RGNNV and SJNNV reassortants produce mortality and replicate in gilthead seabream larvae

Nervous Necrosis Virus (NNV) is one of the most challenging pathogens for aquaculture development nowadays, mainly affecting marine teleost fish of major interest to the aquaculture industry and causing great economic losses. NNV consist in four genotypes, which seem to have a tropism for certain teleost fish species. Among them, gilthead seabream (Sparus aurata) has been considered as a non-susceptible species to the disease produced by traditional NNV genotypes. However, there are some evidences that indicate seabream is able to develop the disease in the presence of certain reassortant strains of NNV, called RGNNV/SJNNV, which possesses the RNA1 segment of the RGNNV genotype and the RNA2 segment of the SJNNV genotype, which may cause a new threat to aquaculture. Therefore, the main objective of this study was to evaluate the susceptibility of gilthead seabream larvae to the reassortant strains RGNNV/SJNNV and SJNNV/RGNNV. For this purpose, larvae were exposed to 104 TCDI50/mL in triplicate tanks with the reassortant strains. Samples of 5 individual larvae were collected at different days post-infection and used for gene expression and infective NNV isolation. Our data show that both reassortants produced mortalities, although the RGNNV/SJNNV was the one which produced the highest mortality and viral gene transcription, which significantly increased from 1 to 7 days post-infection. In conclusion, our study demonstrate that seabream larvae are susceptible to both RGNNV/SJNNV and SJNNV/RGNNV reassortants under laboratory conditions. Further studies should be performed to understand the pathogenicity of the NNV reassortant strains to prevent and control future outbreaks in aquaculture farms

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

Vaccination of Gilthead Seabream After Continuous Xenoestrogen Oral Exposure Enhances the Gut Endobolome and Immune Status via GPER1

In fish culture settings, the exogenous input of steroids is a matter of concern. Recently, we unveiled that in the gilthead seabream (Sparus aurata), the G protein-coupled estrogen receptor agonist G-1 (G1) and the endocrine disruptor 17α-ethinylestradiol (EE2) are potent modulators in polyreactive antibody production. However, the integral role of the microbiota upon immunity and antibody processing in response to the effect of EE2 remains largely unexplored. Here, juvenile seabreams continuously exposed for 84 days to oral G1 or EE2 mixed in the fish food were intraperitoneally (i.p.) immune primed on day 42 with the model antigen keyhole limpet hemocyanin (KLH). A critical panel of systemic and mucosal immune markers, serum VTG, and humoral, enzymatic, and bacteriolytic activities were recorded and correlated with gut bacterial metagenomic analysis 1 day post-priming (dpp). Besides, at 15 dpp, animals received a boost to investigate the possible generation of specific anti-KLH antibodies at the systemic and mucosal interphases by the end of the trial. On day 43, EE2 but not G1 induced a significant shift in the serum VTG level of naive fish. Simultaneously, significant changes in some immune enzymatic activities in the serum and gut mucus of the EE2-treated group were recorded. In comparison, the vaccine priming immunization resulted in an attenuated profile of most enzymatic activities in the same group. The gut genes qPCR analysis exhibited a related pattern, only emphasized by a significant shift in the EE2 group’s il1b expression. The gut bacterial microbiome status underwent 16S rRNA dynamic changes in alpha diversity indices, only with the exposure to oral G1, supporting functional alterations on cellular processes, signaling, and lipid metabolism in the microbiota. By the same token, the immunization elevated the relative abundance of Fusobacteria only in the control group, while this phylum was depleted in both the treated groups. Remarkably, the immunization also promoted changes in the bacterial class Betaproteobacteria and the estrogen-associated genus Novosphingobium. Furthermore, systemic and mucosal KLH-specific immunoglobulin (Ig)M and IgT levels in the fully vaccinated fish showed only slight changes 84 days post-estrogenic oral administration. In summary, our results highlight the intrinsic relationship among estrogens, their associated receptors, and immunization in the ubiquitous fish immune regulation and the subtle but significant crosstalk with the gut endobolome.Versión del edito

Statistical parametric maps of (18)F-FDG PET and 3-D autoradiography in the rat brain: a cross-validation study

PURPOSE: Although specific positron emission tomography (PET) scanners have been developed for small animals, spatial resolution remains one of the most critical technical limitations, particularly in the evaluation of the rodent brain. The purpose of the present study was to examine the reliability of voxel-based statistical analysis (Statistical Parametric Mapping, SPM) applied to (18)F-fluorodeoxyglucose (FDG) PET images of the rat brain, acquired on a small animal PET not specifically designed for rodents. The gold standard for the validation of the PET results was the autoradiography of the same animals acquired under the same physiological conditions, reconstructed as a 3-D volume and analysed using SPM. METHODS: Eleven rats were studied under two different conditions: conscious or under inhalatory anaesthesia during (18)F-FDG uptake. All animals were studied in vivo under both conditions in a dedicated small animal Philips MOSAIC PET scanner and magnetic resonance images were obtained for subsequent spatial processing. Then, rats were randomly assigned to a conscious or anaesthetized group for postmortem autoradiography, and slices from each animal were aligned and stacked to create a 3-D autoradiographic volume. Finally, differences in (18)F-FDG uptake between conscious and anaesthetized states were assessed from PET and autoradiography data by SPM analysis and results were compared. RESULTS: SPM results of PET and 3-D autoradiography are in good agreement and led to the detection of consistent cortical differences between the conscious and anaesthetized groups, particularly in the bilateral somatosensory cortices. However, SPM analysis of 3-D autoradiography also highlighted differences in the thalamus that were not detected with PET. CONCLUSION: This study demonstrates that any difference detected with SPM analysis of MOSAIC PET images of rat brain is detected also by the gold standard autoradiographic technique, confirming that this methodology provides reliable results, although partial volume effects might make it difficult to detect slight differences in small regions

CIBERER : Spanish national network for research on rare diseases: A highly productive collaborative initiative

Altres ajuts: Instituto de Salud Carlos III (ISCIII); Ministerio de Ciencia e Innovación.CIBER (Center for Biomedical Network Research; Centro de Investigación Biomédica En Red) is a public national consortium created in 2006 under the umbrella of the Spanish National Institute of Health Carlos III (ISCIII). This innovative research structure comprises 11 different specific areas dedicated to the main public health priorities in the National Health System. CIBERER, the thematic area of CIBER focused on rare diseases (RDs) currently consists of 75 research groups belonging to universities, research centers, and hospitals of the entire country. CIBERER's mission is to be a center prioritizing and favoring collaboration and cooperation between biomedical and clinical research groups, with special emphasis on the aspects of genetic, molecular, biochemical, and cellular research of RDs. This research is the basis for providing new tools for the diagnosis and therapy of low-prevalence diseases, in line with the International Rare Diseases Research Consortium (IRDiRC) objectives, thus favoring translational research between the scientific environment of the laboratory and the clinical setting of health centers. In this article, we intend to review CIBERER's 15-year journey and summarize the main results obtained in terms of internationalization, scientific production, contributions toward the discovery of new therapies and novel genes associated to diseases, cooperation with patients' associations and many other topics related to RD research

Betanodavirus reassortants replicate and produce mortality in gilthead seabream larvae

Among the pathogens that most affect Mediterranean aquaculture, betanodavirus (NNV), a non-enveloped icosahedral RNA virus with a genome composed of two single-stranded positive-sense RNA segments known as RNA1 and RNA2, stands out. Apart from the traditional NNV genotypes, there are two reassortant genotypes called RGNNV/SJNNV and SJNNV/RGNNV, due to the origin of the RNA genome. It has been confirmed that gilthead seabream, a species resistant to traditional NNV genotypes, is susceptible to developing NNV disease in larval stages when infected by the RGNNV/SJNNV genotype, bringing a new focus to the prevention of infection in aquaculture farms. Therefore, the main objective of this research was to examine the susceptibility of 37 and 86 days post-hatching (dph) seabream larvae against RGNNV/SJNNV an SJNNV/RGNNV reassortants and the infective capacity of both viruses. Our results confirmed that both genotypes induced mortality in seabream larvae of both ages, being higher for RGNNV/SJNNV genotype and in 36 dph larvae. For both ages, the mortality rates were related to the replicative capacity of the virus. In 36 dph larvae, the high mortality can be explained by the high mRNA levels of viral genes from the beginning of the infection, increasing exponentially until the end of the infection, being always higher for RGNNV/SJNNV. In addition, we were able to recover viral particles for both genotypes, again higher for RGNNV/SJNNV. In contrast, the low, but statistically significant, mortality rate for 86 dph larvae might be due to the low replicative capacity of both viruses at this age. This issue together with a low expression level of viral genes and the failure in recovering infective particles seems to indicate that, even when the virus is initially competent to infect cells, its growth is dismissed or even blocked by different immune mechanisms and, therefore, the virus loses its pathogenic capacity. Further studies are needed to understand the host-viral interactions for NNV reassortants since they are threatening marine larviculture. Funded by Ministerio de Ciencia e Innovación-Agencia Estatal de Investigación [MCIN/AEI/10.13039/501100011033, grant PID2019-105522GB-I00 to AC], Ministerio de Economía y Competitividad and FEDER [grant RTI2018-096625-B-C33 to ECP and MA] and Fundación Séneca, Grupo de Excelencia de la Región de Murcia [19883/GERM/15]

Biotechnological tools for the research and control of nodavirus in aquaculture

Introduction. Nodavirus (NNV) is the most threatening marine virus in the Mediterranean area, and it is spreading in the number and type of viral isolates and host fish species, either wild or culture. In our research group we have been studying the immune response of fish against NNV for 2 decades, focusing on gilthead seabream and European sea bass models. While European sea bass is highly susceptible to some classical NNV genotypes the gilthead seabream acts as a resistant and reservoir species. However, their potential vertical transmission, the emergence of new natural NNV recombinants, producing high mortalities to traditionally-resistant species such as gilthead seabream, the climate change and the aquaculture diversification might increase the deleterious effects of NNV infection in the aquaculture sector. Based on this, we are now increasing our knowledge about fish-NNV interactions in the following topics: 1. Generation of biotechnological tools to study NNV and the fish immune response. We have developed several fish cell lines (from seabream, sea bass, European eel or Senegalese sole) susceptible to NNV, antibodies for localization studies and transcriptomic databases, useful as research and diagnostic tools. These tools allow us to increase their applications to other host, marine viruses or, even, other fields. 2. Deepening of the antiviral immune response of fish. We are identifying and characterizing, among others, the involvement of antimicrobial peptides (AMPs) and cell-mediated cytotoxicity as two of the most important antiviral immune mechanisms. Thus, we are able to analyze levels of antibodies and AMPs, or the adaptive cellular cytotoxic activity in fish, novel techniques that are routinely applied in our laboratory. 3. Effect of culture conditions and welfare on susceptibility to NNV. We are characterizing the lethality and presence of the virus in survivors and its success on vertical transmission under different culture conditions, size, or sex of different species susceptible to NNV. We have evidence that survival is more dependent on size than on culture conditions, but the presence of the virus in survivors is not. 4. Design and generation of treatments against NNV. We have developed several effective NNV vaccines in previous projects. We are currently developing the application of sea bass AMPs as antiviral treatments in aquaculture. So far, we have already shown that 4 AMPs derived from sea bass can be applied both as preventive and therapeutics of the disease produced by NNV in sea bass fingerlings. 5. Study of the impact of emerging contaminants on the antiviral immune response. We have evaluated the possible effect that some contaminants, mainly nanoplastics, may have on the immune response of fish and their resistance to NNV infections. Conclusions. In short, our group aims to investigate the fish health status and well-being with interests for the aquaculture sector. We mainly focus on the prevention and improvement of the resistance against nodavirus infections. To this end, we have generated various very interesting biotechnological tools that are exploited through material transfer agreements with research centers and companies, which clearly and decisively adhere to the objectives of the ThinkInAzul programFunding. MCIN/AEI/10.13039/501100011033 (PID2019-105522GB-I00 to A.C.), FPI contract (PRE2020-093771 a L.C.), Juan de la Cierva-Incorporación contract (IJC2020-042733-I a Y.V. y JC2019-040510-I a C.G-F.), OWI TWO (cofunded by EMFF and IEO-CSIC), and the program ThinkInAzul (PRTR-C17.I1 to M.A.E. and E.C-P.)

Construction of different radionuclide templates of rat brains and their use on a new statistic parametric mapping analysis protocol for PET studies. | International WorkshopCNA´10: Bio-medical applications of Micro-PET (20-21 September 2010. Sevilla, Spain)

This work shows the development of protocols to create new 18F‐FDG and 11C‐DTBZ (dyhidrotetrabenazine, a VMAT2 transporter ligand) templates of rat brain for spatial normalisation and definition of standardised areas in images used for setting up SPM analysis of PET data