This dissertation, "Viral Determinants of Influenza A (H5N1) Associated TNF-a Hyper-induction in Human Primary Monocyte-derived Macrophages" by Hing-ki, Charmaine, Wong, 黃馨琦, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled: "Viral determinants of influenza A (H5N1) associated TNF-α hyper-induction in human primary monocyte-derived macrophages" Submitted by Wong Hing Ki Charmaine for the Degree of Master of Philosophy at The University of Hong Kong in August 2006 Abstract Since the first emergence of highly pathogenic H5N1 avian influenza in 1997, the virus has continued to genetically reassort and evolve to become increasingly pathogenic with an expanding host range. Between late 2003 and mid 2006, the newly emerged Z genotype H5N1 avian influenza has spread across ten Asian countries and beyond Southeast Asia, causing high morbidity and mortality in aquatic birds, poultry and humans. The pathogenesis of these highly virulent H5N1 viruses in humans is still largely unknown. Recent findings have suggested that virus-induced cytokine dysregulation may play a crucial role in H5N1 pathogenesis. Investigation of the viral determinants responsible for such cytokine imbalance is critical to the further understanding of the possible mechanisms underlying the disease, and may help define potential therapeutic interventions in human disease associated with H5N1 for better influenza pandemic preparedness. Previously it has been demonstrated that H5N1/97 causes hyper-induction of pro-inflammatory cytokines, most notably TNF-α, in primary human macrophages in vitro. Here, I further explored the TNF-α induction potential of the recent Z genotype H5N1 viruses in a human macrophage model and the viral determinants involved in high TNF-α induction. By measuring mRNA levels using quantitative PCR and protein levels using ELISA, the Z genotype viruses were found to be potent inducers of TNF-α, with some strains inducing up to 4 folds greater than that induced by H5N1/97. In the light of these findings, the viral determinants responsible for the TNF-α hyperinduction were further investigated. I hypothesized that TNF-α hyperinduction in primary human macrophages in vitro may be related to differences in viral morphology, or to certain viral genetic determinants. In this study, it was demonstrated that TNF-α hyperinduction was not associated with the morphology of virus particles, but was closely linked to the presence of certain H5N1 viral gene components. By employing reverse genetics, recombinant viruses containing genes from H5N1 (A/Vietnam/1203/2004) and genes from a low TNF-α inducing virus, H1N1 (A/WSN/33), were constructed to further explore the genetic contribution of the 8 viral segments of Z genotype H5N1 viruses towards TNF-α hyper-induction. Here, it was demonstrated that while recombinants containing the H5 NS, M or NP gene alone in an H1N1 genetic background did not confer high TNF-inducing phenotype, high TNF-α induction was likely a polygenic trait and was observed in recombinants that had specific combinations of H5 genes. One of these combinations involved the H5 replication complex (NP/PA/PB1/PB2), while another combination involved the H5 surface proteins in additional to the H5 matrix proteins (HA/NA/M). Both of these combinations led to significantly increased TNF-α production in primary human macrophages in vitro. DOI: 10.5353/th_b3765902 Subjects: Tumor necrosis factor Avian influenza A virus Viral genetics Macr