Precision Breeding for Viral Resistance

This project deals with selective breeding for resistance to viral diseases in Atlantic salmon. AquaGen delivers eggs to salmon farmers in Norway and the rest of the world; eggs originating from breeding populations that are selected for fast growth as well as robustness and disease resistance. We have previously shown that we can breed the salmon to become more resistant to viral diseases such as infectious pancreatic necrosis (IPN), pancreas disease (PD), cardiomyopathy syndrome (CMS), and heart- and skeletal muscle inflammation (HSMI). In this project, we will fine-tune our genomics-driven breeding program with regards to selection for viral resistance, by employing new and high-resolution methods within genomics and molecular biology. We will use these methods to take on some emerging virally induced disease problems. In the first work package, we will investigate whether there is heritable variation in the salmon's resistance to Salmon Pox, an emerging disease caused by Salmon Gill Poxvirus. Here, we have analysed data from three field outbreaks of Salmon Pox, two coming from AquaGen?s customers and one coming from our own breeding nucleus. Two of these datasets contained mortalities only or survivors only, and on those datasets we applied a so-called Transmission Disequilibrium Test (TDT). The third dataset had both mortalities and survivors. As they stand, the results point to at least one consistent quantitative trait locus (QTL) for resistance to Salmon Pox, albeit of moderate effect. We are now looking for further opportunities to sample both mortalities and survivors, so that genetic correlations between data sets can be calculated, and the ground be set for genomic selection for resistance to Salmon Pox. In the second work package, we will revisit the topic of resistance to IPN. New research has shown that some strains of the virus affect the fish in a different way compared to the virus variant that is most common. We need to establish whether the (presumed) missing effect of the QTL applies for AquaGen's population and methods of selection. Ways to accommodate the new strains of the virus in selection will be sought, should it turn out that these new strains are not affected by the QTL. In 2021, we tested our breeding nucleus to resistance against 1) a newly described isolate of the virus, expected to be unaffected by the QTL and 2) the old strain of the virus, known to be affected by the QTL. Unfortunately, the two experiments were not very informative, because mortality was low in the test with the new virus strain and very low in the test with the old virus strain. Hence, the only thing we found out was that AquaGen's population is somewhat more susceptible to the new virus strain compared to the old virus strain (but still rather resistant). The two experiments will be redone in 2022, and this time we have prepared fish groups with higher frequency of the susceptibility allele at the IPN-QTL. In the last work package, we will investigate whether gene editing (CRISPR-Cas9) can be used to identify salmon genes which the virus needs in order to enter host cells. If such genes are found and if future legislations should permit the use of gene editing beyond research, completely resistant strains of salmon could potentially be produced through CRISPR-induced knock-out of broodstock animals. In this work-package we will carry out a so-called Genome-Scale CRISPR-Cas9 Knockout (GeCKO) screen for genes involved in infection by the IPN virus (both the new and the old strain). In a GeCKO screen, all genes in the genome are knocked out using a cell culture, but in such a way that only one gene is knocked out in each cell. The cells which survive will be those that have had genes knocked out which are essential for viral infection. GeCKO screens have been performed with success in many species, but to our knowledge not in Atlantic salmon so far. At this point, we have identified several obstacles towards the application of GeCKO in salmon, and we are presently working towards solving those.

This project will address three topics related to the Atlantic salmon's resistance to viral diseases, aiming at making the salmon more resistant to viruses. The project will use technologies connected with Precision Breeding; selecting the animals based on a detailed understanding of how genes work and interact. The topics are: 1. Resistance to POX virus: Salmon gill poxvirus (SGP), is an emerging pathogen in Atlantic salmon aquaculture. A pilot study has indicated that resistance is influenced by genetics and that there are intriguing correlations between resistance to poxvirus and resistance to pancreas disease (PD) and heart- and skeletal muscle inflammation (HSMI). The project aims to lay the foundations for marker-assisted- or genomic selection for resistance to SGPV, and to describe and understand these correlations in detail. 2. Resistance to IPN: Marker-assisted selection for IPN resistance, utilizing a major QTL for IPN resistance, has lead to a marked decrease in the number of IPN outbreaks. However, a newly discovered isolate of the virus has been reported to sidestep the QTL. We want to investigate whether this finding also applies to AquaGen’s production of “IPN eggs”, and (if so) to understand the biology underlying these results and make sure that AquaGen’s IPN product covers resistance to all strains of the virus. 3. Gene editing to promote viral resistance: Genome-Scale CRISPR-Cas9 Knockout (GeCKO) screening can be used to identify salmon genes necessary for viral infection. We want to use GeCKO to identify genes necessary for infection by the newly discovered IPN isolates and additionally, by the more well-known isolates. This work package will add to add to our understanding of the relationship between virus isolate and host genetics. The work package will also provide tools and competence which could be applied if gene editing should become a more broadly applied tool it the future. In December 2022 we applied for a one-year extension of the project. In the added year (2024) the project will focus on these tasks: 1. Workpackage H3: GeCKO-screening with other viruses. The GeCKO-screening procedure is, to some extent, the same irrespective of target virus. Other parts of the procedure is unique to each virus. We have now solved some of the biggest technical challenges of the method, and we are keen to exploit this advantagous position by performing GeCKO-screening for another salmon virus, namely the Infectious Salmon Anemia (ISA) virus. 2. Workpackage H1: We have lots of interesting results on the viruses CMS, HSMI, and PD. We would like to extend our research into genetic architecture to those viruses as well, focusing in particular on whole-genome sequencing.