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Viral infections are still a major concern for the aquaculture industry. For salmonid fish, even though breeding strategies and vaccine development have reduced disease outbreaks, viral diseases remain among the main challenges having a negative impact on the welfare of fish and causing massive economic losses for the industry. The main entry port for viruses into the fish is through mucosal surfaces including that of the gastrointestinal tract. The contradictory functions of this surface, both creating a barrier towards the external environment and at the same time being responsible for the uptake of nutrients and ion/water regulation make it particularly vulnerable. The connection between dietary components and viral infections in fish has been poorly investigated and until now, a fish intestinal in vitro model to investigate virus-host interactions has been lacking. Here, we established the permissiveness of the rainbow trout intestinal cell line RTgutGC towards the important salmonid viruses-infectious pancreatic necrosis virus (IPNV), salmonid alphavirus (subtype 3, SAV3) and infectious salmon anemia virus (ISAV)-and explored the infection mechanisms of the three different viruses in these cells at different virus to cell ratios. Cytopathic effect (CPE), virus replication in the RTgutGC cells, antiviral cell responses and viral effects on the barrier permeability of polarized cells were investigated. We found that all virus species infected and replicated in RTgutGC cells, although with different replication kinetics and ability to induce CPE and host responses. The onset and progression of CPE was more rapid at high multiplicity of infection (MOI) for IPNV and SAV3 while the opposite was true of ISAV. A positive correlation between the MOI used and the induction of antiviral responses was observed for IPNV while a negative correlation was detected for SAV3. Viral infections compromised barrier integrity at early time points prior to observations of CPE microscopically. Further, the replication of IPNV and ISAV had a more pronounced effect on barrier function than SAV3. The in vitro infection model established herein can thus provide a novel tool to generate knowledge about the infection pathways and mechanisms used to surpass the intestinal epithelium in salmonid fish, and to study how a virus can potentially compromise gut epithelial barrier functions.

Bacteriocins are emerging as a viable alternative to antibiotics due to their ability to inhibit growth or kill antibiotic resistant pathogens. Herein, we evaluated the ability of the bacteriocin Garvicin KS (GarKS) produced by Lactococcus garvieae KS1546 isolated from cow milk to inhibit the growth of fish and foodborne bacterial pathogens. We found that GarKS inhibited the growth of five fish L. garvieae strains isolated from infected trout and eels. Among fish pathogens, GarKS inhibited the growth of Streptococcus agalactiae serotypes Ia and Ib, and Aeromonas hydrophila but did not inhibit the growth of Edwardsiella tarda . In addition, it inhibited the growth of A. salmonicida strain 6421 but not A. salmonicida strain 6422 and Yersinia ruckeri . There was no inhibition of three foodborne bacterial species, namely Salmonella enterica , Klebsiella pneumoniae , and Escherichia coli . In vitro cytotoxicity tests using different GarKS concentrations showed that the highest concentration of 33 µg/mL exhibited low cytotoxicity, while concentrations ≤3.3 µg/mL had no cytotoxicity on CHSE-214 and RTG-2 cells. In vivo tests showed that zebrafish larvae treated with 33 µg/mL and 3.3 µg/mL GarKS prior to challenge had 53% and 48% survival, respectively, while concentrations ≤0.33 µg/mL were nonprotective. Altogether, these data show that GarKS has a broad inhibitory spectrum against Gram positive and negative bacteria and that it has potential applications as a therapeutic agent for a wide range of bacterial pathogens. Thus, future studies should include clinical trials to test the efficacy of GarKS against various bacterial pathogens in farmed fish.

Here we have studied the impact of lice ( Lepeophtheirus salmonis ) infestation of donor fish on the ability of isolated peripheral blood monocytes (PMBCs) to control the replication of salmonid alphavirus (SAV) ex vivo. PMBC were collected by Percoll gradients at eight and nine weeks post copepodid infestation of Atlantic salmon post smolt. Uninfested fish were controls. PBMCs were then infected ex vivo with SAV (subtype 3), and samples were collected for analysis at two, four, and six days post virus infection. Virus titer in the supernatant was assayed in CHH-1 cells, and in addition, the relative expression of the virus structural protein E2 and selected host antiviral genes, IRF9, ISG15, Mx, and IFIT5, were assayed using real-time PCR. Significantly higher virus replication was detected in cells collected from lice-infested fish compared to controls. Higher virus titer coincided with an inability to upregulate the expression of different immune genes, IFIT5, IRF9, and Mx. These findings point towards compromised ability of PMBCs from lice-infested fish to control virus replication, and, to our knowledge, is the first report showing the direct effect of lice infestation on the interplay between viruses and immune cells. There is a possible impact on the dynamic spread of viral diseases in the aquatic environment.

Tilapia lake virus (TiLV) is a negative-sense single-stranded RNA (-ssRNA) icosahedral virus classified to be the only member in the family Amnoonviridae. Although TiLV segment-1 shares homology with the influenza C virus PB1 and has four conserved motifs similar to influenza A, B, and C polymerases, it is unknown whether there are other properties shared between TiLV and orthomyxovirus. In the present study, we wanted to determine whether TiLV agglutinated avian and piscine erythrocytes, and whether its replication was inhibited by lysosomotropic agents, such as ammonium chloride (NH 4 Cl), as seen for orthomyxoviruses. Our findings showed that influenza virus strain A/Puerto Rico/8 (PR8) was able to hemagglutinate turkey ( Meleagris gallopavo ), Atlantic salmon ( Salmo salar L), and Nile tilapia ( Oreochromis niloticus ) red blood cells (RBCs), while infectious salmon anemia virus (ISAV) only agglutinated Atlantic salmon, but not turkey or tilapia, RBCs. In contrast to PR8 and ISAV, TiLV did not agglutinate turkey, Atlantic salmon, or tilapia RBCs. qRT-PCR analysis showed that 30 mM NH 4 Cl, a basic lysosomotropic agent, neither inhibited nor enhanced TiLV replication in E-11 cells. There was no difference in viral quantities in the infected cells with or without NH 4 Cl treatment during virus adsorption or at 1, 2, and 3 h post-infection. Given that hemagglutinin proteins that bind RBCs also serve as ligands that bind host cells during virus entry leading to endocytosis in orthomyxoviruses, the data presented here suggest that TiLV may use mechanisms that are different from orthomyxoviruses for entry and replication in host cells. Therefore, future studies should seek to elucidate the mechanisms used by TiLV for entry into host cells and to determine its mode of replication in infected cells.

Tilapia is the second most farmed fish species after carp in the world. However, the production has come under threat due to emerging diseases such as tilapia lake virus (TiLV) that causes massive mortalities with high economic losses. It is largely unknown whether different tilapia strains are equally susceptible to TiLV infection. In the present study we compared the susceptibility of gray ( Oreochromis niloticus x O. aureus ) and red tilapia ( Oreochromis spp.) to experimental TiLV infection. Virus was injected intraperitoneally at a concentration of 10 4 TCID 50 /mL. Our findings show that gray tilapia had a lower mortality, 86.44%, but statistically not significantly different ( p = 0.068) from red tilapia (100%). The duration of the mortality period from onset to cessation was similar for the two species, starting at 2-3 days post challenge (dpc) with a median at 10-11 dpi and ending on 20-22 dpi. In addition, there was no difference between species in mean viral loads in brain, liver and headkidney from fish collected soon after death. As for host response, expression levels of IL-1β and TNFα were equally high in brain and headkidney samples while levels in liver samples were low for both red and gray tilapia, which coincides with lower viral loads in liver compared to brain and headkidney for both species. We find that red and gray tilapia were equally susceptible to TiLV infection with similar post challenge mortality levels, equal virus concentration in target organs and similar proinflammatory cytokine responses in target and lymphoid organs at time of death. Nonetheless, we advocate that the search for less susceptible tilapia strains should continue with the view to reduce losses from TiLV infection in aquaculture.

Type I interferons (IFNs) have been shown to play an important role in shaping adaptive immune responses in addition to their antiviral properties in immune cells. To gain insight into the impact of IFN-I-induced pathways involved in early adaptive immune responses, i.e., antigen-presenting pathways, in an Atlantic salmon-derived ( Salmo salar L.) macrophage cell line (TO-cells), we used a comparative de novo transcriptome analysis where cells were treated with IFN-I or kept untreated and concurrently infected with salmonid alphavirus subtype 3 (SAV3). We found that concurrent treatment of TO-cells with IFN-I and SAV3 infection (SAV3/IFN + ) significantly enriched the major histocompatibility complex class I (MHC-I) pathway unlike the non-IFN-I treated TO-cells (SAV3/IFN - ) that had lower expression levels of MHC-I pathway-related genes. Genes such as the proteasomal activator ( PA28 ) and β-2 microglobulin ( β2M ) were only differentially expressed in the SAV3/IFN + cells and not in the SAV3/IFN - cells. MHC-I pathway genes like heat shock protein 90 ( Hsp90 ), transporter of antigen associated proteins ( TAPs ) and tapasin had higher expression levels in the SAV3/IFN + cells than in the SAV3/IFN - cells. There were no MHC-II pathway-related genes upregulated in SAV3/IFN + -treated cells, and cathepsin S linked to the degradation of endosomal antigens in the MHC-II pathway was downregulated in the SAV3/IFN - cells. Overall, our findings show that concurrent IFN-I treatment of TO-cells and SAV3 infection enriched gene expression linked to the MHC-I antigen presentation pathway. Data presented indicate a role of type I IFNs in strengthening antigen processing and presentation that may facilitate activation particularly of CD8+ T-cell responses following SAV3 infection, while SAV3 infection alone downplayed MHC-II pathways.

dsRNA-dependent protein kinase R (PKR) is an interferon-inducible protein that mediates antiviral effects and induces apoptosis. We studied PKR-related apoptosis mechanisms by transfecting wild type pcDNA-carp-wtPKR, a catalytically inactive mutant pcDNA-mut-carpPKR, and empty plasmid in Epithelioma papulosum cyprini (EPC) cells, designated wtPKR, mutPKR, and pcDNA3.1, respectively. PKR was inefficiently expressed from wtPKR unlike mutPKR that produced high PKR levels detected by western blot. eIF2α phosphorylation increased in wtPKR-transfected cells, while for mutPKR, phosphorylation was not different from non-transfected controls. Flow-cytometry revealed high level of apoptosis in wtPKR transfected cells, corresponding with high cytopathic effect. mutPKR and pcDNA3.1 transfection gave significantly less apoptosis and were not different from each other. Caspase-8 and -9 were activated for wtPKR, suggesting death receptor-caspase-8 and mitochondrion-dependent caspase-9 activated pathways, similar to mammalian cells. These findings suggest that the induction of apoptosis via the caspase-8 and -9 pathways are conserved in vertebrate taxa and likely play a role in viral infections of lower vertebrates.

The double-stranded RNA-activated protein kinase R (PKR) is a Type I interferon (IFN) stimulated gene that has important biological and immunological functions. In viral infections, in general, PKR inhibits or promotes viral replication, but PKR-IPNV interaction has not been previously studied. We investigated the involvement of PKR during infectious pancreatic necrosis virus (IPNV) infection using a custom-made rabbit antiserum and the PKR inhibitor C16. Reactivity of the antiserum to PKR in CHSE-214 cells was confirmed after IFNα treatment giving an increased protein level. IPNV infection alone did not give increased PKR levels by Western blot, while pre-treatment with PKR inhibitor before IPNV infection gave decreased eukaryotic initiation factor 2-alpha (eIF2α) phosphorylation. This suggests that PKR, despite not being upregulated, is involved in eIF2α phosphorylation during IPNV infection. PKR inhibitor pre-treatment resulted in decreased virus titers, extra- and intracellularly, concomitant with reduction of cells with compromised membranes in IPNV-permissive cell lines. These findings suggest that IPNV uses PKR activation to promote virus replication in infected cells.

A fundamental step in cellular defense mechanisms is the recognition of "danger signals" made of conserved pathogen associated molecular patterns (PAMPs) expressed by invading pathogens, by host cell germ line coded pattern recognition receptors (PRRs). In this study, we used RNA-seq and the Kyoto encyclopedia of genes and genomes (KEGG) to identify PRRs together with the network pathway of differentially expressed genes (DEGs) that recognize salmonid alphavirus subtype 3 (SAV-3) infection in macrophage/dendritic like TO-cells derived from Atlantic salmon (Salmo salar L) headkidney leukocytes. Our findings show that recognition of SAV-3 in TO-cells was restricted to endosomal Toll-like receptors (TLRs) 3 and 8 together with RIG-I-like receptors (RLRs) and not the nucleotide-binding oligomerization domain-like receptors NOD-like receptor (NLRs) genes. Among the RLRs, upregulated genes included the retinoic acid inducible gene I (RIG-I), melanoma differentiation association 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2). The study points to possible involvement of the tripartite motif containing 25 (TRIM25) and mitochondrial antiviral signaling protein (MAVS) in modulating RIG-I signaling being the first report that links these genes to the RLR pathway in SAV-3 infection in TO-cells. Downstream signaling suggests that both the TLR and RLR pathways use interferon (IFN) regulatory factors (IRFs) 3 and 7 to produce IFN-a2. The validity of RNA-seq data generated in this study was confirmed by quantitative real time qRT-PCR showing that genes up- or downregulated by RNA-seq were also up- or downregulated by RT-PCR. Overall, this study shows that de novo transcriptome assembly identify key receptors of the TLR and RLR sensors engaged in host pathogen interaction at cellular level. We envisage that data presented here can open a road map for future intervention strategies in SAV infection of salmon.

One impediment to the successful oral vaccination in fish is the hostile stomach environment that antigens must cross. Furthermore, uptake of antigens from the gut to systemic distribution is required for induction of systemic immunity, the dynamics of which are poorly understood. In the present study, groups of Atlantic salmon parr were intubated with live or inactivated infectious pancreatic necrosis virus (IPNV), either orally or anally. At 1, 24 and 72 h post infection (p.i.), the fish were sacrificed. Serum was used for assessing IPNV by ELISA, while formalin-fixed head-kidney, spleen, liver and intestine tissues were used for the demonstration of antigens by immunohistochemistry. Both live and inactivated IPNV antigens were observed in enterocytes of the intestines and in immune cells of the head-kidneys and spleens of all groups. In the liver, no antigens were observed in any of the groups. Significantly higher serum antigen OD values (p < 0.04) were observed in orally- compared to anally-intubated fish. By contrast, no difference (p = 0.05) was observed in tissue antigens between these groups by immunohistochemistry. No significant difference (p = 0.05) in serum antigens was observed between groups intubated with live and inactivated IPNV, while in tissues, significantly more antigens (p < 0.03) were observe in the latter compared to the former. These findings demonstrate that both live and inactivated IPNV are taken up by enterocytes in the intestines of Atlantic salmon, likely by receptor-mediated mechanisms. Higher IPNV uptake by the oral compared to anal route suggests that both the anterior and posterior intestines are important for the uptake of the virus and that IPNV is resistant to gastric degradation of the Atlantic salmon stomach.

Inhibition of protein synthesis represents one of the antiviral mechanisms employed by cells and it is also used by viruses for their own propagation. To what extent members of the Birnaviridae family employ such strategies is not well understood. Here we use a type-strain of the Aquabirnavirus, infectious pancreatic necrosis virus (IPNV), to investigate this phenomenon in vitro. CHSE-214 cells were infected with IPNV and at 3, 12, 24, and 48 hours post infection (hpi) before the cells were harvested and labeled with S35 methionine to assess protein synthesis. eIF2α phosphorylation was examined by Western blot while RT-qPCR was used to assess virus replication and the expression levels of IFN-α, Mx1 and PKR. Cellular responses to IPNV infection were assessed by DNA laddering, Caspase-3 assays and flow cytometry. The results show that the onset and kinetics of eIF2α phosphorylation was similar to that of protein synthesis inhibition as shown by metabolic labeling. Increased virus replication and virus protein formation was observed by 12 hpi, peaking at 24 hpi. Apoptosis was induced in a small fraction (1-2%) of IPNV-infected CHSE cells from 24 hpi while necrotic/late apoptotic cells increased from 10% by 24 hpi to 59% at 48 hpi, as shown by flow cytometry. These results were in accordance with a small decline in cell viability by 24hpi, dropping below 50% by 48 hpi. IPNV induced IFN-α mRNA upregulation by 24 hpi while no change was observed in the expression of Mx1 and PKR mRNA. Collectively, these findings show that IPNV induces inhibition of protein synthesis in CHSE cells through phosphorylation of eIF2α with minimal involvement of apoptosis. The anticipation is that protein inhibition is used by the virus to evade the host innate antiviral responses.

The phosphatidylserine receptor (PSR) recognizes a surface marker on apoptotic cells and initiates engulfment. This receptor is important for effective apoptotic cell clearance and maintains normal tissue homeostasis and regulation of the immune response. However, the regulation of PSR expression remains poorly understood. In this study, we determined that interferon regulatory factor-1 (IRF-1) was dramatically upregulated upon viral infection in the fish cell. We observed apoptosis in virus-infected cells and found that both PSR and IRF-1 increased simultaneously. Based on a bioinformatics promoter assay, IRF-1 binding sites were identified in the PSR promoter. Compared to normal viral infection, we found that PSR expression was delayed, viral replication was increased and virus-induced apoptosis was inhibited following IRF-1 suppression with morpholino oligonucleotides. A luciferase assay to analyze promoter activity revealed a decreasing trend after the deletion of the IRF-1 binding site on PSR promoter. The results of this study indicated that infectious pancreatic necrosis virus (IPNV) infection induced both the apoptotic and interferon (IFN) pathways, and IRF-1 was involved in regulating PSR expression to induce anti-viral effects. Therefore, this work suggests that PSR expression in salmonid cells during IPNV infection is activated when IRF-1 binds the PSR promoter. This is the first report to show the potential role of IRF-1 in triggering the induction of apoptotic cell clearance-related genes during viral infection and demonstrates the extensive crosstalk between the apoptotic and innate immune response pathways.