Identification of interferon stimulated genes that restrict cross-species transmission of influenza A virus.

Influenza A virus (IAV) is a major pathogen affecting many species, including humans, pigs and chickens. Seasonal epidemic outbreaks cause significant disease and death in humans, while pandemic outbreaks pose more catastrophic consequences as reflected by the 1918 Spanish Flu outbreak that resulted in more than 40 million deaths. Outbreaks of "bird flu" and "swine flu" result in large scale food production losses, resulting in severe strain on the world economy.

The natural host of IAV is aquatic birds, like ducks, where the virus causes few signs of disease. However, IAV is able to jump to new species, where it can cause more severe symptoms. The occurrence of viral jumps from one species to another is relatively rare due to the difficulty of adapting to a new species. The host interferon (IFN) response represents a major barrier to this occurring.

IFN is a protein released by cells in response to a virus infection. Once secreted, IFN binds to receptors of surrounding cells and triggers production and activation of hundreds of antiviral genes. Viruses that are well adapted to the host cell have evolved multiple mechanisms to counteract the IFN response, enabling efficient virus replication. However, these counter-measures can be less effective when the virus jumps to a new host, due to evolutionary divergence of antiviral genes. For a virus to successfully jump to a new species it must adapt through mutation of its genome to counteract the host IFN response, allowing efficient replication and ultimately spread through the population.

While much work has focused on understanding the IFN system in human cells, less attention has been paid to other species. The flow of IAV between poultry, pigs and humans underlies pandemic outbreaks by aiding exchange of genetic material between viruses adapted to these species. Understanding the IFN response in pigs and chickens and the viral adaptations required when switching between these hosts is therefore crucial to evaluating the pandemic risks associated with specific strains of IAV.

While the antiviral nature of IFN has long been documented, the specific mechanisms of how IFN inhibits different viruses is poorly understood. Recently, more than 500 human genes induced by IFN have been individually cloned, allowing overexpression and investigation of their effects on multiple viruses. This has proven to be a particularly powerful approach, resulting in significant discoveries on the action of IFN in human cells.

In this application, we propose generating similar libraries of pig and chicken genes upregulated by IFN to evaluate their effects on IAV. In addition, we will evaluate the inhibitory effects of these genes in cross-species studies, by overexpressing antiviral genes from one species, in the cells of another, to determine their ability to inhibit strains of IAV adapted to specific host species. We predict that these studies will significantly contribute to our understanding of the IFN response in pigs and chickens, identify genes that are important for inhibiting IAV replication in pigs and chickens and identify IFN genes that represent barriers to IAV jumping from one host species to another. Finally, the generation of these libraries will also be highly valuable for future studies on how the IFN response effects other important pig and chicken pathogens.

Influenza A virus (IAV) causes major epidemic and pandemic outbreaks in important livestock species such as pigs and chickens, as well as human. Interferon (IFN) represents an important early host defence to such viral infections. Furthermore, evolutionary differences in host IFN response in different species represents a major barrier to zoonotic and epizootic IAV infection and adaptation.

Systematic analysis using arrayed Interferon Stimulated Gene (ISG) expression libraries have proven a powerful method for identifying key components of the IFN response in human cells. However, such libraries are not available for pig and chicken, limiting our understanding of the species-specific differences in response and susceptibility to infection. Therefore, we propose generating ISG expression libraries for pig and chicken, using available transcriptomic data, and to use these libraries to define important components of the IFN response against swine and avian IAV.

In addition, we will perform screens expressing the human ISG library in pig and chicken cells, infected with swine and avian IAV respectively, to identify species-specific ISGs, which will be characterised to determine whether they play a role in restricting zoonotic and epizootic infections. we will also investigate the viral adaptations that allow viruses to overcome these species specific barriers.

From this work, we will identify species-specific genetic mechanisms of cellular resistance to flu virus, revealing the molecular architecture against which the virus evolves to achieve host-jumps and defining methods to assess the pandemic potential of flu virus strains.

1. Improve pandemic risk assessment of emerging IAV strains
Pandemic influenza A virus outbreaks constitute a major health threat to humans and livestock species with implications for food security and animal welfare. Zoonotic and epizootic infections underpin the potential threat posed by IAV, providing the conditions for successful adaptation, leading to effective spread of highly pathogenic strains. Our current understanding of the factors required for successful zoonotic infection and adaptation is currently limited. A systematic approach will greatly improve our understanding of which host factors represent an effective barrier against zoonotic infection. Based on this knowledge we can investigate the viral adaptations required to overcome species-specific barriers and therefore predict potential risks associated with emerging virus strains.

2. Generation of valuable data for comparative systematic analysis
Genetic and genomic studies on livestock species has many advantages over equivalent studies in humans, due to detailed historical breeding records, availability of large data sets and significantly fewer ethical hurdles related to access to clinical samples and records. However, livestock research is at a major disadvantage in molecular high-throughput approaches due to the lack of available reagents and expertise. Bridging the gap between genomic studies and high throughput molecular approaches will provide a powerful approach for identifying key genetic factors underlying desirable phenotypes such as resistance to infection and enable generation of improved livestock. In addition, genome-wide CRISPR-Cas9 KO libraries for pig and chicken are currently being generated, funded by a BBSRC Tools and Resources grant (BB/N021738/1). These libraries will be used to generate genome-wide loss of function screens for IAV in the same cell lines used in this study, allowing direct correlation of the two studies. This will provide greater statistical robustness and greater depth to potential global understanding of host virus interactions in these species.

3. Generation of valuable reagents for systematic screens in livestock species
As mentioned above, there is a distinct lack of molecular tools for systematic screens in livestock species. While this application focuses on IAV in pig and chicken cells, the broad potential for application of these libraries is obvious with potential utility against a range of important pig and chicken pathogens. The human libraries have provided a wealth of discoveries in many different important pathogens, as well as discoveries on the fundamental aspects of the human interferon response. We expect the pig and chicken libraries to generate the same level of impact as the human libraries. We believe the interest in using these libraries will be high, as reflected by the supporting letters from potential collaborators who are keen to gain access to the reagents and expertise that this application will provide.

4. Establishment of high throughput gain of function screens in livestock species
We have already generated proof of principle data showing that the lentivirus system is applicable to pig and chicken cell lines. This application will further establish our ability to perform high throughput gain of function screens in these species. While the libraries proposed in this application focus on ISGs, the libraries can be expanded to include most coding and non-coding genes. The possible application of this approach is highlighted by the section in the grant that includes genes identified from large scale genetic and genomic studies on influenza. The same approach could be used for other pathogen infections or for other families of genes.