Activation of Type I and III Interferon Signalling Pathways Occurs in Lung Epithelial Cells Infected with Low Pathogenic Avian Influenza Viruses

The host response to the low pathogenic avian influenza (LPAI) H5N2, H5N3 and H9N2 viruses were examined in A549, MDCK, and CEF cells using a systems-based approach. The H5N2 and H5N3 viruses replicated efficiently in A549 and MDCK cells, while the H9N2 virus replicated least efficiently in these cell types. However, all LPAI viruses exhibited similar and higher replication efficiencies in CEF cells. A comparison of the host responses of these viruses and the H1N1/WSN virus and low passage pH1N1 clinical isolates was performed in A549 cells. The H9N2 and H5N2 virus subtypes exhibited a robust induction of Type I and Type III interferon (IFN) expression, sustained STAT1 activation from between 3 and 6 hpi, which correlated with large increases in IFN-stimulated gene (ISG) expression by 10 hpi. In contrast, cells infected with the pH1N1 or H1N1/WSN virus showed only small increases in Type III IFN signalling, low levels of ISG expression, and down-regulated expression of the IFN type I receptor. JNK activation and increased expression of the pro-apoptotic XAF1 protein was observed in A549 cells infected with all viruses except the H1N1/WSN virus, while MAPK p38 activation was only observed in cells infected with the pH1N1 and the H5 virus subtypes. No IFN expression and low ISG expression levels were generally observed in CEF cells infected with either AIV, while increased IFN and ISG expression was observed in response to the H1N1/WSN infection. These data suggest differences in the replication characteristics and antivirus signalling responses both among the different LPAI viruses, and between these viruses and the H1N1 viruses examined. These virus-specific differences in host cell signalling highlight the importance of examining the host response to avian influenza viruses that have not been extensively adapted to mammalian tissue culture

Biological characterization of RNA polymerase proteins from human 2009 pandemic H1N1 and low pathogenic avian H5N2 and H9N2 influenza viruses

The influenza A genome replication and gene transcription are mediated by polymerase complex composed of three polymerase subunits PA, PB1 and PB2. This polymerase complex is also essential for host adaptation and pathogenesis. To characterize the polymerase complex of 2009 pandemic H1N1 (pH1N1) and low pathogenic avian (LPAI) H5N2 influenza viruses, monoclonal antibodies (mAbs) against these complexes are essential. Therefore, we have challenged mice with purified soluble PA protein (pH1N1/471 and H5N2/F118 strains), insoluble PB2 and PB1 proteins (pH1N1/471 strain). Then, mAbs directed against PA subunit of both strains and PB2 subunit of 2009 pH1N1 strain were successfully generated, while mAb against PB1 subunit was not successful at fusion stage for two attempts. Interestingly, two mAbs-PA(9F5 and 2E2) against pH1N1/471 strain recognized PA protein in virus-infected cells with different staining patterns, which may be explained by their different epitope recognition. In addition, smaller protein products (PA*) were identified by 9F5 in recombinant protein expression, virus-infected cells and mature virus particles. We proved the PA* are PA related and demonstrated the first evidence of their association with the RNP complex. Similar to PA*, PB2* was also revealed by mAb-PB2(4G3), but in the virions, suggesting that PB2* was not virus-associated. However, significance of PB2* remained unclear and it needs further investigation. For the functional analysis of viral polymerase complex, NP and three polymerase subunits of four influenza virus strains have been successfully cloned using a mammalian expression system. H1N1/WSN polymerase complex was found to be the most active than polymerase complexes of other strains tested. In our gene reassortment study, a single reassortant bearing a human-origin PA or PB2 subunit against an avian polymerase background could overcome the host range barrier of avian polymerase in human 293T cells, indicating the PA or PB2 as a major determinant of species tropism and pathogenicity. Again in our RNAi study, potent in vitro inhibition of virus replication was achieved with eight siRNAs against NP and polymerase genes of LPAI H5N2 virus as observed by reduction in mRNA levels, protein expressions and virus titers.DOCTOR OF PHILOSOPHY (SBS

Biochemical and mechanical analysis of decellularized oesophagus for oesophageal tissue engineering

123 p.A number of decellularization methods have been developed to produce acellular xenogenic extracellular matrix (ECM) scaffolds to reconstruct oesophageal defects due to either congenital or acquired diseases. However, the effects of decellularization process on the resulted ECMs have not been fully elucidated yet. The aim of this project is to produce acellular porcine oesophageal scaffolds for oesophageal tissue engineering application. In this study, two different decellularization methods were established; (1) a chemical-based process (using peracetic acid), and (2) a chemical-mechanical based process (combination of peracetic acid and mechanical scraping). The developed decellularization processes were followed by additional hydrogen peroxide wash step. Optimal parameters were based on qualitative (histology and scanning electron microscopy) and quantitative (DNA and collagen assays, mechanical strength tests) studies. Results showed that the chemical-based decellularization process appears to be more effective in removing cellular elements and preserving ECM of porcine oesophagi than the chemical-mechanical based process. Furthermore, mechanical properties were mostly retained in the decellularized ECM scaffolds derived from chemical-based process.Master of Science (Biomedical Engineering

Systems-based approach to examine the cytokine responses in primary mouse lung macrophages infected with low pathogenic avian Influenza virus circulating in South East Asia

Abstract Background Influenza A virus (IAV) is a major public health concern, being responsible for the death of approximately half a million people each year. Zoonotic transmissions of the virus from swine and avian origin have occurred in the past, and can potentially lead to the emgergence of new IAV stains in future pandemics. Pulmonary macrophages have been implicated in disease severity in the lower airway, and understanding the host response of macrophages infected with avian influenza viruses should provide new therapeutic strategies. Results We used a systems-based approach to investigate the transcriptome response of primary murine lung macrophages (PMФ) infected with the mouse-adapted H1N1/WSN virus and low pathogenic avian influenza (LPAI) viruses H5N2 and H5N3. The results showed that the LPAI viruses H5N2 and H5N3 can infect PMФ with similar efficiency to the H1N1/WSN virus. While all viruses induced antiviral responses, the H5N3 virus infection resulted in higher expression levels of cytokines and chemokines associated with inflammatory responses. Conclusions The LPAI H5N2 and H5N3 viruses are able to infect murine lung macrophages. However, the H5N3 virus was associated with increased expression of pro-inflammatory mediators. Although the H5N3 virus it is capable of inducing high levels of cytokines that are associated with inflammation, this property is distinct from its inability to efficiently replicate in a mammalian host

Influenza virus induced changes in STAT1 signalling during virus infection.

<p>(A) A549 cells were infected with either the H1N1/WSN, H9N2, H5N2/F118 or H5N3 viruses or (B) the pH1N1/471, pH1N1/478 or pH1N1/527 virus using an MOI = 4. Then cells were harvested at between 0.2 and 18 hpi in SDS-PAGE boiling mix as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033732#s3" target="_blank">methods</a>. The proteins were transferred on to PVDF membranes by western blotting, and the membranes probed with the appropriate primary and secondary antibodies. The phosphorylated STAT-1 (pSTAT1) and total STAT-1(STAT1) are shown. (A) β-catenin or (B) β-actin provides a loading control. (C) A549 cells were either mock-infected or were infected with the H1N1/WSN, H5N2, H9N2, H5N3, pH1N1/471 or pH1N1/527 viruses using an MOI = 4. At 16 hpi the cells were labelled using anti-MX and goat anti-mouse conjugated to Alexa555. The stained cells were visualised using a Nikon Eclipse 80i Microscope at ×20 magnification using appropriate machine settings.</p

Replication properties of the AIV and pH1N1 viruses in mammalian and avian cell types.

<p>A549, MDCK, and CEF cells were infected either with the H1N1/WSN (♦), H9N2 (▴), H5N2/F118 (▪) or H5N3 (○) viruses using an MOI = 4 and incubated at 37°C. (A) At hourly intervals post infection the cells were harvested and the vRNA levels quantified using qPCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033732#s3" target="_blank">methods</a>. Each value at a specific time point represents the mean of triplicate measurements (p<0.05). The data presented are a representative data set from one of two independent experiments. (B) At 9 hpi mock (M) infected cells or cells infected with either of the four viruses were radiolabelled for 1 hr in DMEM minus methionine (Invitrogen, USA) containing 100 µCi/ml [³⁵S] methionine (Perkin-Elmer, USA). Cells were extracted in boiling mix and analysed by SDS-PAGE. Protein bands corresponding to the neuraminidase protein (NA)/nucleoprotein (NP), the matrix (M) protein and nonstructural 1 (NS1) protein, and the uncleaved heamaggultinin (HA) are indicated. (C) Cells were infected with either the pH1N1/276 (♦), pH1N1/471 (▪), pH1N1/478 (▴), pH1N1/527(X) or H1N1/WSN (+) viruses, and at hourly intervals post infection the cells were harvested and the vRNA levels quantified using qPCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033732#s3" target="_blank">methods</a>. Each value at a specific time point represents the mean of triplicate measurements (p<0.05). The data presented are a representative data set from one of two independent experiments.</p

Comparison of the M gene vRNA levels at 10 hpi in influenza virus-infected A549, MDCK and CEF.

<p>Each cell line was infected with all viruses used in this study at an MOI = 4. The M gene copy numbers are shown per 10⁴ copies of the elongation factor (EF) gene in each host cell line. The vRNA levels are given by a log value. The average value and standard error are shown from triplicate measurements (p<0.05). Representative data from one of two independent experiments is shown.</p

Temporal changes in the expression levels of cytokine and interferon (IFN) and IFN-stimulated genes (ISG) in influenza virus-infected (A) MDCK and (B) CEF cells.

<p>Cells were infected using an MOI = 4, and at between 2 and 10 hpi the host cell mRNA levels compared with that in mock-infected cells. The data were obtained from 3 independent experiments, and probe sets showing either >2 or <−2 fold change (FC) in expression are indicated (p<0.05). In this representation up-regulated (red) or down-regulated (green) refer to the fold changes (FC) in gene expression compared to mock-infected cells in H1N1/WSN, H9N2 or H5N2/F118 virus-infected cells. Also shown are the probe identification (probe ID), accession numbers acquired from GeneBank (Gene symbol) and gene name (Gene title).</p

Temporal changes in (A) cytokine and interferon (IFN)-related gene and (B) IFN-stimulated gene (ISG) expression during influenza virus infection.

<p>A549 cells were infected with H1N1/WSN, pH1N1/527, H9N2 and H5N2/F118 viruses using an MOI = 4, and at between 2 and 10 hpi the host cell mRNA levels compared with that in mock-infected cells. The data were obtained from 3 independent experiments, and probe sets showing either >2 or <−2 fold change (FC) in expression are indicated (p<0.05). Expression profiles of up-regulated (red), down-regulated (green) and genes showing no change in expression (black) in H1N1/WSN, H9N2 and H5N2/F118 virus-infected A549 cells compared to mock-infected cells are shown. Also shown are the probe identification (probe ID), accession numbers acquired from GeneBank (Gene symbol). DEAD box polypeptide 58 is also known as RIG I protein, while mda5 is also known as IFN-induced helicase C domain-containing protein 1. In addition, the MX1 protein is homologous to the MXA protein in humans.</p

Virus titres (pfu/ml) from the tissue culture supernatant of influenza virus-infected MDCK, CEF and A549 cells.

<p>Each cell line was infected with each of the H1N1/WSN, H5N2/F118, H5N3 and H9N2 viruses using an MOI = 0.1 and 0.01 and incubated in DMEM containing 1 µg/ml TPCK trypsin and 0.21% BSA at 37°C. At 48 hpi the virus titres in the tissue culture supernatant were determined by agarose overlay plaque assay on MDCK cells. ND denotes no infectious virus particle detected. Representative data from one of two separate experiments is shown, and the average values are from duplicate measurements (SE<5%).</p