Investigating the role of ANP32A in the replication of Avian Influenza Virus

Influenza viruses infect domestic animals including poultry and pigs, causing loss of income, social and economic disruption. Avian influenza remains one of the major threats to food security. Adaptation of avian influenza in animal hosts leads to viruses that are transmitted in humans and carries the risk of driving emergent influenza pandemics. We have investigated a family of host proteins, ANP32, that are co-opted by influenza virus to support its replication. The difference between ANP32A in chickens and mammalian hosts accounts for one of the host range barriers that protect pigs and humans from zoonotic infections by avian influenza viruses. Avian influenza virus RNA-dependent RNA polymerase is unable to utilise the mammalian ANP32A protein unless it adapts by mutation of the viral PB2 protein. We have previously shown that the form of the protein ANP32A present in flighted birds differs from that in mammals due to presence of an additional exon encoding 33 amino acids that contribute to the mature protein. We have preliminary data suggesting that removal of this exon reduces or avian influenza virus replication in chicken cells. We will identify the specific regions of ANP32A needed for viral protein interactions. We will use new genome editing tools that we have developed to modify chicken cells to test our predictions of which targeted changes to ANP32A will have the most significant effect on avian influenza virus replication. We will generate a series of mutations of ANP32A and test their effects on avian influenza polymerase function and virus replication. We will determine any global changes in the RNA transcriptome of the edited cells containing modified ANP32A protein. We will investigate the function of ANP32A in genome edited animals. This information will inform control strategies for protection of commercial poultry from AIV infection. It will also be of interest to researchers studying influenza virus in humans and livestock.

Influenza viruses infect domestic animals including poultry and pigs, causing loss of income, social and economic disruption. Avian influenza remains one of the major threats to food security. Adaptation of avian influenza in animal hosts leads to viruses that are transmitted in humans and carries the risk of driving emergent influenza pandemics. We have investigated a family of host proteins, ANP32, that are co-opted by influenza virus to support its replication. The difference between ANP32A in chickens and mammalian hosts accounts for one of the host range barriers that protect pigs and humans from zoonotic infections by avian influenza viruses. Avian influenza virus RNA-dependent RNA polymerase is unable to utilise the mammalian ANP32A protein unless it adapts by mutation of the viral PB2 protein. We will first identify the specific regions of ANP32A needed for viral protein interactions. We will use new genome editing tools that we have developed to modify chicken cells, to identify functional regions/amino acids by testing the effectiveness of specific mutations to abrogate these host:viral interactions. We will determine any global changes in the RNA transcriptome of the edited cells containing modified ANP32A protein and characterise any other effects of these modifications, unrelated to avian influenza infection. We will investigate the function of ANP32A in genome edited animals.

Agricultural losses to avian influenza have major impacts through recurring outbreaks in tropical environments mostly in developing countries. Small holder farmers and subsistence farmers lose valuable resources (in terms of livestock) during these outbreaks. The Gates Foundation has invested in promoting scavenger chicken to improve the livelihoods of subsistence farmers, with a strong focus on women who are usually the primary owners of rural chicken. The Gates Foundation is also investing in research at The Roslin Institute (through the Centre for Tropical Livestock Genetics and Health) to develop disease resistance using genetic approaches in poultry, to alleviate the need for vaccination programmes.
Agricultural losses by high pathogenic avian influenza are also severe in developed countries. The 'bird flu' outbreak in the USA in 2015 led to the culling of 48 million layer chickens and turkeys. The economic loss from this outbreak is estimated to be $1.3 billion. Most of these losses were due to the loss of the export market as live poultry products can no longer be exported from affected countries during HPAI incidents.
The major advances in understanding the interaction between specific host genes and influenza virus replication that we propose to investigate here will inform genetic strategies for control of avian influenza in production chickens.
Another primary beneficiary of our research will be the research community, especially those researchers working in avian biology or poultry production. We will exemplify the development and use of new genetic tools for investigating gene function and viral resistance in poultry. This will enable researchers to replace some animal model challenges with cell-based challenges. Poultry researchers will also benefit by the example of creating chicken genetic models that can be used to investigate pathways of infections and resistance.