A chicken primary B cell culture model to study the pathogenesis and improve the control of immunosuppressive viruses of poultry
Abstract
Chicken meat is an important source of dietary protein for people worldwide, and the poultry industry is important for securing enough food to feed the growing human population. Ensuring chickens remain healthy is therefore an economically important issue as well as a welfare issue for the birds. Chickens require a healthy immune system for vaccines to work effectively, and to fight off infection, so if their immune system is suppressed, birds will respond less well to vaccines and be more susceptible to catching infections, some of which can also infect people. The chicken immune system can become suppressed by infection with viruses that target a type of white blood cell known as a B cell. The main viruses that infect B cells in chickens are: Marek's disease virus (MDV), infectious bursal disease virus (IBDV), avian leukosis virus (ALV) and reticuloendotheliosis virus (REV). MDV and IBDV destroy B cells, whereas ALV and REV can cause the B cells to become cancerous and form a tumour. All these viruses can cause immunosuppression. These diseases are poorly controlled: New strains of IBDV have spread and vaccine failures have been reported, and ALV and REV continue to be isolated despite eradication efforts. There is therefore a need for new vaccines and control methods. It might be possible in the future to produce birds that are resistant to these diseases, but we need to better understand how the viruses interact with the cells they infect in order to realise this potential.
Until recently, it was not possible to study how the chicken B cells responded to infection as the cells died when they were taken out of the birds. Therefore, most studies into how these viruses cause disease and immunosuppression had to be conducted using infected chickens that suffered from disease, which could be severe in some cases. Recently, it has become possible to keep chicken B cells alive in the laboratory once removed from birds, and one research group has already used the cells to study MDV infection. We have used the cells to study IBDV infection, and our data suggest that they would be a good model for studying how IBDV interacts with the bird immune system, without the need to infect birds directly. We believe the model has the potential to replace the use of infected birds, and reduce the total number of birds used in this kind of research. The aim of this project is to build on our previous data to establish the chicken B cell model for studying IBDV, and expand it to the study of ALV and REV. In order to do this, we have three objectives: First, we will confirm and validate the use of the model for studying IBDV by comprehensively quantifying changes in the level of gene expression in the B cells following infection compared to what happens in the infected bird. Second, we will expand the model to include ALV and REV. Third, we will use the cells to produce stocks of IBDV as, currently, virus stocks are produced by infecting birds and recovering the virus from organs, so if the B cells can be used for this purpose, it would replace the use of even more infected birds. We will also determine if the cells can be used to screen vaccines for how effective they are against locally circulating and new strains to improve control.
Not only will we be able to replace and reduce the use of infected chickens, but using B cells is actually a better approach than using infected chickens to answer some scientifically important questions, as there is only one cell population as opposed to many, and it is possible to control the amount of virus the cells receive and to know for how long the cells have been infected. In addition, we can apply the model in the future to study how IBDV vaccines interact with the cells, and study co-infection with multiple viruses, so the project not only has a high 3Rs impact, but also a high scientific and translational impact.
Until recently, it was not possible to study how the chicken B cells responded to infection as the cells died when they were taken out of the birds. Therefore, most studies into how these viruses cause disease and immunosuppression had to be conducted using infected chickens that suffered from disease, which could be severe in some cases. Recently, it has become possible to keep chicken B cells alive in the laboratory once removed from birds, and one research group has already used the cells to study MDV infection. We have used the cells to study IBDV infection, and our data suggest that they would be a good model for studying how IBDV interacts with the bird immune system, without the need to infect birds directly. We believe the model has the potential to replace the use of infected birds, and reduce the total number of birds used in this kind of research. The aim of this project is to build on our previous data to establish the chicken B cell model for studying IBDV, and expand it to the study of ALV and REV. In order to do this, we have three objectives: First, we will confirm and validate the use of the model for studying IBDV by comprehensively quantifying changes in the level of gene expression in the B cells following infection compared to what happens in the infected bird. Second, we will expand the model to include ALV and REV. Third, we will use the cells to produce stocks of IBDV as, currently, virus stocks are produced by infecting birds and recovering the virus from organs, so if the B cells can be used for this purpose, it would replace the use of even more infected birds. We will also determine if the cells can be used to screen vaccines for how effective they are against locally circulating and new strains to improve control.
Not only will we be able to replace and reduce the use of infected chickens, but using B cells is actually a better approach than using infected chickens to answer some scientifically important questions, as there is only one cell population as opposed to many, and it is possible to control the amount of virus the cells receive and to know for how long the cells have been infected. In addition, we can apply the model in the future to study how IBDV vaccines interact with the cells, and study co-infection with multiple viruses, so the project not only has a high 3Rs impact, but also a high scientific and translational impact.
Technical Summary
A major cause of immunosuppression in poultry is infection with B-cell tropic viruses such as infectious bursal disease virus (IBDV) and the oncogenic retroviruses avian leukosis virus (ALV) and reticuloendotheliosis virus (REV). In vivo studies, while informative, can cause significant morbidity to infected birds. Recently, it has become possible to culture chicken primary B cells ex vivo. We have shown these cells can support IBDV replication and our data suggest they may be of relevance in vivo. Our aim is to establish a chicken primary B cell culture model to study the pathogenesis and improve the control of immunosuppressive B cell-tropic viruses of importance to the poultry industry that will replace the use of live birds in infection studies and reduce the total number of birds used experimentally in this field. We will achieve this aim by addressing the following objectives:1.) Validate the chicken primary B cell model for studying IBDV-host interactions, 2.) Expand the model to include ALV and REV, 3.) Evaluate the use of chicken primary B cells to test the immunogenicity of vaccine candidates prior to challenge and produce stocks of challenge viruses. Primary B cells will be harvested from chickens, cultured ex vivo, and infected in the laboratory with IBDV. The host-cell transcriptional response to infection will be characterised by RNA-Seq and the relevance of the model will be evaluated by comparing the results to the gene expression profile of infected tissue from in vivo studies. The model will then be expanded to include ALV and REV. The feasibility of using the cells to produce stocks of IBDV will be evaluated by determining the peak virus titre, the pathogenicity, and sequence changes compared to stocks produced in embryonated eggs. Finally, the cells will be used in neutralisation assays to quantify neutralising antibody titres in serum from vaccinated birds against very virulent IBDV strains.
