The role of virus-Induced fatty Acid imbalance in the pathogenesis of salmonid alphavirus infection

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 cytochemistry^1,2. 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

Use of mini-gut model to study host-pathogen interaction and target control of coccidiosis in chicken

Project Outline

Coccidiosis, caused by Eimeria species (Phylum Apicomplexa) is a pervasive and economically significant parasitic disease affecting the poultry industry in the UK and worldwide. This highly contagious protozoan parasite causes severe enteric infections leading to mortality and poor growth in flocks, while predisposing affected birds to secondary infections. The control of coccidia becomes highly problematic due to its complex life cycle extending between the host and environment. Despite advancements in good production practices that doubled poultry production worldwide during the last two decades, the losses due to coccidiosis over the years escalated. In the UK, industry loss increased from £38 million in 1999 to £99.2 million in 2016, whereas it was £10.4 billion worldwide. While financial loss remains a major concern, the use of anticoccidial drugs also called coccidiostat known to develop drug resistance has remained a longstanding concern in terms of food, and environmental safety adding further complexity to the issue. Addressing the growing economic impact of coccidiosis, underscores the need for research and innovation, especially in the field of developing novel treatments and management strategies that are sustainable, cost-effective, and have a minimal environmental impact. The present research methodology exists to assess host-pathogen interaction and response to treatment often requires in vivo pathogen challenge studies, which are costly, time-consuming, and raise ethical concerns. Overcoming these limitations, the research proposed in this PhD aims to leverage chicken intestinal enteroid cultures to study host-pathogen interaction at the mucosal surface in the presence of novel nutraceuticals.

Enteroids, also known as intestinal organoids or enteric organoids, are three-dimensional cell culture models that mimic the structure and function of the intestinal epithelium as an alternative and more ethically sound platform that abides 3R principle. These culture systems are successfully used as a tool for studying various aspects of the digestive system, including nutrient absorption, gut diseases, interactions with pathogens, and the development of new therapeutic and control solutions. Intestinal organoids are successfully used to study apicomplexan parasites (e.g. Crypsoporedia spp) that affect both livestock and humans1. In our laboratory, we have successfully used cultured intestinal cells to study host-pathogen and nutritional absorption studies. In this PhD, we will explore the following objectives to fulfill our aim;

1. Establish a caecal enteroid using 18-day-old embryonated chicken eggs: The cells for the enteroid will be isolated using an established method1. We supplement cells with use of natural growth promotors known to improve intestinal cell integrity 2 and will be assessed using imaging and cell-culture techniques available in the lab.
2. use cecal enteroids to study the infection dynamics: the parasites will be collected as described and sporulated in vitro before infecting enteroids to map parasite invasion, sextual maturation and cell modulation2 using histology, electron microscopy and immunohistochemistry. We will employ cell culture and gene-expression techniques that are already established in our lab to elucidate the host response. From this, we aim to understand potential innate immune mechanisms to define therapeutic targets.
3. assess the suitability of the model for screening studies: to use as a platform to study anticoccidial therapeutics, a panel of novel nutraceuticals to anticoccidial drugs with the potential to be used as feed additives. We will study potential therapeutics that are known to modulate the innate immune capacities of the enteroids.

In completion. The result of this project will establish an in vitro platform that can be used directly in screening feed additives and drug targets in addition to use in studying host-pathogen interaction. The student will benefit from engaging in cutting-edge research with applications to improve the health and welfare of poultry.

References

¹Bhalchandra S, Lamisere H, Ward H. Intestinal organoid/enteroid-based models for Cryptosporidium. Curr Opin Microbiol. 2020 Dec;58:124-129. doi: 10.1016/j.mib.2020.10.002.

²L. Berriman, S.P. Rose, V. Pirgozliev (2017) Effect of dietary phytonutrients in ameliorating coccidiosis challenge, WPSA Annual UK Branch meeting, Chester 25 -27 April, British Poultry Abstracts, p.4

Techniques

• Isolation of Caecal Enteroids: This technique involves isolating caecal enteroids from 18-day-old embryonated chicken eggs, likely utilising established methods.
• Imaging: These techniques will be employed to assess the integrity of intestinal cells, applying microscopy.
• Electron Microscopy: Electron microscopy will provide high-resolution images to study cellular and subcellular structures.
• Parasite culture: parasites will be grown in vitro before infecting enteroids, aiding in the study of infection dynamics.
• Histology and cytology: Histological techniques will be utilised to examine tissue structures and changes in response to infection.
• Immunohistochemistry: This technique involves using antibodies to visualise specific proteins or molecules in tissue sections.
• Gene Expression Analysis: Molecular techniques will be employed to analyse gene expression patterns in response to infection and treatment.