Genetics and Genomics: Chickens and Culicoides midges

Lead Research Organisation: The Pirbright Institute
Department Name: UNLISTED

Abstract

We will initiate the first large-scale de novo genome sequencing project of biting midges, Culicoides spp. This complements ongoing work in the VBD programme. Culicoides are vectors for a number of important pathogens of livestock. The complete genome sequence of these species will be of global significance in creating an entirely new area for Culicoides research with the potential to develop novel control methodologies and elucidate areas such as vector competence that remain poorly understood. The genomic sequence of Culicoides will underpin advances in improving food security by protecting UK agriculture from these potentially catastrophic exotic animal diseases. IAH is the curator of a vital BBSRC resource - 7 inbred and 2 out-bred scientifically important, chicken lines. These lines are a key resource for the AID programme for the study of host-pathogen interactions, especially relating to susceptibility and resistance to disease. They are also available to other groups, providing access to data on other diverse traits, such as gut health and functionality. It is essential that the genetic integrity of the IAH chicken lines is monitored and maintained. Whilst continuing the traditionally microsatellite genotyping we will now be developing a new more comprehensive SNP genotyping method for flock monitoring.
 
Description 'Genetics and Genomics: Chickens and Culicoides midges' has allowed additional research to be conducted that leveraged funding for ongoing research.

This data is now published and being used by the international Culicoides research community.
The use of the new genomic technologies has led to major advances in control of several arboviruses of medical importance such as Dengue. However, the development of tools and resources available for vectors of non-zoonotic arboviruses remains neglected. Biting midges of the genus Culicoides transmit some of the most important arboviruses of wildlife and livestock worldwide, with a global impact on economic productivity, health and welfare. The absence of a suitable reference genome has hindered genomic analyses to date in this important genus of vectors. In the present study, the genome of Culicoides sonorensis, a vector of bluetongue virus (BTV) in the USA, has been sequenced to provide the first reference genome for these vectors. In this study, we also report the use of the reference genome to perform initial transcriptomic analyses of vector competence for BTV.
Results: Our analyses reveal that the genome is 197.4 Mb, assembled in 7,974 scaffolds. Its annotation using the transcriptomic data generated in this study and in a previous study has identified 15,629 genes. Gene expression analyses of C. sonorensis females infected with BTV performed in this study revealed 165 genes that were differentially expressed between vector competent and refractory females. Two candidate genes, glutathione S-transferase (gst) and the antiviral helicase ski2, previously recognized as involved in vector competence for BTV in C. sonorensis (gst) and repressing dsRNA virus propagation (ski2), were confirmed in this study.
Conclusions: The reference genome of C. sonorensis has enabled preliminary analyses of the gene expression profiles of vector competent and refractory individuals. The genome and transcriptomes generated in this study provide suitable tools for future research on arbovirus transmission. These provide a significant resource for these vector lineage, which diverged from other major Dipteran vector families over 200 million years ago. The genome will be a valuable source of comparative data for other important Dipteran vector families including mosquitoes (Culicidae) and sandflies (Psychodidae), and yield potential targets for transgenic modification in vector control and functional studies.

Culicoides biting midges (Diptera: Ceratopogonidae) are a major vector group responsible for the biological transmission of a wide variety of globally significant arboviruses, including bluetongue virus (BTV). In this work we examine current biological resources for the study of this genus, with an emphasis on detailing the history of extant colonies and cell lines derived from C. sonorensis, the major vector ofBTV in the USA. We then discuss the rapidly developing area of genomic and transcriptomic analyses ofbiological processes in vectors and introduce the newly formed Culicoides Genomics and TranscriptomicsAlliance. Preliminary results from these fields are detailed and finally likely areas of future research are discussed from an entomological perspective describing limitations in our understanding of Culicoides biology that may impede progress in these areas.

We have also published the full genome sequence of this species and this has also resulted in additional collaborations and funding for sequencing of further species that are known vectors of animal disease.
Exploitation Route This grant is complete and has successfully produced the first ever genome sequence of the disease insect vector of the Culicoides genus. This will be of significnae to researchers in the vector borne disease area, and beyond. Having published the full genome sequence of this species, this has resulted in additional collaborations and funding for sequencing of further species that are known vectors of animal disease.
Sectors Agriculture, Food and Drink,Communities and Social Services/Policy,Environment,Healthcare,Pharmaceuticals and Medical Biotechnology

URL http://www.pirbright.ac.uk/research/GeneticsGenomics/Default.aspx
 
Description This grant has allowed research to be conducted that leveraged funding for ongoing research. We have also published the full genome sequence of this species and this has also resulted in additional collaborations and funding for sequencing of further species that are known vectors of animal disease.
First Year Of Impact 2012
Sector Agriculture, Food and Drink,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description Contributed to Chief Medical Officers report- Genomics section
Geographic Reach Europe 
Policy Influence Type Implementation circular/rapid advice/letter to e.g. Ministry of Health
URL https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/6310...
 
Description LVIF
Amount $1,500,000 (CAD)
Organisation Bill and Melinda Gates Foundation 
Sector Charity/Non Profit
Country United States
Start 04/2018 
End 09/2020
 
Description Pathfinder BB/R012431/1 Grant holder: Dr Mark Fife. Grant title: chIFITM knockdown/knockout technology as a platform technology for increased vaccine yields in SPF Eggs.
Amount £12,500 (GBP)
Funding ID BB/R012431/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2017 
End 02/2018
 
Title A Versatile Panel of Reference Gene Assays for the Measurement of Chicken mRNA by Quantitative PCR 
Description Quantitative real-time PCR assays are widely used for the quantification of mRNA within avian experimental samples. Multiple stably-expressed reference genes can be used to control random technical variation between samples. It is necessary to select reference genes with the lowest variation in representative samples. The candidate reference gene assays must be reliable. In particular, they should have high amplification specificity and efficiency, and not produce signals from contaminating DNA. Whilst recent research papers identify specific genes that are stable in particular tissues and experimental treatments, here we describe a panel of ten avian gene primer and probe sets that can be used to identify suitable reference genes in many experimental contexts. The panel was tested with TaqMan and SYBR Green systems in two experimental scenarios, a tissue collection, and virus infection of cultured fibroblasts. GeNorm and NormFinder algorithms were able to select appropriate reference gene sets in each case. We show the effects of using the selected genes on the detection of statistically significant differences in expression. The results are compared with those obtained using 28s ribosomal RNA, the present most widely accepted reference gene in chicken work, identifying circumstances where the use of this gene might provide misleading results. Widely used methods for eliminating DNA contamination of RNA reduced, but did not completely remove, detectable DNA. We therefore attached special importance to testing each qPCR assay for absence of signal using DNA template. 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? Yes  
Impact The assays and analyses developed here provide a useful resource for selecting reference genes for investigations of avian biology. 
 
Title IFITM Seq 
Description We have established that chickens encode interferon-inducible transmembrane (IFITM) orthologues that restrict influenza infection in vitro. The aim of our work is to determine the biology and genetic variation of the IFITM locus in chickens, specifically the ability of IFITM genes and alleles to protect the host against both endemic and emerging avian viruses. The output of this project is to identify alleles that correlate with resistance to a number of avian viruses allowing poultry breeding programs to select the beneficial alleles. We have now produced contiguos sequnce for the IFITM Locus that was previously not represented in the Gallus gallus genome. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2016 
Provided To Others? Yes  
Impact We have filed patents for the develpment of vaccines. Patent filed for The Pirbright & Sanger Institutes "Viral resistance genes (IFITM genes) in chickens". International Patent Application No. PCT/GB2014/051693. 
 
Description EBI Culicoides genome project 
Organisation EMBL European Bioinformatics Institute (EMBL - EBI)
Country United Kingdom 
Sector Academic/University 
PI Contribution Performed experiments and provided material for Sequencing and genome build of the Culicoides genome project
Collaborator Contribution Sequencing and genome build for Culicoides genome project.
Impact Publication and public release of genome data for Culicoides genome project
Start Year 2013
 
Description Genetic mapping of vector competence in Culicoides sonorensis. 
Organisation EMBL European Bioinformatics Institute (EMBL - EBI)
Country United Kingdom 
Sector Academic/University 
PI Contribution The primary deliverable of this project will be the elucidation of the genetic basis of vector competence of C. sonorensis for bluetongue virus (BTV) and African horse sickness virus (AHSV). It will also generate the first accurately annotated genome of a Culicoides species worldwide.
Start Year 2012
 
Description Horizon Discovery Ltd Cambridge Research Park, United Kingdom 
Organisation Horizon Discovery Group plc
PI Contribution Verify IFITM knock-out cell by qPCR and immuno-blotting. Verification of further knock-out cell lines by DNA sequencing, immuno-blotting and qPCR Cell infection with Avian Influenza Viruses Assess the viral titre of cells infected with Influenza Viruses in K/O and wildtype cells. Quantitative analysis of viral infection in IFITM K/O cells Demonstrate increased AIV viral titres at lab scale and estimation of what this may equate to with respect to vaccine dose yields. Analysis of IFITM K/O cell lines permissivity to a range of additional animal viruses (Relevant to the LVIF). Milestone 7: Demonstrate permissivity increased and viral titres for a range of LVIF relevant viruses in the edited cell lines.
Collaborator Contribution Develop a CRISPR/cas9 gene editing system to specifically target the chIFITM locus in avian cell culture
Impact Grant funding
Start Year 2017
 
Description ILRI Research Collaboration (Kenya, March 2017) 
Organisation International Livestock Research Institute (ILRI)
Country Kenya 
Sector Charity/Non Profit 
PI Contribution Exchange of ideas and samples for indigenous breeds of livestock
Collaborator Contribution Contributed samples for analysis.
Impact Analysis of indigenous breed genomic sequences for Immune function genes.
Start Year 2017
 
Description Industrial partnership-Aviagen 2018 
Organisation Aviagen Group
Country United States 
Sector Private 
PI Contribution We have established a significant industrial partnership with Aviagen group Ltd. The nature of this work remains confidential.
Collaborator Contribution The nature of this work remains confidential.
Impact The nature of this work remains confidential.
Start Year 2018
 
Description Research Collaboration with Nigeria (23rd - 28th January 2017) 
Organisation University of Nigeria
Country Nigeria 
Sector Academic/University 
PI Contribution Analysis of rare breed indigenous chicken in Nigeria.
Collaborator Contribution Helped with sampling and background information of the breed.
Impact Analysis is ongoing.
Start Year 2017
 
Description Roslin 
Organisation University of Edinburgh
Department The Roslin Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Joint Grant: 'Role of AKT1 & SIVA1 in resistance to avian salmonellosis' Stevens, Kaiser & Fife
Collaborator Contribution Poultry are a key reservoir of human Salmonella infections owing to the ability of some strains to colonise the avian intestines and reproductive tract. Birds often carry the bacteria in the absence of overt symptoms; however some types of Salmonella cause severe typhoid-like diseases in poultry that exert substantial welfare and economic costs. Global population growth and rising affluence are fuelling demand for poultry meat and eggs, and a need exists to enhance the supply and safety of such. Though vaccines are used in layers in some countries, most of the 55 billion chickens reared annually worldwide lack protection against Salmonella infection. We and others have discovered that some chickens exhibit heritable differences in resistance to Salmonella. It may be feasible to selectively breed for birds with improved resilience to Salmonella infection; however this requires the identification of resistance-associated factors and knowledge of how they act. By analysing the genetic material of birds that differ in resistance, we have located a region of the chicken chromosome that confers protection against typhoidal salmonellosis, both in laboratory studies and commercial poultry populations. Recent studies have now resolved the region associated with resistance to just a handful of genes. It is highly plausible that variation affecting two genes in this region (AKT1 and SIVA1) explains why birds react to Salmonella in different ways, as the encoded proteins control host processes that impact on the fate of bacteria. For example, AKT1 and SIVA1 control the death of infected cells and the induction of immune responses, but have opposing activities. It is not possible for us to predict how the genetic changes affecting these genes will alter their expression or activity. Moreover, it is unclear how such factors may control the growth and spread of Salmonella in birds. We therefore propose to: 1. Examine if birds normally respond to Salmonella infection by activating the expression or function of AKT1 and SIVA1. We will examine this in cells cultured from chickens, as well as in intact birds, and associate any differences with host responses and the fate of the bacteria. 2. Examine if lines of chicken known to differ in resistance to Salmonella infection vary in the levels or activation of AKT1 and SIVA1. 3. Use specific inhibitors and bacterial strains to establish that AKT1 activation is necessary for Salmonella to grow and spread in birds. 4. Define the nature, frequency and consequences of genetic changes affecting AKT1 and SIVA1 in commercial poultry populations. This will aid the selective breeding of chickens that show improved resilience to Salmonella infection. We are fortunate to have the support of one of the world's largest poultry breeding companies (Erich Wesjohann Group), who will provide birds, genome sequences, expertise and 10% of total project costs. This reflects the value of the proposed studies to the industry. The consortium has productively collaborated and the proposed studies are a timely, logical and feasible extension of our recent joint research.
Impact SAL1 Patent for Salmonella resistance in chickens"-. PCT/GB2010/000850. The Pirbright Institute.
Start Year 2012
 
Description SANGER IFITM 
Organisation The Wellcome Trust Sanger Institute
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution The Pirbright Institute (TPI) is a world leader in research on viral diseases of livestock with a strong track record in translating this research into effective disease control. The Genetics and Genomics group is interested in understanding the genetics and evolution of animal-pathogen interactions, with the strategic aim of improving the control of viral disease outbreaks. The group's main focus involves the study of the genetic basis of avian viral disease resistance. The virus-restricting IFITM proteins are under investigation in this study, thus contributing directly to the BBSRC key strategic priority on Global Food security.
Collaborator Contribution Wellcome Trust Sanger Institute (WTSI) is a world leader in uncovering the genetic basis of disease including infectious disease, providing results that are translated into diagnostics, treatments or therapies that reduce global health burdens. The virus genomics group investigates human pathogenic virus infections and the molecular genetic determinants of disease pathogenesis and transmission including zoonotic virus infections.
Impact S. Smith, M. S. Gibson, R. Wash, F. Ferrara, E. Wright, N. Temperton, P. Kellam, and M. Fife. (2013) Chicken IFITM3 restricts Influenza viruses and Lyssaviruses in vitro. J. Virology. 87, 12957 Benfield C, Smith SE, Wright E, Wash RS, Ferrara F, Temperton NJ, Kellam P. Bat and pig Interferon-Induced Transmembrane Protein 3 restrict cell entry by influenza virus and lyssaviruses. J Gen Virol. 2015 (in press)
Start Year 2014
 
Description USDA Culicoides 
Organisation U.S. Department of Agriculture USDA
Department National Animal Disease Center
Country United States 
Sector Public 
PI Contribution Combined data for Culicoides genome project to enable publication in high impact journal.
Collaborator Contribution Transcriptomics data for vector competence analysis and genome build.
Impact Combined data for Culicoides genome project to enable publication in high impact journal.
Start Year 2014
 
Title Chicken IFITMs 
Description The present invention provides an avian cell in which the expression or activity of one or more of the following genes, or a homologue thereof: Chicken IFITM1 (SEQ ID No. 1); Chicken IFITM2 (SEQ ID No. 2) and Chicken IFITM3 (SEQ ID No. 3) is reduced. The invention also provides methods for passaging viruses in avian cells, embryos and/or avian cell lines which have reduced expression of one or more IFITM genes and methods which involve investigating the sequence of one or more of the following genes, or a homologue thereof: Chicken IFITM1 (SEQ ID No. 1); Chicken IFITM2 (SEQ ID No. 2) and Chicken IFITM3 (SEQ ID No. 3). 
IP Reference PCT/GB2014/051693 
Protection Patent granted
Year Protection Granted 2018
Licensed Commercial In Confidence
Impact The present invention provides an avian cell in which the expression or activity of one or more of the following genes, or a homologue thereof: Chicken IFITM1 (SEQ ID No. 1); Chicken IFITM2 (SEQ ID No. 2) and Chicken IFITM3 (SEQ ID No. 3) is reduced. The invention also provides methods for passaging viruses in avian cells, embryos and/or avian cell lines which have reduced expression of one or more IFITM genes and methods which involve investigating the sequence of one or more of the following genes, or a homologue thereof: Chicken IFITM1 (SEQ ID No. 1); Chicken IFITM2 (SEQ ID No. 2) and Chicken IFITM3 (SEQ ID No. 3). This patent has enabled us to leverage funding for continued work.
 
Title SAL1 Patent for Salmonella resistance in chickens 
Description SAL1 Patent for Salmonella resistance in chickens 
IP Reference PCT/GB2010/000850 
Protection Patent application published
Year Protection Granted
Licensed No
Impact Secured further funding and allowed Link grant with commercial company.
 
Description Chair of organising Committee for Wellcome Trust-Animal Genetics and Diseases 2017 
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 This meeting brought together specialists working on the interface between genomics, genetic engineering and infectious disease with the aims of improving animal and human health and welfare.

Scientific sessions included:
Genetics of immune responses and disease resistance
Genetically engineered livestock (including genome editing)
Quantitative genetics and epigenetics applied to disease
Epidemiology and pathogen evolution
Bioinformatics, comparative and functional genomics
Precision medicine of animal companions
Year(s) Of Engagement Activity 2017
URL https://coursesandconferences.wellcomegenomecampus.org/events/item.aspx?e=635&dm_i=2SUU,HOGH,4R4AW1,...
 
Description Encouraging women into science and engineering STEM. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact MH Visited Warwick School, Redhill to talk to secondary students about careers in STEM. A very positive outcome and well received.
Year(s) Of Engagement Activity 2018
 
Description Holt School Surrey. Bee meadow & A-level outreach Talk. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact I discussed the importance of pollinators for biodiversity and food security with the younger students. I also discussed careers in science with A-Level students. There was good interaction and many questions surrounding both topics.
Year(s) Of Engagement Activity 2018
 
Description Innovate Guildford Outreach (12th March) 
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 With amazing exhibits, activities, workshops, talks and competitions, it's FREE to attend. Explore the four zones: Engineering, One Health, Digital Media and Future Living, experience interactive performances, hear inspiring speakers (including leading gaming pioneer Peter Molyneux and animal health pioneer Professor Nick Bacon) and take in an amazing atmosphere. Theatre, music and comedy will feature through the day, with everything from how technology might have affected Shakespeare's plays to risky recycling and photographic secrets.
Year(s) Of Engagement Activity 2017
URL https://www.guildford.gov.uk/innovateguildford
 
Description Innovate Guildford Outreach (12th March) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Innovate Guildford Outreach (12th March).
Year(s) Of Engagement Activity 2016
 
Description Kingdown School Warminster Wiltshire talk to 6th form students about career in science. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact I presented my career path to the students to engage them on STEM careers. I had a good level of discussion and many questions.
Year(s) Of Engagement Activity 2018
 
Description Organiser of the Animal Genetics and Diseases conference 08 - 10 May 2019 Wellcome Genome Campus, UK. Highlighting recent advances in animal genetics and genomic technologies. 
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 I am on the organising committee for this conference. The second conference in this series will highlight recent advances in animal genetics and genomic technologies. It will bring together specialists working on the interface of genomics, genetic engineering and infectious disease with the aims of improving animal and human health and welfare.

Novel genomic technologies, mathematical modelling and quantitative genetics approaches, applied to host animals, as well as their pathogens, have transformed the understanding of animal diseases, host-pathogen interactions and epidemiology and their effects on productivity of farmed animal species and food supply chains.

This year's conference will not only put the spotlight on the immune response of host animals and epidemiology but also cover the genetics and genomics of pathogens and the impact of animal-human relationships.

We encourage registrations from researchers, breeders and technical specialists interested in learning and disseminating the latest cutting-edge techniques and methodologies across model species, wildlife, farmed animals and companion animals.
Year(s) Of Engagement Activity 2019
URL https://coursesandconferences.wellcomegenomecampus.org/our-events/animal-genetics-diseases-2019/?dm_...
 
Description STEM Outreach Event (Farnborough, 9th November) 
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 See web link
Year(s) Of Engagement Activity 2016
 
Description Taiwan -UK Partnering Award: Surveillance of influenza viruses 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Attended the International Flu Virus Symposium for Celebrating IPM 47th Anniversary" in Tri-Service General Hospital in Taipei, Taiwan.

Tri-Service General Hospital address:
No. 325, Sec. 2, Chenggong Rd., Neihu Dist., Taipei City 114, Taiwan (R.O.C.)

Invited speaker for medical staff and research scientists.
Year(s) Of Engagement Activity 2019
URL https://www.cdc.gov.tw/rwd/english
 
Description Tillingbourne bug hunt. New Rd, Chilworth, Guildford GU4 8NB 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Stimulating increased interest in science and research. Good interest and engagement.
Year(s) Of Engagement Activity 2018
 
Description Winston Churchill Careers Fair. Winston Churchill School, Hermitage Road, St Johns, Woking, Surrey, GU21 8TL 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact AS attended and presented at this event to stimulate increased interest in science and research.
Year(s) Of Engagement Activity 2018