Effect of ß-glucans on rainbow trout (Oncorhynchus mykiss) IgM+ B cells.

ß-glucans are carbohydrates present in the cell wall of many fungi, which are often used as immunostimulants in feeds for farmed species. Their capacity to activate innate immune responses directly acting on innate cell populations has been widely documented in fish. However, whether they can affect the functionality of adaptive immune cells has been scarcely explored. In this context, in the current work, we have determined the effects of ß-glucans on rainbow trout blood IgM+ B cells in the presence or absence of 2,4,6-trinitrophenyl hapten conjugated to lipopolysaccharide (TNP-LPS), a model antigen. For this, rainbow trout peripheral blood leukocytes were incubated with different doses of ß-glucans or media alone in the presence or absence of TNP-LPS for 48 h. The size, levels of expression of surface MHC II, antigen processing and phagocytic capacities and proliferation of IgM+ B cells were then studied by flow cytometry. The number of IgM-secreting cells in the cultures was also estimated by ELISpot. ß-glucans significantly decreased the levels of surface MHC II expression and the antigen processing capacities of these cells, especially in the presence of TNP-LPS, while they increased their phagocytic activity. On their own, ß-glucans slightly activated the proliferation of IgM+ B cells but reduced that induced by TNP-LPS. In contrast, ß-glucans significantly increased the number of cells secreting IgM in the cultures. This effect of ß-glucans on the IgM-secreting capacity of B cells was also confirmed through a feeding experiment, in which the IgM-secreting capacity of blood leukocytes obtained from fish fed a ß-glucan-supplemented diet for one month was compared to that of leukocytes obtained from fish fed a control diet. Altogether, these findings contribute to increase our knowledge regarding the effects of ß-glucans on fish adaptive responses.

Yersinia ruckeri infection activates local skin and gill B cell responses in rainbow trout.

Teleost fish lack organized structures in mucosal tissues such as those of mammals, but instead contain dispersed B and T cells with the capacity to respond to external stimuli. Nonetheless, there is still a great lack of knowledge regarding how B cells differentiate to plasmablasts/plasma cells in these mucosal surfaces. To contribute to a further understanding of the mechanisms through which fish mucosal B cells are activated, in the current study, we have studied the B cell responses in the skin and gills of rainbow trout (Oncorhynchus mykiss) exposed to Yersinia ruckeri. We have first analyzed the transcription levels of genes related to B cell function in both mucosal surfaces, and in spleen and kidney for comparative purposes. In a second experiment, we have evaluated how the infection affects the presence and size of B cells in both skin and gills, as well as the presence of plasmablasts secreting total or specific IgMs. The results obtained in both experiments support the local differentiation of B cells to plasmablasts/plasma cells in the skin and gills of rainbow trout in response to Y. ruckeri. Interestingly, these plasmablasts/plasma cells were shown to secrete specific IgMs as soon as 5 days after the exposure. These findings contribute to a further understanding of how B cells in the periphery respond to immune stimulation in teleost fish.

Role of the melanocortin system in zebrafish skin physiology.

The agouti-signaling protein (ASIP) acts as both a competitive antagonist and inverse agonist of melanocortin receptors which regulate dorsal-ventral pigmentation patterns in fish. However, the potential role of ASIP in the regulation of additional physiological pathways in the skin is unknown. The skin plays a crucial role in the immune function, acting as a physical limitation against infestation and also as a chemical barrier due to its ability to synthesize and secrete mucus and many immune effector proteins. In this study, the putative role of ASIP in regulating the immune system of skin has been explored using a transgenic zebrafish model overexpressing the asip1 gene (ASIPzf). Initially, the structural changes in skin induced by asip1 overexpression were studied, revealing that the ventral skin of ASIPzf was thinner than that of wild type (WT) animals. A moderate hypertrophy of mucous cells was also found in ASIPzf. Histochemical studies showed that transgenic animals appear to compensate for the lower number of cell layers by modifying the mucus composition and increasing lectin affinity and mucin content in order to maintain or improve protection against microorganism adhesion. ASIPzf also exhibit higher protein concentration under crowding conditions suggesting an increased mucus production under stressful conditions. Exposure to bacterial lipopolysaccharide (LPS) showed that ASIPzf exhibit a faster pro-inflammatory response and increased mucin expression yet severe skin injures and a slight increase in mortality was observed. Electrophysiological measurements show that the ASIP1 genotype exhibits reduced epithelial resistance, an indicator of reduced tissue integrity and barrier function. Overall, not only are ASIP1 animals more prone to infiltration and subsequent infections due to reduced skin epithelial integrity, but also display an increased inflammatory response that can lead to increased skin sensitivity to external infections.

Mucosal and systemic immune effects of Bacillus subtilis in rainbow trout (Oncorhynchus mykiss).

Bacillus spp. are well known for their probiotic properties. Hence, the long-term feeding of Bacillus spp. strains to different fish species has been proved to confer beneficial effects regarding growth or pathogen resistance, among others. However, whether these strains could function as mucosal adjuvants, up-regulating immune responses after a single administration, has not yet been investigated in fish. Thus, in the current work, we have performed a series of experiments in rainbow trout (Oncorhynchus mykiss) aimed at establishing the potential of two Bacillus subtilis spore-forming strains, designated as ABP1 and ABP2, as oral adjuvants/immunostimulants. As an initial step, we evaluated their transcriptional effects on the rainbow trout intestinal epithelial cell line RTgutGC, and in gut tissue explants incubated ex vivo with the two strains. Their capacity to adhere to RTgutGC cells was also evaluated by flow cytometry. Although both strains had the capacity to modulate the transcription of several genes related to innate and adaptive immune responses, it was the ABP1 strain that led to stronger transcriptional effects, also exerting a higher binding capacity to intestinal epithelial cells. Consequently, we selected this strain to establish its effects on splenic B cells upon in vitro exposure as well as to determine the transcriptional effects exerted in the spleen, kidney, and gut after a single oral administration of the bacteria. Our results showed that B. subtilis ABP1 had the capacity to modulate the proliferation, IgM secreting capacity and MHC II surface expression of splenic B cells. Finally, we confirmed that this strain also induced the transcription of genes involved in inflammation, antimicrobial genes, and genes involved in T cell responses upon a single oral administration. Our results provide valuable information regarding how B. subtilis modulates the immune response of rainbow trout, pointing to the usefulness of the ABP1 strain to design novel oral vaccination strategies for aquaculture.

Comprehensive transcriptome profiling and functional analysis of the meagre (Argyrosomus regius) immune system.

Meagre (Argyrosomus regius) belongs to the family Sciaenidae and is a promising candidate for Mediterranean aquaculture diversification. As a relatively recent species in aquaculture, the physiological consequences of the immune system activation in meagre are understudied. Spleen, as a primary lymphoid organ has an essential role in meagre immune and inflammatory responses. In this study, we have evaluated the in vivo effects of lipopolysaccharide (LPS) on the spleen transcriptome of meagre by RNA-seq analysis at 4 and 24 h after injection.

Evaluation of the Potential of Marine Algae Extracts as a Source of Functional Ingredients Using Zebrafish as Animal Model for Aquaculture.

Research on immunotherapeutic agents has become a focus for the treatment of fish diseases. The ability of algae to produce secondary metabolites of potential interest as immunotherapeutics has been documented. The present research intended to assess antiviral and antibacterial activities of macro- and microalgae extracts against viral and bacterial pathogens and explore their immunomodulatory potential using zebrafish (Danio rerio) larvae as a model organism. The cytotoxicity and antiviral activity of eight methanolic and ethanolic extracts from two macroalgae (Fucus vesiculosus, Ulva rigida) and two microalgae (Nannochloropsis gaditana, Chlorella sp.) were analyzed in established fish cell lines. Six extracts were selected to evaluate antibacterial activity by disk diffusion and growth inhibition assays. The three most promising extracts were characterized in terms of fatty acid composition, incorporated at 1% into a plant-based diet, and evaluated their effect on zebrafish immune response and intestinal morphology in a short-term feeding trial. All extracts exhibited in vitro antiviral activity against viral hemorrhagic septicemia and/or infectious pancreatic necrosis viruses. Methanolic extracts from F. vesiculosus and U. rigida were richer in saturated fatty acids and exhibited in vitro antibacterial action against several bacteria. Most promising results were obtained in vivo with F. vesiculosus methanol extract, which exerted an anti-inflammatory action when incorporated alone into diets and induced pro-inflammatory cytokine expression, when combined with the other extracts. Moreover, dietary inclusion of the extracts improved intestinal morphology. In summary, the results obtained in this study support the potential of algae as natural sources of bioactive compounds for the aquaculture industry.

Temporary protection of rainbow trout gill epithelial cells from infection with viral haemorrhagic septicaemia virus IVb.

The branchial epithelium is not only a primary route of entry for viral pathogens, but is also a site of viral replication and subsequent shedding may also occur from the gill epithelium. This study investigated the potential of agents known to stimulate innate immunity to protect rainbow trout epithelial cells (RTgill-W1) from infection with VHSV IVb. RTgill-W1 cells were pretreated with poly I:C, FuGENE(®) HD + poly I:C, lipopolysaccharide (LPS), LPS + poly I:C or heat-killed VHSV IVb and then infected with VHSV IVb 4 days later. Cytopathic effect (CPE) was determined at 2, 3, 4, 7 and 11 days post-infection. Virus in cells and supernatant was detected using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). All of the treatments delayed the onset of CPE (per cent of monolayer destruction), compared with untreated controls; however, killed VHSV or poly I:C combined with LPS was the most effective. Similarly, the detection of viral RNA in the supernatant was delayed, and the quantity was significantly (P < 0.05) reduced by all treatments with the exception of LPS alone (4 days). Unlike many of the other treatments, pretreatment of RTgill-W1 with heat-killed VHSV did not upregulate interferon 1, 2 or MX 1 gene expression.

Molecular characterization of three novel chemokine receptors in rainbow trout (Oncorhynchus mykiss).

Chemokines signal through a family of seven-transmembrane domain G-coupled receptors in order to regulate both leukocyte mobilization and activate the recruited cells. Although many chemokines have been identified in rainbow trout (Oncorhynchus mykiss), only a few chemokine receptors have been reported to date. In this work, we have cloned three novel chemokine receptors in rainbow trout. One of these receptors seems to be a clear orthologue of CCR6, while the second one constitutes a novel CCR9 gene different from the previous CCR9 reported in this species. This gene, which we have designated as CCR9B, represents another lineage of fish CCR9 genes, not previously identified. Finally, a deeper phylogenetic analysis of the third novel chemokine receptor gene, which had been identified on the basis of sequence similarity to CCR3, constitutes a novel lineage of CCR receptors which has no equivalent in humans and that may be teleost-specific. We have designated this novel gene as CCR13, to avoid any possible ascription to mammalian genes. Further transcriptional studies revealed that CCR6 was constitutively transcribed in thymus, gills, hindgut and peripheral blood leukocytes (PBLs), while CCR9B was strongly transcribed in thymus and PBLs but also in spleen, gills, hindgut and brain at lower levels. CCR13, on the other hand, was strongly detected in spleen, head kidney and PBLs and faintly in thymus, gills, brain and gonad. The data provided constitutes a step forward the identification of novel chemokine receptors that may contribute to a future understanding of chemokine signalling in fish.

Differential effects of viral hemorrhagic septicaemia virus (VHSV) genotypes IVa and IVb on gill epithelial and spleen macrophage cell lines from rainbow trout (Oncorhynchus mykiss).

The two most prominent genotypes of viral hemorrhagic septicemia virus (VHSV) are -I in the Northeastern Atlantic region and -IV in North America, but much more is known about the cellular pathogenesis of genotype -I than -IV. VHSV genotype -IV is divided into -IVa from the Northeast Pacific Ocean and -IVb from the Great Lakes and both of which are less virulent to rainbow trout than genotype -I. In this work, infections of VHSV-IVa and -IVb have been studied in two rainbow trout cell lines, RTgill-W1 from the gill epithelium, and RTS11 from spleen macrophages. RTgill-W1 produced infectious progeny of both VHSV-IVa and -IVb. However, VHSV-IVa was more infectious than -IVb toward RTgill-W1: -IVa caused cytopathic effect (CPE) at a lower viral titre, elicited CPE earlier, and yielded higher titres. By contrast, no CPE and no increase in viral titre were observed in RTS11 cultures infected with either genotype. Yet in RTS11 all six VHSV genes were expressed and antiviral genes, Mx2 and Mx3, were up regulated by VHSV-IVb and -IVa. However, replication appeared to terminate at the translational stage as viral N protein, presumably the most abundant of the VSHV proteins, was not detected in either infected RTS11 cultures. In RTgill-W1, Mx2 and Mx3 were up regulated to similar levels by both viral genotypes, while VHSV-IVa induced higher levels of IFN1, IFN2 and LGP2A than VHSV-IVb.

Immune responses elicited in rainbow trout through the administration of infectious pancreatic necrosis virus-like particles.

Virus like particles (VLPs) against viral pathogens not only constitute a novel approach for the development of antiviral vaccines for an specific virus, but also for the creation of multivalent vaccines in which antigens from other pathogens may be expressed on the surface of these VLPs. Despite positive results on protection for many of these VLPs in both fish and mammals, not many studies have focused on the immune response triggered by these particles; studies that may provide hints for the identification of immune mechanisms responsible for antiviral protection, which are mostly unknown in fish. In the current work, we have studied the levels of transcription of several immune genes in the spleen of rainbow trout (Oncorhynchus mykiss) intraperitoneally injected with VLPs from infectious pancreatic necrosis virus (IPNV) focusing on the chemokine response as well as the response of genes related to interferon (IFN) production. Surprisingly, the capacity of VLPs to induce chemokines differed from that of live IPNV, suggesting a direct effect of viral replication on the chemokine response in this organ. While VLPs up-regulated the transcription of CK3, CK10 and CXCd and down-modulated CK5B, CK6 and CK9 transcription, a previous study in which the transcription of γIP, CXCd, CK1, CK3, CK5B, CK6, CK7A, CK9 and CK12 had been studied demonstrated that IPNV only significantly up-regulated CK6 and down-modulated CK3 in the spleen. On the other hand, the administration of VLPs produced a strong mobilization to the peritoneum of CD4(+), IgM(+), IgT(+) and CD83(+) leukocytes similar to that induced by the live viral infection. In both cases, this leukocyte recruitment seemed to be greatly mediated through CK3, CK5B, CK9 and CK10 chemokine production. These results together with the fact that VLPs strongly induced non-specific lymphocyte proliferation and specific anti-IPNV antibody production point to VLPs as excellent candidates for vaccine development.

Viral hemorrhagic septicaemia virus (VHSV) up-regulates the cytotoxic activity and the perforin/granzyme pathway in the rainbow trout RTS11 cell line.

A survey of immune-relevant genes that might be up-regulated in response to viral hemorrhagic septicaemia virus (VHSV) in the rainbow trout monocyte-macrophage cell line, RTS11, unexpectedly revealed an increased expression of perforin (PRF) and granzyme (GRZ) genes, which represent components of the major cytotoxic pathway. The natural killer-enhancing factor (NKEF), also known to modulate cytotoxic activity, was up-regulated at the gene but strikingly down-regulated at protein level. The expression of these genes was not affected in head kidney leukocytes (HKLs) infected with VHSV, leading us to evaluate the potential cytotoxic activity of RTS11 and HKLs. For the first time, the cytotoxic activity of RTS11 against xenogeneic targets has been demonstrated, although this was modest relative to HKLs. Yet the activity in RTS11 was significantly increased by VHSV, as in HKLs. This cytotoxic activity elicited by viral infection appeared to require viral gene expression because inactivated VHSV failed to increase RTS11 cytotoxic activity. As for other immune functions, RTS11 cells provide a model for further studying cytotoxic activities of fish monocyte-macrophages.

The introduction of multi-copy CpG motifs into an antiviral DNA vaccine strongly up-regulates its immunogenicity in fish.

The protection conferred by antiviral DNA vaccines in fish is known to rely greatly on innate immune responses. Since oligodeoxynucleotides (ODNs) containing unmethylated CpG dinucleotides (CpG motifs) have been shown to induce potential innate immune responses, we have introduced several copies (either two or four) of a fragment containing multiple CpG sequences of known immunostimulatory effects into a DNA vaccine against viral hemorrhagic septicemia virus (VHSV). We have determined the effects of this introduction on the vaccine immunogenicity, measured as immune gene induction, serum neutralizing activity and antigen-dependent proliferation. When comparing the effects of the vaccine containing 2 copies of this CpG fragment (pVHSV-2CpG) or that containing 4 copies of the fragment (pVHSV-4CpG) with the original VHSV DNA vaccine (pVHSV), we found that the levels of expression of type I interferon (IFN) were significantly up-regulated in muscle and spleen when the CpG fragments were introduced. An up-regulation in the levels of MHC-I expression in spleen were also observed in response to the modified vaccines, whereas, the levels of transcription of interleukin 1ß (IL-1ß) were strongly reduced in comparison to the original vaccine. Important but very variable differences were also observed concerning the vaccine induction of IFN-γ. Moreover, the serum neutralizing capacity was strongly increased as fish were vaccinated with plasmids containing more CpG fragments. Taken together, all these results demonstrate a strongly increased immunogenicity of the VHSV DNA vaccine, through the introduction of this multicopy CpG fragment.

An active DNA vaccine against infectious pancreatic necrosis virus (IPNV) with a different mode of action than fish rhabdovirus DNA vaccines.

Although there are some commercial vaccines available against infectious pancreatic necrosis virus (IPNV), the disease still continues to be a major problem for aquaculture development worldwide. In the current work, we constructed a DNA vaccine against IPNV (pIPNV-PP) by cloning the long open reading frame of the polyprotein encoded by the viral RNA segment A. In vitro, the vaccine is properly translated giving the functional IPNV polyprotein since preVP2, VP2 and VP3 proteins were detected because of the VP4-protease cleavage. EPC cells transfected with the vaccine plasmid expressed the viral proteins and induced the expression of type I interferon (IFN)-induced Mx genes. Furthermore, IPNV synthesized proteins seemed to assemble in virus-like particles as evidenced by electron microscopy. In vivo, rainbow trout specimens were intramuscularly injected with the vaccine and expression of immune-relevant genes, the presence of neutralizing antibodies and effect on viral load was determined. The pIPNV-PP vaccine was expressed at the injection site and up-regulated MHC Ialpha, MHC IIalpha, type-I interferon (IFN), Mx, CD4 and CD8alpha gene expression in the muscle, head kidney or spleen, although to a much lower extent than the up-regulations observed in response to an effective DNA vaccine against viral hemorrhagic septicaemia virus (VHSV). However, the IPNV vaccine was also very effective in terms of acquired immunity since it elicited neutralizing antibodies (in 6 out of 8 trout fingerlings) and decreased 665-fold the viral load after IPNV infection. The effectiveness of this new IPNV DNA vaccine and its possible mechanism of action are discussed and compared to other viral vaccines.

Identification of six novel CC chemokines in gilthead seabream (Sparus aurata) implicated in the antiviral immune response.

Chemokines are key regulators of migration and consequent activation of migrating leukocytes. CC chemokines constitute the largest chemokine group with 24-28 members in mammalian species, and even more in teleost fish, with up to 81 members in zebrafish Danio rerio. Further studies concerning fish chemokine genes will help elucidate the complexity of this chemokine group which has considerably expanded in some teleosts. In the current work, we have identified six novel CC chemokine genes within previously generated gilthead seabream (Sparus aurata) EST databases. The six novel chemokine sequences all show characteristic features of CC chemokines, such as the 4 conserved cysteine residues and a signal peptide. The nomenclature for chemokines in different fish species is not in concordance to mammalian nomenclature as it is difficult to establish true mammalian orthologs, and therefore a different nomenclature has been established for each fish species. In this work, we have named the seabream genes according to the rainbow trout CC chemokine with which they have the highest identity, therefore we have designated the novel seabream CC chemokines as CK1, CK3, CK5, CK7, CK8 and CK10. Expression analysis have also been performed with these new chemokines, as well as with the previously identified seabream chemokine designated as CCL4, which according to our proposed nomenclature should be renamed CK5B. In this sense, we have determined the pattern of constitutive chemokine expression in different seabream tissues. The effect that different immune non-replicative stimuli had in the levels of expression of the chemokines in head kidney leukocytes showed many strong suppressive effects in their transcription levels, and up-regulations mainly in response to mitogens. In vivo, however, when non-replicative virus or heat-killed bacteria were injected, the viral particles up-regulated chemokine expression in the spleen and not in head kidney. Finally, in the context of a real infection such as that of nodavirus in the brain, all the CC chemokines studied were significantly induced. This study constitutes a further step towards the elucidation of an immunological and/or physiological role for fish chemokines.

New tools to study RNA interference to fish viruses: Fish cell lines permanently expressing siRNAs targeting the viral polymerase of viral hemorrhagic septicemia virus.

Previous studies have indicated that low transfection efficiency can be a major problem when gene inhibition by the use of small interfering RNAs (siRNAs) is attempted in fish cells. This may especially be true when targeting genes of viruses which are fast replicating and which can still infect cells that have not been transfected with the antiviral siRNAs. To increase the amount of antiviral siRNAs per cell a different strategy than transfection was taken here. Thus, we describe carp epithelioma papulosum cyprinid (EPC) cell clones expressing siRNAs designed to target the L polymerase gene of the viral hemorrhagic septicemia virus (VHSV), a rhabdovirus affecting fish. Eight siRNA sequences were first designed, synthesized and screened for inhibition of in vitro VHSV infectivity. Small hairpin (sh) DNAs corresponding to three selected siRNAs were then cloned into pRNA-CMV3.1/puro plasmids, transfected into EPC cells and transformed clones were obtained by puromycin selection. Sequence-specific interference with VHSV could only be observed with EPC clones transformed with a mixture of the three shDNAs, rather than with those clones obtained with individual sh DNAs. However, interference was not specific for VHSV as infection with an heterologous fish rhabdovirus, was also reduced to a similar extent. It was shown that this reduction was not due to an Mx response in the transformed cell clones. Here, we discuss some of the possible reasons for such data and future work directions. EPC clones stably expressing rhabdoviral specific siRNA sequences could be a strategy to further investigate the use of RNA interference for targeting costly fish pathogenic viruses.

The immunogenicity of viral haemorragic septicaemia rhabdovirus (VHSV) DNA vaccines can depend on plasmid regulatory sequences.

A plasmid DNA encoding the viral hemorrhagic septicaemia virus (VHSV)-G glycoprotein under the control of 5' sequences (enhancer/promoter sequence plus both non-coding 1st exon and 1st intron sequences) from carp beta-actin gene (pAE6-G(VHSV)) was compared to the vaccine plasmid usually described the gene expression is regulated by the human cytomegalovirus (CMV) immediate-early promoter (pMCV1.4-G(VHSV)). We observed that these two plasmids produced a markedly different profile in the level and time of expression of the encoded-antigen, and this may have a direct effect upon the intensity and suitability of the in vivo immune response. Thus, fish genetic immunisation assays were carried out to study the immune response of both plasmids. A significantly enhanced specific-antibody response against the viral glycoprotein was found in the fish immunised with pAE6-G(VHSV). However, the protective efficacy against VHSV challenge conferred by both plasmids was similar. Later analysis of the transcription profile of a set of representative immune-related genes in the DNA immunized fish suggested that depending on the plasmid-related regulatory sequences controlling its expression, the plasmid might activate distinct patterns of the immune system. All together, the results from this study mainly point out that the selection of a determinate encoded-antigen/vector combination for genetic immunisation is of extraordinary importance in designing optimised DNA vaccines that, when required for inducing protective immune response, could elicit responses biased to antigen-specific antibodies or cytotoxic T cells generation.

Transfection improvements of fish cell lines by using deacylated polyethylenimine of selected molecular weights.

A new tool for DNA transfer to fish cell lines such as epithelioma papulosum cyprini (EPC) and rainbow trout gonad (RTG2), has been optimized by testing commercially available polyethylenimine (PEI) polymers as transfectant reagents. Deacylated 25 kDa PEI polymers were selected amongst the most active and then low toxicity deacylated PEIs fractions around 15 kDa were obtained by gel filtration chromatography to increase 3-4-fold their initial in vitro transfection efficiency. The EPC and plasmids coding for reporter genes were first used to optimize variable values for best expression by transfection with deacylated low toxicity PEI while both EPC/RTG2 and a plasmid coding for the glycoprotein G gene of the fish pathogen, viral haemorrhagic septicemia virus (VHSV) were then used to demonstrate some of their practical applications. Due to its relatively low price, defined chemical composition and availability, low toxicity deacylated PEI might be used for numerous applications for all those studying fish cell immunology in vitro as well as in vivo.

Transcription of immune genes upon challenge with viral hemorrhagic septicemia virus (VHSV) in DNA vaccinated rainbow trout (Oncorhynchus mykiss).

Even though DNA vaccination has proven as one of the most effective methods in controlling fish rhabdoviruses, the immune mechanisms responsible for protection are still unknown. Many studies have focused on studying which cytokines and immune genes are triggered in response to the vaccine at different times post-vaccination. However, to elucidate the mechanism(s) responsible for protection, to our understanding it is also of great relevance to study the immune response to the virus in fish that have been previously vaccinated and compare it to the effects that the virus might have on non-vaccinated fish. This type of study has never been performed to date in fish. Thus, in the current work, we vaccinated rainbow trout (Oncorhynchus mykiss) with a DNA vaccine against viral hemorrhagic septicemia virus (VHSV), and 30 days post-vaccination we challenged the fish with a virulent VHSV. It was then, that we studied the immune response to the virus at very early times post-infection in fish, in order to compare the effects of VHSV on vaccinated or non-vaccinated trout. We studied the levels of expression of interleukin 1beta (IL-1beta), major histocompatibility complex (MHC) class Ialpha and IIalpha genes, immunoglobulin M (IgM), CD8alpha, type I interferon (IFN), Mx, IFN-gamma and natural killer enhancing factor (NKEF) in head kidney, spleen and blood. When we compared the effect that VHSV had on vaccinated fish to the effect that the virus produced in fish vaccinated with the empty plasmid, the genes that were significantly up-regulated were IL-1beta and MHC IIalpha in the spleen at day 1 post-infection, MHC Ialpha in all organs at day 1 post-infection, and IFN and Mx in the spleen and blood at days 1 and 3 post-infection, respectively. Genes that correlate with an increased specific immune response were not significantly increased in response to VHSV in these vaccinated animals. The results suggest that DNA vaccination induces a memory state in fish that, on the contrary to what would occur in mammals, primes the non-specific immune responses upon a later encounter with the virus.

In vitro search for alternative promoters to the human immediate early cytomegalovirus (IE-cMV) to express the G gene of viral haemorrhagic septicemia virus (VHSV) in fish epithelial cells.

Present DNA vaccines against fish rhabdoviruses require intramuscular injection (fish-to-fish vaccination) of their G-protein gene under the control of the human immediate early cytomegalovirus (IE-CMV) promoter, while immersion delivery (mass DNA vaccination), for instance, by using fish epithelial-specific promoters, would be more practical for aquaculture. To find fish epithelial-specific promoters alternative to the IE-CMV, a comparative study of the effectiveness of different fish promoters constitutively expressing the G gene of the viral haemorrhagic septicemia virus (VHSV) in the epithelial papulosum cyprini (EPC) cell line was performed. The study included MCV1.4 (an alternative IE-CMV promoter version), AE6 (a version of the carp beta-actin promoter), long terminal repeats (LTR) of zebrafish or walleye retroviruses, trout Mx1, carp myosin-heavy-chain and flatfish pleurocidin promoters and salmonid sleeping beauty (SB)/medaka Tol2 transposon repeats. The G-protein expression in transfected EPC cells was studied by estimating the number of cells expressing the G-protein in their membrane and the average expression level per cell. In addition, in an attempt to reduce their sizes, some regions of the MCV1.4 and AE6 promoters were deleted and expression levels compared to those observed for full-length promoters. Since both zebrafish LTR and carp AE6 promoters were the most effective regulatory sequences for expressing the VHSV G-protein in EPC cells, these sequences might be candidates for new DNA vaccine vectors for fish epithelial tissues avoiding the IE-CMV promoter. Furthermore, known transcription factor binding sites (TFBS) common to most of the fish G-expressing promoters, might enable the future design of fully synthetic or hybrid promoters with improved efficacy of VHSV G-protein expression in epithelial fish cells.