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Adaptive evolutionary pathways of highly pathogenic avian H5N1 influenza in humans

Periodic Reporting for period 1 - HAAIV (Adaptive evolutionary pathways of highly pathogenic avian H5N1 influenza in humans)

Reporting period: 2015-11-01 to 2017-10-31

- What is the problem/issue being addressed?
Highly pathogenic H5N1 influenza viruses are endemic in wild birds and poultry across several Asian countries, and continue to cause human infections with high mortality. Global concern persists that these avian influenza viruses will evolve into viruses that can efficiently transmit between humans and may then cause a severe influenza pandemic. However, estimating the real risk of such a pandemic scenario is difficult due to limited understanding of the evolutionary requirements of H5N1 viruses to cross the species barrier and adapt to humans. There is therefore an urgent need to increase our insight into H5N1 influenza evolution within the human host and identify and characterize previously unrecognized adaptive evolutionary pathways.

- Why is it important for society?
In view of the continuing widespread circulation amongst birds and occurrences of severe human infections, global concerns persist of an imminent devastating H5N1 pandemic if these viruses were to evolve towards efficient transmission between humans. This concern is reinforced by recent experimental research in ferrets showing that airborne transmission of H5N1 viruses only would require a limited number of genetic changes. The focus of the proposed project is to better understand the pandemic risk of HPAI H5N1 viruses by detailed analyses of viral evolution during actual human infections in Indonesia and Vietnam and phenotypic characterization of viral variants observed during these infections.
Society and in particular policy makers and public health authorities can benefit from this research by using the results of the research to improve our ability to predict, prevent and intervene in outbreaks of HPAI H5N1.

- What are the overall objectives?
The aim of the proposed research is to identify evolutionary pathways involved in adaptation of avian influenza viruses to humans. This aim will be achieved by using whole genome virus sequence analyses from clinical specimens obtained from birds and H5N1-infected patients (Aim 1), followed by phenotypic characterization of mutations potentially relevant for human adaptation using recombinant viral proteins and viruses (Aim 2)
Aim 1. Identify and characterize viral evolutionary pathways of potential relevance for human adaptation of HPAI H5N1 viruses at quasispecies levels
To identify evolutionary pathways towards human adaptation, we will characterize the complexity of viral populations and viral evolution of avian influenza viruses in humans and in birds. By comparing frequencies of specific mutations between human and avian HPAI H5N1 infection and by analyzing the dynamics of specific variants during the course of human infection in serially obtained clinical specimens, mutations potentially relevant for human adaptation will be identified.

Aim 2. Phenotypic characterization of evolutionary changes of HPAI H5N1 in humans and their effect on pathogenesis and human adaptation
To determine the biological relevance of specific mutations identified under aim 1, these will be further characterized by phenotypic assays. We will introduce single mutations and combinations thereof in H5N1 reference viruses and investigate the effects on protein function and virus replication. It is well known that both receptor specificity as well as polymerase activity of influenza viruses have been implicated to play an important role in human adaptation. We will therefore focus on characterization of receptor specificity of the hemagglutinin protein of H5N1 and polymerase activity of variants of the polymerase subunits PB2, PB1 and PA.
The expected results will yield new insights in the common evolutionary processes of the Highly pathogenic avian influenza H5N1 hemagglutinin and polymerase complex and new knowledge on the possible routes of human adaptation. These findings will contribute to improving epidemic and pandemic risk assessment, clinical impact assessment and provide important new insights into the design of novel antiviral drugs and outbreak control measures.
Figure 1. Schematic representation of infection of humans with avian influenza (zoonosis).