Response of human primary monocyte-derived macrophages to infection with highly pathogenic human influenza a virus subtype H5N1

by Cheung, Chung-yan

(Uncorrected OCR) Abstract of thesis entitled: Response of human primary monocyte-derived macrophages to infection with highly pathogenic human influenza A virus subtype H5N1 Submitted by Cheung Chung Yan for the degree of Doctor of Philosophy at The University of Hong Kong in December 2004 Abstract The ability to affect adversely the host cell, host organism or population of host organisms determines the virulence of a virus. Influenza A viruses have been responsible for three pandemics of severe human respiratory disease this century. Avian species harbour a reservoir of influenza viruses of diverse subtypes, which can contribute genes to generate new pandemic human strains through genetic reassortment with prevailing human influenza strains. Since 1997, purely avian influenza viruses of subtypes H5N1, H7N7 and H9N2 have caused human disease, with fatal consequences in the instance of H5N1 and H7N7 influenza viruses. The biological basis for severe human disease associated with influenza A H5N1 remains unclear. This study addresses the hypothesis that cytokine dysregulation is a significant factor in severe human disease caused by H5N1 infection. In this study, we investigate the gene expression profile of human primary monocyte-derived macrophages in response to influenza virus infection in vitro. Macrophages are a biologically relevant model as they are one of the major cell types in the respiratory system and a major source of cytokines. This study document that H5N1 virus genotypes associated with severe human disease, but not human H1N1 or H3N2 viruses, or other H5N1 virus genotypes, induce excessive levels of pro-inflammatory cytokines in macrophages infected in vitro. Cytokines such as tumour necrosis factor (TNF)-a, interferon (IFN)-P, and interleukin (IL)- 1P were greatly induced early after infection, with a later phase induction of chemokines including CXCL-10/Interferon-inducible protein (IP)-10, CCL2/macrophage chemotactic protein (MCP)-1, CCL3/Macrophage inflammatory protein (MIP)-1a, and CCL4/MIP-1p. These cytokines may contribute to the pathogenesis of the disease on their own or through downstream cascades. Microarray analysis and the use of specific inhibitors of different kinases suggest that the p38 MAP signaling pathway mediates excessive pro-inflammatory cytokine production in H5N1 infected macrophages. Work on naturally occurring reassortant H5N1 and related viruses suggest that the virus internal genes mediate the high induction of pro-inflammatory cytokines in vitro. Experiments performed with viruses created by reverse genetics suggest that the nonstructural gene of the H5N1 viruses from 1997 may contribute to hyper-induction of proinflammatory cytokines. However, it appears that multiple genes of a particular gene constellation determine the ability to induce high pro-inflammatory cytokines in macrophages. Defining the biological basis for the hyper-induction of pro-inflammatory cytokines provide avenues for the development of new therapeutic intervention in human disease associated with highly pathogenic human influenza viruses. Such understanding is of particular importance since H5N1 influenza A virus has spread through poultry across many countries in Asia and has real potential to become a major threat to human health. Word count : 418