Vaccines for chronic viral pathogens in salmon- generation of interferon attenuated cell lines. Acroym: SalVacCell Project goal / key aims. There is a major requirement to generate new vaccines for viral pathogens in the salmon aquaculture industry. Viruses represent one of the major economic losses to the salmon industry, which is a direct reflection of the lack of highly protective vaccines. In order to improve vaccine design and testing high quantities of viruses are required, but at present this is not possible. This project will use cell lines that have been edited by CRISPR/cas9 to be highly permissive for growth of difficult to grow viruses. We will knockout the type I interferon pathway which is the major antiviral mechanism in animals. Specifically we will target key genes that regulate interferon induced cellular responses to viral infection. These cell lines will additionally be used to explain the underlying function of genes associated with natural viral resistance / susceptibility in salmon that is of direct importance to breeding programs. We have secured Industrial partners Benchmark (vaccine company) and Landcatch (fish breeding). Consortium agreements will clearly define roles and confidentiality of IP. OBJECTIVES 1. Development of IFN-deficient fish cell lines by knock-out of IFN function 2. Evaluation KO cell lines to mount an antiviral response to confirm phenotype 3. Use the cell lines to explain naturally occurring resistance / susceptibility to fish viruses 4. Comparison of yield for viral particle production and viral diagnostic turn-over time between traditional cell lines and newly developed cell lines KEY CHALLENGES The overarching challenge is to be able to produce high titres of virus for vaccine companies to be able to improve design and protection to economically important chronic viral pathogens. Many labs have attempted to knock down genes in salmonid cells, but to date this has not been achieved. To our knowledge we are the first to create such cell lines and as such our unfunded preliminary work has already overcome a major hurdle. The challenge of this project is to fully exploit our cell line technology for both disease management and also to greatly improve the basis of genetic selection. Deliverables- to be used by partners a. Engineered cell lines that are deficient in antiviral responses. b. Cells lines that can be used to produce high titre of viruses for vaccines c. Capacity to upscale viral production in industrial environment. d. Improved precision of selection for disease resistance in salmon breeding Project duration: 24 months Total project cost: £354622 Contribution requested from (100%) BBSRC/NERC £249,622 (£199697.60 80%) Contribution from MSS: secured £65K Contribution from Benchmark secured £20K Contribution from Landcatch secured £20K

Farmed salmon is a major source of high quality protein and fatty acids essential for human health. Salmon aquaculture is worth approximately £1Bn to the UK economy, and supports many rural and coastal communities. However, disease outbreaks have a major negative effect on salmon production and animal welfare. Infectious salmon anaemia (ISA) is one such disease, and is sometimes dubbed 'salmon flu' because it is caused by a virus (ISAV) that is similar in to influenza. At present, ISA is a notifiable disease in the UK, meaning farmers are obliged to cull their stock in the event of an outbreak. Vaccination and biosecurity cannot fully prevent outbreaks, and developing disease resistant strains of salmon is high priority. Selective breeding can result in moderate improvements in disease resistance of salmon stocks and may take many generations. However, a revolutionary approach known as genome editing has potential to rapidly increase the rate at which disease resistant salmon can be produced. Genome editing involves the use of "gene scissors" to precisely cut the genome at a specific location, leading to small-scale targeted changes in the DNA sequence. In this proposal, genome editing technology will be used to investigate genes underlying resistance to ISAV, and potentially to produce a disease-resistant salmon. The first stage of the project is to identify target genes that will be edited. This will be achieved by measuring the ISAV resistance in a selective breeding program. Genetic markers dispersed throughout the salmon genome will then be used to map individual genes that contribute to variation in resistance in the population. Salmon from resistant and susceptible families will also be sequenced and to identify candidate genes and mutations causing this genetic effect on resistance to ISAV. In parallel to the 'forward genetic' approach described above, a 'reverse genetic' approach to identifying ISAV resistance candidates will be employed using cell culture models. A genome editing method known as CRISPR-Cas9 will be applied to destroy the function of key candidate ISAV resistance genes in the cell lines. Two methods of choosing candidate genes will be used. The first is based on prior knowledge of the biology of the interaction between the virus and the host cell, partly harnessing extensive research which has been performed on ISAV's close relative influenza. The second is to use the genes affecting natural resistance identified in the forward genetic screen described above. These edited cell lines will be infected with ISAV, and the impact of the edited gene on ISAV resistance and cellular response to infection will be assessed. This will build on an ongoing project to develop genome editing for salmon cell lines. Finally, genome editing will be used in Atlantic salmon embryos to test the highest priority ISAV resistance genes, especially where knockout of the gene has an impact on resistance in cell culture. Targeted editing of the genes will be performed by microinjecting newly fertilised embryos, which will be reared until the freshwater fry stage. These edited embryos, and unedited controls from the same family, will be challenged with ISAV. The nature and frequency of the edited genes in the resistant and susceptible salmon will be measured. This proposal has potential to create Atlantic salmon with resistance to a problematic viral disease (ISA) using a novel breeding technology. As such, it could have major animal welfare and economic impacts via prevention of outbreaks and subsequent culling of stocks. The approaches will be directly relevant to other viral disease in fish aquaculture. While the regulatory landscape for application of edited animals in food production is uncertain, a successful outcome of this proposal will provide a high profile example of the power of this technology to understand biology and to improve food security and animal health.