Project Outline

Atlantic salmon is prised for its high omega-3 polyunsaturated fatty acids, offering health benefits for humans, especially cardiovascular and neurological health. Salmon aquaculture boosts the UK's export economy and global food security with a low carbon footprint. However, it faces challenges, primarily due to infectious diseases like salmon pancreas disease (PD), caused by salmonid alphavirus (SAV). Vaccination is the primary method for SAV control, but its efficacy across virus genotypes can be inconsistent. Alternative approaches, like functional feeds that enhance host immunity, are gaining traction. However, the precise mechanisms behind nutritional immune modulation still need to be better understood. Manufacturers are producing modified lipid-profile feeds to manage viral infections in salmon, yet the mechanisms at play remain unclear.

For SAV, functional feeds with altered lipid compositions have been reported to reduce heart inflammation and pathology. Improved understanding of viral perturbation of lipid metabolism in cultured salmon, particularly that occurring as a consequence of SAV infections, can thus be considered a strategic priority, offering opportunities to develop new approaches to the management of viral disease in fish, including insights into how feeds might be optimised to increase the resistance of fish against SAV infection. Our understanding of how SAV manipulates host machinery during replication is limited. Enveloped viruses, like SAV, heavily depend on lipids for their life cycle1. They create replication complexes (RCs) that protect against the immune response by rearranging intracellular membranes. Our previous research has shown SAV-related membrane rearrangement in salmon cell lines².

Understanding the interplay between viral pathogens and host lipid metabolism is crucial for advancing scientific knowledge and practical applications in aquaculture disease management. This research aims to examine viral modulation of host lipid metabolism in a range of cultured salmon cells, specifically focusing on how changes in lipid metabolism affect SAV replication and infectivity. We will use two established cell lines, TO (head kidney derived immune competent cells) and CHH-1 (chum salmon heart), to assess the following objectives

1. the impact of fatty acid supplements on SAV replication and infectivity in vitro. We will culture salmonid cells TO and CHH-1 in media supplemented with LC-PUFAs to establish cells with a fish-like composition before infecting them with SAV and testing infection kinetics (TCID50 and qPCR).

2. the structural and compositional changes in fish-like cells challenge with SAV. The changes to sub-cellular membrane structures in infected and non-infected fish-like cells will be studied using our established protocols for TEM and immune cytochemistry1,2. The total lipid content, lipid class composition, and lipid droplet distribution will be studied in infected and non-infected fTO and fCHH-1 cells using mass spectrophotometry.

3. to characterise the perturbation of lipid metabolism and innate immunity to viral infectivity. To study the impact of lipid homeostasis, we will measure viral infectivity in cells treated with hypolipidemic agents (e.g. statin and Niacin). We will knock down RIG-I (Retinoic Acid-Inducible Gene-I) employing CRISPR/Cas9 to study the interaction between innate immune mechanism and lipid metabolism. RIG is a key sensor of viral RNA in the cytoplasm and plays a critical role in initiating the innate immune response to alphaviruses4. By knocking down RIG-I, you can investigate its specific role in the antiviral response against alphaviruses in cells deprived of lipids.

Our research takes a multidisciplinary approach, including state-of-the-art molecular biology, electron microscopy, and gene expression analysis, to study how SAV affects salmon cells. We'll monitor lipid metabolism changes, viral replication, and the impact of lipid supplementation on viral infectivity. This integrated approach helps us study virus-lipid interactions for disease management, aiding the development of innovative disease management strategies. The knowledge gained from this study could have broader applications in understanding host-pathogen interactions, potentially influencing approaches to managing viral diseases in other contexts, including human health.

References

¹Chan RB, Tanner L, Wenk MR. Implications for lipids during replication of enveloped viruses. Chem Phys Lipids. 2010 Jun;163(6):449-59. doi:

²T K. Herath, H W. Ferguson, K.D. Thompson, A. Adams, R. H. Richards (2012). The ultra-structural morphogenesis of Salmon Alphavirus, Journal of Fish Diseases 35(799 – 808)

³T.K. Herath, H W. Ferguson, J E. Bron, K D. Thompson, A. Adams Adams, M. Weidmann, K. Muir, R H. Richards (2016). Pathogenesis of experimental salmonid alphavirus infection in vivo: an ultrastructural insight. Veterinary Research 47:7

⁴Thoresen D, Wang W, Galls D, Guo R, Xu L, Pyle AM. The molecular mechanism of RIG-I activation and signaling. Immunol Rev. 2021 Nov;304(1):154-168. doi: 10.1111/imr.13022.

Techniques

• Cell culture
• Viral infection assay and kinetics
• Fluorescent Microscopy
• Scanning and transmission electron microscopy
• Molecular biology - CRISPR/Cas9 and RT-qPCR
• Bio-informatics
• Mass spectrophotometry
• Immune Cytochemistry