Host cell determinants of BoHV-1 pathogenesis: a genome wide analysis.

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

Abstract

Infectious bovine rhinotracheitis (IBR) is highly contagious respiratory and genital tract disease of cattle caused by a herpesvirus, Bovine herpesvirus -1 (BoHV-1). Clinical symptoms of the respiratory disease include high fever, weight loss, coughing, excessive salivation, nasal discharge, conjunctivitis, inflamed nasal passages, and breathing difficulties. Abortions can also occur in pregnant cattle. IBR results in significant distress to the animal and significant economic losses to the cattle industry.
Like most viruses BoHV-1 relies on the host cell to provide the machinery need to replicate the virus. However, infected cells also make proteins which inhibit virus replication and the outcome of infection depends on the balance between pro and anti-virus cell factors. Cells contain about 20-30,000 genes and little is known about which of these genes encode proteins that inhibit, or are essential for, BoHV-1 replication. An understanding of the host factors involved in viral replication is important in developing not only genetic approaches to disease resistance, but also new vaccines and treatments.
Previously we were only able to study the effects of one or two genes at a time, but new technologies allow us to investigate the function of all genes in a cell in a small number of experiments. Genome editing technology allows us to precisely target single genes within a cell and either block the ability of that gene to make a protein (turn off the gene), or make the gene produce more protein than normal (turn on the gene). We will create two populations (libraries) of cells; in one library we will use genome editing to produce a population of cells in which each individual cell has a different single gene turned off, and in the second library a population in which each individual cell has a different single gene turned on.
We will use these libraries to look at how cells in which individual genes are turned on or off respond to infection with BoHV-1. We will infect the libraries with BoHV-1 and measure how the infection proceeds compared to unmodified cells. We can select populations of cells in which the virus replicates better or worse and identify which genes have been turned on or off in that population. We predict that if a gene makes a protein which is essential for virus replication, then the virus will replicate less well in the turned off library, but it will replicate better in the turned on library. If a gene makes a protein which inhibits virus replication, then the virus will will replicate better in the turned off library, but it will replicate less well in the turned on library. This approach will allow us to better understand how the virus and cell interact and how the cells control virus infection. It has the potential to identify new genes that inhibit BoHV-1 infection, which could be included in breeding programmes to increase resistance to this important animal pathogen or provide the basis for better vaccines or drugs.

Technical Summary

BoHV-1, a herpesvirus, is the causative agent of infectious bovine rhinotracheitis (IBR) and causes significant economical losses to the cattle industry. Little is known about the cellular factors which inhibit, or are essential, for BoHV-1 replication. An understanding of the host factors involved in viral replication is key to developing not only genetic approaches to disease resistance but also new vaccines and therapeutics. We will develop two genome wide CRISPR libraries for cattle: 1) Loss of function library (LOF) where the expression of individual genes is knocked down: 2) Gain of function library (GOF) where the expression of individual genes is switched on. We will screen both libraries following infection with BoHV-1 to identify host genes essential for replication or resistance. We will infect cells at both low and MOI. For low MOI infection we will harvest surviving cells after 4 days. Using PCR for the CRISPR sgRNAs and sequencing we will identify the genes targeted in these cells. Genes required for BoHV-1 replication will be overrepresented in LOF cells; genes that restrict BoHV-1 replication will be overrepresented in GOF cells. For high MOI we will harvest cells at 3 times and FACs sort into High, Median and Low BoHV-1 antigen expressing pools Genes targeted in these pools will be identified as above. In the LOF pools genes required for BoHV-1 replication will be overrepresented in the low pool; genes that restrict BoHV-1 replication will be overrepresented in the high pool. In the GOF pools genes required for BoHV-1 replication will be overrepresented in the high pool; genes that restrict BoHV-1 replication will be overrepresented in the low pool. We will validate a number of candidate genes by creating cell lines in which only the candidate gene is affected. This study will provide detailed information on cellular factors essential for replication or resistance to BoHV-1 and will inform future studies on the development of resistant animals.

Planned Impact

Impact Summary
(i) The academic community. Scientifically, the project constitutes a step change in the approach to investigating host pathogen interactions at the cellular level. It will bring together virologists whose expertise lies in understanding pathogenesis with geneticists whose expertise lies in genome manipulation to understand cell and organism phenotype. This impact will be delivered via publication in journals, presentations at conferences, seminars, and by making data and the bovine sgRNA libraries available. Little is known about bovine genes in contrast to e.g. human and mouse. The reagents and data produced will therefore also benefit colleagues in the wider bovine research community by providing a better understanding of bovine gene function.
(ii) The entire chain of users of animal products, including meat packers, milk processors, retailers and consumers will benefit because the knowledge generated will increase our understanding of the pathogenesis of this economically important disease leading to new intervention strategies including possible future scientific breeding programmes aimed at developing resistant animals. This will lead to an improvement in animal welfare with decreased production costs to producers, increased production yields and food security.
iii) UK Treasury will benefit from increased tax revenues through increased profitability of the farm animal supply chain.
(iv) UK science infrastructure and capacity. The proposed methods and resultant materials will provide a platform for increased research capabilities in the UK, maintaining its scientific reputation and associated institutions, with increased capability for sustainable agricultural production. The post-docs working on the project will have the opportunity to be trained at a world-class institute in a cutting edge area of research that unifies two fields.
(v) Society. This project will demonstrate the value of genome editing for identifying the genetic basis of disease resistance, which will help the general public to understand and appreciate the great value of genome editing technology. The knowledge will also feed into educational programs.
(vi) International. The impact of BoHV1 is felt worldwide. The international scientific and agriculture sector will benefit from the knowledge generated in this project.
(vii) Commercial. The opportunity for commercial follow-up on targets identified in this project offers the opportunity for entrepreneurial activity.

Publications

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Proudfoot C (2019) Genome editing for disease resistance in pigs and chickens. in Animal frontiers : the review magazine of animal agriculture

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Whitelaw B (2019) We have entered the era of genome-edited farmed animals. in Emerging topics in life sciences

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LILLICO S (2020) Livestock breeding for the 21st century: the promise of the editing revolution in Frontiers of Agricultural Science and Engineering

 
Description We have successfully designed and generated the GECKO and SAM libraries. Both libraries have been successfully used in Screens using BoHV-1. The GECKO screen identified a number of novel anti-viral and proviral candidates and several of these have been successfully validated. Several manuscripts outlining these findings are currently in preparation. The SAM screen has been carried out and we are currently awaiting the results. We have produced a number of cell lines expressing variants of the CAs9 protein which will be of use to those working on bovine pathogens and biology.
Exploitation Route Yes . we have made the TRIM5 KO cells available to collaborators, Drs D Bailey and T, Tuthill , Pirbright Institute and Prof M. Palmarini CVR Glasgow. We have agreed to collaborate with Dr Bailey and Prof Palmarini to use the library to investigate host factors involved in the viruses they work on. We have established collaborations with Dr E Gault (The Roslin Institute) and Dr P Beard (Pirbright Institute/Roslin) to use te GECKO libray in screes for Bovine rotavirus and Lumpy Skin disease virus respectively.
Sectors Agriculture, Food and Drink,Education,Manufacturing, including Industrial Biotechology

 
Description We designed and delivered three hands on workshops entitled "Coughs, Coughs and CRISPR" for Higher and Advanced Higher shoal pupils. This workshop has also been delivered to overseas High school students. We also presented a lecture on Genome Editing to High school teachers as part of a CPD course.
Sector Education
Impact Types Societal

 
Title CRISPR / Cas9 gene knockout library 
Description We have built a bovine specific CRISPR / Cas9 gene knockout library. 
Type Of Material Technology assay or reagent 
Year Produced 2020 
Provided To Others? Yes  
Impact Validated in bovine cells for genes involved in BoHV1 cell infection with manuscripts reporting this submitted for publication. 
 
Description A Workshop , " DNA Profiling: The Great Escape" aimed at HIgh school students 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact A Workshop , " DNA Profiling: The Great Escape" aimed at HIgh school students. Organised by the Social Mobility Foundation with students from North Ayrshire Council, South Lanarkshire Council , Midlothian Council, and Glasgow City Council, Wide ranging , pupil directed discussion on science as a career . Pupils reported that this changed their view of what was possible fro them to achieve.
Year(s) Of Engagement Activity 2018
 
Description 36th International Society for Animal Genetics Conference (2017) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact The ISAG conferences is one of the biggest animal genetics conferences in the world and was attended by more than 750 people from academia, industry, and policy making organizations from around the world at Dublin. I won a travel award to attend this conference, presented the project to the entire attendees via a poster and was chosen to give a presentation to an audience of around 100 people.
Year(s) Of Engagement Activity 2017
URL https://www.isag.us/2017/
 
Description Animal Biotechnology Workshop, Charlottesville 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact Other audiences denotes scientists. Attended an international workshop on animal biotechnology regulations where plans for international harmonisation of regulations was proposed.
Year(s) Of Engagement Activity 2017
 
Description Animal Genetics and Diseases Conference Wellcome Genome Campus, Hinxton, Cambridge, UK : Genome wide CRISPR knockout screen identifies host factors involved in Bovine Herpes Virus Type 1 infection. 
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 an international conference on Animal genetics and diseases. Reported the use of CRIPSRi screens in cattle, raising awareness of the feasibility of using this approach in the study of host:pathogen interactions in livestock. Feed back from participants was positive and generated discussion.
Year(s) Of Engagement Activity 2019
 
Description Annual Roslin Institute Open Doors Day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact The annual Roslin Institute Open Doors day was held on 14 October 2017 and was attended by 491 people. It involved 81 members of staff and students and showcased a wide range of research arising from BBSRC strategic investment, including (but not limited to), food safety, influenza, genetic improvement, DNA, imaging and the ethical treatment of animals in research.
Year(s) Of Engagement Activity 2017
 
Description Biology Teachers CPD Event 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Talk and discussions with 20 Higher Biology teachers about science, specifically about next generation sequence analysis technology and how that is being applied to address many different scientific questions
Year(s) Of Engagement Activity 2018
 
Description CPD event for High School Biology teachers 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact SSERC CPD Summer School for 22 high school teachers. A lecture on the principles and application of genome editing and delivery of parts of a workshop we were developing on "Cows, Coughs and CRISPR". The participants reported an increased understanding of the topic and their feedback on the workshop allowed us identify an appropriate level of content and to further tailor it to the needs of the school curriculum.
Year(s) Of Engagement Activity 2018
 
Description Chair of Session 3A: Large Animal Transgenesis, TT2020 Conference, Israel 
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 Chair of the above online conference which sparked questions and discussion. Chance also to join in other sessions online. Most of the audience were fellow scientists
Year(s) Of Engagement Activity 2020
 
Description Collaborators' meeting, India 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Indian partnerships in industry and science meeting
Year(s) Of Engagement Activity 2019
 
Description DBT Indian visit to Roslin 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Delegated visit to discuss joint development of research programmes in livestock sector for enhancing health and productivity of farm animals.
Year(s) Of Engagement Activity 2020
 
Description Easter Bush Campus annual open day 
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 The annual Roslin Institute Open Doors day was held on 13 October 2018 and was attended by over 600 people. It showcased a wide range of research arising from BBSRC strategic investment, including (but not limited to), gene editing food safety, influenza, genetic improvement, DNA, imaging and the ethical treatment of animals in research.
Year(s) Of Engagement Activity 2018
 
Description EuroScience Open Forum Meeting 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Genome editing involves altering some of the individual letters that make up an organism's genetic code at precise points. The technology can be used to introduce beneficial characteristics into plants and animals, such as resistance to a specific disease or improved adaptation to different environments. The changes introduced are the same as those that could occur spontaneously in nature. In this session, we will showcase how genome editing is being researched in institutions across Europe for treatment or prevention of animal diseases. The goal of the research presented here is to improve the health and welfare of farmed animals around the world, and to improve the security of food supplies in low and middle-income countries. We will start by introducing the technology and how it works, and explore how it is being used to manage a range of diseases in salmon, pigs, and cattle. A representative from a company specialised in providing genetic solutions to the farm industry will speak about how the sector is looking at the problem. Presentations will be followed by a panel discussion and Q&A session in which the audience will be asked to consider the perspective of different groups (e.g. farmers, consumers, policy makers, researchers, low and middle income countries) and will have the opportunity to put questions to the speakers.
Year(s) Of Engagement Activity 2020
 
Description Food Evolution Film 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Other audiences
Results and Impact Participated in academic film on food biotechnology
Year(s) Of Engagement Activity 2017
 
Description Gene Editing in Livestock, Washington DC 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Attendance and participation in the above event talk on Genome Editing Livestock
Year(s) Of Engagement Activity 2019
 
Description Genome Wide CRISPR Knockout Screen Identifies Host Factors Important for Bovine Herpes Virus Type 1 Replication. Plant and Animal Genomics PAG 2020 San Diego, USA 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact PAG is the major international conference on plant and animal genetics. Reported the use of CRIPSRi screens in cattle, raising awareness of the feasibility of using this approach in livestock to study genetic basis of susceptibility or resistance to pathogens. Feed back from participants was positive and generated discussion.
Year(s) Of Engagement Activity 2020
 
Description Genome Wide CRISPR Knockout Screen Identifies Host Factors Involved in Bovine Herpes Virus Type 1 Infection. Genome Science Edinburgh, UK Invited speaker ( Dr W. Tan) 
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 an international genetics conference. Reported the use of CRIPSRi screens in cattle, raising awareness of the feasibility of using this approach in the study of host:pathogen interactions in livestock. Feed back from participants was positive and generated discussion.
Year(s) Of Engagement Activity 2019
 
Description Highland Show 
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 Institute stand at the Royal Highland Show. Demonstrating focussed activity and discussing the work of the University and the institute in relation to food safety, farm animal welfare, production efficiency, disease resistance etc.
Year(s) Of Engagement Activity 2018
 
Description International Society for Animal Genetics Conference, Lleida, Spain 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Other audiences denotes scientists. Scientific symposium of 400 academic and students
Year(s) Of Engagement Activity 2019
 
Description International Sympoisum on Animal Geneticw, Dublin 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact Other audiences denotes scientists. Presented at scientific symposium to 400 students and students.
Year(s) Of Engagement Activity 2017
 
Description Laboratory based workshop for High school students 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact As part of the funding of BB/P003966/1 we developed a hands on , full day, workshop, cows, coughs and CRIPSR, for High school pupils based on genome editing. This approach aligned with the Biology curriculum and was designed to a) give pupils hands on experience of common laboratory techniques, b) to introduce them to scientific method and the approach to developing a research project, c) to introduce them to the work done in the Institute and in this grant in particular and d) to increase there understanding of the life of a scientist and the opportunities available to them. 16 Pupils and3 staff from schools in North Lanarkshire , all from areas of social deprivation, attended the course. The pupils enjoyed the course and all felt that they had gained a better understanding of the science we do and of the potential careers open to them. Likewise the staff were very positive about the workshop and the effect on their pupils.
Year(s) Of Engagement Activity 2018
 
Description MSc Lecture on Genome Modification of Livestock 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact MSc lecture fo 100 students on Genome Modificatio of Livestock
Year(s) Of Engagement Activity 2019
 
Description Meet The Scientists 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Visit to a local primary school to engage with a questions and answers session about science and science careers
Year(s) Of Engagement Activity 2018
 
Description National Academy of Science Colloquium Workshop, LA 
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 National Academy of Scinces Colloquium Life 2.0, The Promise and Challenge of a CRISPR Path to a Sustainable Plant, Attended conference and gave talk on genome editing which I agreed could be made public
Year(s) Of Engagement Activity 2019
 
Description Plant and Animal Genomics Workshop, USA 
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 Workshop Lead in the above workshop/conference, leading on Animal Genetics.
Year(s) Of Engagement Activity 2020
 
Description Primary School "Big Science" visit 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Visit to local primary school as part of their Big Science activity. Discussing science and scientific careers with the pupils and presenting awards to them based on a science project they had recently completed.
Year(s) Of Engagement Activity 2018
 
Description Question of Taste PCR Masterclass 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Practical workshop run by the Roslin Institute public engagement outreach team to allow High School pupils to run PCRs and investigate the genetics of a specific taste receptor.
Year(s) Of Engagement Activity 2018
 
Description Roslin Institute Open Day 2017 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact I volunteered at the open day and organized activities and games that a lot of pupils and parents engaged in. These activities sparked interests in science from the pupils and also facilitated knowledge exchange.
Year(s) Of Engagement Activity 2017
 
Description School workshop at The Roslin Institute 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact "Cows, coughs and CRISPRs" full day practical workshop introducing 15 pupils from High Schools on the Scottish Mobility Foundation list to the concepts of genome editing and practical aspects molecular biology. The pupils reported not only that they had learned from, and enjoyed the session but that they had also gained an insight into the possibilities open to them in Higher education.
Year(s) Of Engagement Activity 2019
 
Description Scientific Conference, Utrecht 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Scientific event to identify with other scientists a road map for animal biotechnology in Europe
Year(s) Of Engagement Activity 2017
 
Description Transgenic Animal Research Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Attendance at above conference as an invited speaker on Genome Modification of Livestock.
Year(s) Of Engagement Activity 2019
 
Description Transgenic Animal Research Conference, Tahoe, US 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Other audiences denotes scientist. Presented at scientific symposium for 100 students and scientists
Year(s) Of Engagement Activity 2017
 
Description U3A Talk - Rewriting Livestock, Biggar Library 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Invited talk to general public on genome editing of livestock
Year(s) Of Engagement Activity 2019
 
Description University Delegation to Bangalore India 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact University delegation to visit scientists in India to establish collaborative activities
Year(s) Of Engagement Activity 2017
 
Description University of Kent, invited speaker 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Invited speaker on animal biotechnology
Year(s) Of Engagement Activity 2019
 
Description Visit to Roslin Institute by Owen Thompson MP for UK Parliament for Midlothian 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Policymakers/politicians
Results and Impact Socially distanced visit to Roslin Institute by Owen Thompson MP. This visit was to see the work of the Institute in respect of their COVID-19 research. This involved a tour of the lab. Other research of the Institute was promoted including animal bioscience, breeding and biotechnology
Year(s) Of Engagement Activity 2020