Understanding the function and formation of infectious bronchitis virus membrane rearrangements

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. Specifically we 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.

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.

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 vaccines and 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.