signal transduction, metabolism, transcription, development and transport. Furthermore, the most significant pathways switched to PPAR signaling pathway and complement and coagulation cascades, showing that the host might start to repair excessive tissue damage by anti-inflammatory functions. These results on PID 7 demonstrated beneficial turnover for host to prevent excessive inflammatory damage and recover the normal state by activating these clusters of genes.ConclusionsThis study shows how the target organ responds to H1N1 swine influenza virus infection in pigs. The observed gene expression profile could help to screen the potential host agents for reducing the prevalence of swine influenza virus and further understand the molecular pathogenesis associated with H1N1 infection in pigs.

Seroprevalence and risk factors of swine influenza in Spain

Swine influenza is caused by type A influenza virus. Pigs can be infected by both avian and human influenza viruses; therefore, the influenza virus infection in pigs is considered an important public health concern. The aims of present study were to asses the seroprevalence of swine influenza subtypes in Spain and explore the risk factors associated with the spread of those infections. Serum samples from 2151 pigs of 98 randomly selected farms were analyzed by an indirect ELISA for detection of antibodies against nucleoprotein A of influenza viruses and by the hemagglutination inhibition (HI) using H1N1, H1N2 and H3N2 swine influenza viruses (SIV) as antigens. Data gathered in questionnaires filled for each farm were used to explore risk factors associated with swine influenza. For that purpose, data were analyzed using the generalized estimating equations method and, in parallel by means of a logistic regression. By ELISA, 92 farms (93.9%; CI95%: 89.1–98.7%) had at least one positive animal and, in total, 1340/2151 animals (62.3%; CI95%: 60.2–64.3%) were seropositive. A total of 1622 animals (75.4%; CI95%: 73.6–77.2%) were positive in at least one of the HI tests. Of the 98 farms, 91 (92.9%; CI95%: 87.7–98.1%) had H1N1 seropositive animals; 63 (64.3%; CI95%: 54.6–73.9%) had H1N2 seropositive pigs and 91 (92.9%; CI95%: 87.7–98.1%) were positive to H3N2. Mixed infections were detected in 88 farms (89.8; CI95%: 83.7–95.9%). Three risk factors were associated with seroprevalences of SIV: increased replacement rates in pregnancy units and, for fatteners, existence of open partitions between pens and uncontrolled entrance to the farm.

Restricted Infectivity of a Human-Lineage H3N2 Influenza A Virus in Pigs Is Hemagglutinin and Neuraminidase Gene Dependent

Influenza A viruses cause pandemics at sporadic intervals. Pandemic viruses can potentially be introduced into the human population through in toto transfer of an avian influenza virus or through reassortment between avian and human strains. Pigs are believed to play a central role in the creation of pandemic viruses through reassortment because of their susceptibility to infection with both avian and human influenza viruses. However, we recently found that a human-lineage H3N2 influenza virus was highly restricted in its ability to infect pigs after intranasal inoculation. We hypothesized that this restricted infectivity phenotype was controlled by the hemagglutinin (HA) and neuraminidase (NA). To test this, we infected pigs with reverse genetics-created HA plus NA reassortant viruses. Specifically, introduction of the HA and NA genes of a contemporary H3N2 swine virus into the genetic background of the wholly human virus resulted in a significant increase in virus shedding and pathogenicity. These data indicate that the HA/NA can play important roles in controlling human influenza virus infectivity in pigs. The results further support the premise that a barrier exists to human influenza virus infection in pigs, which may limit the role of pigs in pandemic virus creation through reassortment of human and avian influenza viruses.

Effects of DNA dose, route of vaccination, and coadministration of porcine interleukin-6 DNA on results of DNA vaccination against influenza virus infection in pigs.

To examine the effects of DNA dose, site of vaccination, and coadministration of a cytokine DNA adjuvant on efficacy of H1-subtype swine influenza virus hemagglutinin (HA) DNA vaccination of pigs. 24 eight-week-old mixed-breed pigs. 2 doses of DNA were administered 27 days apart by use of a particle-mediated delivery system (gene gun). Different doses of HA DNA and different sites of DNA administration (skin, tongue) were studied, as was coadministration of porcine interleukin-6 (pIL-6) DNA as an adjuvant. Concentrations of virus-specific serum and nasal mucosal antibodies were measured throughout the experiment, and protective immunity was assessed after intranasal challenge with homologous H1N1 swine influenza virus. Increasing the dose of HA DNA, but not coadministration of pIL6 DNA, significantly enhanced virus-specific serum antibody responses. Pigs that received DNA on the ventral surface of the tongue stopped shedding virus 1 day sooner than pigs vaccinated in the skin of the ventral portion of the abdomen, but none of the vaccinated pigs developed detectable virus-specific antibodies in nasal secretions prior to challenge, nor were they protected from challenge exposure. Vaccinated pigs developed high virus-specific antibody concentrations after exposure to the challenge virus. Co-administration of pIL-6 DNA did not significantly enhance immune responses to HA DNA vaccination or protection from challenge exposure. However, HA DNA vaccination of pigs, with or without coadministration of pIL-6 DNA, induced strong priming of the humoral immune system.

Virologic and serologic surveillance for human, swine and avian influenza virus infections among pigs in the north-central United States.

Summary. Influenza virus infection in pigs is both an animal health problem and a public health concern. As such, surveillance and characterization of influenza viruses in swine is important to the veterinary community and should be a part of human pandemic preparedness planning. Studies in 1976/1977 and 1988/1989 demonstrated that pigs in the U.S. were commonly infected with classical swine H1N1 viruses, whereas human H3 and avian influenza virus infections were very rare. In contrast, human H3 and avian H1 viruses have been isolated frequently from pigs in Europe and Asia over the last two decades. From September 1997 through August 1998, we isolated 26 influenza viruses from pigs in the north-central United States at the point of slaughter. All 26 isolates were H1N1 viruses, and phylogenetic analyses of the hemagglutinin and nucleoprotein genes from 11 representative viruses demonstrated that these were classical swine H1 viruses. However, monoclonal antibody analyses revealed antigenic heterogeneity among the HA proteins of the 26 viruses. Serologically, 27.7% of 2,375 pigs tested had hemagglutination-inhibiting antibodies against classical swine H1 influenza virus. Of particular significance, however, the rates of seropositivity to avian H1 (7.6%) and human H3 (8.0%) viruses were substantially higher than in previous studies.

Systemic and mucosal immune responses to H1N1 influenza virus infection in pigs

Influenza is a common respiratory disease in pigs, and since swine influenza viruses are zoonotic pathogens, they also pose human health risks. Pigs infected with influenza virus mount an effective immune response and are protected from subsequent challenge, whereas the currently available, inactivated-virus vaccine does not consistently confer complete protection to challenge. To develop and evaluate new vaccination strategies, it is imperative to fully understand the immune responses that are associated with protection following natural infection. Therefore, we have evaluated the phenotype and kinetics of immune responses to primary and re-challenge infection with H1N1 swine influenza virus in the pig. Through the use of isotype-specific antibody secreting cell ELISPOT assays, interferon-γ ELISPOT assays and isotype-specific ELISAs on serum, nasal wash and bronchoalveolar lavage samples, we defined the humoral and cellular immune responses, both locally in the respiratory tract and systemically, to this viral infection. Virus-specific serum IgG, IgA, and HI titers all peaked 2–3 weeks after primary infection and did not substantially increase after re-challenge. The predominant virus-specific isotype in serum was IgG. Pigs responded with virus-specific IgG and IgA in both the upper (nasal washes) and lower (bronchoalveolar lavages) airways; IgA was the predominant isotype in both sites. Despite the fact that the pigs were completely protected from re-challenge, the antibody titers in the nasal washes increased. Results of the antibody-secreting cell ELISPOT assays demonstrated that the numbers of both IgG and IgA secreting cells in the nasal mucosa were dramatically higher than in any other tissue examined. In contrast, IFN-γ secreting cells were predominantly localized to the spleen and tracheobronchial lymph nodes. These data will be helpful in the future development and evaluation of novel vaccines.