Aquaculture Europe 2023

September 18 - 21, 2023


Add To Calendar 20/09/2023 12:00:0020/09/2023 12:15:00Europe/ViennaAquaculture Europe 2023UBIQUITINATION IN RESPONSE TO INFECTIOUS SALMON ANAEMIA VIRUS AND INFECTIOUS PANCREATIC NECROSIS VIRUS IN ATLANTIC SALMON Salmo salarStolz 0The European Aquaculture Societywebmaster@aquaeas.orgfalseDD/MM/YYYYaaVZHLXMfzTRLzDrHmAi181982


Robert Stewart1,*, Maeve Ballantyne1, Ophélie Gervais1, Sam Martin2, Beatriz Orosa3, Diego Robledo1


1The Roslin Institute, The Royal (Dick) School of Veterinary Study, The University of Edinburgh, Edinburgh, United  Kingdom

2 Scottish Fish Immunology Research Centre , University of Aberdeen, Aberdeen, United Kingdom

3 Institute of Molecular Plant Sciences, University of Edinburgh, Edinburgh, United Kingdom




Atlantic salmon (Salmo salar) is one of the most globally economically important  marine aquaculture species. Improving our  understanding of the  teleost  immune response to pathogens could help combat the high disease burden  in the aquaculture  sector. Ubiquitination  is an essential post-translational modification , known to play critical roles in the initiation, regulation, and termination of the innate immune system in mammalian species. Ubiquitin is present in all eukaryotic cells and is highly evolutionary conserved suggesting  that its  role in immunity may be conserved in other vertebrates. Despite evidence of many virus-inducible ubiquitination-related genes in fish4 and the observed up-regulation of the ubiquitination pathway in infected fish5, the role of ubiquitination in  response to  infection is  still  poorly understood. To identify the network of ubiquitin-related responses to viral infection this study combine s  analysis of the ubiquitinated proteome  and gene expression of an Atlantic salmon cell line  post-viral infection , using mass spectroscopy and RNA sequencing .


Materials and Methods

 Salmon Head Kidney cells (SHK-1) were inoculated with Infectious salmon anaemia virus (ISAV) or Infectious pancreatic necrosis virus (IPNV). Samples were collected at 24 and 48  hours post-infection (hpi), along with time-matched controls (4 biological replicates per condition ). For proteomics, c ells  were ly sed by freeze-thaw and ubiquitinated proteins were enriched using HaloTagged Ubiquilin. Ubiquitinated protein content was measured via western blot, then submitted for Mass spectroscopy analysis.  Mass spectroscopy was performed with data-dependent scanning , selection based on the host and viral proteome.  RNA was extracted using TRIzol,  and  polyA RNA-seq libraries prepared using standard Illumina protocols and  sequenced  on a Novaseq 6000 as 150PE reads. Low-quality reads were removed, and gene expression was estimated using Kallisto6 and the Atlantic salmon reference transcriptome ( Ssal_v3.1, GCA_905237065.2)7.


Infection of SHK-1 cells with ISAV induced a global up- regulation of ubiquitin, whilst IPNV infection induced an overall decrease in cellular ubiquitin (Figure 1A) .  This is consistent with the mass spectroscopy data, where  the total ubiquitination profile of ISAV samples incurred a net positive fold change , whilst IPNV induced an overall  negative fold change in ubiquitinated proteins , especially at 24 hpi (Figure 1B). A small number of proteins (4 -50 depending on the condition)  showed significant differences in  ubiquitination  in response to infection, including several relevant immune genes. For example , a  TRIM ubiquitin ligase, TRIM25-like protein, showed significantly increased ubiquitination  in response to ISAV at 24 hpi .  Interestingly, all viral proteins were identified  in the mass spectrometry data, and both  the viral polymerase basic protein 2 and Matrix Protein 1 of ISAV contained di-glycine remnants consistent with ubiquitination.  

RNA sequencing of the same samples revealed a massive up-regulation of gene expression in response to IPNV, both at 24 hpi and 48 hpi, while the response to ISAV was considerably more restricted (1033 vs 157 differentially expressed genes at 24 hpi).


 Our results demonstrate  a differential regulation of ubiquitination upon viral infection of different viruses in Atlantic salmon cells. We have found a TRIM25-like protein amongst the proteins with increased ubiquitination in response to ISAV. However, IPNV infection mainly led to decreased levels of ubiquitination in a small number of proteins. This lack of ubiquitination response contrasts with the large dysregulation of gene expression, and may suggest IPNV has the ability to regulate ubiquitination to enable infection. Additionally, multiple viral proteins contained ubiquitination sites upon digestion, namely  ISAV  viral polymerase basic protein 2 and matrix protein 1.


1                       FAO. The State of World Fisheries and Aquaculture 2022. Towards Blue Transformation. . doi: (2022).

2                       Zinngrebe, J., Montinaro, A., Peltzer, N. & Walczak, H. Ubiquitin in the immune system. EMBO Rep 15, 28-45, doi:10.1002/embr.201338025 (2014).

3                       Zuin, A., Isasa, M. & Crosas, B. Ubiquitin signaling: extreme conservation as a source of diversity. Cells 3, 690-701, doi:10.3390/cells3030690 (2014).

4                       van der Aa, L. M. et al. FinTRIMs, fish virus-inducible proteins with E3 ubiquitin ligase activity. Dev Comp Immunol 36, 433-441, doi:10.1016/j.dci.2011.08.010 (2012).

5                       Gervais, O. et al. Understanding host response to infectious salmon anaemia virus in an Atlantic salmon cell line using single-cell RNA sequencing. BMC Genomics 24, 161, doi:10.1186/s12864-023-09254-z (2023).

6                       Bray, N. L., Pimentel, H., Melsted, P. & Pachter, L. Near-optimal probabilistic RNA-seq quantification. Nat Biotechnol 34, 525-527, doi:10.1038/nbt.3519 (2016).

7                       Lien, S. et al. The Atlantic salmon genome provides insights into rediploidization. Nature 533, 200-205, doi:10.1038/nature17164 (2016).