# Dietary Methionine Supplementation Improves Rainbow Trout (Oncorhynchus mykiss) Immune Responses Against Viral Haemorrhagic Septicaemia Virus (VHSV)

## TL;DR

Adding methionine to rainbow trout diets boosts their immune response to a deadly virus, potentially improving fish health in aquaculture.

## Contribution

This study is the first to show that dietary methionine enhances antiviral immunity in fish, with viperin as a key mediator.

## Key findings

- Methionine supplementation increased antiviral gene expression, including viperin, in rainbow trout infected with VHSV.
- Fish on methionine-enriched diets showed improved immune recovery and reduced viral load after infection.
- Transcriptomic analysis revealed enhanced immune-related pathways in the skin and gills of methionine-fed fish.

## Abstract

This study explored the impact of dietary methionine supplementation on immune modulation and resistance to the viral haemorrhagic septicaemia virus (VHSV) in rainbow trout (Oncorhynchus mykiss). Juvenile fish were fed for four weeks with either a control diet meeting standard amino acid requirements or a methionine-supplemented diet containing twice the normal DL-methionine level. Following infection with VHSV, samples were collected at multiple time points to assess immune responses, oxidative stress, viral load, and gene expression. Methionine supplementation influenced leukocyte and monocyte activity, oxidative balance, and the expression of antiviral genes, particularly at 72 h post-infection when viral activity peaked. Transcriptomic analysis revealed the enhanced activation of immune-related pathways in the skin and gills of supplemented fish, including the upregulation of TLR3, NF-κB, IRF3/7, and VIG1 (viperin). The strong induction of VIG1, a S-adenosylmethionine dependent antiviral protein linked to methionine metabolism, indicates a key role in host defence. Overall, methionine-enriched diets improved immune recovery and activity against the virus, suggesting that dietary methionine may act as a nutritional immunostimulant and potential future prophylactic strategy to enhance fish health and resilience in aquaculture.

Several studies have demonstrated that methionine supplementation in fish diets enhances immune status, inflammatory response, and resistance to bacterial infections by modulating for DNA methylation, aminopropylation, and transsulfuration pathways. However, the immunomodulatory effects of methionine in viral infections remain unexplored. This study aimed to evaluate the effect of methionine supplementation on immune modulation and resistance to the viral haemorrhagic septicaemia virus (VHSV) in rainbow trout (Oncorhynchus mykiss). Two diets were formulated and fed to juvenile rainbow trout for four weeks: a control diet (CTRL) with all nutritional requirements, including the amino acid profile required for the species, and a methionine-supplemented diet (MET), containing twice the normal requirement of DL-methionine. After feeding, fish were bath-infected with VHSV, while control fish were exposed to a virus-free bath. Samples were collected at 0 (after feeding trial), 24, 72, and 120 h post-infection for the haematological profile, humoral immune response, oxidative stress, viral load, RNAseq, and gene expression analysis. In both diets, results showed a peak in viral activity at 72 h, followed by a reduction in viral load at 120 h, indicating immune recovery. During the peak of infection, leukocytes, thrombocytes, and monocytes migrated to the infection site, while oxidative stress biomarkers (superoxide dismutase glutathione S-transferase, and glutathione redox ratio) suggested a compromised ability to manage cellular imbalance due to intense viral activity. At 120 h, immune recovery and homeostasis were observed due to an increase in the amount of nitric oxide, GSH/GSSG levels, leukocyte replacement, monocyte influx, and a reduction in the viral load. When focusing on the infection peak, gene ontology (GO) analysis showed several exclusively enriched pathways in the skin and gills of MET-fed fish, driven by the upregulation of several key genes. Genes involved in recognition/signalling, inflammatory response, and other genes with direct antiviral activity, such as TLR3, MYD88, TRAF2, NF-κB, STING, IRF3, -7, VIG1, caspases, cathepsins, and TNF, were observed. Notably, VIG1 (viperin), a key antiviral protein, was significantly upregulated in gills, confirming the modulatory role of methionine in inducing its transcription. Viperin, which harbours an S-adenosyl-L-methionine (SAM) radical domain, is directly related to methionine biosynthesis and plays a critical role in the innate immune response to VHSV infection in rainbow trout. In summary, this study suggests that dietary methionine supplementation can enhance a more robust fish immune response to viral infections, with viperin as a crucial mediator. The improved antiviral readiness observed in MET-fed fish underscores the potential of targeted nutritional adjustments to sustain fish health and welfare in aquaculture.