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HAVBRUK2-Stort program for havbruksforskning

Synthetic biology and rational virus attenuation - a study on Salmonid pancreas disease virus

Alternative title: Syntetisk biologi og fremstilling av svekkede virus - en studie på Salmonid pancreas disease virus (SPDV)

Awarded: NOK 2.1 mill.

For the salmon aquaculture industry to be sustainable and profitable, it is important that virus infections are reduced. Viruses such as Salmon alphavirus-3 (SAV3) impact the health of salmon by causing pancreas disease (PD). Unfortunately, existing vaccines against SAV3 are not effective enough, and this motivated us to use advanced synthetic biology approaches to design a better vaccine against SAV3. We did this by altering the genome sequence of SAV3, and incorporating those changes into a 're-designed? SAV3 virus. When we tested the re-designed SAV3 in salmon, we found that it could not produce disease typical of SAV3, and this was an indication that we had achieved our aim of making SAV3 less virulent in salmon. Normally SAV3 causes >80% mortality in salmon fry, but the re-designed SAV3 caused no mortality. In fact, after salmon fry initially infected with the re-designed SAV3 were re-infected with virulent SAV3, again, no mortality was observed. This is an indication that we have made a potential virus vaccine against PD. But how did our project get to these results? To begin with, we used the DNA sequence of a SAV3 isolate (which we call 'wild-type SAV3') obtained from a fish with PD, and we changed the DNA sequence of it. We did this in a way that the DNA changes would affect an important biological property of the DNA sequence. By making these changes we were able to make a new 're-designed SAV3', based on the sequence of the wild-type SAV3. We then made additional changes to the re-designed SAV3, and ended up with seven slightly similar versions of the original re-designed SAV3 virus. Before testing the wild-type SAV3 and the seven version of the re-designed SAV3 in salmon, we decided to test them in salmon cell lines. What we were interested in finding out is whether there were any differences in the ability of all these viruses to replicate in salmon cell lines. Very good replication would mean that the viruses are quite virulent and able to replicate. Very poor replication would mean that the viruses were weak and not so virulent in fish cells. The results of this test showed that the wild-type SAV3 replicated very well in fish cells, which is what we expected since this virus was isolated directly from a salmon with severe PD. The seven versions of the re-designed SAV3 differed in their ability to replicate in fish cells. This means that they differed in the degree to which they were weakened, as result of the changes to the DNA we had initially made. Some of them did not replicate at all, whereas for four of them there was a gradient in their ability to replicate, which is a good property when designing virus vaccines. We began testing the wild-type SAV3 and three versions of the re-designed SAV3 in salmon fry instead of salmon fish cell lines in January 2020. For the trial, we exploited the fact that the three weakened viruses cover a broad range of attenuation in fish cells to test the impact of this on the development of a successful vaccine against pancreas disease. While conducting the fish trial, we made observations of how salmon fry responded when injected with each of the three attenuated viruses, and we compared that with how they responded to injection with the wild-type SAV3, or when they were not injected with any virus at all. Various organ samples were obtained from fish during the trial, and these samples will be used for further analyses that will show the extent to which the attenuated viruses are weakened in fish, and how the fish immune system responds to them. After 80 days of running the fish trial, the evidence clearly showed that two of the attenuated viruses produced no mortality in fish, whereas the third attenuated virus produced up to 60% mortality. In comparison, all the fish infected with the wild-type SAV3 died as a result of pancreas disease, and none of the uninfected fish died. These results suggest that the attenuated viruses potentially function as a vaccine. To test this, surviving fish were re-injected with wild-type SAV3 to start the second phase of the fish trial. If in the first phase of the fish trial the attenuated viruses successfully 'trained' the immune system of these fish to fight off an infection with wild-type SAV3 (an indication of vaccine effect), then the fish would be protected against wild-type SAV3 during the second phase of the fish trial. Results obtained by the end of the fish trial indicated that this was indeed the case because whereas there was no mortality among fish that were previously injected with the attenuated viruses, 25% mortality was observed in fish not previously injected with these attenuated viruses.

We designed a Salmon alphavirus virus subtype 3 (SAV3) that is attenuated in salmon. Participants in this project have gained experience in synthetic biology and the design of potential virus vaccines. Research programs other than ours have benefited from the work conducted in this project by including our findings in their research programs. The project has also benefited our partners in France and South Africa, and further cemented our collaboration due to the synergy of our work. The sustainability of salmon aquaculture necessitates good fish health, and this will have welfare as well as economic benefits. The attenuated SAV3 can be used as research tools to study the immune system of salmon, so as to ultimately improve fish health. However, our use of synthetic biology may be controversial to members of society. We expect the scientific knowledge gained in this research to contribute to daily discussion of how our society should engage with advances in biotechnology.

The SYBIATT project is based on the hypothesis that a live variant of SPDV will be attenuated by changing either its codon-pair bias, CpG content, or codon:anticodon pairing energy, and that such a variant will still elicit a protective antibody and cellular immune response in salmon. The long-term goal is to use this live-attenuated SPDV and systems biology to study in greater detail the dynamics of complex immune interaction networks in salmon that are protected from PD. The project activities fall into four main parts: - First, attenuated variants of SPDV will be designed using three different strategies and subsequently synthesised as infective clones. The design will be done in close collaboration with Dr Darren Martin (UCT). - Second, the performance of the infective clones (variants) will be tested, e.g. for their infectivity, attenuation and genetic stability. This work will be done in collaboration with Prof Bremont and Dr Biacchesi (INRA), partly in France. - Third, the variants will be applied for (in-vivo) studies of virus-host interactions, development of immunity, genetic stability etc. Prof Bremont and Dr Biacchesi (INRA) and Dr Sundaram (NSC) will be closely collaborating and some of the studies will be done in France. - Fourth, systems biological software tools and approaches will be applied to explore and interpret RNAseq data from above studies. This work will be done in close collaboration with Dr Sundaram (NSC). The SYBIATT project will be an integrated part of the on-going MucoPath project at NVI. In context of MucoPath and in collaboration with Prof Bremont and Dr Biacchesi, Drs Monjane and Grove have designed and synthesised the first SPDV variants and are currently initiating the testing of these. While this preliminary work indeed supports the feasibility of the current project, SYBIATT will be a most vital extension and strengthening that will help the on-going activities to reach their final goal.

Funding scheme:

HAVBRUK2-Stort program for havbruksforskning