Characterization of the interferon pathway in the teleost fish gonad against the vertically transmitted viral nervous necrosis virus

One of the most powerful innate immune responses against viruses is mediated by type I IFN. In teleost fish, it is known that virus infection triggers the expression of ifn and many IFN-stimulated genes, but the viral RNA sensors and mediators leading to IFN production are scarcely known. Thus, we have searched for the presence of these genes in gilt-head sea bream (Sparus aurata) and European sea bass (Dicentrarchus labrax), and evaluated their expression after infection with viral nervous necrosis virus (VNNV) in the brain, the main viral target tissue, and the gonad, used to transmit the virus vertically. In sea bream, a fish species resistant to the VNNV strain used, we found an upregulation of the genes encoding MDA5 (melanoma differentiation-associated gene 5), TBK1 (TANK-binding kinase 1), IRF3 (IFN regulatory factor 3), IFN, Mx [myxovirus (influenza) resistance protein] and PKR (dsRNA-dependent protein kinase receptor) proteins in the brain, which were unaltered in the gonad and could favour the dissemination by gonad fluids or gametes. Strikingly, in European sea bass, a very susceptible species, we also identified, transcripts coding for LGP2 (Laboratory of Genetics and Physiology 2), MAVS (mitochondrial antiviral signalling), TRAF3 (TNF receptor-associated factor 3), TANK (TRAF family member-associated NFkappaB activator) and IRF7 (IFN regulatory factor 7), and found that all the genes analysed were upregulated in the gonad, but only mda5, lgp2, irf3, mx and pkr were upregulated in the brain. These findings supported the notion that the European sea bass brain innate immune response is unable to clear the virus and pointed to the importance of gonad immunity to control the dissemination of VNNV to the progeny - an aspect that is worth investigating in aquatic animals.Postprin

NK-lysin peptides ameliorate viral encephalopathy and retinopathy disease signs and provide partial protection against nodavirus infection in European sea bass

Antimicrobial peptides (AMP) comprise a wide range of small molecules with direct antibacterial activity and immunostimulatory role and are proposed as promising substitutes of the antibiotics. Additionally, they also exert a role against other pathogens such as viruses and fungi less evaluated. NK-lysin, a human granulysin orthologue, possess a double function, taking part in the innate immunity as AMP and also as direct effector in the cell-mediated cytotoxic (CMC) response. This molecule is suggested as a pivotal molecule involved in the defence upon nervous necrosis virus (NNV), an epizootic virus provoking serious problems in welfare and health status in Asian and Mediterranean fish destined to human consumption. Having proved that NK-lysin derived peptides (NKLPs) have a direct antiviral activity against NNV in vitro, we aimed to evaluate their potential use as a prophylactic treatment for European sea bass (Dicentrarchus labrax), one of the most susceptible cultured-fish species. Thus, intramuscular injection of synthetic NKLPs resulted in a very low transcriptional response of some innate and adaptive immune markers. However, the injection of NKLPs ameliorated disease signs and increased fish survival upon challenge with pathogenic NNV. Although NKLPs showed promising results in treatments against NNV, more efforts are needed to understand their mechanisms of action and their applicability to the aquaculture industry.This work was supported by Ministerio de Economía y Competitividad (MINECO; grant AGL2016-74866-C3-1-R with FEDER co-funds), Agencia Espanola ˜ de Investigacion ´ (PID2019-105522 GB-I00), Fundacion ´ S´eneca, Grupo de Excelencia de la Regi´ on de Murcia (grant 19883/GERM/15) and National Commission for Scientific & Technological Research Chile (grant FONDECYT N◦ 1170379)

Vacunas DNA frente a nodavirus. Administración oral y transferencia materna para proteger los estadios larvarios más vulnerables de lubina

Nodavirus (NNV) es el agente causal de la retinopatía y encefalopatía viral (VER) que genera elevadas mortalidades en larvas y juveniles de lubina y de otras especies potencialmente importantes para la acuicultura Mediterránea. Por ello, en este trabajo hemos evaluado dos formas distintas de administración de una vacuna DNA: (i) la vacunación oral de una vacuna encapsulada en partículas de quitosano y (ii) la vacunación de hembras para potenciar la transferencia materna de inmunidad a la descendencia. Hemos obteniendo resultados favorables en ambos procedimientos, aunque estudios más exhaustivos serían necesarios para determinar los mecanismos implicados en estas respuestas y poder así mejorar la eficacia de los tratamientos

BEI Inactivated Vaccine Induces Innate and Adaptive Responses and Elicits Partial Protection upon Reassortant Betanodavirus Infection in Senegalese Sole

Nervous necrosis virus (NNV), the causative agent of viral encephalopathy and retinopathy (VER), is one of the most threatening viruses affecting marine and freshwater fish species worldwide. Senegalese sole is a promising fish species in Mediterranean aquaculture but also highly susceptible to NNV and VER outbreaks, that puts its farming at risk. The development of vaccines for aquaculture is one of best tools to prevent viral spread and sudden outbreaks, and virus inactivation is the simplest and most cost-effective method available. In this work, we have designed two inactivated vaccines based on the use of formalin or binary ethylenimine (BEI) to inactivate a reassortant NNV strain. After vaccination, the BEI-inactivated vaccine triggered the production of specific IgM-NNV antibodies and stimulated innate and adaptive immune responses at transcriptional level (rtp3, mx, mhcii and tcrb coding genes). Moreover, it partially improved survival after an NNV in vivo challenge, reducing the mid-term viral load and avoiding the down-regulation of immune response post-challenge. On the other hand, the formalin-inactivated vaccine improved the survival of fish upon infection without inducing the production of IgM-NNV antibodies and only stimulating the expression of herc4 and mhcii genes (in head-kidney and brain, respectively) during the vaccination period; this suggests that other immune-related pathways may be involved in the partial protection provoked. Although these vaccines against NNV showed encouraging results, further studies are needed to improve sole protection and to fully understand the underlying immune mechanismThis work was supported by grant RTI2018-094687-B-C21 from MICIU (Spain) co-funded by FEDERS

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

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

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