Transcriptomics in lung tissue upon respiratory syncytial virus infection reveals aging as important modulator of immune activation and matrix maintenance.

Aging poses an increased risk of severe infection by respiratory syncytial virus (RSV). The many different biological pathways comprising the response to infection in lungs that are influenced by aging are complex and remain to be defined more thoroughly. Towards finding new directions in research on aging, we aimed to define biological pathways in the acute response to RSV that are affected in the lungs by aging. We therefore profiled the full transcriptome of lung tissue of mice prior to and during RSV infection both at young and old age. In the absence of RSV, we found aging to downregulate genes that are involved in constitution of the extracellular matrix. Moreover, uninfected old mice showed elevated expression of pathways that resemble injury, metabolic aberrations, and disorders mediated by functions of the immune system that were induced at young age only by an exogenous trigger like RSV. Furthermore, infection by RSV mounted stronger activation of anti-viral type-I interferon pathways at old age. Despite such exaggerated anti-viral responses, old mice showed reduced control of virus. Altogether, our findings emphasize important roles in aging-related susceptibility to respiratory disease for extracellular matrix dysfunctions and dysregulated immune activation in lungs

18S rRNA is a reliable normalisation gene for real time PCR based on influenza virus infected cells

Background: One requisite of quantitative reverse transcription PCR (qRT-PCR) is to normalise the data with an internal reference gene that is invariant regardless of treatment, such as virus infection. Several studies have found variability in the expression of commonly used housekeeping genes, such as beta-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), under different experimental settings. However, ACTB and GAPDH remain widely used in the studies of host gene response to virus infections, including influenza viruses. To date no detailed study has been described that compares the suitability of commonly used housekeeping genes in influenza virus infections. The present study evaluated several commonly used housekeeping genes [ACTB, GAPDH, 18S ribosomal RNA (18S rRNA), ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide (ATP5B) and ATP synthase, H+ transporting, mitochondrial Fo complex, subunit C1 (subunit 9) (ATP5G1)] to identify the most stably expressed gene in human, pig, chicken and duck cells infected with a range of influenza A virus subtypes. Results: The relative expression stability of commonly used housekeeping genes were determined in primary human bronchial epithelial cells (HBECs), pig tracheal epithelial cells (PTECs), and chicken and duck primary lung-derived cells infected with five influenza A virus subtypes. Analysis of qRT-PCR data from virus and mock infected cells using NormFinder and BestKeeper software programmes found that 18S rRNA was the most stable gene in HBECs, PTECs and avian lung cells. Conclusions: Based on the presented data from cell culture models (HBECs, PTECs, chicken and duck lung cells) infected with a range of influenza viruses, we found that 18S rRNA is the most stable reference gene for normalising qRT-PCR data. Expression levels of the other housekeeping genes evaluated in this study (including ACTB and GPADH) were highly affected by influenza virus infection and hence are not reliable as reference genes for RNA normalisation

Identification of Novel Avian Influenza Virus Derived CD8+ T-Cell Epitopes

Avian influenza virus (AIV) infection is a continuing threat to both humans and poultry. Influenza virus specific CD8+ T cells are associated with protection against homologous and heterologous influenza strains. In contrast to what has been described for humans and mice, knowledge on epitope-specific CD8+ T cells in chickens is limited. Therefore, we set out to identify AIV-specific CD8+ T-cell epitopes. Epitope predictions based on anchor residues resulted in 33 candidate epitopes. MHC I inbred chickens were infected with a low pathogenic AIV strain and sacrificed at 5, 7, 10 and 14 days post infection (dpi). Lymphocytes isolated from lung, spleen and blood were stimulated ex vivo with AIV-specific pooled or individual peptides and the production of IFNγ was determined by ELIspot. This resulted in the identification of 12 MHC B12-restricted, 3 B4-restricted and 1 B19-restricted AIV- specific CD8+ T-cell epitopes. In conclusion, we have identified novel AIV-derived CD8+ T-cell epitopes for several inbred chicken strains. This knowledge can be used to study the role of CD8+ T cells against AIV infection in a natural host for influenza, and may be important for vaccine development

Highly pathogenic avian influenza virus infection in chickens but not ducks is associated with elevated host immune and pro-inflammatory responses

Highly pathogenic avian influenza (HPAI) H5N1 viruses cause severe infection in chickens at near complete mortality, but corresponding infection in ducks is typically mild or asymptomatic. To understand the underlying molecular differences in host response, primary chicken and duck lung cells, infected with two HPAI H5N1 viruses and a low pathogenicity avian influenza (LPAI) H2N3 virus, were subjected to RNA expression profiling. Chicken cells but not duck cells showed highly elevated immune and pro-inflammatory responses following HPAI virus infection. HPAI H5N1 virus challenge studies in chickens and ducks corroborated the in vitro findings. To try to determine the underlying mechanisms, we investigated the role of signal transducer and activator of transcription-3 (STAT-3) in mediating pro-inflammatory response to HPAIV infection in chicken and duck cells. We found that STAT-3 expression was down-regulated in chickens but was up-regulated or unaffected in ducks in vitro and in vivo following H5N1 virus infection. Low basal STAT-3 expression in chicken cells was completely inhibited by H5N1 virus infection. By contrast, constitutively active STAT-3 detected in duck cells was unaffected by H5N1 virus infection. Transient constitutively-active STAT-3 transfection in chicken cells significantly reduced pro-inflammatory response to H5N1 virus infection; on the other hand, chemical inhibition of STAT-3 activation in duck cells increased pro-inflammatory gene expression following H5N1 virus infection. Collectively, we propose that elevated pro-inflammatory response in chickens is a major pathogenicity factor of HPAI H5N1 virus infection, mediated in part by the inhibition of STAT-3

Visualisation and characterisation of mononuclear phagocytes in the chicken respiratory tract using CSF1R-transgenic chickens

Additional file 2. Location of B cells, T cells and follicular dendritic cells (FDC) in the lung of MacReporter chickens. The BALT region of 5 to 7 week old non-vaccination animals were analysed for B, T and FCD cells. Isotype controls were used to standardise the microscope and examine aspecific binding before acquiring images (A-B). The GC of MacReporter animals are tightly packed with Bu1-CSF1R-eGFP+ FDC cells and Bu1+CSF1R-eGFP- B cells (C) with few Bu1+ B cells found in the parabronchi (F). CD3+ T cells are disperse within and outside the GC (D) and parabronchi (G). CSF1R-eGFP+ FDC cells express Fc receptors and trap immunoglobulin by expressing IgY (E) and CSF1R-eGFP+ IgY+ FDC are rarely detected out with the GC, BALT region of the lung. GC are indicated by white dashed lines

Novel AIV-specific CD8+ T cell epitopes within the LPAI H7N1 virus.

<p>Epitope mapping resulted in 11 novel CD8+ T-cell epitopes in the nucleoprotein (A) and 5 epitopes in the matrix 1 protein (B) of the LPAI H7N1 virus. In black, B12-restricted epitopes, in grey, B4-restricted epitopes (A) or B19-restricted epitopes (B).</p

CD8+ T-cell frequencies in tissues of LPAIV infected chickens.

<p>Percentages of CD8αα+CD3+ T cells (A–C) and CD8βα+CD3+ T cells (D–F) were analysed by flowcytometry in lung, spleen and PBMC at several days post infection. Absolute numbers were calculated by multiplying the percentage of CD8αα+CD3+ T or CD8βα+CD3+ T cells with the total number of cells isolated from lung (G, I) and spleen (H, J). Mean plus SEM is shown. In white: uninfected controls (UNINF, n = 12), in grey infected birds (n = 3 per time point).</p

Identification of MHC B12-restricted CD8+ T-cell epitopes using peptide pools.

<p>Lung cells were stimulated with B12-restricted peptide pools and IFNγ-producing cells were determined by IFNγ ELIspot analysis. Results for three individuals birds are shown at 5 dpi (A–C), 7 dpi (D–F), 10 dpi (G–I) and 14 dpi (J–L). Mean plus SEM is shown, n = 3 per group. Positive responses (*) and “significant” peptides inducing a positive response in 2 out of 3 chickens (↓) are indicated.</p

Epitope prediction for MHC B12 based on anchor residues.

<p>Predicted epitopes and their localization based on anchor residues that have been described for B12 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031953#pone.0031953-Kaufman1" target="_blank">[32]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031953#pone.0031953-Wallny1" target="_blank">[33]</a>. X represents any amino acid. Anchor residues specific for the different MHC types are indicated in bold. A variable number of amino acids between the anchor residues is indicted with (X).</p

Screening of MHC B12-restricted CD8+ T-cell epitopes using individual peptides.

<p>Lung cells were isolated, <i>in vitro</i> re-stimulated with B12-restricted peptide pools and IFNγ-producing cells were determined by IFNγ ELIspot analysis at 7 dpi (A) and 10 dpi (B). Mean plus SEM is shown, n = 4 per group. Positive responses (*) and “significant” peptides inducing a positive response in 2 out of 3 chickens (↓) are indicated.</p