Influenza is a viral respiratory disease having a major impact on public health. Influenza A virus (IAV) usually causes mild transitory disease in humans. However, in specific groups of individuals such as severely obese, the elderly, and individuals with underlying inflammatory conditions, IAV can cause severe illness or death. In this review, relevant small and large animal models for human IAV infection, including the pig, ferret, and mouse, are discussed. The focus is on the pig as a large animal model for human IAV infection as well as on the associated innate immune response. Pigs are natural hosts for the same IAV subtypes as humans, they develop clinical disease mirroring human symptoms, they have similar lung anatomy, and their respiratory physiology and immune responses to IAV infection are remarkably similar to what is observed in humans. The pig model shows high face and target validity for human IAV infection, making it suitable for modeling many aspects of influenza, including increased risk of severe disease and impaired vaccine response due to underlying pathologies such as low-grade inflammation. Comparative analysis of proteins involved in viral pattern recognition, interferon responses, and regulation of interferon-stimulated genes reveals a significantly higher degree of similarity between pig, ferret, and human compared with mice. It is concluded that the pig is a promising animal model displaying substantial human translational value with the ability to provide essential insights into IAV infection, pathogenesis, and immunity.

Actinobacillus pleuropneumoniae causes pleuropneumonia in pigs, a disease which is associated with high morbidity and mortality, as well as impaired animal welfare. To obtain in-depth understanding of this infection, the interplay between virulence factors of the pathogen and defense mechanisms of the porcine host needs to be elucidated. However, research has traditionally focused on either bacteriology or immunology; an unbiased picture of the transcriptional responses can be obtained by investigating both organisms in the same biological sample. Host and pathogen responses in pigs experimentally infected with A. pleuropneumoniae were analyzed by high-throughput RT-qPCR. This approach allowed concurrent analysis of selected genes encoding proteins known or hypothesized to be important in the acute phase of this infection. The expression of 17 bacterial and 31 porcine genes was quantified in lung samples obtained within the first 48 hours of infection. This provided novel insight into the early time course of bacterial genes involved in synthesis of pathogen-associated molecular patterns (lipopolysaccharide, peptidoglycan, lipoprotein) and genes involved in pattern recognition (TLR4, CD14, MD2, LBP, MYD88) in response to A. pleuropneumoniae. Significant up-regulation of proinflammatory cytokines such as IL1B, IL6, and IL8 was observed, correlating with protein levels, infection status and histopathological findings. Host genes encoding proteins involved in iron metabolism, as well as bacterial genes encoding exotoxins, proteins involved in adhesion, and iron acquisition were found to be differentially expressed according to disease progression. By applying laser capture microdissection, porcine expression of selected genes could be confirmed in the immediate surroundings of the invading pathogen. Microbial pathogenesis is the product of interactions between host and pathogen. Our results demonstrate the applicability of high-throughput RT-qPCR for the elucidation of dual-organism gene expression analysis during infection. We showed differential expression of 12 bacterial and 24 porcine genes during infection and significant correlation of porcine and bacterial gene expression. This is the first study investigating the concurrent transcriptional response of both bacteria and host at the site of infection during porcine respiratory infection.

Streptococcus suis is a zoonotic pathogen of swine involved in arthritis, polyserositis, and meningitis. Colonization of piglets by S. suis is very common and occurs early in life. The clinical outcome of infection is influenced by the virulence of the S. suis strains and the immunity of the animals. Here, the role of innate immunity was studied in cesarean-derived colostrum-deprived piglets inoculated intranasally with either virulent S. suis strain 10 (S10) or non-virulent S. suis strain T15. Colonization of the inoculated piglets was confirmed at the end of the study by PCR and immunohistochemistry. Fever (≥40.5 °C) was more prevalent in piglets inoculated with S10 compared to T15 at 4 h after inoculation. During the 3 days of monitoring, no other major clinical signs were detected. Accordingly, only small changes in transcription of genes associated with the antibacterial innate immune response were observed at systemic sites, with S10 inducing an earlier response than T15 in blood. Local inflammatory response to the inoculation, evaluated by transcriptional analysis of selected genes in nasal swabs, was more sustained in piglets inoculated with the virulent S10, as demonstrated by transcription of inflammation-related genes, such as IL1B, IL1A, and IRF7. In contrast, most of the gene expression changes in trachea, lungs, and associated lymph nodes were observed in response to the non-virulent T15 strain. Thus, S. suis colonization in the absence of systemic infection induces an innate immune response in piglets that appears to be related to the virulence potential of the colonizing strain.

MicroRNAs (miRNAs) are a class of short regulatory RNA molecules which are implicated in modulating gene expression. Levels of circulating, cell-associated miRNAs in response to influenza A virus (IAV) infection has received limited attention so far. To further understand the temporal dynamics and biological implications of miRNA regulation in circulating leukocytes, we collected blood samples before and after (1, 3, and 14 days) IAV challenge of pigs. Differential expression of miRNAs and innate immune factor mRNA transcripts was analysed using RT-qPCR. A total of 20 miRNAs were regulated after IAV challenge, with the highest number of regulated miRNAs seen on day 14 after infection at which time the infection was cleared. Targets of the regulated miRNAs included genes involved in apoptosis and cell cycle regulation. Significant regulation of both miRNAs and mRNA transcripts at 14 days after challenge points to a protracted effect of IAV infection, potentially affecting the host’s ability to respond to secondary infections. In conclusion, experimental IAV infection of pigs demonstrated the dynamic nature of miRNA and mRNA regulation in circulating leukocytes during and after infection, and revealed the need for further investigation of the potential immunosuppressing effect of miRNA and innate immune signaling after IAV infection.

The innate immune system is paramount in the response to and clearance of influenza A virus (IAV) infection in non-immune individuals. Known factors include type I and III interferons and antiviral pathogen recognition receptors, and the cascades of antiviral and pro- and anti-inflammatory gene expression they induce. MicroRNAs (miRNAs) are increasingly recognized to participate in post-transcriptional modulation of these responses, but the temporal dynamics of how these players of the antiviral innate immune response collaborate to combat infection remain poorly characterized. We quantified the expression of miRNAs and protein coding genes in the lungs of pigs 1, 3, and 14 days after challenge with swine IAV (H1N2). Through RT-qPCR we observed a 400-fold relative increase in IFN-λ3 gene expression on day 1 after challenge, and a strong interferon-mediated antiviral response was observed on days 1 and 3 accompanied by up-regulation of genes related to the pro-inflammatory response and apoptosis. Using small RNA sequencing and qPCR validation we found 27 miRNAs that were differentially expressed after challenge, with the highest number of regulated miRNAs observed on day 3. In contrast, the number of protein coding genes found to be regulated due to IAV infection peaked on day 1. Pulmonary miRNAs may thus be aimed at fine-tuning the initial rapid inflammatory response after IAV infection. Specifically, we found five miRNAs (ssc-miR-15a, ssc-miR-18a, ssc-miR-21, ssc-miR-29b, and hsa-miR-590-3p)–four known porcine miRNAs and one novel porcine miRNA candidate–to be potential modulators of viral pathogen recognition and apoptosis. A total of 11 miRNAs remained differentially expressed 14 days after challenge, at which point the infection had cleared. In conclusion, the results suggested a role for miRNAs both during acute infection as well as later, with the potential to influence lung homeostasis and susceptibility to secondary infections in the lungs of pigs after IAV infection.

Streptococcus suis is a porcine and zoonotic pathogen in the upper respiratory tract, expressing different capsular serotypes and virulence-associated factors. Given its genomic and phenotypic diversity, the virulence potential of S. suis cannot be attributed to a single factor. Since strong inflammatory response is a hallmark of S. suis infection, the objective of this study was to investigate the differences in transcriptional host responses to two serotype 2 and one serotype 9 strains. Both serotypes are frequently found in clinical isolates. We infected porcine precision-cut lung slices (PCLSs) with two serotype 2 strains of high (strain S10) and low (strain T15) virulence, and a serotype 9 strain 8067 of moderate virulence. We observed higher expression of inflammation-related genes during early infection with strains T15 and 8067, in contrast to infection with strain 10, whose expression peaked late. In addition, bacterial gene expression from infected PCLSs revealed differences, mainly of metabolism-related and certain virulence-associated bacterial genes amongst these strains. We conclude that the strain- and time-dependent induction of genes involved in innate immune response might reflect clinical outcomes of infection in vivo, implying rapid control of infection with less virulent strains compared to the highly virulent strain S10.