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

Lead Research Organisation: University of Edinburgh
Department Name: The Roslin Institute

Abstract

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.

Technical Summary

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.

Planned Impact

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'.

Publications

10 25 50
 
Description Our has been to understand why some birds, such as the chicken are very susceptible to avian influenza, whilst others, such as the mallard duck are resistant. In this work we discovered that ducks have a gene called IFITM1 that may confer increased resistance to avian influenza. Chickens are not able to use IFITM1 to fend off flu in the same way as ducks can.
Data analysis is still ongoing for this project, but final outcomes will include:
-A profile of the host response to avian influenza infection in 6 bird species (each showing different susceptibilities to infection)
-An analysis of infection over time in 3 tissues (lung, ileum and brain)
-Comparison of the effects of low path flu infection with high path infection
-Validation of candidate genes by in vitro assays
- A list of genes which can be examined in the future for variants associated with viral resistance
Exploitation Route Knowledge of genes involved in resistance to avian influenza may be used to search for genetic markers in these genes to select for resistance.
Sectors Agriculture, Food and Drink

URL http://www.ncbi.nlm.nih.gov/pubmed/26238195
 
Title India flu - in vitro assays 
Description Development of in vitro assays for assessment of candidate gene function in response to flu infection 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact These assays allow us to verify the effects of candidate genes upon flu infection, without using whole animal experiments 
URL https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4523026/
 
Title India flu - chicken transcriptome 
Description Transcriptomes from brain, lung and ileum tissue from chicken in response to high and low path avian flu 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact Identification of genes involved in resistance/susceptibility to avian flu 
 
Title India flu - crow transcriptome 
Description Transcriptomes from brain, lung and ileum tissue from crow in response to high and low path avian flu 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact Identification of genes involved in resistance/susceptibility to avian flu 
 
Title India flu - duck transcriptome 
Description Transcriptomes from brain, lung and ileum tissue from duck in response to high and low path avian flu 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact Identification of genes involved in resistance/susceptibility to avian flu 
 
Title India flu - goose transcriptome 
Description Transcriptomes from brain, lung and ileum tissue from goose in response to high and low path avian flu 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact Identification of genes involved in resistance/susceptibility to avian flu 
 
Title India flu - pigeon transcriptome 
Description Transcriptomes from brain, lung and ileum tissue from pigeon in response to high and low path avian flu 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact Identification of genes involved in resistance/susceptibility to avian flu 
 
Title India flu - turkey transcriptome 
Description Transcriptomes from brain, lung and ileum tissue from turkey in response to high and low path avian flu 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact Identification of genes involved in resistance/susceptibility to avian flu 
 
Description India DBT collaboration 
Organisation Indian Council of Agricultural Research
Department National Institute of High Security Animal Diseases
Country India 
Sector Public 
PI Contribution Using the tissues collected in Bhopal, we have sequenced and analysed the avian transcriptomic response to avian influenza challenge in 6 bird species (chicken, turkey, crow, goose, pigeon, duck).
Collaborator Contribution All influenza challenge experiments were carried out by the team in Bhopal, tissues collected and sent over to the UK for processing and analysis
Impact Identification of candidate genes for resistance to avian flu.
Start Year 2014
 
Description Annual conference of Microbiology society 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Annual conference of Microbiology society -April 2017 (Edinburgh, UK) IFITM talk
Year(s) Of Engagement Activity 2017
 
Description Avian genome evolution and the origins of species diversity. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited lecture: Avian genome evolution and the origins of species diversity. In "Molecular evolution of avian species", SMBE2016, Queensland's Gold Coast, Australia, 3-7 July, 2016.
Year(s) Of Engagement Activity 2016
 
Description BBSRC-DBT Livestock Health and Disease workshop (2012), Delhi, India in Feb 2012 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact BBSRC-DBT Livestock Health and Disease workshop (2012), Delhi, India in Feb 2012

no actual impacts realised to date
Year(s) Of Engagement Activity 2012
 
Description Bhopal visit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk and visit: Avian genomics (2015) 11-13 Feb, Bhopal, India.
Year(s) Of Engagement Activity 2015
 
Description Edinburgh India Institute Inaugural conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact "Identification of the molecular basis of differential host responses to rapidly evolving Avian Influenza viruses in different avian species" in Edinburgh India Institute Inaugural conference -"Innovative Engagement for Sustainable Development: the Edinburgh-India Story" held in 15-16 MAY 2014 at University of Edinburgh, UK
Year(s) Of Engagement Activity 2014
 
Description Edinburgh India Institute, Inaugural conference on 15-16 May 2014 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact Gave a short talk about avian influenza BBSRC-DBT project and how it came about, joint talk with our India collaborators. Edinburgh India Institute, Inaugural conference on 15-16 May 2014. John McIntyre Conference Centre, Edinburgh. Conference theme: Innovative Engagement for Sustainable Development: the Edinburgh-India Story.
Year(s) Of Engagement Activity 2014
 
Description Flu Fighters 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact The Roslin Institute Open day October 2017; Flu Fighters (Lonneke Vervelde)
Year(s) Of Engagement Activity 2017
URL https://www.ed.ac.uk/roslin/news-events/latest-news/easter-bush-campus-open-day
 
Description Flu talk - journalists 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Presentation of our work on avian flu to a selection of Indian and Japanese journalists. Government representatives were also present. Interest in future Scottish/Indian proposals was shown.
Year(s) Of Engagement Activity 2015
 
Description Influenza update meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Influenza Update meeting - Nov 2017 (Edinburgh, UK) Talk on India/Tripura M segment morphology work
Year(s) Of Engagement Activity 2017
 
Description Meeting of the Indian Society of Animal Genetics and Breeding 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A. Mishra, P Vijayakumar ,AA(2017) Meta-analysis approach for deciphering the molecular pathogenesis of Avian Influenza in susceptible and tolerant avian species. XIII Annual Meeting of the Indian Society of Animal Genetics and Breeding & National Conference on "Challenges in Quantitative Genetics for Improvement of Indigenous Animal Genetic Resources (AnGR)" held on January 19-20, 2017
Year(s) Of Engagement Activity 2017
 
Description Munster flu meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact 5th international influenza meeting. IFITM talk. Munster, Germany. Sep 2016
Year(s) Of Engagement Activity 2016
 
Description New tools and prospects for chicken genomics 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk at Genetics Society of Australia (GSA), July 2015, University of Adelaide, Australia
Year(s) Of Engagement Activity 2015
 
Description PAG 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited speaker at Plant and Animal Genome conference 2018 (San Diego, USA) 'Avian flu: the winners and the losers' - Jan 2018
Year(s) Of Engagement Activity 2018
 
Description Past, Present and Future of Genomics in Poultry Breeding. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk: Past, Present and Future of Genomics in Poultry Breeding. 25th World's Poultry Congress, Beijing, 5-9 September, 2016.
Year(s) Of Engagement Activity 2016
 
Description Poultry Breeders Roundtable 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact • Invited speaker at National poultry breeders roundtable meeting (St Louis, USA) - JS May 2017
Year(s) Of Engagement Activity 2017
 
Description Production Animal Health and Welfare Research: Impacts and Opportunities. 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk at Production Animal Health and Welfare Research: Impacts and Opportunities. 19-20 Feb, 2015, Indian Council for Agricultural Research (ICAR), New Delhi, India
Year(s) Of Engagement Activity 2015
 
Description Reception hosted by Jim Eadie MSP 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Reception hosted by Jim Eadie MSP. Guest: Dr Gopalkrishna Gandhi, who will deliver a lecture on: Independence: Dream and Reality. Venue: Committee Room 2, The Scottish Parliament. Date and time: 30 September 2014, 6.00pm
Year(s) Of Engagement Activity 2014
 
Description Roslin flu meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact 'Avian flu: the winners and the losers' at Annual influenza meeting, Roslin Nov 2017
Year(s) Of Engagement Activity 2017
 
Description Statesman article 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Article: David W. Burt (2014) Genome sequencing of poultry and other birds presents new opportunities for breeding, and combating avian influenza and other endemic diseases. "The Statesman Feb 2014".
Year(s) Of Engagement Activity 2014
 
Description Talk to CEVA 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Talk to CEVA about avian flu - (Sep 2017 Roslin)
Year(s) Of Engagement Activity 2017
 
Description Veterinary Advances in Animal Health and Welfare Research 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Development of New Tools for Genetic Selection for a Sustainable Poultry Industry (2014), Veterinary Advances in Animal Health and Welfare Research - Impact and Opportunities, Feb 20-24, 2014, NIANP Auditorium, Adugodi, Bangalore, India
Year(s) Of Engagement Activity 2014