Expression of ssa-miR-155 during ISAV infection in vitro: Putative role as a modulator of the immune response in Salmo salar.

Multiple cellular components are involved in pathogen-host interaction during viral infection; in this context, the role of miRNAs have become highly relevant. We assessed the expression of selected miRNAs during an in vitro infection of a Salmo salar cell line with Infectious Salmon Anemia Virus (ISAV), the causative agent of a severe disease by the same name. Salmon orthologs for miRNAs that regulate antiviral responses were measured using RT-qPCR in an in vitro time-course assay. We observed a modulation of specific miRNAs expression, where ssa-miR-155-5p was differentially over-expressed. Using in silico analysis, we identified the putative mRNA targets for ssa-miR-155-5p, finding a high prevalence of hosts immune response-related genes; moreover, several mRNAs involved in the viral infective process were also identified as targets for this miRNA. Our results suggest a relevant role for miR-155-5p in Salmo salar during an ISAV infection as a regulator of the immune response to the virus.

Transcriptome profiling in head kidney of rainbow trout (Oncorhynchus mykiss) after infection with the low-virulent Nagano genotype of infectious hematopoietic necrosis virus.

Infectious hematopoietic necrosis virus (IHNV) causes clinical diseases and mortality in a wide variety of salmonid species. Here, we studied transcriptional responses in rainbow trout infected by the IHNV-Nagano strain isolated in Korea. RNA-seq-based transcriptome analysis of head kidney tissues cataloged differentially expressed genes. Enrichment analysis of gene ontology annotations was performed, and a total of fifteen biological process terms were commonly identified at all time points. In the Kyoto Encyclopedia of Genes and Genomes pathway analysis, pathogen recognition receptor (PRR) signaling pathways such as the retinoic-acid-inducible gene-I-like receptor signaling pathway and the Toll-like receptor signaling pathway were identified at all time points. The nucleotide-binding oligomerization-domain-like receptor signaling pathway and cytosolic DNA-sensing pathway were identified at days 1 and 3. Protein-protein interaction network and centrality analyses revealed that the immune system, signaling molecules, and interaction pathways were upregulated at days 1 and 3, with the highest centrality of tumor necrosis factor. Cancer, cellular community, and endocrine system pathways were downregulated, with the highest centrality of fibronectin 1 at day 5. STAT1 was upregulated from days 1 to 5 with a high centrality. The reproducibility and repeatability of the transcriptome analysis were validated by RT-qPCR. IHNV-Nagano infection dynamically changed the transcriptome profiles in the head kidney of rainbow trout and induced a defense mechanism by regulating the immune and inflammatory pathways through PRR signaling at an early stage. Downregulated pathways involved in extracellular matrix formation and focal adhesion at day 5 indicated the possible failure of wound healing, which is important in the pathogenesis of IHNV infection.

Seawater transmission and infection dynamics of pilchard orthomyxovirus (POMV) in Atlantic salmon (Salmo salar).

The Tasmanian salmon industry had remained relatively free of major viral diseases until the emergence of pilchard orthomyxovirus (POMV). Originally isolated from wild pilchards, POMV is of concern to the industry as it can cause high mortality in farmed salmon (Salmo salar). Field observations suggest the virus can spread from pen to pen and between farms, but evidence of passive transmission in sea water was unclear. Our aim was to establish whether direct contact between infected and naïve fish was required for transmission, and to examine viral infection dynamics. Atlantic salmon post-smolts were challenged with POMV by either direct exposure via cohabitation or indirect exposure via virus-contaminated sea water. POMV was transmissible in sea water and direct contact between fish was not required for infection. Head kidney and heart presented the highest viral loads in early stages of infection. POMV survivors presented low viral loads in most tissues, but these remained relatively high in gills. A consistent feature was the infiltration of viral-infected melanomacrophages in different tissues, suggesting an important role of these in the immune response to POMV. Understanding POMV transmission and host-pathogen interactions is key for the development of improved surveillance tools, transmission models and ultimately for disease prevention.

Differential immunogene expression profile of European sea bass (Dicentrarchus labrax, L.) in response to highly and low virulent NNV.

European sea bass is highly susceptible to the nervous necrosis virus, RGNNV genotype, whereas natural outbreaks caused by the SJNNV genotype have not been recorded. The onset and severity of an infectious disease depend on pathogen virulence factors and the host immune response. The importance of RGNNV capsid protein amino acids 247 and 270 as virulence factors has been previously demonstrated in European sea bass; however, sea bass immune response against nodaviruses with different levels of virulence has been poorly characterized. Knowing the differences between the immune response against both kinds of isolates may be key to get more insight into the host mechanisms responsible for NNV virulence. For this reason, this study analyses the transcription of immunogenes differentially expressed in European sea bass inoculated with nodaviruses with different virulence: a RGNNV virus obtained by reverse genetics (rDl956), highly virulent to sea bass, and a mutated virus (Mut247+270Dl956, RGNNV virus displaying SJNNV-type amino acids at positions 247 and 270 of the capsid protein), presenting lower virulence. This study has been performed in brain and head kidney, and the main differences between the immunogene responses triggered by both viruses have been observed in brain. The immunogene response in this organ is stronger after inoculation with the most virulent virus, and the main differences involved genes related with IFN I system, inflammatory response, cell-mediated response, and apoptosis. The lower virulence of Mut247+270Dl956 to European sea bass can be associated with a delayed IFN I response, as well as an early and transitory inflammation and cell-mediated responses, suggesting that those can be pivotal elements in controlling the viral infection, and therefore, their functional activity could be analysed in future studies. In addition, this study supports the role of capsid amino acids at positions 247 and 270 as important determinants of RGNNV virulence to European sea bass.

A chimeric recombinant infectious hematopoietic necrosis virus induces protective immune responses against infectious hematopoietic necrosis and infectious pancreatic necrosis in rainbow trout.

Infectious pancreatic necrosis virus (IPNV) and infectious hematopoietic necrosis virus (IHNV) are two common viral pathogens that cause severe economic losses in all salmonid species in culture, but especially in rainbow trout. Although vaccines against both diseases have been commercialized in some countries, no such vaccines are available for them in China. In this study, a recombinant virus was constructed using the IHNV U genogroup Blk94 virus as a backbone vector to express the antigenic gene, VP2, from IPNV via the reverse genetics system. The resulting recombinant virus (rBlk94-VP2) showed stable biological characteristics as confirmed by virus growth kinetic analyses, pathogenicity analyses, indirect immunofluorescence assays and western blotting. Rainbow trout were immunized with rBlk94-VP2 and then challenged with the IPNV ChRtm213 strain and the IHNV Sn1203 strain on day 45 post-vaccination. A significantly higher survival rate against IHNV was obtained in the rBlk94-VP2 group on day 45 post-vaccination (86%) compared with the PBS mock immunized group (2%). Additionally, IPNV loads decreased significantly in the rBlk94-VP2 immunized group in the liver (28.6-fold to 36.5-fold), anterior kidney (21.7-fold to 44.2-fold), and spleen (14.9-fold to 22.7-fold), as compared with the PBS mock control group. The mRNA transcripts for several innate and adaptive immune-related proteins (IFN-γ, IFN-1, Mx-1, CD4, CD8, IgM, and IgT) were also significantly upregulated after rBlk94-VP2 vaccination, and neutralizing antibodies against both IHNV and IPNV were induced on day 45 post-vaccination. Collectively, our results suggest that this recombinant virus could be developed as a vaccine vector to protect rainbow trout against two or more diseases, and our approach lays the foundations for developing live vaccines for rainbow trout.

Salmon cells SHK-1 internalize infectious pancreatic necrosis virus by macropinocytosis.

We have previously shown that infectious pancreatic necrosis virus (IPNV) enters the embryo cell line CHSE-214 by macropinocytosis. In this study, we have extended our investigation into SHK-1 cells, a macrophage-like cell line derived from the head kidney of Atlantic salmon, the most economically important host of IPNV. We show that IPNV infection stimulated fluid uptake in SHK-1 cells above the constitutive macropinocytosis level. In addition, upon infection of SHK-1 cells, IPNV produced several changes in actin dynamics, such as protrusions and ruffles, which are important features of macropinocytosis. We also observed that the Na+/H+ pump inhibitor EIPA blocked IPNV infection. On the other hand, IPNV entry was independent of clathrin, a possibility that could not be ruled out in CHSE 214 cells. In order to determine the possible role of accessory factors on the macropinocytic process, we tested several inhibitors that affect components of transduction pathways. While pharmacological intervention of PKI3, PAK-1 and Rac1 did not affect IPNV infection, inhibition of Ras and Rho GTPases as well as Cdc42 resulted in a partial decrease in IPNV infection. Further studies will be required to determine the signalling pathway involved in the macropinocytosis-mediated entry of IPNV into its target cells.

Functional characterization of IL-10 and its receptor subunits in a perciform fish, the mandarin fish, Siniperca chuatsi.

Interleukin (IL)-10 is an immune-regulatory cytokine with multiple functions. In the current study, IL-10 and its two receptors, IL-10R1 and IL-10R2 were identified in mandarin fish, Siniperca chuatsi. The inhibitory effect of mandarin fish IL-10 was investigated on pro-inflammatory cytokine expression and the ligand-receptor relationship. This IL-10 possesses conserved cysteine residues, predicted α-helices and a typical IL-10 family signature motif, similar to its mammalian orthologue, and IL-10R1 harbours predicted JAK1 and STAT3 binding sites in the intracellular region. The fish IL-10 and IL-10R1 exhibit high expression levels in several immune-related organs/tissues, such as spleen, trunk kidney and head kidney, and IL-10R2 possesses a constitutive expression pattern. The expression of IL-10 shows significant increase in spleen from infectious spleen and kidney necrosis virus (ISKNV) infected mandarin fish, where the two receptors also exhibit different levels of induced expression. Mandarin fish IL-10 also exhibits significant response to the stimulation of LPS, PHA and PMA, with the two receptors exhibiting an interesting decrease in expression following the treatment of PMA. The pro-inflammatory cytokines, IL-6, IL-1ß, IL-8, TNF-α, show diminished up-regulation in LPS-stimulated splenocytes pre-incubated with IL-10, indicating the anti-inflammatory roles of mandarin fish IL-10. In EPC cells transfected with different combinations of receptors, IL-10 can enhance the expression of suppressor of cytokine signalling 3 (SOCS3) only when IL-10R1 and IL-10R2 are both expressed, suggesting the participation of the two receptors in signal transduction of mandarin fish IL-10. Similar results are observed with the usage of chimeric receptors, IL-10R1/CRFB1 and IL-10R2/CRFB5. Overall, mandarin fish IL-10 shares conserved ligand-receptor system and the prototypical inhibitory activities on pro-inflammatory cytokine expression with mammalian IL-10, implying the evolutionary conservation of this cytokine.

New cell lines for efficient propagation of koi herpesvirus and infectious salmon anaemia virus.

The production of piscine viruses, in particular of koi herpesvirus (KHV, CyHV-3) and infectious salmon anaemia virus (ISAV), is still challenging due to the limited susceptibility of available cell lines to these viruses. A number of cell lines from different fish species were compared to standard diagnostic cell lines for KHV and ISAV regarding their capability to exhibit a cytopathic effect (CPE) and to accumulate virus. Two cell lines, so far undescribed, appeared to be useful for diagnostic purposes. Fr994, a cell line derived from ovaries of rainbow trout (Oncorhynchus mykiss), produced constantly high ISA virus (ISAV) titres and developed a pronounced CPE even at high cell passage numbers, while standard cell lines are reported to gradually loose these properties upon propagation. Another cell line isolated from the head kidney of common carp (Cyprinus carpio), KoK, showed a KHV induced CPE earlier than the standard cell line used for diagnostics. A third cell line, named Fin-4, established from the fin epithelium of rainbow trout did not promote efficient replication of tested viruses, but showed antigen sampling properties and might be useful as an in vitro model for virus uptake or phagocytosis.

Vaccination with UV-inactivated nodavirus partly protects European sea bass against infection, while inducing few changes in immunity.

Developing viral vaccines through the ultraviolet (UV) inactivation of virus is promising technique since it is straightforward and economically affordable, while the resulting viruses are capable of eliciting an adequate antiviral immune response. Nodavirus (NNV) is a devastating virus that mainly affects European sea bass juveniles and larvae, causing serious economic losses in Mediterranean aquaculture. In this work, a potential vaccine consisting on UV-inactivated NNV (iNNV) was generated and administered to healthy juveniles of European sea bass to elucidate whether it triggers the immune response and improves their survival upon challenge. First, iNNV failed to replicate in cell cultures and its intraperitoneal administration to sea bass juveniles also failed to produce fish mortality and induction of the type I interferon (IFN) pathway, indicating that the NNV was efficiently inactivated. By contrast, iNNV administration induced significant serum non-specific antimicrobial activity as well as a specific antiviral activity and immunoglobulin M (IgM) titres against NNV. Interestingly, few changes were observed at transcriptional level in genes related to either innate or adaptive immunity, suggesting that iNNV could be modulating the immune response at protein or functional level. In addition, the iNNV vaccinated group showed improved survival, reaching a relative survival percentage of 57.9%. Moreover, challenged fish that had been vaccinated presented increased serum antibacterial, antiviral and IgM titres, as well as the higher transcription of mhc1a, ifn, isg15 and cd8a genes in brain, while in the head-kidney the transcription of mhc1a, mhc2b and cd8a was down-regulated and mx, isg15 and tcrb was up-regulated. Although the UV-inactivated vaccine against NNV showed promising results, more effort should be addressed to improving this prophylactic method by increasing our understanding of its action mechanisms, thus enabling the mortality rate of NNV to be further reduced.

Immune-modulation of two BATF3 paralogues in rainbow trout Oncorhynchus mykiss.

Basic leucine zipper transcription factor ATF-like (BATF) -3 is a member of the activator protein 1 (AP­1) family of transcription factors and is known to play a vital role in regulating differentiation of antigen-presenting cells in mammals. In this study, two BATF3 homologues (termed BATF3a and BATF3b) have been identified in rainbow trout (Oncorhynchus mykiss). Both genes were constitutively expressed in tissues, with particularly high levels of BATF3a in spleen, liver, pyloric caecae and head kidney. BATF3a was also more highly induced by PAMPs and cytokines in cultured cells, with type II IFN a particularly potent inducer. In rIL-4/13 pre-stimulated cells, the viral PAMPS polyI:C and R848 had the most pronounced effect on BATF3 expression. BATF3 expression could also be modulated in vivo, following infection with Yersinia ruckeri, a bacterial pathogen causing redmouth disease in salmonids, or with the rhabdovirus IHNV. The results suggest that BATF3 may be functionally conserved in regulating the differentiation and activation of immune cells in lower vertebrates and could be explored as a potential marker for comparative investigation of leucocyte lineage commitment across the vertebrate phyla.

Differential expression and functional diversification of diverse immunoglobulin domain-containing protein (DICP) family in three gynogenetic clones of gibel carp.

Diverse immunoglobulin (Ig) domain-containing protein (DICP) family is a novel bony fish-specific multi-gene family encoding diversified immune receptors. However, their function and the implication of binding partners remain unknown. In this study, we first identified 28 DICPs from three gibel carp gynogenetic clones and revealed their high variability and clone-specific feature. After crucian carp herpesvirus (CaHV) infection, these DICPs were significantly upregulated in head kidney, kidney and spleen. The up-regulation folds in clone A+, F and H were related to the susceptibility to CaHV, progressively increasing from resistant clone to susceptible clone. Overexpression of gibel carp DICPs inhibited interferon (IFN) and viperin promoter-driven luciferase activity. The additions of E. coli extracts and lipid A significantly enhanced the inhibition effect. In addition, gibel carp DICPs can interact with SHP-1 and SHP-2. These findings suggest that gible carp DICPs, as inhibitory receptors, might specifically recognize lipid A, and then interact with SHP-1 and SHP-2 to inhibit the induction of IFN and ISGs.

The first report of siglec-3/CD33 gene in a teleost (rock bream, Oplegnathus fasciatus): An analysis of its spatial expression during stimulation to red seabream iridovirus (RSIV) and two bacterial pathogens.

Siglec-3/CD33 is a myeloid-specific inhibitory receptor that is expressed on cells of the immune system, where it is believed to play a regulatory role, modulating the inflammatory and immune responses. We characterized CD33 (RbCD33) in rock bream which is a transmembrane protein with two IG-like domains and a cytoplasmic tail. It has a deduced amino acid sequence of 390 residues and has tyrosine-based signaling motifs in the cytoplasmic tail. The RbCD33 mRNA was highly expressed in peripheral blood leukocytes and was also detected in the muscle, spleen, skin, head kidney, gills, trunk kidney, heart, stomach, brain, intestine and liver by quantitative real-time PCR. A temporal variation in expression of RbCD33 was observed in different tissues after stimulating with E. tarda, S. iniae and red seabream iridovirus (RSIV). In the head kidney tissue, E. tarda and S. iniae induced RbCD33, while a down regulation was observed with RSIV. In addition, in spleen tissue, S. iniae caused a very high induction of RbCD33 in comparison with an E. tarda and RSIV challenge. In the liver and gill tissues, all three pathogens induced a high expression of RbCD33. The expression pattern in various tissues and its high induction after pathogen stimulation suggests that RbCD33 plays an important role in initiating the immune response via the inhibition of signal transduction of the myeloid lineage cells.

Atlantic salmon adapted to seawater for 9 weeks develop a robust immune response to salmonid alphavirus upon bath challenge.

Pancreas disease (PD) caused by salmonid alphavirus (SAV) is the most serious viral disease in Norwegian aquaculture. Study of the immune response to SAV will aid preventative measures including vaccine development. The innate immune response was studied in Atlantic salmon infected by either bath immersion (BI) or by intra-muscular (i.m.) injection (IM) with SAV subtype 3, two and nine weeks after seawater transfer (Phases A and B respectively). Phase A results have been previously published (Moore et al., 2017) and Phase B results are presented here together with a comparison of results achieved in Phase A. There was a rapid accumulation of infected fish in the IM-B (IM Phase B) group and all fish sampled were SAV RNA positive by 7 dpi (days post infection). In contrast, only a few SAV RNA positive (infected) fish were identified at 14, 21 and 28 dpi in the BI-B (BI Phase B) group. Differences in the transcription of several immune genes were apparent when compared between the infected fish in the IM-B and BI-B groups. Transcription of the analysed genes peaked at 7 dpi in the IM-B group and at 14 dpi in the BI-B group. However, this latter finding was difficult to interpret due to the low prevalence of SAV positive fish in this group. Additionally, fish positive for SAV RNA in the BI-B group showed higher transcription of IL-1ß, IFNγ and CXCL11_L1, all genes associated with the inflammatory response, compared to the IM-B group. Histopathological changes in the heart were restricted to the IM-B group, while (immune) cell filtration into the pancreas was observed in both groups. Compared to the Phase A fish that were exposed to SAV3 two weeks after seawater transfer, the Phase B fish in the current paper, showed a higher and more sustained innate immune gene transcription in response to the SAV3 infection. In addition, the basal transcription of several innate immune genes in non-infected control fish in Phase B (CT-B) was also significantly different when compared to Phase A control fish (CT-A).

Experimental infection of Tilapia Lake Virus (TiLV) in Nile tilapia (Oreochromis niloticus) and red tilapia (Oreochromis spp.).

Since 2015, a novel orthomyxo-like virus, tilapia lake virus (TiLV) has been associated with outbreaks of disease and massive mortality of cultured Nile and red tilapia (Oreochromis niloticus and Oreochromis spp., respectively) in Thailand. In this study, TiLV was isolated from field samples and propagated in the permissive E-11 cell line, with cytopathic effect (CPE) development within 3-5days post-inoculation. Electron micrographs of infected E-11 cells and fish tissues confirmed the rounded, enveloped virions of 60 to 80nm with characteristics very similar to those of Orthomyxoviridae. In vivo challenge studies showed that high mortality in Nile (86%) and red tilapia (66%) occurred within 4-12days post-infection. The virus was re-isolated from challenged fish tissues in the permissive cell line, and PCR analysis confirmed TiLV as a causative pathogen. The distinct histopathology of challenged fish included massive degeneration and inflammatory cell infiltration in the liver and brain as well as the presence of eosinophilic intracytoplasmic inclusions in hepatocytes and splenic cells. Our results fulfilled Koch's postulates and confirmed that TiLV is an etiologic agent of mass mortality of tilapia in Thailand. The emergence of this virus in many countries has helped increase awareness that it is a potential threat to tilapia aquacultured in Thailand, Asia, and worldwide.

Bivalent DNA vaccine induces significant immune responses against infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus in rainbow trout.

Infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are important pathogens of salmon and trout. An active bivalent DNA vaccine was constructed with the glycoprotein gene of Chinese IHNV isolate Sn1203 and VP2-VP3 gene of Chinese IPNV isolate ChRtm213. Rainbow trout (5 g) were vaccinated by intramuscular injection with 1.0 µg of the bivalent DNA vaccine and then challenged with an intraperitoneal injection of IHNV, IPNV, or both, at 30 and 60 days post-vaccination (d.p.v.). High protection rates against IHNV were observed, with 6% and 10% cumulative mortality, respectively, compared with 90-94% in the mock-vaccinated groups. IPNV loads (531-fold and 135-fold, respectively) were significantly reduced in the anterior kidneys of the vaccinated trout. Significant protection against co-infection with IHNV and IPNV was observed, with cumulative mortality rates of 6.67% and 3.33%, respectively, compared with 50.0% and 43.3%, respectively, in the mock-vaccinated groups. No detectable infective IHNV or IPNV was recovered from vaccinated trout co-infected with IHNV and IPNV. The bivalent DNA vaccine increased the expression of Mx-1 and IFN-γ at 4, 7, and 15 d.p.v, and IgM at 21 d.p.v., and induced high titres (≥160) of IHNV and IPNV neutralizing antibodies at 30 and 60 d.p.v.

Transcriptomic analysis of common carp anterior kidney during Cyprinid herpesvirus 3 infection: Immunoglobulin repertoire and homologue functional divergence.

Cyprinid herpesvirus 3 (CyHV-3) infects koi and common carp and causes widespread mortalities. While the virus is a significant concern for aquaculture operations in many countries, in Australia the virus may be a useful biocontrol agent for pest carp. However, carp immune responses to CyHV-3, and the molecular mechanisms underpinning resistance, are not well understood. Here we used RNA-Seq on carp during different phases of CyHV-3 infection to detect the gene expression dynamics of both host and virus simultaneously. During acute CyHV-3 infection, the carp host modified the expression of genes involved in various immune systems and detoxification pathways. Moreover, the activated pathways were skewed toward humoral immune responses, which may have been influenced by the virus itself. Many immune-related genes were duplicated in the carp genome, and often these were expressed differently across the infection phases. Of particular interest were two interleukin-10 homologues that were not expressed synchronously, suggesting neo- or sub-functionalization. The carp immunoglobulin repertoire significantly diversified during active CyHV-3 infection, which was followed by the selection of high-affinity B-cells. This is indicative of a developing adaptive immune response, and is the first attempt to use RNA-Seq to understand this process in fish during a viral infection.

Characterization of rock bream (Oplegnathus fasciatus) complement components C1r and C1s in terms of molecular aspects, genomic modulation, and immune responsive transcriptional profiles following bacterial and viral pathogen exposure.

The complement components C1r and C1s play a crucial role in innate immunity via activation of the classical complement cascade system. As initiators of the pathogen-induced signaling cascade, C1r and C1s modulate innate immunity. In order to understand the immune responses of teleost C1r and C1s, Oplegnathus fasciatus C1r and C1s genes (OfC1r and OfC1s) were identified and characterized. The genomic sequence of OfC1r was enclosed with thirteen exons that represented a putative peptide with 704 amino acids (aa), whereas eleven exons of OfC1s represented a 691 aa polypeptide. In addition, genomic analysis revealed that both OfC1r and OfC1s were located on a single chromosome. These putative polypeptides were composed of two CUB domains, an EGF domain, two CCP domains, and a catalytically active serine protease domain. Phylogenetic analysis of C1r and C1s showed that OfC1r and OfC1s were evolutionary close to the orthologs of Pundamilia nyererei (identity = 73.4%) and Oryzias latipes (identity = 58.0%), respectively. Based on the results of quantitative real-time qPCR analysis, OfC1r and OfC1s transcripts were detected in all the eleven different tissues, with higher levels of OfC1r in blood and OfC1s in liver. The putative roles of OfC1r and OfC1s in response to pathogenic bacteria (Edwardsiella tarda and Streptococcus iniae) and virus (rock bream iridovirus, RBIV) were investigated in liver and head kidney tissues. The transcription of OfC1r and OfC1s was found to be significantly upregulated in response to pathogenic bacterial and viral infections. Overall findings of the present study demonstrate the potential immune responses of OfC1r and OfC1s against invading microbial pathogens and the activation of classical signaling cascade in rock bream.

ISAV infection promotes apoptosis of SHK-1 cells through a ROS/p38 MAPK/Bad signaling pathway.

The impact that the infectious salmon anemia virus (ISAV) has on the immune response of Salmo salar, from the perspective of activating/inactivating cellular processes, is currently unknown. Therefore, the present study evaluated this interaction and found that SHK-1 cells infected with ISAV resulted in respiratory burst activation and the induction of a strong pro-apoptotic imbalance through an increased expression of the Bad protein and decreased transcripts of Bcl-xl. Interestingly, the pharmacological inhibition of the p38 MAPK protein through SB203580 blocked the production of reactive oxygen species, the activity of caspase 3, and the formation of apoptotic nuclei in SHK-1 cells. Additionally, when the NADPH oxidase complex, a producer of superoxide anions, was blocked through apocynin, decreased apoptotic activity was observed in infected cells without significant modifications to viral amplification. These results, together with bioinformatics analysis performed for the Bad gene of fugu, suggest that the ISA virus triggers a strong production of oxygen radicals capable of activating transduction signaling pathways and mediating the expression and activation of pro-apoptotic proteins through the p38 MAPK pathway, all of which results in the apoptosis of ISAV infected cells.

Flow cytometry detection of infectious pancreatic necrosis virus (IPNV) within subpopulations of Atlantic salmon (Salmo salar L.) leucocytes after vaccination and during the time course of experimental infection.

In the present study, intracellular infectious pancreatic necrosis virus (IPNV) in salmon leucocytes was detected by flow cytometry after experimental cohabitant challenge. IPNV vaccinated, non-vaccinated and intraperitoneally (i.p.) infected salmon (virus shedders) were analysed at different times throughout the period when mortality occurred. Fish that had survived 61 days post challenge (carriers) were also analysed. In particular, we analysed the presence of IPNV in B-cells (C7G7+cells) and in neutrophils (E3D9+ cells) in head kidney leucocytes (HKL) and in peripheral blood leucocytes (PBL). IPNV was present in HKL and PBL from all challenged fish groups at all samplings, including carriers. IPNV was also found intracellular in other leucocytes than B-cells and neutrophils. During the time course of infection there were changes in proportion of B-cells and neutrophils and in proportions of IPNV+ cells. In vaccinated fish, a delay in the changes observed in the proportion of IPNV+ cells and in the proportions of the two subpopulations was identified. The vaccinated fish were protected against disease as no fish died compared to 30.8% of non-vaccinated cohabitant fish. All i.p. infected fish, except one, survived the challenge. This is consistent with previous studies and confirmed that the routes of infection can influence mortality. The analyses in this study could not identify any factors enlightening this absence of mortality in i.p. infected fish, but both flow cytometry and qRT-PCR showed that i.p. infected fish were carriers of IPNV. The present study also found that IPNV was present in both B-cells and neutrophils as well as in other leucocytes in all carriers after cohabitant challenge. These fish had survived 9 weeks post challenge and 4 weeks after mortality has ceased. The fish harbouring virus within their leucocytes might become life long carriers and represent a risk for disease outbreaks, being virus shedders. Such fish are protected from later infections if the virus exposure has resulted in protective immunity. Flow cytometry was found to be very suitable for detection of intracellular virus after in vivo challenge and the sensitivity was demonstrated by the detection of virus in carriers.

A NK-lysin from Cynoglossus semilaevis enhances antimicrobial defense against bacterial and viral pathogens.

NK-lysin is an effector protein of cytotoxic T lymphocytes and natural killer cells. Mammalian NK-lysin is known to possess antibacterial property and antitumor activity. Homologues of NK-lysin have been identified in several teleost species, but the natural function of fish NK-lysin remains essentially unknown. In this study, we identified a NK-lysin, CsNKL1, from half-smooth tongue sole (Cynoglossus semilaevis) and analyzed its expression, genetic organization, and biological effect on pathogen infection. CsNKL1 is composed of 135 residues and shares 33.1-56.5% overall sequence identities with other teleost NK-lysin. CsNKL1 possesses a Saposin B domain and six conserved cysteine residues that in mammals are known to form three intrachain disulfide bonds essential to antimicrobial activity. The genomic sequence of the ORF region of CsNKL1 is 1240bp in length and, like human NK-lysin, contains five exons and four introns. Expression of CsNKL1 occurred in multiple tissues and was upregulated by bacterial and viral infection in a time dependent manner. When CsNKL1 was overexpressed in tongue sole, significant upregulation of interleukin-1 and chemokines was observed in spleen and head kidney. Following bacterial and viral infection, the pathogen loads in the tissues of CsNKL1-overexpressing fish were significantly lower than those in control fish. These results indicate that CsNKL1 possesses the novel capacities of immunomodulation and enhancing antimicrobial defense against pathogens of both bacterial and viral nature.