Developing Rapid Responses to Emerging Virus Infections of Poultry (DRREVIP)

Lead Research Organisation: Imperial College London
Department Name: Dept of Medicine

Abstract

The study of viruses of poultry made important contributions to the development of the modern science of virology, due to the ready availability of infectious materials and experimental subjects and by the fact that the majority of the poultry viruses posed no threat to humans. Subsequently the same factors allowed development of techniques for the assay and propagation of viruses in the laboratory, using eggs then tissue culture. These same techniques then played important roles in mammalian virology. Nowadays poultry virology helps protect us from emerging zoonotic viruses such as H5N1 and West Nile virus. It also helps protect the supply of the most important and universally acceptable sources of animal protein to feed a growing world population. That supply has to be protected throughout the sophisticated modern industry, from elite founder stocks in worldwide (including UK and European) breeder companies right down to the massive, obvious level of broiler and egg production. Infectious threats arise regularly, sometimes from previously unknown viruses.
Less obviously, poultry virology plays major roles in human health and in the Biotechnology and Pharmaceutical industries. Isolation and diagnosis of viruses is often conducted in eggs or avian cells, and some important vaccines (notably seasonal and pandemic influenza vaccines) are produced in them. Diagnosis and isolation of viruses that remain unknown, and their production to make new vaccines, will probably require the development of genetically modified eggs and chicken cells.
The UK has traditionally been strong in poultry virus research, arguably leading to the development of the influenza vaccine and to the discovery of antiviral interferon. It has also contributed extensively to work on emerging viruses. BBSRC plans increased effort in poultry virology in the new National Centre for Livestock Virology at IAH Pirbright, working alongside a new National Avian Research Facility at Roslin, Edinburgh. However, until those facilities are completed, the field faces difficulties in recruitment, retention, succession and critical mass.
This proposal addresses important scientific challenges in the design and development of modified cells to allow better isolation and diagnosis of emerging viruses as well as faster and better production of vaccines against them (to protect global food supply but also to benefit the Biotechnology and Pharmaceutical industries). It will cover endemic and exotic viruses as, in the poultry sector, new viruses rapidly cross national and continental boundaries to become global problems.
More importantly, it will help secure effective capacity in UK poultry virus research in advance of the new facilities. It establishes a small, focussed and focal network of university teams, with considerable experience of poultry virology, to recruit young post-doctoral scientists. It will be supported by a leading, representative researcher each from Roslin and IAH, offering network access to those facilities and assisting in managing the programme. Recruits will be trained in the demands of the academic sector, alongside and in collaboration with biomedical scientists, to publish in high impact journals and regularly aim for academic fellowships, yet they will receive strong guidance in poultry virology (and related disciplines) from their teams and the network, with strong support from the IAH and Roslin. The programme cannot cover all aspects of poultry virology and related disciplines but it is hoped that other centres of excellence with interests in poultry virology (e.g. Edinburgh, Belfast, Liverpool, Warwick, DEFRA's AHVLA) will participate in training and collaborating with those who will form the core of the next generation of UK poultry virologists. At the end, some recruits are expected to join the national poultry research facilities while others will remain in universities, but in strong positions to collaborate with those in the facilities.

Technical Summary

Whether emerging viruses present as clinical or sub-clinical, acute or persistent, affects how far they might spread before they are recognised. In either situation, interactions between the virus and cell will be crucial, as they will dictate whether or not the virus will be able to replicate and whether or not it induces the expression of host responses that might be used as key signals of otherwise inapparent infection.

These interactions will be affected by host genetic factors that influence innate antiviral responses, particularly the "cell-autonomous" or "intrinsic" responses, and by virus countermeasures. Consequently, this programme focuses on:

(i) characterizing innate and intrinsic responses, and the influence of different host genetic backgrounds;
(ii) isolating new substrates for virus diagnosis, analysis and propagation (by modifying cells and by producing transgenic chickens);
(iii) studying interactions between known viruses and the innate/intrinsic responses. MDV, ALV-J, IBDV and CAV are good examples of viruses causing sub-clinical and persistent infections. AIV and Newcastle disease virus (NDV) will also be studied because they have well recognised modulators of the mammalian innate/intrinsic responses that are likely to target the chicken (and other avian) responses in broad, but distinct, ways. If AIV were to return to poultry from mammalian reservoirs to which it has become adapted (rather than from ducks, as is usual) it might also be limited by its altered receptor tropism; and
(iv) streamlining recombinant vaccine development by quickly identifying appropriate epitopes and antigens to incorporate into the vaccine (particularly for pathogens with large genomes) that induce suitable responses across different chicken breeds.

In doing so, the programme will create also a cadre of new generation poultry virologists well trained and equipped to tackle the threat posed to global food security by emerging viruses of poultry.

Planned Impact

Potential beneficiaries of this research would be:

Commercial vaccine producers.
The work, though directly relevant to producers of poultry vaccines will have ramifications for the vaccine industry in the human sector. Benefits of the work, if it leads (directly or indirectly) to improved conventional live, killed, recombinant or subunit vaccines will be across the board: increased food security, wealth creation (vaccine producers, poultry producers), quality-of-life (reducing risk of pandemic flu, protecting supply of poultry meat and eggs - a key source of nutrition worldwide, protecting from infection).

Poultry Breeders.
Breeders will have new approaches and materials for diagnostic of incipient latent infections that might threaten their flocks.

Poultry Farmers.
Farmers will benefit from access to improved recombinant vaccines. Initially this will be via relatively high technology commercial producers. However, improvements in yield could reduce vaccine costs making them more accessible in developing countries and smaller-scale operations.

Replacement, Refinement and Reduction of Animals in Research (3Rs).
Most of the programme aims to improve and develop cell lines to allow more work with viruses and vaccines in vitro. In particular the technology to be developed in Goal 5 should to reduce animal usage and severity of procedures by early elimination of some vaccine candidates using just a few animals without the need for challenge experiments, leaving only the best vaccine candidates to receive the full and careful analysis using challenge experiments.

Publications


10 25 50
 
Description As the award enters its final year, the various components of the project are on track for their targets. A key finding last year was the ground-breaking demonstration, reported by Long et al in Nature (Species difference in ANP32A underlies influenza A virus polymerase host restriction. Long, J. et al. Nature, 2016; 529 (7584): 101 DOI: 10.1038/nature16474) of the identity of the gene that limits avian influenza virus replication to avian cells, unless they acquire specific mutations to allow them to replicate in mammalian cells, including human. Humans have the same gene but it is longer in all birds except ratites like the ostrich. Consistent with this discovery is the observation by others that avian influenza H5N1 isolated from ostriches looks just like a virus isolated from humans rather than one from chickens.
Last year we also published a manuscript that constitutes a key resource for the relevant community, describing the chicken interferome (Giotis, ES et al. Chicken
interferome: avian interferon-stimulated genes identified by microarray and RNA-seq of primary chick embryo fibroblasts treated with a chicken type I interferon (IFN-alpha). Vet Res. 2016 Aug 5;47(1):75. doi: 10.1186/s13567-016-0363-8).
The study of viruses of poultry made important contributions to the development of the modern science of virology, due to the ready availability of infectious materials and experimental subjects and by the fact that the majority of the poultry viruses posed no threat to humans. Subsequently the same factors allowed development of techniques for the assay and propagation of viruses in the laboratory, using eggs then tissue culture. These same techniques then played important roles in mammalian virology. Nowadays poultry virology helps protect us from emerging zoonotic viruses such as H5N1 and West Nile virus. It also helps protect the supply of the most important and universally acceptable sources of animal protein to feed a growing world population. That supply has to be protected throughout the sophisticated modern industry, from elite founder stocks in worldwide (including UK and European) breeder companies right down to the massive, obvious level of broiler and egg production. Infectious threats arise regularly, sometimes from previously unknown viruses. Less obviously, poultry virology plays major roles in human health and in the Biotechnology and Pharmaceutical industries. Isolation and diagnosis of viruses is often conducted in eggs or avian cells, and some important vaccines (notably seasonal and pandemic influenza vaccines) are produced in them. Diagnosis and isolation of viruses that remain unknown, and their production to make new vaccines, will probably require the development of genetically modified eggs and chicken cells. The UK has traditionally been strong in poultry virus research, arguably leading to the development of the influenza vaccine and to the discovery of antiviral interferon. It has also contributed extensively to work on emerging viruses. BBSRC plans increased effort in poultry virology in the new National Centre for Livestock Virology at IAH Pirbright, working alongside a new National Avian Research Facility at Roslin, Edinburgh. However, until those facilities are completed, the field faces difficulties in recruitment, retention, succession and critical mass. This proposal addresses important scientific challenges in the design and development of modified cells to allow better isolation and diagnosis of emerging viruses as well as faster and better production of vaccines against them (to protect global food supply but also to benefit the Biotechnology and Pharmaceutical industries). It will cover endemic and exotic viruses as, in the poultry sector, new viruses rapidly cross national and continental boundaries to become global problems. More importantly, it will help secure effective capacity in UK poultry virus research in advance of the new facilities. It establishes a small, focussed and focal network of university teams, with considerable experience of poultry virology, to recruit young post-doctoral scientists. It will be supported by a leading, representative researcher each from Roslin and IAH, offering network access to those facilities and assisting in managing the programme. Recruits will be trained in the demands of the academic sector, alongside and in collaboration with biomedical scientists, to publish in high impact journals and regularly aim for academic fellowships, yet they will receive strong guidance in poultry virology (and related disciplines) from their teams and the network, with strong support from the IAH and Roslin. The programme cannot cover all aspects of poultry virology and related disciplines but it is hoped that other centres of excellence with interests in poultry virology (e.g. Edinburgh, Belfast, Liverpool, Warwick, DEFRA's AHVLA) will participate in training and collaborating with those who will form the core of the next generation of UK poultry virologists. At the end, some recruits are expected to join the national poultry research facilities while others will remain in universities, but in strong positions to collaborate with those in the facilities. Extend and develop the scientific base in poultry virus research to maintain capacity pending commissioning of new national facilities. Aims: (1) address recognition of emerging viruses before wide dissemination of inapparent infections; (2) address need to prepare for emergence of new subtypes of avian influenza. Better substrates for production of vaccine viruses from (3) transgenic chickens or (4) designer cell lines; (5) faster identification of antigenic epitopes for recombinant vaccines. GOAL A: To create a cadre of new researchers who will form the core of the next generation of UK poultry virologists. GOAL 1 is the development of systems to recognize the pre-symptomatic presence of novel pathogens. Firstly spontaneously or artificially immortalized cell lines will be isolated from CEFs produced from chickens that differ in their genetic susceptibility to viral pathogens. Transcriptomic and proteomic analysis will be conducted on control and induced, or infected CEFs or cell lines, probing for signatures of altered cellular expression and metabolism, for which rapid assays will be developed to detect 'signatures' in culture or in infected embryonated eggs. These same 'omic approaches will also be applied elsewhere to support the other goals. 1.1 Development of spontaneously or artificially immortalized cell lines for the propagation of chicken pathogens 1.2 Identifying innate immune responses to persistent viruses using transcriptomics and proteomics of infected CEFs and cell lines. 1.3 Identifying innate immunity genes activated by virus infection of eggs 1.4 Development of qPCR macro-array for the detection of infection signatures 1.5 Development of indicator cell lines for the detection of chicken pathogens GOAL 2 (overlapping with generation of cell lines in Goal 1) will be the direct generation of cell lines more permissive to vaccine virus propagation, by expression of viral modulators or cellular regulators of the innate responses. 2.1 Modification of immortalised chicken cell lines to express putative virus and host-encoded antagonists of innate immunity 2.2 Modification of immortalised chicken cell lines with altered expression of cellular innate response genes 2.2(i) Development of a chicken-specific RNAi screening pipeline. 2.2(iii) Derivation of chicken cell lines more permissive to avian viruses. 2.2(iv) Identification of chicken host factors that enhance antiviral responses. GOAL 3 is the identification of viral and host determinants affecting the outcome of infection with AIV, including HPAI subtypes H5 and H7, to assist in evaluation of whether we need prepare for emergence of novel subtypes into poultry. 3.1 Construction of cell lines to investigate host-range factors for influenza virus 3.2 Generation of rg AIV to study the role of NS1. 3.3 Generation of rgAIV encoding GFP to study in vivo tropism in chickens. 3.4 Generation of rg reassortants of pH1N1 and AIV to test chicken infectivity. GOAL 4 will be the production of trangenic chicken lines in which innate immune suppressor genes are induciblyexpressed, to significantly increase the yield of vaccine viruses produced in embryonated eggs, and in CEFs derived from them. 4.1 Virus in vivo dynamics studies. 4.2 "Super-eggs" enhanced vaccine production vehicles. 4.2.1 Construction of inducible expression plasmids for combinations of IIS genes and chicken ST6GAL1. 4.2.2 Construction of transgenic chickens. 4.3 Comparison of yields and isolation efficiency in transgenic eggs. GOAL 5 is the development and evaluation of a new high-throughput method of determining in vivo T cell responses, to assist recombinant vaccine production, starting with IBDV as a model before tackling the large genome of MDV. 5.1 Developing class I and class II pipelines using selected peptides from IBDV 5.2 Validating both peptide pipelines using the genome of IBDV 5.3. Scanning the MDV proteome using overlapping peptide
Exploitation Route The approaches to cell engineering that we are developing will be of use to the veterinary vaccine industry
Sectors Agriculture, Food and Drink,Environment,Government, Democracy and Justice,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
URL https://www.sciencedaily.com/releases/2016/01/160106143025.htm
 
Description We are in the first year of the project, so we have not finished developing the reagents for outside use.
 
Description Chair of Health & Safety Executive's (HSE) Scientific Advisory Committee on Genetic Modification (Contained Use) (SACGM(CU)
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
URL http://webcommunities.hse.gov.uk/connect.ti/SACGM/grouphome
 
Description Global Challenges Research Fund Strategy Advisory Group
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description Prof Wendy Barclay: Member of Department of Health's New and Emerging Respiratory Virus Threats Advisory Group (NERVTAG)
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
Impact The New and Emerging Respiratory Virus Threats Advisory Group (NERVTAG) is an expert committee of the Department of Health (DH), and advises the Chief Medical Officer (CMO) and, through the CMO, to ministers, DH and other Government departments on the threat posed by new and emerging respiratory viruses. It provides scientific risk assessment and mitigation advice on the threat posed by new and emerging respiratory virus threats and on options for their management
URL https://www.gov.uk/government/groups/new-and-emerging-respiratory-virus-threats-advisory-group
 
Description Small Project Grant
Amount £9,937 (GBP)
Funding ID E Giotis 
Organisation The Houghton Trust 
Sector Charity/Non Profit
Country Unknown
Start 11/2014 
End 10/2015
 
Description Studentship Identification of genetic markers in the haemagglutinin glycoprotein critical for antigenic activity of H9N2
Amount £118,000 (GBP)
Funding ID BBS/E/I/00001759 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 10/2015 
End 09/2019
 
Title ArrayExpress Accession E-MTAB-3711 
Description Affymetrix GeneChip CEF +/- IFN 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact Giotis et al Veterinary Research (2016) 
URL http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-3711/
 
Title ArrayExpress Accession E-MTAB-3712 
Description ST Array CEF +/- IFN 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact Giotis et al Vet Res (2016) 
URL http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-3712
 
Title E-MTAB-3643 
Description Affymetrix Chicken Gene 1.0 ST Arrays "The identification of positive chicken factors for influenza polymerase activity" 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
Impact Long et al. Nature (2016) 
URL https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-3643
 
Title Chicken TRIM15/"IgY precipitating reagent"/ ZNF777 
Description Chicken IgY binding protein By Frederic Sorgeloos & Ian Goodfellow (University of Cambridge) 
IP Reference  
Protection Protection not required
Year Protection Granted
Licensed Yes
Impact Valuable research tool
 
Description Cabaret of Dangerous Ideas: Dolly the Sheep: major discovery or minor distraction? Presenter, Edinburgh Festival Fringe. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Presentation with two colleagues, one a stem cell biologist and one a histrian of science in society, discussing the impact on research and society of the birth of Dolly the Sheep 20 years ago. Potential of GM technologies to confer resistance to diseases in farmed animals was discussed. Plenty of opportunity for audience contributions to debate.
Year(s) Of Engagement Activity 2016
URL http://www.youtube.com/watch?v=LClmL5C9YJE&feature=youtu.be
 
Description Come Dine With The Future 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact What will we be eating in 50 years time? H Sang and 4 other presenters described what will be on their dinner menu, considering advances in technology and challenges of sustainability
Year(s) Of Engagement Activity 2016
URL http://www.youtube.com/watch?v=Bkta-s7z9DY
 
Description Genome Editing and the Future of Farming 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact On the 6th September The Roslin Institute opened its doors to an array of delegates from the worlds of research, industry and policy to discuss genome editing and its role in the future of farming, with many international colleagues including from USDA and OECD.
The global challenge of food security is time critical as we will need to produce 70% more food by 2050 without destroying the environment. Innovations in food production techniques are urgently required. Editing the genomes of crops and livestock offers new possibilities to address this complex issue. The meeting discussed the status of the technologies in crops and farmed animals and the associated regualtory and societal challenges were debated, lead by presenters with a broad array of relevant expertise. My role was as chair of a session.
Year(s) Of Engagement Activity 2016
URL http://www.nib.ac.uk/reporting-on-the-first-nib-specialist-meeting-genome-editing-and-the-future-of-...
 
Description Interview for the magazine Science & Vie (Science & Life, the n°1 science magazine in France) 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Interview for an article on genetically modified farm animals resistant to infectious diseases.
Year(s) Of Engagement Activity 2017
 
Description NERC Public Engagement Strategy information event "Public engagement: discussing GM animals" 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Presented on "Public engagement: discussing GM animals" to illustrate the benefits, interest and challenges of public engagement on a potentailly controversial topic
Year(s) Of Engagement Activity 2016
 
Description Planned, introduction and chairing SGM debate on Influenza "Gain-of-Function" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Wendy Barclay planned & introduced debate on Influenza "Gain-of-Function" controversy at Society for General Microbiology Annual Conference, Birmingham 30 Mar-1 Apr 2015. Mike Skinner chaired the debate which was attended by >300 scientists (including many PhD students) and regulators.
Year(s) Of Engagement Activity 2015
URL http://www.microbiologysociety.org/all-microsite-sections/microbiology-today/index.cfm/article/8D4A5...