SUMMARY. Highly pathogenic avian influenza viruses (HPAIVs) (“bird flu”) devastate the poultry industry, threaten wildlife, damage economies, and constitute a permanent pandemic threat. HPAIVs emerge from low pathogenic avian influenza viruses (LPAIVs) upon transmission from wild waterfowl (e.g., ducks, geese, gulls), their main reservoir, to terrestrial poultry (e.g., chickens, turkeys). The transition from LPAIV to HPAIV results from the insertion of nucleotides coding for multiple basic amino acids in the protease cleavage site of the viral hemagglutinin (HA) gene during replication of the viral genome by the influenza virus polymerase. This change in HA leads to systemic virus dissemination characterized by an endotheliotropism in poultry with mortality rates up to 100%. In contrast, systemic virus dissemination, severe disease and endotheliotropism upon HPAIV infection are rare or absent in most species of duck, wild and domestic. Interestingly, the transition from LPAIV to HPAIV has only been observed in influenza viruses of the H5 and H7 subtypes. Moreover, although LPAIVs circulate extensively in wild waterfowl, there is no evidence that they can evolve into HPAIVs in these species. HPAIV emergence is currently unpredictable because the mechanisms of initial emergence through nucleotide insertion by the influenza virus polymerase, and subsequent process of natural selection in avian hosts remain poorly understood. To understand the molecular mechanism of nucleotide insertion, we have recently predicted subtype-specific RNA stem-loop structures at the HA cleavage site. Here, we hypothesize that the stem of the stem-loop structure refolds during viral RNA replication leading to the template closing on itself, trapping the polymerase in the loop and causing it to stutter and insert nucleotides. Additionally, we hypothesize that specific RNA sequences present in H5 and H7 stem-loops determine why insertions only occur in these subtypes. To test these hypotheses, we successfully developed in vitro polymerase assays, including single-molecule assays, with which nucleotide insertions in HA RNA can be reliably detected with high throughput via circular resequencing. Secondly, we hypothesize that intrinsic differences in the ability of HPAIV to spread systemically in poultry versus waterfowl determine the process of natural selection of HPAIVs from LPAIVs and explain the host species-specificity of HPAIV emergence. More specifically, we hypothesize that HPAIV are selected in poultry and not in waterfowl because of their endotheliotropism in poultry supporting their systemic dissemination. To test this hypothesis, we designed competition experiments between LPAIV and HPAIV to study the natural selection at the host level in chickens (as a model for poultry) and ducks (as a model for waterfowl) and at the cellular level using newly developed in vitro transwell co-culture models of primary respiratory and intestinal epithelial and endothelial cells of chickens and ducks. Increased knowledge about HPAIV emergence will fill crucial knowledge gaps on influenza and may provide a point of action to predict – and thus possibly control - HPAIV emergence and subsequent outbreaks that are threatening animal and human health.

PROJECT NARRATIVE Highly pathogenic bird flu viruses are a major burden for the poultry industry, animal welfare, ecology and a threat to human health. We will study the mechanisms underlying their emergence from low pathogenic forms. This research will fill major gaps in our knowledge and ultimately help to control bird flu outbreaks.