Identification of the molecular basis of differential host responses to rapidly evolving Avian Influenza viruses in different avian species

Avian Influenza, caused by the highly pathogenic avian influenza virus (HPAIV) H5N1, is taking a huge toll on the Indian poultry industry since the first outbreak in 2006. Despite eradication and confirmation of disease free status, re-emergence continues. Since each outbreak needs eradication of all poultry within a 3km radius, every episode has a major economic and social impact on small and marginal poultry farmers of rural India. This is particularly acute in India where backyard poultry is popular and thickly populated villages usually lie within the eradication zone. As a notifiable disease, Avian Influenza can have serious implications for international trade and further harm the poultry industry of India. Due to its zoonotic potential it has public health concerns and owing to the co-habitation of poultry and humans with a culture of live bird markets, there is always a fear of the emergence of pandemic flu.

HPAI H5N1 affects several avian species, including domesticated chickens, turkeys, quails, guinea fowl as well as wild birds; however the response to infection varies widely. Ducks and waterfowl are often resistant i.e. they become infected but are capable of clearing the virus or carry the virus without symptoms, and act as reservoirs. In contrast, poultry are highly susceptible i.e. they become infected and are not able to clear the virus, which results in high mortality. Since 2006 Avian Influenza outbreaks in India have mainly affected domestic chickens. Strikingly in the 2011 outbreak, high mortality was also observed in crows and ducks, previously assumed to be resistant to H5N1 infection. The mechanisms that promote high pathogenicity of these recent clades of AIV in normally resistant species are not completely understood, but may be caused by viral mutations that facilitate virus replication and dysregulation of the host immune response.

Though the pathogenesis of AIV in chickens is known to some extent, little is understood about mechanisms of resistance in other birds, which can act as reservoirs of infection to poultry. At the molecular level, resistant species may have pathways able to check disease progression and/or ameliorate immunopathology. With many avian genomes sequenced and with the availability of cutting edge tools, like RNAseq, it is now possible to examine global responses to viral infection in any bird. We propose a comparative transcriptomic approach to identify molecular signatures responsible for the differences in host susceptibility to H5N1 seen between avian species. We propose an integrated approach, combining viral pathology, transcriptome analysis, bioinformatics and functional tests of genes in vitro and in vivo, complemented by corresponding analyses of the differences in viral genes that confer higher virulence in the resistant species to investigate the molecular mechanisms underlying these species differences.

Six avian species, chickens/turkeys (highly susceptible with heavy mortality), geese/pigeons (carriers with sporadic mortality) and ducks/crows (resistant to most AIV infections but having differential response to virus of different clades), will be infected with H5N1 from different clades, as well as LPAIV H9N2. Global responses to infection will be studied by RNAseq, bioinformatics and cytohistochemistry of critical tissues. Comparison of pathways involved in host responses in susceptible/resistant species and different AIV clades will be used to identify putative host resistant genes. Their roles will be tested in knockdown and over-expression experiments. The roles of sequence differences between virus isolates that show differential pathogenicity in normally resistant birds will be probed by reverse genetics and other functional assays. The knowledge gained from these comparisons can be used to develop sustainable strategies to control Avian Influenza infections in domestic poultry.

We will use an integrated approach that combines virology, genomics and bioinformatics to investigate the molecular nature of differences in viral resistance between avian species and the pathogenicity of viral clades. The overall strategy is to compare groups of birds that differ in their resistance/susceptibility to clades of avian influenza viruses.

Two experimental comparisons will be made. The first will compare host responses between chickens/turkeys (susceptible) vs. ducks/geese/crows/pigeons (resistant) against HPAI (H5N1 clade 2.2). Controls will be mock and LPAI (H9N2) infections. The second experiment will compare host responses between ducks/crows (susceptible) vs. geese/pigeons (resistant) against HPAI (H5N1 clade 2.3.2.1). Controls will be mock, LPAI (H9N2) and HPAI (H5N1 clade 2.2).

The differential patterns of gene expression detected in these comparisons will be further investigated by network analyses to define gene expression modules. These in turn will be classified for enrichment for gene classes, gene ontology terms, KEGG pathways, etc. Dysregulation of genetic regulatory networks will be analysed from a joint analysis of the expression of gene regulators (transcription factors, cofactors, and miRNAs) and predicted targets. Using coding sequences predicted from the sequences of 48 avian genomes we will examine the rates of evolution of these genes and associate any differences between species using branch and branch-site models of evolutionary change (PAML).

From a meta-analysis of results combined with other studies on avian influenza virus (RNAi, yeast2hhybrids, etc.) we will define host genes (and viral mutations in different clades) that are likely to play a role in host resistance. The expression of these genes will be confirmed by qPCR, in situ hybridisation and immunocytochemistry. The role of these genes will tested further in over-expression and knockdown studies in experimental avian cells.

Though the pathogenesis of Avian Influenza virus in chickens and ducks is known to some extent, little is understood about the mechanisms of resistance in other birds, in which each species can act as reservoirs of infection for poultry and human populations. This project will directly address the lack of knowledge of the genes and mechanisms controlling resistance to Avian Influenza virus in different avian species by exploiting the latest sequencing and gene expression technologies. Together with access to novel resources and facilities (multiple avian genome sequences, access to high containment facilities for avian influenza disease challenge, high throughput sequencing facilities, bioinformatics skills and resources, etc.) this will allow us to identify candidate genes, pathways and ultimately genetic differences between species that control resistance to this virus.

Beneficiaries from this research will therefore include academics interested in the genetics and evolution of disease resistance, host-pathogen interactions and the immune system. Results will be published, when appropriate, in peer-reviewed scientific journals and will be presented at various national and international conferences. Transcribed sequences and gene expression data will be submitted to the appropriate international databases for future access.

The grant will also facilitate the training and development of the postdoctoral researchers involved, both from the UK (e.g. genomics and bioinformatics) and Indian (virology and disease containment) sides, with the exchange of personnel between the laboratories helping foster longer term collaboration with a strategically important overseas trading partner.

Effective industry uptake of the outcomes of our research will be facilitated by close links with Biosciences KTN, who identified disease resistance and animal health as the top priorities for UK breeding industries. Also, previously-established contacts with companies such as Pfizer, Aviagen and HyLine will ease this transfer of knowledge from the academic environment to industry. This work has the potential to impact on the sustainability of the poultry industry in the UK, and thus to inform DEFRA policy. It also therefore falls within the remit of the BBSRC Food Security Strategy Board.

In scientific terms this project directly addresses the Joint BBSRC-DBT Call for Collaborative Proposals to support Farmed Animal Disease and Health (FADH) Research, in particular in the Priority Areas (Genetics of Host Disease Resistance and Novel Diagnostic Tools) and Cross-cutting Themes (Pathogen Biology).

Investigating Pathogen Biology at the Molecular Level: A basic understanding of the biology of pathogens is essential for development of effective prevention and intervention strategies. For example:

"Better knowledge of pathogens, their (intermediate) hosts, diversity, virulence and drug sensitivity (including genomic approaches to understand the expression pattern and role of individual genes in colonisation, pathogenesis and transmission; host's immune response; transmission pathways and host specificity).

"Basic immunological research to understand susceptibility and resistance to infection."

It also falls within the remit of the BBSRC 'Animal systems, health and wellbeing' Research Committee and matches the BBSRC scientific priority of 'Food Security' in terms of 'Animal Health' and 'Livestock Production'.