Understanding the function and formation of infectious bronchitis virus membrane rearrangements

Lead Research Organisation: The Pirbright Institute
Department Name: Nidovirus-cell Interactions

Abstract

Poultry products are a major source of animal protein for human consumption with approximately 55 billion chickens raised globally every year. The UK poultry industry contributes approximately £3.4 billion to the economy annually but infectious diseases, including viruses, are a continual threat to animal welfare and productivity. The infectious disease that results in the largest economic losses to the UK poultry industry is caused by infectious bronchitis virus (IBV). IBV infection results in animal welfare costs and significant economic losses due to poor meat quality, poor egg production and poor egg quality. It is estimated that IBV affects 22 million chickens and costs the UK poultry industry £23 million every year. Currently available vaccines against IBV are costly to produce and do not protect against all of the different circulating viral strains. Therefore, new vaccination strategies are required. By understanding how IBV replicates itself and how it manipulates the host cell to allow replication, it will be possible to develop alternative and potentially more efficient control strategies, benefitting animal welfare, the poultry industry and the UK economy.

IBV is a coronavirus with a positive strand RNA (+RNA) genome. A critical step during the life cycle of coronaviruses is RNA synthesis, the process of copying the viral genome for packaging into new virus particles and to allow viral proteins to be produced. IBV RNA synthesis, as for all +RNA viruses, is closely linked with the rearrangement of cellular membranes, providing a platform for the assembly of the viral proteins responsible for RNA synthesis, known as replication-transcription complexes (RTCs), and protecting viral RNA from host defences. However, IBV induces more than one type of rearranged membrane and the role of these different structures in the virus life cycle is not known.

This proposal aims to understand the formation of IBV RTCs responsible for viral RNA synthesis. Specificallye will identify where in the cell viral RTCs are located and RNA synthesis takes place. We will also identify which viral and cellular proteins are important for the formation of rearranged membranes. The information gained will not only be useful for understanding how IBV and other coronaviruses replicate but will provide insight into potential ways to alter or prevent virus replication for future vaccine and anti-viral development.

Technical Summary

Infectious bronchitis virus (IBV), an avian coronavirus, is a highly contagious avian pathogen prevalent throughout the world and is the cause of an economically important disease causing losses to the global poultry industry and a threat to food security. Like all positive strand RNA viruses, IBV induces the rearrangement of cellular membranes during infection to support assembly of viral RNA replication-transcription complexes (RTCs). These membrane rearrangements play an important role in the critical step of viral RNA synthesis during the virus life cycle. The aim of this project is to understand the formation of infectious bronchitis virus replication-transcription complexes responsible for viral RNA synthesis. This will be achieved by:

1. Determining the cellular location of IBV RTCs and RNA synthesis during infection.
Using a variety of bioimaging techniques, the location in the cell of RTCs and nascent viral RNA will be determined, including which membrane rearrangements are involved.

2. Defining the viral determinants involved in induction of membrane rearrangements.
By expressing viral proteins in the absence of other viral components, the proteins involved in the formation of membrane rearrangements will be identified. Using virology and molecular biology techniques, important protein-protein interactions will be characterised. In addition, an anti-viral drug known to inhibit membrane rearrangements will be used to study the role of viral proteins in rearrangement of membranes.

3. Assessing the role of cellular proteins in the formation of membrane rearrangements.
Cellular proteins that interact with viral proteins responsible for membrane rearrangements will be identified by mass spectroscopy. A role for these proteins will be validated using bioimaging, virology and cell biology techniques, including RNAi.

Planned Impact

Poultry products are a major source of protein for human consumption with around 55 billion chickens raised globally. The UK poultry industry contributes approximately £3.4 billion per year to the economy but is constantly under threat from infectious diseases. The major cause of loss to the UK poultry industry with significant animal welfare impacts is infectious bronchitis virus, costing £23 million per year. Infectious bronchitis accounted for the largest segment (24.3%) of the poultry diseases market in 2012 and it has been estimated that every 10% reduction in IBV would be worth around £654 million globally. Currently available vaccines are inactivated virus or live attenuated virus produced by serial passage through chicken eggs. These vaccines are costly to produce and attenuation mechanisms are not understood. Furthermore, vaccines often do not protect against the multiple virus strains. Chickens are given multiple vaccines and are often not fully protected against all circulating strains. Developing more efficient control strategies against IBV will benefit the UK economy, as well as farmers and animal welfare. The work described in this proposal will have future impacts on the poultry industry by providing vital information that is needed in the development of novel vaccine production strategies, through rational virus attenuation, by academic scientists and vaccine producers.

Academia and human and livestock health: Coronaviruses, like IBV, are significant causes of disease in animals including pigs, cattle and domestic animals, as well as humans. Recent SARS-coronavirus and MERS-coronavirus outbreaks also highlight zoonotic potential for this family of viruses. The step of virus replication studied in this proposal is critical and common among all coronaviruses. By using IBV as a model, valuable knowledge can be gained that will inform academics in other fields, furthering scientific research and underpinning and facilitating work to design vaccinend particularly anti-viral therapies for other economically important viruses or those posing a threat to human health.

BBSRC: Food security and animal welfare are research areas of strategic importance to BBSRC. Results from this work will provide background knowledge essential for designing novel therapeutic strategies, required for securing UK and global food security.

TPI: Characterising virus-host interactions for economically important livestock pathogens, like IBV, is a strategic aim of TPI. This work will characterise in detail how IBV replicates within the host cell and manipulates the cellular environment to allow efficient virus replication. In addition, cellular proteins important in this will be identified. This work not only helps fulfil TPIs scientific aims, but will also benefit the reputation of TPI in academia.

Students and public: The work proposed here will be of general interest to farmers, veterinarians, students and the public. Information will be disseminated via public engagement and STEM outreach events, TPIs website and press releases, where appropriate.

Training and development: In addition to these impacts, the project will have a direct impact on career development and training of a PDRA. The researcher will be trained in a variety of specialist and transferable skills in bioimaging, general molecular biology and virology techniques. The researcher will have access to a variety of professional development training courses run by TPI and will be actively encouraged to gain experience of public engagement, report writing and presentation of data through posters and oral presentations. Finally the outputs of this proposal will directly impact the PI (HM). As a New Investigator, HM will gain an invaluable opportunity to manage a project, strengthen existing collaborations and generate data required for scientific development and for submission of future grant applications needed to become a successful independent researcher.
 
Description Like many viruses, infectious bronchitis virus changes the structure of host cell membranes during replication to form new compartments in the cell called the replication organelle. This is thought of as a virus factory because it is thought that this is where the viral machinery required for making new copies of the viral genetic material assembles. The viral and cellular proteins required for formation of replication organelles are not known and the precise role of different parts of the replication organelle are also unknown. Using advanced microscopy experiments, we have identified that expression one viral protein results in the formation of sheets of paired endoplasmic reticulum membranes, a key feature of replication organelles in infected cells. This provides the first piece of information about how IBV rearranges cellular membranes to form replication organelles. However, other structures that form part of the replication organelle, structures known as double membrane spherules and double membrane vesicles, were not produced by expression of any of the combinations of viral proteins tested. Therefore, this demonstrates that additional factors are required for complete reconstruction of the replication organelle and further work will be required to identify these. Interestingly, we have also identified that there are differences in how IBV proteins work together to form replication organelles when compared with other closely related viruses.

In other parts of the project, we have identified suitable markers for imaging replication organelles and have developed methods for detection of viral RNA synthesis. Using these techniques, we have studied in more detail physical characteristics about the location of IBV RNA synthesis and how nascent RNA moves within the cell. Through two independent routes we are working to identify the precise location of viral RNA synthesis on the replication organelle membranes. This is been long debated within the research field. To facilitate this, we have established a correlative light electron microscopy (CLEM) protocol.

Finally, cellular proteins located at the replication organelle are being investigated through a proteomics informed by transcriptomics approach, in collaboration with A, Davidson and D. Matthews at University of Bristol. We have optimised a protocol for this and data analysis is underway. Results will be validated in the last few months of the project.
Exploitation Route Results from this project are being used directly to support follow on funding applications to allow continued research on this topic. Within Pirbright, technical knowledge gained through this project is being passed onto several other research groups to allow protocols we have developed or established to be used by others working in other research areas. Finally, data has been published allowing other researchers working in related fields to follow up on our observations and incorporate our findings into their work.
Sectors Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology

 
Description Data from generated during this project has been used to support a further funding application.
First Year Of Impact 2019
 
Description Andrew Davidson - Proteome of viral replication organelles 
Organisation University of Bristol
Department Avon Longitudinal Study of Parents and Children (ALSPAC)
Country United Kingdom 
Sector Academic/University 
PI Contribution Projects are being initiated to study the cellular proteins associated with coronavirus replication organelle membranes and a funding application has been submitted to BBSRC.
Collaborator Contribution Andrew has provided assistance with experimental planning and drafting of a grant application. He has also provided protocols to allow preliminary data to be generated. Once protocols have been established, Andrew will facilitate mass spectrometry data generation and will analyse the resulting data.
Impact No outcomes to date, projects being initiated and funding applications in progress.
Start Year 2018
 
Description Ben Neuman - Study of IBV induced membrane rearrangements 
Organisation Texas A&M University–Texarkana
Country United States 
Sector Academic/University 
PI Contribution My team provide samples for electron microscopy analysis. In addition, we provide expertise in molecular virology and cell biology and experience of membrane rearrangements induced by infectious bronchitis virus. I contribute intellectual input in data analysis and project development.
Collaborator Contribution Ben Neuman and his team perform electron microscopy sample processing and imaging as well as experience in data processing. Ben Neuman also contributes intellectual input in data analysis and project development.
Impact Maier et al. Extensive coronavirus induced membrane rearrangements are not a determinant of pathogenicity. Sci. Rep. 6: 27126.
Start Year 2014
 
Description Ben Neuman - Study of IBV induced membrane rearrangements 
Organisation University of Reading
Country United Kingdom 
Sector Academic/University 
PI Contribution My team provide samples for electron microscopy analysis. In addition, we provide expertise in molecular virology and cell biology and experience of membrane rearrangements induced by infectious bronchitis virus. I contribute intellectual input in data analysis and project development.
Collaborator Contribution Ben Neuman and his team perform electron microscopy sample processing and imaging as well as experience in data processing. Ben Neuman also contributes intellectual input in data analysis and project development.
Impact Maier et al. Extensive coronavirus induced membrane rearrangements are not a determinant of pathogenicity. Sci. Rep. 6: 27126.
Start Year 2014
 
Description David Matthews - Proteomics informed by transcriptomics 
Organisation University of Bristol
Department Proteomics Facility
Country United Kingdom 
Sector Academic/University 
PI Contribution Projects are being initiated to study the cellular proteins associated with coronavirus replication organelle membranes and a funding application has been submitted to BBSRC.
Collaborator Contribution David has assisted in project design and drafting of a funding application to BBSRC. We are in the process of generating transcriptomics data, which David will process to provide an expressed protein database for peptide identification following mass spectrometry.
Impact No outcomes to date, a funding application has been submitted to BBSRC.
Start Year 2018
 
Description Montse Barcena - Studying the role of coronavirus membrane rearrangements 
Organisation Leiden University Medical Center
Country Netherlands 
Sector Academic/University 
PI Contribution My team has provided reagents and biological samples for the study of the role of IBV induced membrane rearrangements. In addition we have provided expertise and knowledge for the set up of protocols and assisted in data analysis.
Collaborator Contribution M Barcena has provided extensive experience in electron microscopy including use of specialised protocols. She has provided expertise in data analysis and protocol development.
Impact Several presentation of data have been made at international conferences.
Start Year 2014
 
Description Paul Verkade - Electron tomography and CLEM 
Organisation University of Bristol
Department Avon Longitudinal Study of Parents and Children (ALSPAC)
Country United Kingdom 
Sector Academic/University 
PI Contribution Nicole Doyle has performed electron tomography to analyse the three dimensional structure of membrane rearrangements induced by the expression of IBV non-structural proteins 4 or 3 and 4 in the absence of other viral components. Sample set up was performed at Pirbright and analysis was subsequently performed at University of Bristol followed by additional analysis at Pirbright.
Collaborator Contribution Paul has assisted with electron tomography data generation providing access to electron microscopes in Bristol as well as training for Nicole Doyle in performing experiments and analysing and modelling data. He is also advising Nicole while establishing correlative light electron microscopy at Pirbright.
Impact doi: 10.3390/v10090477
Start Year 2018
 
Description Coronavirus lecture MSc students (Surrey) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact A lecture was presented on the replication, cellular interactions and pathogenesis of coronaviruses to 15-20 MSc students from University of Surrey. The students were engaged and interested, answering and asking questions during the session.
Year(s) Of Engagement Activity 2016,2018
 
Description Diamond Light Source Open day - HJM 
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 A public open day for Diamond Light Source, which Pirbright attended as a user of the facility to showcase research being performed at Pirbgith in collaboration with Diamond. There was a lot of interest in the fundamental research and sicussion about future plans and how it fit with the facility at Diamond.
Year(s) Of Engagement Activity 2019
 
Description Farnborough futures careers event - HJM 
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 The careers event aimed to introduce the range of careers available at Pirbright. In addition I discussed my own career progression and highlighted the range of possible careers that I could have entered. Finally, I discussed some details of the research with interested students. The students had a wide range of backgrounds and career aims but were interested to hear about the Institute and the sort of work opportunities available. Several of them planned to follow up by investigating future job opportunities at Pirbright.
Year(s) Of Engagement Activity 2019
 
Description Innovate Guildford, ND 
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 Presented at the Pirbright Institute stand discussing science performed by the Institute with the general public. The stand focussed on an exhibit on avian influenza virus but more general virology topics were also discussed including our research.
Year(s) Of Engagement Activity 2017
 
Description Lecture on entereic coronaviruses to MSc Students (Surrey) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact A lecture was presented on the pathology, diagnosis and detection of enteric coronaviruses, including virus replication and interaction with the host. The students were engaged and asked/answered question throughout the session. We were invited to repeat the lecture for the subsequent student intakes.
Year(s) Of Engagement Activity 2015,2016,2018
 
Description Presentation at Avian coronaviruses meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Data was presented at the Avian coronaviruses and other complicating pathogens meetings in 2014 and 2016. Data was then discussed with other scientists in the field.
Year(s) Of Engagement Activity 2014,2016
 
Description Presentation at Avian viruses focussed meeting - Sept 2018 ND 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Presentation of data as an offered paper at the molecular biology and pathogenesis of avian viruses meeting, Oxford Sept 2018.
Year(s) Of Engagement Activity 2018
 
Description Presentation at Bioimaging day event 
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 Other audiences
Results and Impact I was asked to present data at a bioimaging day event held at Pirbright to showcase recent studies using bioimaging facilities. I presented and discussed my data with other researchers.
Year(s) Of Engagement Activity 2016
 
Description Presentation at Inter-institute Bioimaging networking meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact An event was held to enable improved collaboration opportunities between scientists working with bioimaging techniques at the different research institutes within the UK. I presented data from the last 10 years describing our work characterising replication organelles induced by IBV, describing the different techniques and approaches we have used and the outlook for the future. The talk aimed to showcase the bioimaging facilities at Pirbright to encourage new collaborations.
Year(s) Of Engagement Activity 2018
 
Description Presentation at RIVR meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact Oral presentation of my work to other researchers at the Recently Independent Virology Researchers meeting. I discussed my data with other researchers and potential for future collaboration.
Year(s) Of Engagement Activity 2016,2017,2019,2020
 
Description Presentation by ND at Nidovirus symposium 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact The post-doctoral researcher presented our data at the 14th international Nidovirus symposium. She discussed data and potential collaboration with other scientisits from the field.
Year(s) Of Engagement Activity 2017
 
Description Presentation of data at Microbiology Society Annual conference 2019 - ND 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact Presentation of data to scientists at the annual Microbiology Society Conference, Belfast. Following the presentation there were questions and discussion with other scientists.
Year(s) Of Engagement Activity 2019
 
Description Q&A statement on coronaviruses provided to Science Media Centre - HJM 
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 A Q&A statement on coronaviruses was provided to the science media centre, who distributed it to media outlets. Excerpt have been used in 1420 articles incuding the Daily Mail and syndicate articles. The Q&A was also used in a Pirbright Institute media statment.
Year(s) Of Engagement Activity 2020
 
Description Reverse genetics seminar to MSc students 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact A seminar was presented describing reverse genetics of viruses, including infectious bronchitis virus and the application of reverse genetics in research. The students were interested and engaged, participating in activities and asking questions. We were invited to present the seminar in subsequent years for new student intakes.
Year(s) Of Engagement Activity 2013,2014,2015,2016,2018,2019
 
Description STEAMfest - HJM 
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 A large open day event at hosted at University of Surrey for students from a number of schools in the region to explore different scientific questions and areas of work.
Year(s) Of Engagement Activity 2019
 
Description Science Enrichment day at Park Mead School - ND 
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 An engagment event at a local school teachin year 5 and 6 students about mosquitos and some of the work done at Pirbright.
Year(s) Of Engagement Activity 2019
 
Description Teentech Guildford - ND 
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 Teentech is an event held for students from a range of local school who have an interest in science to experience the range of scientific activities that could be open to them for future careers. Students in small groups rotate around stands at the event to perform activities at each station, guided by scientists. The Pirbright stand was demonstrating the principle of genetic modification of mosquitos. The children were very interested in the exhibit and asked lots of questions.
Year(s) Of Engagement Activity 2013,2019
 
Description The Hurst careers event - HJM 
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 The Hurst school held a careers event for GCSE students to attend throughout the day. The Pirbright stand contained a variety of engagement activities to demonstrate the research we perform and to stimulate discussion. The students seemed interested in the activities and those more interested in a science career asked a variety of questions about the research and entry into scientific research as a career.
Year(s) Of Engagement Activity 2018
 
Description Tillingbourne School bug hunt - ND 
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 Primary school students were involved in an event to identify insects in the school grounds.
Year(s) Of Engagement Activity 2018
 
Description Tillingbourne School science event - ND 
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 Tillingbourne School held a science event for year 3 students to explore different aspects of science including smell, acid & alkali, magnetism etc. Nicole was based at the microscopy station helping children to view samples under the microscope, explain what they were looking at and answer any questions to children asked. The children were highly engaged and interested in the exhibit.
Year(s) Of Engagement Activity 2018,2019
 
Description Winston Churchill School Career Event - ND 
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 A careers event at a local school for yeat 7 to 11 students about Pirbright, what we do, the kinds of careers they can get into and routes to those careers.
Year(s) Of Engagement Activity 2019