Fish granzymes and their role in the innate cell-mediated cytotoxicity against nodavirus-infected cells

Granzymes (Gzm) are granule-associated serine proteases, which are important effector molecules in cell-mediated cytotoxicity, classified by its proteolytic activity in: tryptase (GzmA and GzmK), Asp-ase (GzmB), Met-ase (GzmM) or chymase (others). In fish, even at gene level, very few studies have partially identified and characterize them. In this work we have identified the activity of these four granzymes, using colorimetric assays, in leucocytes from gilthead seabream (Sparus aurata) and European sea bass (Dicentrarchus labrax), and how they are increased upon cytotoxic activity against nodavirus-infected tumor cells. The results also showed that GzmA was the most abundant and GzmB the lowest. Deeper characterization of the GzmB led us to identify that seabream leucocytes under cytotoxic response against nodavirus-infected cells showed significantly increased GzmB activity whilst this pattern failed to do so in sea bass leucocytes. Moreover, flow cytometry studies also suggested the presence of GzmB into the cytoplasm of effector cells. At gene level, seabream GzmB gene was found to be up-regulated in leucocytes under cytotoxic activity, mainly if the target cells were infected with nodavirus. These data will through some light in the importance of granzymes in the cytotoxic response of fish and their defense against nodavirus infection

Transcription of histones H1 and H2B is regulated by several immune stimuli in gilthead seabream and European sea bass

Histones (H1 to H4) are the primary proteins which mediate the folding of DNA into chromatin; however, and in addition to this function, histones have been also related to antimicrobial peptides (AMPs) activity in vertebrates, in fact, mammalian H1 is mobilized as part as the anti-viral immune response. In fish, histones with AMP activity have been isolated and characterized mainly from skin and gonads. One of most threatening pathogens for wild and cultured fish species nowadays is nodavirus (NNV), which target tissues are the brain and retina, but it is also able to colonize the gonad and display vertical transmission. Taking all this into account we have identified the h1 and h2b coding sequences in Eu- ropean sea bass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata) fish species and studied their pattern of expression under naïve conditions and NNV in vivo infection. The data obtained prompted us to study their role on the immune response of gonad and head-kidney leucocytes upon viral (NNV), bacteria (Vibrio anguillarumor Photobacteriumdamselae), pathogen-associated molecular patterns (PAMPs) or mitogens stimulation. The h1 and h2b genes are expressed in a wide range of tissues and their expression is modify by infection or other immune stimuli, but further studies will be needed to determine the significance of these changes. These results suggest that h1 expression is related to the immune response against NNV in the brain, while h2b transcription seems to be more important in the head-kidney. Moreover, the potential role of histones as anti-viral agents is suggested and further characterization is in progress

Antimicrobial response is increased in the testis of European sea bass but not in gilthead seabream upon nodavirus infection

Antimicrobial peptides (AMPs) have a crucial role in the fish innate immune response, being considered a fundamental component of the first line of defence against pathogens. Moreover, AMPs have not been studied in the fish gonad since this is used by some pathogens as a vehicle or a reservoir to be transmitted to the progeny, as occurs with nodavirus (VNNV), which shows vertical transmission through the gonad, and/or gonadal fluids but no study has looked into the gonad of infected fish. In this framework, we have characterized the antimicrobial response triggered by VNNV in the testis of European sea bass, a very susceptible species of the virus, and in the gilthead seabream, which acts as a reservoir, both in vivo and in vitro, and compared with that present in the serum and brain (target tissue of VNNV). First, our data show a great antiviral response in the brain of gilthead seabream and in the gonad of European sea bass. In addition, for the first time, our results demonstrate that the antimicrobial activities (complement, lysozyme and bactericidal) and the expression of AMP genes such as complement factor 3 (c3), lysozyme (lyz), hepcidin (hamp), dicentracin (dic), piscidin (pis) or β-defensin (bdef) in the gonad of both species are very different, but generally activated in the European sea bass, probably related with the differences of susceptibility upon VNNV infection, and even differs to the brain response. Furthermore, the in vitro data suggest that some AMPs are locally regulated playing a local immune response in the gonad, while others are more dependent of the systemic immune system. Data are discussed in the light to ascertain their potential role in viral clearance by the gonad to avoid vertical transmission.Postprin

Characterization of the interferon pathway in the European sea bass after nodavirus infection

One of the most powerful innate immune responses against virus is mediated by type I interferon (IFN). We searched the presence of genes involved in the IFN pathway in European sea bass (Dicentrarchus labrax) and evaluated their regulation by nodavirus (VNNV) infection in brain and gonad of infected specimens as well as in a new sea bass cell line (DLB-1), derived from brain. We identified genes encoding MDA5 (Melanoma Differentiation Associated gene 5), LGP2 (Laboratory of Genetics and Physiology 2), MAVS (mitochondrial antiviral signaling protein), TRAF3 (tumour necrosis factor receptor-associated factor 3), TANK (TRAF family member-associated NF-kB activator), TBK1 (TANK binding kinase 1), IRF3 (IFN regulatory factor 3), IRF7 and PKR (dsRNA dependent protein kinase receptor). Thus, in the susceptible species European sea bass, we found that mda5, lgp2 or irf3 gene expression in brain was up-regulated by VNNV infection, as ifn, mx and pkrgenes at different sampling times. Strikingly, most of the genes were up-regulated in gonad. In DLB-1 cell line, most of them were up-regulated by VNNV infection but in lower level than the induction provoked by poly I:C treatment. Further studies will be conducted to characterize the IFN pathway in sea bass and their role in the immune response against VNNV

Recombinant nodavirus vaccine produced in bacteria and administered without purification elicits humoral immunity and protects European sea bass against infection

Viral necrosis virus (NNV) or nodavirus causes fish viral encephalopathy and retinopathy worldwide. In some cases, mortalities in aquaculture industry can reach up to 100%, some species being especially sensitive as is the case of European sea bass (Dicentrarchus labrax), one of the main cultured species in the Mediterranean, with the consequent economical loses. Development of new vaccines against NNV is in the spotlight though few re- searches have focused in European sea bass. In this study we have generated a recombinant NNV (rNNV) vaccine produced in Escherichia coli expressing the capsid protein and administered it to European sea bass juveniles by two different routes (intraperitoneal and oral). The last being considered non-stressful and desired for fish farming of small fish, which in fact are the most affected by NNV. Oral vaccine was composed of feed pellets containing the recombinant whole bacteria, and injected vaccine was composed of recombinant bacteria pre- viously lysed. Our results revealed production of specific anti-NNV IgM following the two vaccination proce- dures, levels that were further increased in orally-vaccinated group after challenge with NNV. Genes related to interferon (IFN), T-cell and immunoglobulin markers were scarcely regulated in head-kidney (HK), gut or brain. Vaccination by either route elicited a relative survival response of 100% after NNV challenge. To our knowledge, this is the first report of a recombinant vaccine followed by no purification steps which resulted in a complete protection in European sea bass when challenged with NNV.Versión del edito

Potential Impacts in the Gilthead Seabream Larviculture by Nodavirus

The nervous necrosis virus (NNV) leads to viral encephalopathy and retinopathy (VER) disease in more than 170 fish species, mainly from marine habitats. It replicates in the central nervous tissues, reaching up to 100% mortalities after a few days of infection, mainly in the larvae and juvenile stages. This is continuously spreading and affecting more species, both wild and cultured, posing a risk to the development of the aquaculture industry. In the Mediterranean Sea, it mainly affects European sea bass (Dicentrarchus labrax) and some grouper species (Epinephelus spp.). Interestingly, in the gilthead seabream (Sparus aurata), typically resistant to common NNV strains, great mortalities in hatcheries associated with typical clinical signs of VER have been confirmed to be caused by RGNNV/SJNNV reassortants. Thus, we have evaluated the susceptibility of seabream larvae to either RGNNV/SJNNV or SJNNV/RGNNV reassortants, as well as the larval immunity. Based on our results we can conclude that: (i) gilthead seabream larvae are susceptible to infection with both NNV reassortant genotypes, but mainly to RGNNV/SJNNV; (ii) virus replicated and infective particles were isolated; (iii) larval immunity was correlated with larval survival; and (iv) larval resistance and immunity were correlated with age of the larvae. Further investigations should be carried out to ascertain the risks of these new pathogens to Mediterranean larviculture

Diferencias sexuales en la respuesta inmune innata de la gónada en lubinas

Nodavirus (NNV) es un virus con probada transmisión sexual tanto en hembras como en machos. En este trabajo hemos analizado la respuesta citotóxica, fagocítica y de producción de reactivos de oxigeno (ROIs) por primera vez en gónada, tanto en ovario como en testículo, de lubinas (Dicentrarchus labrax). Los resultados demuestran que la actividad citotóxica y producción de ROIs es semejante en ambos sexos, no así la actividad fagocítica ya que hay más proporción de células fagocíticas en el testículo que en el ovario de lubina. Este estudio representa un estudio preliminar que demuestra ciertos niveles de actividad citotóxica en la gónada de lubina, la cual es inducible tras una infección por NNV

Inorganic arsenic causes apoptosis cell death and immunotoxicity on European sea bass (Dicentrarchus labrax)

Inorganic arsenic (As) is one of the most toxic pollutants in the water. We have studied their effects on the marine teleost European sea bass (Dicentrarchus labrax) at 2 and 10 days of 5 μMofAs2O3 (sub-lethal doses) waterborne exposure. Arsenic accumulates in liver and gill tissues. The expression profile of five genes (bax, blc2, casp3, casp8 and casp9) involved in apoptosis cell death confirmed apoptotic effects in liver, slight changes in gill and no effects in skin according with the histopathology findings. Total IgM level and peroxidase activities were increased at 2 and 10 days, respectively. The bactericidal activity was decreased at 2 days after As exposure. A general decrease of cellular immune activities with significant differences in the case of respiratory burst activity was observed after 2 and 10 days of exposure. This work describes for the first time the effects of As exposure on European sea bass.Versión del editor2,35

Immune response against intravitreally injected VNNV in European sea bass specimens

Viral diseases are responsible for many economic losses in modern aquaculture producing high mortalities. The viral nervous necrosis virus (VNNV), a single stranded RNA virus, produces the viral encephalopathy and retinopathy (VER) disease that is considered one of the most serious viral diseases in marine aquaculture. The virus mainly affects the central nervous system provoking necrosis and vacuolation of the cells of the spinal cord, brain and retina. Although some studies localized VNNV in the inner nuclear cell layer and ganglion layer adjacent to the circumferential germinal zone at the ciliary margin towards the iris, no studies deal with how the virus affects the vision function and whether the immune response is orchestrated in the retina. Thus, we intravitreally infected healthy specimens of European sea bass (Dicentrarchuslabrax) with VNNV and analysed the pattern of expression of relevant immune-related genes after 1, 4, 24, 72 h and 7days. In addition, eyes were extracted, and its retinas were dissected and processed for transmission-electron-microscopy study. From the first day, structural changes were found in the retina. The more relevant changes observed were swollen cone pedicles (hydropic degeneration), on the third day and a diffuse vacuolation in the inner plexiform layer, concurrent with disrupted myelin sheaths, in the optic nerve fibers layer

Estimulación de la respuesta citotóxica en cerebro y gónada de lubina tras una infección con nodavirus

En este trabajo hemos analizado algunos genes efectores de la respuesta citotóxica en el cerebro, riñón cefálico y testículo de lubinas (Dicentrarchus labrax) tras una infección experimental con nodavirus (NNV), un virus con probada transmisión sexual tanto en hembras como en machos. Este estudio representa un estudio preliminar que demuestra por primera vez en peces, una inducción de varios genes implicados en la citotoxicidad mediada por leucocitos tras una infección viral del testículo y sugiere la búsqueda de tratamientos que estimulen la actividad citotóxica con el fin de bloquear la colonización de la gónada por NNV y su posterior transmisión vertical