Precision Breeding for Viral Resistance

In this project, we will "fine-tune" the breeding work AquaGen is doing on virus resistance by utilizing new and even more precise methods in genomics/molecular genetics. We will use these methods to address some new problems that have arisen in the industry in recent years or that may become problems in the future. In the first work package, we will examine whether resistance to salmon pox is genetically determined and whether we can breed salmon to become more resistant to this disease. We have already collected data from three different outbreaks of salmon pox, one of which occurred in a fish group belonging to AquaGen’s breeding nucleus and two of which happened sites belonging to clients of AquaGen. The results suggested that we had identified a region on the genome (a QTL) that has a consistent effect on salmon pox. In the project, we have procured an additional dataset, and we have thoroughly examined all the datasets. Unfortunately, the mentioned QTL was not validated in the fourth dataset, and we also discovered an error in one of the previous datasets. Consequently, the result of this work package is that there is no QTL with a significant effect on resistance to salmon pox; rather, the genetic control of this trait is governed by a large number of genes spread across the salmon genome. The heritability of the resistance was estimated at 0.35, which means that about one-third of the variation in resistance is hereditary. It is therefore possible to select for increased resistance, but there is no "simple solution" in the form of a QTL with a large effect. We are working on developing a future strategy for further efforts. In work package number two, we are looking at resistance to IPN. Recent research has shown that there are variants of the virus that act differently compared to the most common variant. In the project, we conducted challenge tests against both 1) a new isolate of the virus suspected not to be affected by the known IPN QTL and 2) an old isolate known to be affected by the IPN QTL. Such tests were carried out in both 2021 and 2022. The results clearly showed that the QTL does not work on the new variant of the virus, also in AquaGen's salmon strain. On the old variant of the virus, the QTL still had the same strong effect. The heritability of resistance to the new variant of the virus was low: 0.08 in 2021 and not significantly different from zero in 2022. Genomic selection for increased resistance through will therefore be challenging, making the next workpackage even more important. In the final work package, we aim to find out if gene editing (CRISPR-Cas9) can be used to identify genes in salmon that are necessary for a virus to infect salmon cells. If we identify such genes and future legislation allows it, we could make the salmon more resistant by "knocking out" these genes using CRISPR. In this work package, we are using a so-called Genome-Scale CRISPR-Cas9 Knockout (GeCKO) screen in Atlantic salmon. In a GeCKO screen, all genes in a cell culture from the organism are knocked out, but in such a way that only one gene is knocked out in each individual cell. After the genes are knocked out, the cell culture is infected with a virus. The method can be used to identify genes that are essential for viral infection, as only cells with such genes knocked out will survive. We have encountered many obstacles along the way, most of which are related to the fact that the method was developed for mammals and not for fish. We have therefore had to modify many of the method's individual steps. The main problem is getting the CRISPR machinery into the salmon cells in a reproducible way and getting it to perform the "prescribed" gene editing. We believe we have now solved most of the problems, and we think we will be able to complete the GeCKO screen within the project's time frame (which has been extended). Our main goal is to perform a GeCKO screen for new variants of the IPN virus. While the project has been ongoing, there have been outbreaks of IPN among AquaGen’s customers in both Norway and Chile. Some of the outbreaks have been atypical. For example, one customer in Chile experienced a very high (50%) proportion of "losers," i.e., fish that stop growing. A customer in Norway experienced relatively high mortality, even though the sequence of the virus isolate indicated that it was of the "old" type. We want to study some of the relevant virus isolates further. Infection tests against new virus isolates will therefore be conducted in the first quarter of 2025. We are also working on developing an ex vivo test to determine whether IPN virus isolates are "sensitive" to the QTL or not. As of today, infection tests are required to determine this with certainty, and infection tests are expensive and not ideal in terms of fish welfare.

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.