Fish antimicrobial peptides (AMPs) are small peptides whose actions have great implications in the innate immune response of teleost fish, being considered critical as the first line of defence against a wide spectrum of pathogens. Antibiotics have been widely used in aquaculture, resulting in bacterial resistance. The potential of AMPs as alternative to antibiotics makes them a very interesting tool as they represent lower‐risk peptides to develop resistances, or even in the fight against other pathogens such as viruses. Beyond their antimicrobial function, fish AMPs possess several relevant but little‐known characteristics and capabilities such as host‐defence peptides, antioxidants, active compounds of immunogenic drugs and as adjuvants. Moreover, their antitumour properties make them potential drugs to be used in oncological treatments in human subjects. Novel methodologies as clustered regularly interspaced short palindromic repeats associated proteins system (CRISPR‐Cas), or even their use as nutraceutics, seem to be promising instruments to enlarge the functions of fish AMPs. This review discusses these new methodologies, as well as the latest research on fish AMP functions, newly discovered properties of AMPs and their future applications in both the aquaculture industry and human health care.
Viruses are threatening pathogens for fish aquaculture. Some of them are transmitted through gonad fluids or gametes as occurs with nervous necrosis virus (NNV). In order to be transmitted through the gonad, the virus should colonize and replicate inside some cell types of this tissue and avoid the subsequent immune response locally. However, whether NNV colonizes the gonad, the cell types that are infected, and how the immune response in the gonad is regulated has never been studied. We have demonstrated for the first time the presence and localization of NNV into the testis after an experimental infection in the European sea bass (Dicentrarchus labrax), and in the gilthead seabream (Sparus aurata), a very susceptible and an asymptomatic host fish species, respectively. Thus, we localized in the testis viral RNA in both species using in situ PCR and viral proteins in gilthead seabream by immunohistochemistry, suggesting that males might also transmit the virus. In addition, we were able to isolate infective particles from the testis of both species demonstrating that NNV colonizes and replicates into the testis of both species. Blood contamination of the tissues sampled was discarded by completely fish bleeding, furthermore the in situ PCR and immunocytochemistry techniques never showed staining in blood vessels or cells. Moreover, we also determined how the immune and reproductive functions are affected comparing the effects in the testis with those found in the brain, the main target tissue of the virus. Interestingly, NNV triggered the immune response in the European sea bass but not in the gilthead seabream testis. Regarding reproductive functions, NNV infection alters 17β-estradiol and 11-ketotestosterone production and the potential sensitivity of brain and testis to these hormones, whereas there is no disruption of testicular functions according to several reproductive parameters. Moreover, we have also studied the NNV infection of the testis in vitro to assess local responses. Our in vitro results show that the changes observed on the expression of immune and reproductive genes in the testis of both species are different to those observed upon in vivo infections in most of the cases.
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 NFkB 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.
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 mechanism.
Nervous necrosis virus (NNV) causes high mortalities in several marine species. We aimed to evaluate the innate cell-mediated cytotoxic (CMC) activity of head-kidney leucocytes (HKLs) isolated from naïve European sea bass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata), a very susceptible and resistant fish species to NNV, respectively, against fish cell lines infected with NNV. Seabream HKLs showed significantly increased innate CMC activity against NNV-infected cells, compared to those uninfected, while sea bass HKLs failed to do so. Thus, we performed a RNA-seq study to identify genes related to the CMC activity of sea bass leucocytes. Thus, we found that sea bass HKLs incubated with DLB-1 cells alone (CMC_DLB1) or with NNV-infected DLB-1 cells (CMC_DLB1-NNV) showed very similar transcriptomic profiles and the GO analysis revealed that most of the up-regulated genes were related to immunity. Strikingly, when the CMC samples with and without NNV were compared, GO analysis revealed that most of the up-regulated genes in CMC_DLB1-NNV samples were related to metabolism and very few to immunity. This is also in agreement with the functional data. These data point to the escape of CMC activity by NNV infection as an important factor involved in the high susceptibility to nodavirus infections of European sea bass.
Peroxiredoxins (Prxs) are a family of antioxidant enzymes that protect cells from oxidative damage. In addition, Prxs may act as modulators of inflammation, protect against cell death and tumour progression, and facilitate tissue repair after damage. The most studied roles of Prx1 and Prx2 are immunological. Here we present a review on the effects of some immunostimulant treatments and bacterial, viral, or parasitic infections on the expression of fish Prxs at the gene and/or protein level, and point to their important role in immunity. The Prxs show antioxidant activity as well as a protective effect against infection. Some preliminary data are presented about the role of fish Prx1 and Prx2 in virus resistance although further studies are needed before the role of fish Prx in immunity can be definitively defined.
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