Bilateral BBSRC-FAPESP Research Grant: Dissecting Essential Roles of Chicken Interferon Stimulated Genes in the Pathobiology of Poultry Viruses

Poultry sectors of the UK and Brazil are contributing significantly to the cheapest and the most accessible source of meat protein. The UK poultry industry is contributing around £4 billion to the national economy whereas Brazil is the largest producer of poultry and poultry products, after United States and China. Despite this high production, infectious diseases are a major hurdle in achieving increasing demands of poultry products, around the globe. The total losses caused by infectious disease are enormous. For example, only Marek's disease virus (MDV), a cancer-causing virus of poultry, accounts for £2 billion losses worldwide. Massive vaccines application remained major disease-controlling tool against viruses. Despite widespread vaccinations against viruses (e.g. 22 billion vaccine doses for MDV only per year), the threat from poultry diseases is on rise and highlights the gaps in our fundamental understanding of poultry responses against virus natural infections or in immunization (vaccines). Such gaps has left us indebted on the molecular mechanism of chicken's involvement in influenza zoonoses and as a result quarantine and slaughtering of infected birds, as only choice, cost the poultry industry billions of dollars. Interferons, produced following viral infection of avian cells, stimulate the expression of hundreds of genes (more than 350 in humans) called interferon stimulated genes (ISGs). Exploitation of these ISGs could potentially be applied in favour of vaccine production, better vaccination protocols and controlling infections. In this proposal, we will initially identify the nature and magnitude of chicken ISGs (chISGs) and study their effects against five avian viruses causing infections in poultry (chicken, duck, turkey) ranging from mild to severe in terms of clinical manifestation. These viruses are of immense commercial and zoonotic importance not only for UK and Brazil but also throughout the world. Using broad-range profiling systems, we will identify those chISGs in the chicken cells that inhibit or enhance virus infections. Once identified, the factors that enhance virus infection will be overexpressed in established chicken cell line and the ones that inhibit virus infection will be removed or silenced within chicken cells. This modified cell line will be used to increase the replication of viruses for both industrial vaccine production and laboratory scale virus isolation. Given the fact that the current global poultry vaccine market is high, even a ten-fold increase in virus yield, which is potentially achievable, would have significant impact on the cost for vaccine. Beside industrial relevance, this new proposal, to be carried out jointly between The Pirbright Institute, UK and the State University of Campinas, Brazil, is crucial to understand the mechanism by which viruses induce interferon system of the chicken, some of which will be applicable to other species such as duck or turkeys. Finally, the findings from the project will be very valuable in examining the ability of the chISGs to protect the chickens against viruses of both poultry importance and those that can switch from poultry to human.

To initiate successful virus replication cycle, viruses have to breach a cascade of host cellular responses. Among these barriers, innate immune responses are most potent and independent to nature of pathogens. The objectives of this work are to define the interactions between chicken interferon stimulated genes (chISGs) and diverse poultry viruses that are of economic importance both in Brazil and UK. Our preliminary transcriptomic and expression analysis indicate that chISGs are the most prominent immune system genes expressed in Marek's disease virus (MDV) infected chickens, indicating that chISGs essentially mediate pathobiology of viruses. In this proposal, we will dissect the significance and breadths of chISGs against poultry viruses using large-scale, genome-wide and high throughput screen platforms. Specifically, we will measure the temporal expression of ISGs that synergistically or antagonistically regulate specific virus replication using a pre-established lentivirus-based ISG library. We will combine these data with results gain through shRNA mediated gene silencing to catalogue chISGs with broad-spectrum or virus specific actions. These data will be integrated within mechanistic studies to provide an incredible rich data on how viruses interact with host immune responses, essentially contributed by ISGs, and the ways viruses have adapted to circumvent these responses. These experimentations will be performed with avian influenza virus, avian paramyxovirus and MDV at The Pirbright Institute, UK and with avian metapneumoviruses and infectious bursal disease virus at the State University of Campinas, Brazil using homogeneous system in two laboratory settings. Finally, the gained information will be utilized in determining markers of enhanced viral resistance and modifying a previously characterized cell line (DF-1) to increase the growth kinetics of avian viruses which otherwise are not growing in cell culture or grow at lower titer.

Several factors such as climate changes, decline in agriculture land use and deforestation are seriously threatening global food security. In this scenario, poultry industry continues to expand at a dramatic rate in many parts of the worlds including the UK and Brazil, providing an economical source of proteins compared to other sectors of livestock. The poultry production in the UK fulfills 90% of the national demands and contributes annually around £4 billion to the UK economy supporting 60,000 associated jobs. Similarly, Brazil is an emerging economy with a large and increasing poultry industry for both egg (31.5 billion dozens annually) and poultry meat production (13.058 million tons, annually), which places Brazil among the three largest producers worldwide, along with United States and China. However, one of the major challenges to sustainable poultry production is the growth in infectious diseases, particularly caused by avian viruses. Therefore, understanding the complex interplay in host-pathogen interactions is crucial not only to devise control strategies but also to ascertain the role of chicken in the emergence of zoonotic pathogens. One of the aims of this proposal will be the academic work necessary to underpin and facilitate a range of benefits for different stakeholders including poultry industry, vaccine companies and scientific communities. The discovery of the role of chicken interferon stimulated genes (chISGs) will provide new insights into the avian innate immunity, which can be utilized to counter viral pathogens of poultry. There have been significant advancements in developing broad-scale and genome-wide platforms to understand the pathobiology of several pathogens in mammals, however, such state-of-the-art techniques in avian species are currently lacking. Knowledge with respect to molecular bases of host responses to virus infections, as the current proposal aims to achieve, will benefit avian research community to map chISGs expression against wide range of environmental stress that compromise the poultry production. Due to current importance of global poultry vaccine market (currently $1,128 million and is expected to rise to $2,040 million by 2018) and lack of model cell line for poultry viruses (e.g. for MDV), the ultimate aim of this proposal is to utilize the information gained for the modification of a previously characterized cell lines which would have superior properties for enhanced virus replication both for vaccine production and vector delivery. These findings would have significant impact on vaccine doses per cell-passage, reduce the cost of vaccine and subsequently create opportunities to engage vaccine industry in the project. Outreach activities will be conducted to efficiently disseminate the information directly to stakeholders. The proposed studies will provide opportunities for the professional developments of scientists, and staff employed on this project through cutting edge scientific methods and technological advances both at The Pirbright Institute, UK and the State University of Campinas, Brazil. We will engage with the general public and scientific communities through continuous updating the information on websites of the consortium and partner's institutions, presentation of results in scientific meetings, and provision of press releases to media outlets. Scientists employed during the project will also be encouraged to take part in public engagement. In conclusions, improved understanding of the impact of viral infections and how the host responses contribute to the viral pathobiology will build bases to be used for devising better control measures, vaccine design and to underpin the crucial role of chicken in emergence of zoonotic influenza viruses. Collectively, the gained information, as described in this project, will have positive knock-on benefit both socially and for the national economies.