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

Characterization of avian H9N2 and human 2009 pandemic influenza viruses isolated in Singapore

Influenza A virus infection imposes major public health problem. In the past, there were several influenza pandemic outbreaks that killed millions of people. One of the most notable is the 1918 "Spanish Flu", followed by "Asian Flu" in 1957 and "Hong Kong Flu" in 1968. Since then, highly pathogenic avian influenza virus H5Nl outbreaks have been highlighted for the potential of the pandemic emergence. The emergence of high pathogenic avian influenza (HP AI) strains was resulted from the mutation of low pathogenic avian influenza (LPAI) strains. Similarly, H9N2 influenza viruses also have spread around the world, from wild birds to domestic poultry, especially in Asia. There is a documented evidence of the infection of H9N2 influenza viruses in humans, thus posing them as potential pandemic risks. Recently the 2009 pandemic swine influenza virus also emerged in April 2009, which has reached pandemic level, and co-circulates with seasonal influenza virus.DOCTOR OF PHILOSOPHY (SBS

A System Based-Approach to Examine Cytokine Response in Poxvirus-Infected Macrophages

The poxviruses are large, linear, double-stranded DNA viruses about 130 to 230 kbp, that have an animal origin and evolved to infect a wide host range. Variola virus (VARV), the causative agent of smallpox, is a poxvirus that infects only humans, but other poxviruses such as monkey poxvirus and cowpox virus (CPXV) have crossed over from animals to infect humans. Therefore understanding the biology of poxviruses can devise antiviral strategies to prevent these human infections. In this study we used a system-based approach to examine the host responses to three orthopoxviruses, CPXV, vaccinia virus (VACV), and ectromelia virus (ECTV) in the murine macrophage RAW 264.7 cell line. Overall, we observed a significant down-regulation of gene expressions for pro-inflammatory cytokines, chemokines, and related receptors. There were also common and virus-specific changes in the immune-regulated gene expressions for each poxvirus-infected RAW cells. Collectively our results showed that the murine macrophage RAW 264.7 cell line is a suitable cell-based model system to study poxvirus host response

A systems-based approach to analyse the host response in murine lung macrophages challenged with respiratory syncytial virus

Background: Respiratory syncytial virus (RSV) is an important cause of lower respiratory tract infection in young children. The degree of disease severity is determined by the host response to infection. Lung macrophages play an important early role in the host response to infection and we have used a systems-based approach to examine the host response in RSV-infected lung-derived macrophage cells. Results: Lung macrophage cells could be efficiently infected (>95%) with RSV in vitro, and the expression of several virus structural proteins could be detected. Although we failed to detect significant levels of virus particle production, virus antigen could be detected up until 96 hours post-infection (hpi). Microarray analysis indicated that 20,086 annotated genes were expressed in the macrophage cells, and RSV infection induced an 8.9% and 11.3% change in the global gene transcriptome at 4 hpi and 24 hpi respectively. Genes showing up-regulated expression were more numerous and exhibited higher changes in expression compared to genes showing down-regulated expression. Based on gene ontology, genes with cytokine, antiviral, cell death, and signal transduction functions showed the highest increases in expression, while signalling transduction, RNA binding and protein kinase genes showed the greatest reduction in expression levels. Analysis of the global gene expression profile using pathway enrichment analysis confirmed that up-regulated expression of pathways related to pathogen recognition, interferon signalling and antigen presentation occurred in the lung macrophage cells challenged with RSV. Conclusion: Our data provided a comprehensive analysis of RSV-induced gene expression changes in lung macrophages. Although virus gene expression was detected, our data was consistent with an abortive infection and this correlated with the activation of several antivirus signalling pathways such as interferon type I signalling and cell death signalling. RSV infection induced a relatively large increase in pro-inflammatory cytokine expression, however the maintenance of this pro-inflammatory response was not dependent on the production of infectious virus particles. The sustained pro-inflammatory response even in the absence of a productive infection suggests that drugs that control the pro-inflammatory response may be useful in the treatment of patients with severe RSV infection.Published versio

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

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

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