# Functional MDA5 knockout in CHSE-214 cells decreases the response to long dsRNA and enhances Chum Salmon Reovirus susceptibility.

Shayne Oberhoffner, Mathilde Peruzzi, Bertrand Collet, Stephanie Dewitte-Orr

► To cite this version:

Shayne Oberhoffner, Mathilde Peruzzi, Bertrand Collet, Stephanie Dewitte-Orr. Functional MDA5 knockout in CHSE-214 cells decreases the response to long dsRNA and enhances Chum Salmon Reovirus susceptibility. PubMed, 2025, 172, pp.105466-105466. 10.1016/j.dci.2025.105466. hal-05299997

HAL Id: hal-05299997

https://hal.science/hal-05299997v1

Submitted on 30 Oct 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>
<figure><img src="image_2.png" /></figure>

# Functional MDA5 knockout in CHSE-214 cells decreases the response to long dsRNA and enhances Chum Salmon Reovirus susceptibility

Shayne Oberhoffner<sup>a</sup>, Mathilde Peruzzi<sup>b</sup>, Bertrand Collet<sup>b</sup>, Stephanie DeWitte-Orr<sup>a,c,*</sup>

## ARTICLE INFO

### Keywords:

dsRNA
CHSE
MDA5
CSV
Mx1
vig-3
vig-4

## ABSTRACT

Many organisms express sensors that detect long dsRNA, a nucleic acid produced by almost all viruses during replication. These subcellular compartment-specific pattern recognition receptors (PRRs) detect viral replication and rapidly induce a generalist immune response mediated by type I interferon and interferon-stimulated genes (ISGs). In this study, the role of melanoma differentiation-associated protein (MDA)5, a cytoplasmic sensor of long dsRNA, was measured in CHSE-214 cells that have been genetically modified for MDA5 to either be non-functional (frameshift mutation; MDA5C1) or have a silent point mutation (MDA5C2). These cells were transfected with long dsRNA of either 659bp or 334bp in length, and their ability to induce ISG transcript expression (*Mx1*, *vig-3* and *vig-4*) was measured by RT-qPCR. MDA5C1 produced significantly less *Mx1*, *vig-3* and *vig-4* transcripts compared to MDA5C2 when treated with the 659bp dsRNA molecule, while there was no difference between cell lines in *Mx1* expression levels when induced by a 334 bp dsRNA molecule. MDA5C1 was more susceptible to CSV than MDA5C2, and dsRNA treatment was able to protect both cell lines from CSV infection. However, there were no length dependent effects observed, nor were there differences in dsRNA-mediated protection between cell lines. This data suggests that salmonid MDA5 has a length preference similar to mammals, with longer dsRNA molecules being better ligands for this receptor. It also suggests that MDA5 may play a role in the antiviral response against CSV but the antiviral response is not dependent on MDA5. These are important findings for understanding the fundamental importance of MDA5 in the antiviral response of salmonids.

## 1. Introduction

Activation of the innate immune system is dependent on pattern recognition receptors (PRRs) binding pathogen-associated molecular patterns (PAMPs), such as the long double-stranded (ds)RNA (>30bp) (DeWitte et al., 2010a) produced by nearly all viruses at some point during their replication cycle (DeWitte et al., 2010a). This recognition event induces a signaling cascade that culminates in the production of type I interferons (IFN-I), induction of interferon-stimulated genes (ISGs), and establishment of the antiviral state in the infected cell and nearby uninfected cells (Jenik et al., 2024). The use of long dsRNA as a prophylactic antiviral therapeutic is appealing because its stimulation of an interferon-mediated response is consistent and independent of sequence, virus genome type, or virus infectivity (Jacobs and Langland, 1996). As different classes of viruses replicate some or all their genome

in different subcellular compartments (Chinchar, 1999), the existence of different compartment-specific PRRs ensures the innate immune response is not restricted by the absence of a receptor (DeWitte et al., 2010a). Some of the most studied PPRs are in the endosome and cytoplasm: the endosome, as the destination of endocytosed materials, is monitored by toll-like receptor (TLR)3, while the cytoplasm is surveyed by melanoma differentiation-associated protein (MDA)5 and retinoic acid-inducible gene I (RIG-I). The activity of these PRRs is supplemented by other cytoplasmic and nuclear PRRs as well (DeWitte et al., 2010a).

The existence of multiple PRRs for the same ligand throughout the cell invites evaluation of their respective roles in protecting the host against viral infection. These investigations need to be species-specific, for example in birds, as ducks express RIG-I but chickens naturally lack RIG-I and subsequently can have their response to RNA ligands abolished by MDA5 knockout (Lee et al., 2020). In humans, which

<footnote><sup>a</sup> Department of Biology, Wilfrid Laurier University, Canada</footnote>

<footnote><sup>b</sup> INRAE, UVSQ, Université Paris-Saclay, Jouy-en-Josas, France</footnote>

<footnote><sup>c</sup> Department of Health Sciences, Wilfrid Laurier University, Canada</footnote>

<footnote>* Corresponding Author. Wilfrid Laurier University 75 University Avenue West Waterloo, ON, N2L 3C5, Canada.
E-mail address: sdewitteorr@wlu.ca (S. DeWitte-Orr).</footnote>
possess TLR3, the loss of MDA5 leads to persistent viral infection (Asgari et al., 2017). The innate immune system response in fish is not as well characterized as in mice and humans, despite their usefulness as model organisms. A key reason for studying the antiviral innate immune response in salmonids is their vital contribution to the global food chain (FAO, 2024) and economy (Statistics Canada, 2025); without effective antiviral treatments, disease outbreaks leave fish farms vulnerable to substantial losses (Behringer et al., 2020). The Chinook salmon (Oncorhynchus tshawytscha) embryonic cell line (CHSE-214) is known to be unable to respond to extracellular dsRNA (Jensen et al., 2002), providing an opportunity to study the protective response from two different cytosolic PRRs, MDA5 and RIG-I, when transfected with dsRNA. As described in Collins et al., 2024, two derivatives of CHSE-214 were developed using CRISPR/cas9; MDA5C1, which has a deletion of 2 nucleotides that result in a frameshift and premature stop codon, and MDA5C2, which has a silent point mutation R20S (Collins et al., 2024). The present study investigates the effects of varying lengths of dsRNA on ISG expression at the transcript and antiviral state levels in MDA5C1 compared to MDA5C2. DsRNA length is an important factor in human PRR detection, where RIG-I senses dsRNA molecules under 500 bp in length (Im et al., 2023), while MDA5 detects dsRNA greater than 500 bp (Peisley et al., 2012). To test if this length dependent recognition was present in fish, cells were treated with 334bp and 694bp *in vitro* transcribed dsRNA. This work represents an initial investigation of dsRNA-induced responses in salmonid cells lacking MDA5, where the upregulation of ISGs and protection against infection by Chum Salmon Reovirus (CSV), used in this study as a model aquatic virus, was tested.

## 2. Materials and methods

### 2.1. Salmonid cell culture

The chinook salmon embryonic salmon cell line CHSE-214 was maintained in 75 cm² plugged flasks (Corning, Corning, NY) at 18 °C in Leibovitz's (L)-15 media (Corning) supplemented with 2.05 mM l-glutamine (Cytiva, Westborough, MA), 10 % (v/v) fetal bovine serum (FBS, Cytiva), 1 % (v/v) penicillin/streptomycin (10 mg/ml streptomycin and 10,000 U/ml penicillin; Gibco [Thermo Fisher Scientific], Mississauga, ON). MDA5C1 and MDA5C2 were maintained similarly to CHSE-214, but with the addition of 500 µg/ml Geneticin (G418, Gibco), and 30 µg/ml Hygromycin (Gibco) to the media.

### 2.2. dsRNA synthesis

The focus of this study was on dsRNA length, since interferon induction does not depend on sequence but is affected by length. As such we chose a 659bp region of the Viral Hemorrhagic Septicemia virus (VHS)-IVB nucleocapsid (N) gene was purchased from Integrated DNA Technologies as a DNA gBLOCK® (Oberhoffner et al., 2025) and used for the synthesis of both the 659bp and 334bp dsRNA molecules. This Each dsRNA synthesis primer (Table 1) contained a T7 polymerase promoter

sequence added to the 5' end. PCR reactions were performed in a BioRad T100 thermal cycler (BioRad, Hercules, CA): Initial denaturation was performed at 95 °C for 5 min, followed by 35 cycles of 95 °C denaturation for 30 s, 58 °C annealing for 30 s, and 72 °C extension for 1 min, completed with a final extension at 72 °C for 4 min. PCR reactions were purified using the MinElute PCR purification kit (Qiagen, Aarhus, Denmark) following the manufacturer instructions and used as a template to make dsRNA using the MEGAscript RNAi Kit (Thermo Fisher Scientific), following the manufacturer instructions. The length of the resulting dsRNA was confirmed by agarose gel electrophoresis and quantified using a NanoDrop Lite Spectrophotometer (Thermo Fisher Scientific).

### 2.3. Transfection of long dsRNA for qPCR analysis of ISG expression

MDA5C1 and MDA5C2 cells were seeded at $4 \times 10^5$ cells/well in a 6-well plate (Corning) in complete media and allowed to attach overnight at 18 °C. Treatments using 334 or 659bp dsRNA were prepared using FuGENE 6 (Promega, Madison, WI) in a 1.5:1 (v/m) ratio of FUGENE to dsRNA following manufacturer's instructions and previous study (Monjo et al., 2017). The media was removed from the cells, which were then transfected with the dsRNA-FuGENE combination in basal media for 24, 48, or 72hrs with 2 µg/mL 659bp dsRNA or 48hrs with 2 µg/mL 334bp dsRNA. Media alone was used as a negative control. After the treatment time, the media was removed, and cells were rinsed with 1X DPBS (Thermo Fisher Scientific) and then lysed with TRIzol (Invitrogen [Thermo Fisher Scientific]). RNA extraction was performed following manufacturer instructions, and 500 ng total RNA was incubated with DNase I (Thermo Fisher Scientific) for 30 min before reverse transcription using iScript (BioRad) following manufacturer protocols. qPCR was performed with a SensiFast SYBR No-ROX Kit (Meridian Bioscience, Cincinnati, OH) with the primers in Table 1. Initial denaturation was performed at 95 °C for 5 min, followed by 40 cycles of: 95 °C for 5 s and 60 °C for 30 s. Single-product amplification and primer efficiency were validated via melt curve analysis. Data is presented as the mean fold change calculated using the ΔΔCt method of at least three independent experiments with the standard error of the mean (SEM).

### 2.4. Antiviral assay

#### 2.4.1. Virus propagation and tissue culture infectious dose (TCID₅₀)

Chum salmon reovirus (CSV) was selected for this study due to its distinctive cytopathic effect, characterized by syncytia formation, which allows viral replication to be readily quantified. CSV was propagated on monolayers of CHSE-214 cells for 5 days in L-15 containing 2 % FBS at 17 °C until syncytia developed. Cells were frozen to -80 °C and then thawed. Media was filtered with a 0.22 µm syringe filter, aliquoted, and kept at -80 °C. To determine the viral titer, CHSE-214 cells were plated at $2 \times 10^4$ cells/well in a 96-well plate (Corning) using L-15 media containing 10 % (v/v) FBS and 1 % (v/v) penicillin/streptomycin (10 mg/ml streptomycin and 10,000 U/ml penicillin) and left to attach

<caption>**Table 1**</caption>

Primer Sequences used for *in vitro* transcription of VHS-IVB N-Gene dsRNA (bolded letters indicate T7 promoter sequence) and for qPCR analysis of ISG expression.

<table><thead><tr><th>Sequence</th><th>5'-3' Sequence</th><th>Product length (bp)</th><th>Reference/ Accession number</th></tr></thead><tbody><tr><td>659bp dsRNA Forward</td><td>AATACGACTCACTATAGGGGATCACCAAGGCCCTCTACG</td><td>659</td><td>MH6655270.1</td></tr><tr><td>659bp dsRNA Reverse</td><td>AATACGACTCACTATAGGGTTCTCCTGCTCTTTCCCCCCT</td><td></td><td></td></tr><tr><td>334bp dsRNA Forward</td><td>AATACGACTCACTATAGGGGATGAGTCGGATGCTGGGAG</td><td>334</td><td></td></tr><tr><td>334bp dsRNA Reverse</td><td>AATACGACTCACTATAGGGCGGGCTTTGGGATCTGACTT</td><td></td><td></td></tr><tr><td>efla Forward</td><td>CGCACAGTAACACCGAAACTAATTAAGC</td><td>131</td><td>XM_024396038.2</td></tr><tr><td>efla Reverse</td><td>GCCTCCGCACTTGTAGATCAGATG</td><td></td><td></td></tr><tr><td>vig-3 Forward</td><td>ACCCAGTTCAAAGCCAAGGT</td><td>70</td><td>15</td></tr><tr><td>vig-3 Reverse</td><td>CCCTCGTGAATCAGCCTCTG</td><td></td><td></td></tr><tr><td>Mx1 Forward</td><td>CGGAGTTCGTCTCAACGTCT</td><td>140</td><td></td></tr><tr><td>Mx1 Reverse</td><td>CCCTTCCACGGTACGTCTTC</td><td></td><td></td></tr><tr><td>vig-4 Forward</td><td>CAGCTCTGGAGAAGTGACGG</td><td>83</td><td>XM_021575340.2</td></tr><tr><td>vig-4 Reverse</td><td>GCAGAAGAGGAAAGGGGGAC</td><td></td><td></td></tr></tbody></table>
overnight. A CSV aliquot was serially diluted tenfold in L-15 containing 2 % FBS and added to the wells of the plate. The cells were incubated at 17 °C for 5 days to allow syncytia to develop, and infected wells were counted to determine the TCID₅₀/mL using the Reed and Muench method (Reed and Muench, 1938).

### 2.4.2. CSV antiviral assay

MDA5C1 and MDA5C2 cells were plated and transfected with 2 µg/mL 334 or 659bp dsRNA as described in Section 2.3. The media was then changed to L-15 + 2 % FBS and the cells were infected with CSV (MOI = 0.05) for 5 days. MOI was determined as per Monjo et al., 2017 (Monjo et al., 2017). Untreated cells infected with CSV were used as a control. Media was collected from cells and viral titers were measured using the protocol outlined in Section 2.4.1, with each treatment condition being serially diluted on its own 96-well plate of CHSE-214 and CPE being evaluated after 7 days. Cells were not frozen prior to virus collection to determine the number of virus particles released from lysed cells, representing the population that could disseminate to other cells (or be shed by fish in an in vivo model). TCID₅₀/mL was calculated using the Reed and Muench method (Reed and Muench, 1938) and the titer results are reported as log₁₀ transformed TCID₅₀/mL. Due to the difference in viral titers between MDA5C1 and MDA5C2 cell lines, when comparing effects with dsRNA, the data was normalized as a fold change to the virus only control.

## 2.5. Data analysis

Data are representative of the mean ± standard error of the mean of three or more individual experimental replicates. Statistical analyses were performed using GraphPad Prism 10 (GraphPad, San Diego, CA) and Python (Python Software Foundation, Wilmington, DE). Due to the small sample size (n = 3–5) statistical testing was performed using a non-parametric Mann-Whitney U Test, where a p-value <0.05 was considered significant. Effect sizes were calculated using a Cliff's delta analysis to quantify the magnitude of differences independent of sample size.

# 3. Results and discussion

## 3.1. ISG transcript expression changes with dsRNA treatment

CHSE-214 is an interesting cell line in which to study dsRNA signaling, as it lacks functional class A scavenger receptors (Monjo et al., 2017) required for the recognition and internalization of extracellular dsRNA (DeWitte et al., 2010b). This provides an opportunity to analyze the role of the remaining PRRs, including MDA5. Derivatives of CHSE-214 cells were engineered to possess one of two mutations to their MDA5 receptor: a frameshift truncation (MDA5C1) of the protein or a silent substitution (MDA5C2) (Collins et al., 2024). To measure the effects of long dsRNA on ISG expression in MDA5C1 versus MDA5C2, the cells were transfected with either a 659 or 334 bp dsRNA molecule and ISG expression was measured at 24, 48, or 72hrs post treatment, as human MDA5 preferentially binds dsRNA >500 bp¹³. There was no significant increase in ISG expression in MDA5C1 relative to untreated controls (Fold change = 1, data not shown) while MDA5C2 demonstrated significant increased levels of *Mx1* at 48, *vig-3* at 24, and *vig-4* at 24hrs after treatment (Fig. 1). There were no significant increases in ISG expression at 72hrs in any group relative to untreated control cells (data not shown). When using a 334bp dsRNA molecule there was not a statistically significant difference in *Mx1* transcript expression between MDA5C1 and MDA5C2 or relative to untreated control cells, suggesting the 334bp dsRNA molecule did not signal through MDA5. The difference in kinetics of ISG expression, with *vig-3* being induced earlier than *Mx1*, is well documented in salmonids (Alkie et al., 2019), and so 48hrs was selected as the transfection duration for antiviral studies. It should be noted that fish cell lines such as CHSE-214 are challenging to transfect

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Lack of MDA5 alters ISG expression profiles to two different dsRNA lengths. MDA5C1 and MDA5C2 cells were transfected with 2 µg/mL 659bp dsRNA for 24 or 48hrs (A-C) or 2 µg/mL 334bp dsRNA for 48hrs (D). *Mx1*, *vig-3* and *vig-4* transcript expression levels were measured by RT-qPCR and reported as a fold change from the untreated control using the ΔΔCT method. Data were analyzed using a Mann-Whitney *U* test. Data with different letters indicate statistical significance at *p* < 0.05.</figcaption></figure>

with lipid-mediated protocols, producing very low transfection efficiencies (Collet et al., 2018). In the present study, transfection efficiency was ~10% across cell lines, as determined by preliminary experiments using a GFP-expression vector. These low rates likely contributed to the relatively modest ISG induction observed (5–10 fold). However, it is important to note that GFP plasmid transfection is not directly comparable to dsRNA transfection. Unlike GFP, dsRNA induces interferon, which acts in a paracrine manner to stimulate ISG expression in surrounding non-transfected cells. As a result, the impact of low dsRNA transfection rates within a monolayer is more complex to interpret.

## 3.2. CSV antiviral assays

Next, the impact of the differences in ISG expression between MDA5C1 and MDA5C2 were assessed at the antiviral level. When comparing CSV replication in MDA5C1 and MDA5C2, while there was a lack of statistical significance; however, Cliff's delta analysis revealed a large effect size (δ = −0.889), indicating that nearly all TCID₅₀ values in MDA5C1 exceeded those in MDA5C2, suggesting a biologically meaningful difference in viral susceptibility between the two cell lines with MDA5C1 being more susceptible (Fig. 2A). This result corelates well with studies in mice, where MDA5 knockout alone had a minor role in reovirus replication (Loo et al., 2008), and in humans MDA5 and RIG-I sensing of reoviruses have compensatory and redundant roles (Abad and
<figure><img src="image_4.png" /><figcaption>Fig. 2. The effect of MDA5 on CSV replication, in the presence and absence of dsRNA treatment.</figcaption></figure>

(A) Viral titers were measured from MDA5C1 and MDA5C2 cells infected with CSV (MOI = 0.05) for 5 days. Viral titers were measured as TCID₅₀/mL. (B) Viral titers were measured in cells treated with 2 µg/mL of 334bp or 659bp dsRNA for 48hrs followed by infection with CSV (MOI = 0.05) for 5 days. Data are reported as a fold change over untreated/infected control cells. All data represent 3 independent experiments. Statistical analysis was between groups was performed using a non-parametric Mann-Whitney U Test. Viral titer effect sizes were analyzed using Cliff's delta analysis.

Danthi, 2020). Conversely, it has recently been shown that chickens infected with avian reovirus experience a rapid induction of MDA5 to combat infection (Xie et al., 2019). Salmonids therefore may be similar to mammals in the role of MDA5 in their anti-reovirus IFN response.

The 48hr prophylactic treatment of MDA5C1 and MDA5C2 with 334 or 659bp dsRNA provided protection against CSV as determined by Cliff's delta analysis (δ = -1.00 for both MDA5C1 and MDA5C2; Fig. 2B), which reflects complete ordinal separation despite the limited sample size (n = 3). However, this effect did not reach statistical significance when assessed by Mann-Whitney U Test. Additionally, there were not significant differences in the fold change of virus concentration between dsRNA lengths (334bp vs. 659bp) or between cell lines (MDA5C1 vs MDA5C2) treated with the same dsRNA length. This suggests that the length effects indicated by differences in ISG transcript levels did not translate to differences in the antiviral state during infection with CSV. It is possible MDA5 may play a larger role in anti-reovirus immunity with higher MOIs in CHSE, as a high MOI of mammalian reovirus outer capsid protein μ1 inhibited expression of MDA5 and directly bound MDA5 in the cell, targeting it for degradation in human embryonic kidney cells (Wu et al., 2024). This suggests that results in the present work may be different at a higher MOI.

## 4. Conclusion

Despite the threat of aquatic viruses to the health of wild and commercial fish populations, there is little known about the role of individual key proteins that recognize viral dsRNA and initiate an innate immune response. By using dsRNA whose lengths are above and below the recognition threshold of mammalian MDA5, it was demonstrated that CHSE cells not expressing MDA5 showed less responsiveness to 659bp dsRNA but equal responsiveness to 334bp dsRNA compared with CHSE producing MDA5 with a point mutation, and that Chum Salmon Reovirus produced slightly higher titers in MDA5 knockout cells, suggesting this PRR has a role in limiting reovirus replication. This appears to be consistent with other vertebrates, as recent findings in live chickens and human cells show that MDA5 function is attenuated by reoviruses during infection (Xie et al., 2019; Wu et al., 2024). Future studies involving other reoviruses that greatly impact salmonids, such as *Piscine orthoreovirus*, should be tested to determine if a loss of MDA5 is impactful to infection progression. Additionally, using a single MOI and a single dsRNA treatment time point are limitations of this study and thus studying the role of MDA5 in limiting reovirus infection under a

range of MOIs and at different dsRNA treatment times will be pursued next. DsRNA was able to reduce the effect size of the CSV titre, but no difference was observed in the level of protection induced by 334bp and 659bp dsRNA. Since there are other PRRs in the cell than MDA5 and RIG-I, it's possible these other antiviral pathways were contributing to the equal antiviral effects between dsRNA lengths. While the current work examined three ISGs at different post-transfection time points, dsRNA can induce hundreds of ISGs and different regulatory pathways that were not analyzed in the present study that could impact antiviral defense. Future work using RNAseq would provide a more thorough insight into the importance of MDA5 in antiviral protection. This work highlights the role of MDA5 in dsRNA sensing and reovirus antiviral responses in salmonids, emphasizing an important role for this sensor in detecting long dsRNA in fish.

## CRediT authorship contribution statement

**Shayne Oberhoffner:** Writing – review & editing, Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Data curation.
**Mathilde Peruzzi:** Writing – review & editing, Methodology, Data curation, Conceptualization.
**Bertrand Collet:** Writing – review & editing, Validation, Methodology, Formal analysis, Data curation, Conceptualization.
**Stephanie DeWitte-Orr:** Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Funding acquisition, Formal analysis, Conceptualization.

## Acknowledgments

This project was supported by an NSERC Discovery grant held by SDO.

## Data availability

Data will be made available on request.

## References

Abad, A.T., Danthi, P., 2020. Recognition of reovirus RNAs by the innate immune system. Viruses 12 (6). https://doi.org/10.3390/v12060667.

Alkie, T.N., et al., 2019. Enhancing innate antiviral immune responses in rainbow trout by double stranded RNA delivered with cationic phytotglycogen nanoparticles. Sci. Rep. 9 (1). https://doi.org/10.1038/s41598-019-49931-2.

Asgari, S., et al., 2017. Severe viral respiratory infections in children with IFIH1 loss-of-function mutations. Proc. Natl. Acad. Sci. U. S. A 114 (31), 8342–8347. https://doi.org/10.1073/pnas.1704259114.

Behringer, D.C., Wood, C.L., Krkošek, M., Bushek, D., 2020. Disease in fisheries and aquaculture. In: Marine Disease Ecology. Oxford University Press, pp. 183–210. https://doi.org/10.1093/oso/9780198821632.003.0010.

Chinchar, V.G., 1999. Replication of viruses. In: Encyclopedia of Virology. Elsevier, pp. 1471–1478. https://doi.org/10.1006/rwvi.1999.0245. https://linkinghub.elsevier.com/retrieve/pii/B0122270304002454.

Collet, B., Collins, C., Lester, K., 2018. Engineered cell lines for fish health research. Dev. Comp. Immunol. 80, 34–40. https://doi.org/10.1016/j.dci.2017.01.013.

Collins, C., et al., 2024. Effect of a loss of the mda5/ifih1 gene on the antiviral resistance in a Chinook salmon Oncorhynchus tshawytscha cell line. PLoS One 19 (10), e0311283. https://doi.org/10.1371/journal.pone.0311283.

DeWitte-Orr, S.J., Mossman, K.L., 2010a. dsRNA and the innate antiviral immune response. Future Virol. 5 (3), 325–341. https://doi.org/10.2217/fvl.10.18.

DeWitte-Orr, S.J., et al., 2010b. An accessory to the “Trinity”: SR-As are essential pathogen sensors of extracellular dsRNA, mediating entry and leading to subsequent type I IFN responses. PLoS Pathog. 6 (3). https://doi.org/10.1371/journal.ppat.1000829.

FAO, 2024. Brief to the state of world fisheries and aquaculture 2024. Blue transformation in action. https://doi.org/10.4060/cd0690en.

Im, J.H., et al., 2023. Mechanisms of length-dependent recognition of viral double-stranded RNA by RIG-I. Sci. Rep. 13 (1). https://doi.org/10.1038/s41598-023-33208-w.

Jacobs, B., Langland, J., 1996. When two strands are better than one: the mediators and Modulators of the Cellular responses to double-stranded RNA. Virology 219 (2), 339–349. https://doi.org/10.1006/viro.1996.0259. https://linkinghub.elsevier.com/retrieve/pii/S0042682296902597.

Jenik, K., Oberhoffner, S., DeWitte-Orr, S.J., 2024. Response to pathogens - innate immunity. In: Encyclopedia of Fish Physiology. Elsevier, pp. 334–345. https://doi.org/10.1016/b978-0-323-90801-6.00093-8.
Jensen, I., Larsen, R., Robertsen, B., 2002. An antiviral state induced in Chinook salmon embryo cells (CHSE-214) by transfection with the double-stranded RNA poly I:C. Fish Shellfish Immunol. 13 (5), 367-378. https://doi.org/10.1006/fsim.2002.0412.

Lee, S Bin, et al., 2020. Targeted knockout of MDA5 and TLR3 in the DF-1 chicken fibroblast cell line impairs innate immune response against RNA ligands. Front. Immunol. 11. https://doi.org/10.3389/fimmu.2020.00678.

Loo, Y.-M., et al., 2008. Distinct RIG-I and MDA5 signaling by RNA viruses in innate immunity. J. Virol. 82 (1), 335-345. https://doi.org/10.1128/jvi.01080-07.

Monjo, A.L., Poynter, S.J., DeWitte-Orr, S.J., 2017. CHSE-214: a model for studying extracellular dsRNA sensing in vitro. Fish Shellfish Immunol. 68, 266-271. https://doi.org/10.1016/j.fsi.2017.07.025.

Oberhoffner, S.J., et al., 2025. Using cationic liposomes as carriers for long dsRNA to trigger an antiviral response in rainbow trout cell lines. In: In Vitro Cellular & Developmental Biology - Animal. https://doi.org/10.1007/s11626-024-01002-1. htt ps://link.springer.com/10.1007/s11626-024-01002-1.

Peisley, A., et al., 2012. Kinetic mechanism for viral dsRNA length discrimination by MDA5 filaments. Proc. Natl. Acad. Sci. U. S. A 109 (49). https://doi.org/10.1073/ pnas.1208618109.

Reed, L.J., Muench, H., 1938. A simple method of estimating fifty per cent endpoints. Am. J. Epidemiol. 27 (3), 493-497. https://doi.org/10.1093/oxfordjournals.aje. a118408. https://academic.oup.com/aje/article-lookup/doi/10.1093/oxfordjo urnals.aje.a118408.

Statistics Canada, 2025. Table 32-10-0107-01 Aquaculture, production and value. https://doi.org/10.25318/3210010701-eng.

Wu, B., et al., 2024. Mammalian reovirus μ1 protein attenuates RIG-I and MDA5-mediated signaling transduction by blocking IRF3 phosphorylation and nuclear translocation. Mol. Immunol. 170, 131-143. https://doi.org/10.1016/j. molimm.2024.04.010.

Xie, L., et al., 2019. Altered gene expression profiles of the MDA5 signaling pathway in peripheral blood lymphocytes of chickens infected with avian reovirus. Arch. Virol. 164 (10), 2451-2458. https://doi.org/10.1007/s00705-019-04340-8.