Author: marina02

Experimental infection of rainbow trout with Piscine orthoreovirus 2 (PRV-2)

Piscine orthoreoviruses (recently renamed Orthoreovirus piscis by ICTV) have emerged as a challenge for salmonid aquaculture worldwide.

Piscine orthoreovirus (PRV) belongs to the family Reoviridae, has a double stranded RNA genome consisting of 10 segments and double protein capsid with icosahedral symmetry.

Three subtypes of PRV have been reported: PRV-1, PRV-2, and PRV-3.

PRV-1 has caused heart and skeletal muscle inflammation (HSMI) in Atlantic salmon (Salmo salar) in Norway since 1999. The major histopathological findings are in located in the heart and the red skeletal muscle. Affected fish show pancarditis, and severely affected fish also have red muscle inflammation.

PRV-3 was discovered in 2013 following a thorough investigation of unexplained mortalities in rainbow trout (Oncorhynchus mykiss) farmed in fresh water in Norway. Experimental trials showed that PRV-3 replicated in rainbow trout blood and efficiently transmitted to naïve host in a cohabitation trial. Pathology occurs predominantly in heart.

PRV-2 was shown to cause erythrocytic inclusion body syndrome (EIBS) in coho salmon (Oncorhynchus kisutch) in 2016 in Japan. The anaemic condition denoted as EIBS was first described in 1977 in rainbow trout and in 1987 in chinook salmon (Oncorhynchus tshawytscha) from the Pacific Northwest of North America. To date, PRV-2 like virus has been described in Alaska. Little is known about the capability of the virus to infect Rainbow trout.

Specific pathogen free rainbow trout were included in a cohabitation challenge trial. Briefly, PRV-2 infected tissue from Coho salmon was homogenized in EMEM supplemented with antibiotic. Shedder fish were tagged by adipose fin clipping and IP injected with supernatant from the aforementioned homogenate. Fish were cohabited in a tank with naïve non-tagged cohabitants. A group of mock- injected fish was included. The ratio of shedders to cohabitants  was set at 50:50.

At selected time points, 6 shedders and 6 cohabitants were euthanized and organ samples collected to assess viral load and heart histopathological changes. Blood was collected to measure heamatocrit (Hct). Fish were inspected daily over the 7 weeks trial to assess the presence of clinical signs and morbidity.

No morbidity nor reduced survival was observed in the experiment. Infected fish developed severe anemia, yielding Hct value of 10% in the lowest cases.

Efficient transfer of virus from shedders to cohabitants occurred starting from 3 weeks post challenge. Histopathological changes observed in heart were predominantly epicarditis.

Rainbow trout can be considered a susceptible host for PRV-2.

1. VENDRAMIN, NICCOLÓ, DTU AQUA, Presenter
2. Sørensen, Juliane, DTU AQUA, Author
3. Teena, Klinge, DTU AQUA, Author
4. Cuenca, Argelia, DTU AQUA, Author

Toll-like receptor 3 function in a chinook salmon Oncorhynchus tshawytscha salmonid cell line

Introduction Nucleic acids represent a class of pathogen-associated molecular patterns (PAMPs) that indicate the presence of a pathogen. They are detected by of pattern-recognition receptors including six subfamilies of Toll-like receptors (TLR). There are 10 to 12 TLR in mammals and even more in diverse fish species. Among these receptors, TLR3 is located on the endosomal or plasma membrane and binds to double-stranded RNA (dsRNA) derived from viruses, as well as poly(I:C), its synthetic analogue. Upon dsRNA binding, TRIF is recruited to the TLR3 dimer and leads to the activation of NF-κB/IRF which induces theproduction of pro-inflammatory cytokines type-I interferons (IFN1). CHSE-214 is an epithelioid cell line initially isolated from the Chinook salmon Oncorhynchus tshawytscha embryos and is being extensively used for diagnosing viral diseases in fish. This cell line became a biological material used to study the function of genes involved in the immune response. Yet, this cell line has been found unable to respond naturally to extra cellular poly I:C unlike other available salmonid fish cell lines, such as RTG2, RTS11 or TO. This deficiency also extends to the CHSE-214 -derived recombinant cell line CHSE-EC expressing GFP permanently. In the present study, we demonstrate that the tlr3 gene is missing from the CHSE-derived cell lines genome and we show that the extracellular dsRNA responsiveness can be restored by complementation in a novel stable clonal cell line, TLR3-Pur-C4. The characterisation of this cell line shed light on the regulation of the dsRNA/tlr3 activation in salmonid fish.

Methodology The genome of CHSE-EC was sequenced using nanopore technology and analysed using minimap2/IGV. The chinook salmon tlr3 expression vector was obtained by gene synthesis. Clonal tlr3-expressing CHSE-EC cells was obtained by electroporation, puromycin selection and manual cloning (TLR3-Pur-C4 cell line). Responsiveness to dsRNA was evaluated by measuring the induction of mx and other Interferon Stimulated Genes (ISGs) by qPCR.

Results and Conclusions In the CHSE-EC genome 70 % of the chromosome 34, including the tlr3 gene, is missing. RNAseq data analysis showed that CHSE-EC cells express tlr1, tlr5, tlr18 and 5 members of the tlr11/12/13/19/21/22/23 subgroup. In addition, all the genes encoding tlr-related signalling molecules are present in the genome and expressed in CHSE-EC cells. dsRNA binding and IFN1 activation are fully restored in the TL3-Pur-C4 cell line. Loss-of-function studies are currently under way to understand precisely the dsRNA-induced activation of IFN1 pathway.

1. COLLET, BERTRAND, INRAE, Presenter
2. PERUZZI, MATHILDE, INRAE, Author
3. COLLINS, CATHERINE, UCC, Author
4. REBL, ALEXANDER, FBN, Author
5. BOUDINOT, PIERRE, INRAE, Author
6. DE WITT-ORR, STEPHANIE, WLU, Author
7. CATHERINE-MEZERAY, JUSTINE, INRAE, Author

Diverse lineages of Piscine orthoreovirus (PRV) in Icelandic salmonids

Introduction: Piscine orthoreovirus (PRV) is associated with persistent heart and skeletal muscle inflammation (HSMI) in multiple salmonid species, affecting both wild and farmed populations globally. The chronic nature of this disease has significant implications for ecosystem health, animal welfare, and economic losses in aquaculture. In Iceland, PRV-1 is widespread in farmed salmon (Salmo salar), both in freshwater and marine environments. Although Arctic charr (Salvelinus alpinus) is also farmed in Iceland, there was no information on PRV occurrence in this species. This context provides a valuable opportunity to screen for PRV and investigate its genetic variability and evolutionary potential across the three indigenous salmonid species in Iceland: Atlantic salmon, Arctic charr, and brown trout (Salmo trutta), in both wild and aquaculture settings.

Methodology: Wild and farmed specimens of Atlantic salmon (N=331), Arctic charr (N=251), and brown trout (N=79) were screened for PRV using qPCR, followed by histopathological examinations. At least one genomic segment (S1) from PRV-positive individuals was sequenced, and additional segments previously linked to virulence were sequenced in a subset of samples to further explore genomic variability. Phylogenetic trees were constructed for each segment using additional publicly available PRV sequence data.

Results: For the first time we report the presence of PRV in both wild and farmed Icelandic Arctic charr, with sequences showing similarity to PRV2. This variant was found in farmed Arctic charr with high viral loads, while both incidence and load were low in the wild fish. Histopathological examination showed signs of HSMI that could be linked to PRV-2 infection. Additionally, we document the presence of PRV-3 in brown trout from at least two Icelandic lakes. In Atlantic salmon, PRV-1 was found at high prevalence and viral load in salmon populations of wild origin, farmed for restocking purposes, while lower values were observed in wild salmon. Sequence variation was relatively low among sea-grazing salmon, but greater genetic diversity was observed in wild populations.

Conclusions: These findings expand the known host range of PRV revealing for the first time its presence in Arctic charr worldwide, and brown trout in Iceland. They also show distinct patterns of prevalence, viral load, and sequence variability across species and environments, underscoring the virus’s evolutionary potential.

1. de la Cámara, Marina, Institute for Experimental Pathology at Keldur, Presenter
2. Kristmundsson, Árni, Institute for Experimental Pathology at Keldur, Author
3. Sigurðardóttir, Heiða, Institute for Experimental Pathology at Keldur, Author
4. Bragason, Birir Þór, Institute for Experimental Pathology at Keldur, Author

Atlantic salmon trim25 is virus-inducible and protects Salmon Head Kidney cells from ISAV infection

TRIM25 is an E3-ubiquitin ligase that has been identified in multiple viral and viral mimic challenges as a virus-induced gene, putatively part of the type I interferon pathway. In model species, TRIM25 acts as both a sensor of viral RNA and a signal transducer, acting via the RIG-like receptor pathway. However, this mechanism remains uncharacterised in the global economic heavyweight Atlantic salmon (Salmo salar), a marine-raised commercial species constantly threatened and damaged by viral outbreaks. The purpose of this project is to characterize the function of the two Salmo salar trim25 ohnologues (ssTRIM25 – ENSSSAG00000054152 and ENSSSAG00000046838) in order to better understand the host response to viruses, whilst also attempting to explore the role of the highly expanded and diversified trim repertoire in teleost fish. By utilising CRISPR-Cas9 genome editing, we have been able to specifically knock-out each ohnologue of ssTRIM25, and thus have shown the ohnologue specific induction and protection of ssTRIM25 in an Atlantic salmon head kidney cell line (SHK-1). For the first time, we have demonstrated the redundant functionalization of one duplicate of trim25 and also shown how overexpression of ssTRIM25 leads to protection from viral infection. This research may lead to the production of more efficient vaccine production cell lines and potentially to genome-engineered Atlantic salmon with increased resistance to virus, in addition to improving understanding of trim genes in fish.

1. STEWART, ROB, The University of Edinburgh, Presenter
2. Perez, Noelia, 2 University of Santiago de Compostela, Author
3. Clark, Thomas, The University of Edinburgh, Author
4. Ballantyne, Maeve, The University of Edinburgh, Author
5. Martin, Samuel, Scottish Fish Immunology Research Centre, Author
6. Beatriz, Orosa-Puente, The University of Edinburgh, Author
7. Diego, Robledo, The University of Edinburgh, Author

Occurrence of Persistence of Infectious Hematopoietic Necrosis Virus in Rainbow Trout Surviving in Experimental Infection

Rainbow trout

Oncorhyncus mykiss 

is the most commonly produced trout species in Japan. Infectious hematopoietic necrosis (IHN) caused by IHNV is one of the most serious diseases in rainbow trout aquaculture. After recovering from IHN, the fish becomes apparently healthy with a normal appetite, and, consequently, IHNV cannot be isolated from the fish by the routine virus isolation procedure using cell culture. However, virus persistence in the fish is suspected. In this study, we tried to examine the relationship between fish immune responses and the occurrence of virus persistence in surviving fish.

Four IHNV strains with different virulence isolated in Japan in 2013: YN1311, NT1301, TV1308, and SO1304 (abbreviated as YN, NT, TV, and SO, respectively) were used. Rainbow trout (11.8g) were infected by bathing with the virus strains and reared at 15C. The cumulative mortality rated in YN, NT, TV, and SO infected groups were 80%, 20%, 10%, and 10%, respectively. Virus was re-isolated in the YN, NT, TV, and SO groups up to 20, 20, 9, and 12 days post-infection (dpi), respectively, in the routine virus isolation procedure using EPC cell line. Surviving fish received intraperitoneal injection with immunosuppressants (dexamethasone and cyclosporine) every 2 days for a total of 5 times to reactivate IHNV for allowing virus isolation on EPC cells. After immunosuppression, the virus was re-isolated in the TV, NT, and SO groups until 30, 37, and 44 dpi, respectively, but it could not be isolated in the YN group even at 30 dpi. The results suggest that IHNV persistence occurs frequently in surviving fish with a lower mortality and hardly in survivors with a higher mortality rate, which can be related to the strength of the immune response induced by different virus kinetics and loads in fish. In a further infection experiment, mortality ended at 20 dpi in the fish (8.0g) infected with YN and SO with cumulative mortality rate of 95% and 10%, respectively. The virus was re-isolated up to 30 and 83 dpi from the survivors of the YN and SO groups, respectively, after immunosuppression, showing persistent infection occurred in the SO group but not in the YN group. Transcriptome analyses using the kidney of fish periodically sampled from both groups were conducted. Immune-related gene expression levels will be discussed in relation to the occurrence of IHNV persistence in rainbow trout.

1. VERGEL, Joseph Carlo, Tokyo University of Marine Science and Technology Japan, Presenter
2. TSUBOI, Gosuke, Tokyo University of Marine Science and Technology Japan, Author
3. SHIBATA, Yuki, Tokyo University of Marine Science and Technology Japan, Author
4. SEIDA, Hakuto, Tokyo University of Marine Science and Technology Japan, Author
5. KIUCHI, Yusuke, Tokyo University of Marine Science and Technology Japan, Author
6. MATSUMOTO, Megumi, Tokyo University of Marine Science and Technology Japan, Author
7. KATO, Goshi, Tokyo University of Marine Science and Technology Japan, Author
8. KONDO, Hidehiro, Tokyo University of Marine Science and Technology Japan, Author
9. SANO, Motohiko, Tokyo University of Marine Science and Technology Japan, Author

Cell classification and characterization of gill-epithelial antigen sampling cell population based on gene expression patterns in rainbow trout

 Immersion vaccination drastically reduces the labor cost of vaccination to farmed fish. However, only limited number of immersion vaccine are available in aquaculture. Previously, we showed that gill-epithelial antigen sampling (GAS) cells take up vaccines during immersion, degrade them in their cytoplasm, and probably evoke local immune responses in rainbow trout. May-Grünwald Giemsa staining of sorted GAS cells showed that the cell population was composed of two morphologically distinct cell types. One had a large nucleus (LN-type), the other had a condensed nucleus (CN-type). This study aimed to classify and characterize GAS cell population based on gene expression patterns using single-cell RNA sequencing (scRNA-seq) and fluorescence in situ hybridization (FISH).

Epithelial cells of the gills were dispersed using EDTA and fluorescently stained with monoclonal antibody 2B4-1 and Ulex europaeus agglutinin (UEA)-1. GAS cell population (2B4-1⁺/UEA-1⁺) was sorted by flow cytometry and subjected to scRNA-seq by Drop-seq using a microfluidic device. Gene expression profiles of 3,461 cells were obtained by Illumina NextSeq 2000. Of these, 1854 cells that passed the quality check, were used for clustering analysis by Uniform Manifold Approximation and Protection (UMAP) with the Seurat package.FISH of sorted GAS cells was performed targeting candidate marker genes including tryptase, ferric chelate reductase1 (frrs1), zymogen granule membrane protein 16 (zg16), ribosomal protein S20. (rps20), ribosomal protein L7 (rl7) and S6 ribosomal protein (rps6) in each cell group detected by UMAP analysis.

UMAP analysis revealed that there were two major cell groups in the GAS cell population: A and B. Cell group A specifically expressed MHC classⅡ, tryptase, frrs1 and zg16 genes. Cell group B expressed various ribosomal protein (RP) genes such as rps20, rl7, rps6 genes. FISH showed that fluorescent signals of tryptase, frrs1 and zg16 were detected in LN-type GAS cells, while these signals were barely detected in CN-type GAS cells. Fluorescent signals of rl7, rps20 and rps6 were detected in both LN- and CN-type GAS cells. These data showed that cell group A and B correspond to LN-type and CN-type GAS cells, respectively. LN-type GAS cells are probably transcriptionally active cells expressing MHC classⅡ, tryptase, frrs1 and zg16, while CN-type GAS cells expressing only RP genes might approach to cell death in the turnover of the epithelial cells.

1. Saito, Nagisa, Tokyo University of Marine Science and Technology, Presenter
2. Harada, Masachika, Tokyo University of Marine Science and Technology, Author
3. Kusaka, Hiroki, Tokyo University of Marine Science and Technology, Author
4. Yuasa, Hajime, Tokyo University of Marine Science and Technology, Author
5. Konishi, Kayo, Tokyo University of Marine Science and Technology, Author
6. Matsumoto, Megumi, Tokyo University of Marine Science and Technology, Author
7. Sano, Motohiko, Tokyo University of Marine Science and Technology, Author
8. Koiwai, Keiichiro, Tokyo University of Marine Science and Technology, Author
9. Kato, Goshi, Tokyo University of Marine Science and Technology, Author

Italian IHNV low and highly pathogenic to rainbow trout displays different infection strategies

Introduction

In recent years, Infectious Hematopoietic Necrosis has been displaying an increase in its distribution and incidence in Europe. In Italy, IHNV is characterized by a fast evolutionary rate and displacement phenomena have been observed with highly pathogenic IHNV strain replacing low pathogenic ones in some areas. The replacements resulted in severe and persistent IHN outbreaks with relevant impact on rainbow trout productions.

Methodology

A low pathogenic IHNV and a highly pathogenic IHNV were characterized through in vitro and in vivo assays to investigate the mechanisms at the base of the different virulence profile. Viral replication steps on RTG-2 cells were compared through both quantitative RT-qPCR and end-point titration methods. In vivo challenges were performed to identify the ID50 and the LD50 of both IHNV isolates. In addition, disease progression was investigated at histological level coupled with immunohistochemistry assays.

Results

The highly pathogenic IHNV isolate displayed a lower adsorption efficiency coupled with a higher total viral production compared to the low pathogenic IHNV isolate. The low pathogenic isolate showed a lower ID50 compared to the highly pathogenic one but conversely it had been not possible to identify the LD50 due to low mortality level observed. At histology, low pathogenic isolate induced mild lesions in target organs analysed at earlier timepoints but the same were not present in later ones. Conversely, in fish exposed to the highly pathogenic isolate severe histological lesions appeared later but were persistent. A similar behaviour was observed through immunohistochemistry.

Conclusions

Differences in the virulence profile of the tested IHNV isolates corresponded to different infection strategies. The highly pathogenic isolate induced slower and long lasting infection with severe histological lesions that could explain the virulence observed. Conversely, the low pathogenic isolate induced a quick and transient infection with a mild effect on the target organs. The characterization of more IHNV isolates and the investigation of the host immune response to infection will be required to shed light on the mechanisms responsible for the different behaviour observed.

Funded by the Italian Ministry of Health project IZSVe RC 13/19.

1. MARSELLA, ANDREA, IZSVE, Presenter
2. PASCOLI, FRANCESCO, IZSVE, Author
3. LUCON XICCATO, ROMY, IZSVE, Author
4. ABBADI, MIRIAM, IZSVE, Author
5. BURATIN, ALESSANDRA, IZSVE, Author
6. BIASINI, LORENA, IZSVE, Author
7. BERTO, PAOLA, IZSVE, Author
8. PRETTO, TOBIA, IZSVE, Author
9. TOSON, MARICA, IZSVE, Author
10. TOFFAN, ANNA, IZSVE, Author

In cold water: impacts of acute cold-stress and Vibrio harveyi challenge on the microRNA and mRNA response of barramundi (Lates calcarifer).

Introduction:

Barramundi (Lates calcarifer), also known as Asian seabass, are emerging globally as an important warmwater aquaculture species. However, disease outbreaks caused by Vibrio species, such as Vibrio harveyi, remain a major impediment to sustained industry growth. Climate change exacerbates these challenges, as extreme weather events like cyclones and tropical storms can trigger rapid temperature drops, increasing the risk of infections by opportunistic bacteria. This study investigated the combined impact of acute cold-stress and V. harveyi infection on the early mRNA and microRNA (miRNA) responses of juvenile barramundi to better understand host-pathogen interactions.

Methodology:

Using a challenge model established in Samsing et al. (2023), juvenile barramundi received a skin-wound and were immersed for 1 hour in either V. harveyi (~6 × 10⁸ CFU mL⁻¹) or phosphate-buffered saline at 30°C. Fish were then transferred to aquaria maintained at either optimal (30°C) or cold-stress (22°C) conditions for both Vibrio and Control groups. Spleen tissue was collected 24 hours post-challenge for total RNA extraction, followed by mRNA and miRNA sequencing. Custom bioinformatic pipelines were developed to analyse the transcriptome response and to quantify the miRNA response of barramundi to bacterial infection for the first time.

Results:

Cold-stressed, V. harveyi infected fish showed the strongest transcriptional response with 3,231 differentially expressed genes (DEGs) compared to fish infected at 30°C (1,361 DEGs) or cold-stressed controls (346 DEGs). The majority of DEGs in cold-stressed infected fish were associated with a pronounced pro-inflammatory response including significant upregulation of cytokines and interleukins. In contrast to the strong mRNA response, miRNA expression was highest in fish infected at 30°C with 36 differentially expressed miRNAs (both up- and down-regulated) compared to just 8 up-regulated in cold-stressed V. harveyi infected fish. MiRNAs are potent mediators of mRNA expression and are known to impact the immune-regulatory capacity of teleosts.

Conclusions:

The substantial differential expression of miRNAs in barramundi kept at 30°C, and the lack of miRNAs in cold stressed fish, suggests that cold stress impaired the immune-regulatory capacity of affected fish. This likely contributed to an unchecked hyper-inflammatory response in cold-stressed fish infected with V. harveyi which ultimately may have led to the much higher mortality observed in cold-stressed infected fish (73%) compared to fish housed at 30°C (15%).

1. Sullivan, Roisin, School of Life and Environmental Sciences, Faculty of Science, The University of Sydney, Camden, NSW, Australia, Presenter
2. Becker, Joy, School of Life and Environmental Sciences, Faculty of Science, The University of Sydney, Camden, NSW, Australia, Author
3. Samsing, Francisca, Sydney School of Veterinary Science, Faculty of Science, The University of Sydney, Camden, NSW, Australia, Author

Holding Back the Tide of Aquatic Pathogens: How Virus Genomics Can Protect the Aquaculture Industry

Salmon aquaculture is one of the fastest-growing food production system worldwide, driven by rising global demand for high-quality protein and sustainable seafood. However, the industry faces substantial challenges, with nearly 17% of salmon dying during the marine phase of production, and 38% of these deaths attributed to infectious diseases. Viral infections not only result in high mortality but also compromise fish welfare, increase environmental impact, and raise production costs.

Among these viral diseases, infectious salmon anemia (ISA), caused by the infectious salmon anemia virus (ISAV), is one of the most devastating, with major outbreaks reported in Norway, the Faroe Islands, Chile, and Canada. Understanding the emergence, evolution and transmission dynamics of ISAV is critical for developing effective disease control strategies. Althoughgenomic epidemiology provides powerful tools to for investigating virus transmission and evolution, previous studies have been constrained by the limited availability of sequence data, which has predominantly been confined to partial genomes.

In this study, we developed and applied robust whole-genome sequencing protocols to generate nearly 170 complete ISAV genomes. We also utilized a comprehensive surveillance framework that incorporated detailed metadata from hundreds of salmon farms across Norway and the United Kingdom. By integrating genomic and epidemiological data, we estimated ISAV evolutionary dynamics and reconstructed transmission pathways between fish farms, as well as between farmed and wild Atlantic salmon populations. Whole-genome sequencing is particularly critical for ISAV, a segmented virus, as it enables the detection and characterization of reassortment events that may contribute to viral evolution and emergence of new variants. We further assessed the factors driving ISAV transmission, including geographic proximity farm connectivity via wellboat movements, and other farm characteristics. Additionally, we examined genetic variations in segments 5 and 6 to investigate how pathogenic variants (HPRdel) arise from non-pathogenic precursors (HPR0).

This study provides novel insights into the evolutionary dynamics and transmission pathways of ISAV, highlighting how whole-genome sequencing enables the detection of reassortment events and the emergence of new variants. It also emphasizes the values of integrating genomic epidemiology with detailed metadata to identify factors driving viral spread. These findings underscore the values of using genomic epidemiology in informing targeted biosecurity measures and outbreak management strategies. Continued investment in whole-genome surveillance is critical for limiting viral transmission, enhancing disease control, and minimizing the economic losses associated with ISAV infections in salmon aquaculture.

1. Zhao, Mingli, ROYAL VETERINARY, UK COLLEGE, Presenter
2. Duault, Helene, INRAE, VetAgro Sup, UMR EPIA, Université Clermont Auvergne, French, Author
3. Solheim, Mari Aas, PHARMAQ AS, Norway, Author
4. Madhun, Abdullah, Institute of Marine Research, Norway, Author
5. Soares, Silvia, Marine Directorate, Marine Laboratory, UK, Author
6. Filipe, Ana Da Silva, MRC-University of Glasgow Centre for Virus Research, University of Glasgow, UK, Author
7. Smollett, Katherine, MRC-University of Glasgow Centre for Virus Research, University of Glasgow, UK, Author
8. Matthews, Chris, PHARMAQ Analytiq Limited, UK, Author
9. Alexandersen, Svein, PHARMAQ AS, Norway, Author
10. Fournié, Guillaume, INRAE, VetAgro Sup, UMR EPIA, Université Clermont Auvergne, French, Author
11. Karlsen, Marius, PHARMAQ AS, Norway, Author
12. Hill, Sarah, ROYAL VETERINARY COLLEGE, UK, Author

Role of European sea bass proteins in the course of betanodavirus infections

Introduction

Fish rtp3, belonging to the receptor-transporting protein (RTP) family, has been described as a viral responsive gene. In previous studies, we have characterized two European sea bass (Dicentrarchus labrax) rtp3 genes (rtp3X1, rtp3X2). In addition, an in vivo study revealed induction of rtp3X1 and X2 after LPS and poly I:C inoculation; however, the highest transcriptional level was recorded after nervous necrosis virus (NNV, Betanodavirus genus) inoculation. This virus is the causative agent of the viral nervous necrosis, the main viral disease affecting European sea bass. It is genetically composed of two single-stranded, positive-sense segments (RNA1, RNA2), and is classified into four genotypes, although only RGNNV causes high mortalities in European sea bass.

Aim

In vitro evaluation of the European sea bass RTP3X1 and RTP3X2 activity following RGNNV inoculation.

Material and Methods

Three E11 cell lines permanently expressing the sea bass RTP3 proteins were stablished (Dlrtp3X1-E11, Dlrtp3X2-E11 and Dlrtp3X1+X2-E11). The activity of both RTP3 proteins was evaluated by quantifying: (i) cellular survival rate after viral inoculation (MTT reduction assay and violet crystal staining), (ii) viral genome (absolute qPCR), (iii) extracellular infective viral particles (TCID₅₀ method), (iv) immuno-related gene transcription (relative qPCR), and (v) apoptosis (TUNEL assay).

Results

The survival rate of RGNNV-inoculated RTP3X1-expressing cells was lower than the survival rate of controls cells, which is in concordance with the results of viral genome quantification. On the contrary, the expression of RTP3X2 increased the cellular survival following viral inoculation, which is supported by the significant reduction in the copy number of both viral segments recorded in these cells. These results were corroborated by viral titration. Differences regarding the survival rate or the viral genome copy number between Dlrtp3X1+X2-E11 cells and control cells were not recorded. Modulation of immuno-gene transcription was observed. Thus, the expression of RTP3X1 resulted in an earlier transcription of mx in response to poly I:C (24 h p.i.) whereas RTP3X2 expression reduced mx transcription at all sampling times. Regarding tnf-α, both proteins decreased the transcription of this gene triggered by bacterial extracts. Finally, the TUNEL assay showed significant increase of apoptotic cells in inoculated and non-inoculated RTP3X1-expressing cells.

Conclusions

This study contributes to further understand the European sea bass response against betanodavirus, concluding that sea bass RTP3 proteins may exert an antiviral role by interfering different cellular mechanisms, and suggesting a mayor role of RTP3X2 over RTP3X1 in controlling NNV infections.

Funding: Agencia Estatal de Investigación. Ref. PID2020-115954RB-I00

1. MORENO, PATRICIA, UNIVERSITY OF MALAGA, Presenter
2. ALVAREZ-TORRES, DANIEL, UNIVERSITY OF MALAGA, Author
3. GARCIA-ROSADO, ESTHER, UNIVERSITY OF MALAGA, Author
4. BEJAR, JULIA, UNIVERSITY OF MALAGA, Author
5. ALONSO, M. CARMEN, UNIVERSITY OF MALAGA, Author