Regla Medina-Gali

Autophagy-inducing peptides from mammalian VSV and fish VHSV rhabdoviral G glycoproteins (G) as models for the development of new therapeutic molecules.

It has not been elucidated whether or not autophagy is induced by rhabdoviral G glycoproteins (G) in vertebrate organisms for which rhabdovirus infection is lethal. Our work provides the first evidence that both mammalian (vesicular stomatitis virus, VSV) and fish (viral hemorrhagic septicemia virus, VHSV, and spring viremia carp virus, SVCV) rhabdoviral Gs induce an autophagic antiviral program in vertebrate cell lines. The transcriptomic profiles obtained from zebrafish genetically immunized with either Gsvcv or Gvhsv suggest that autophagy is induced shortly after immunization and therefore, it may be an important component of the strong antiviral immune responses elicited by these viral proteins. Pepscan mapping of autophagy-inducing linear determinants of Gvhsv and Gvsv showed that peptides located in their fusion domains induce autophagy. Altogether these results suggest that strategies aimed at modulating autophagy could be used for the prevention and treatment of rhabdoviral infections such as rabies, which causes thousands of human deaths every year.

Zebra Fish Lacking Adaptive Immunity Acquire an Antiviral Alert State Characterized by Upregulated Gene Expression of Apoptosis, Multigene Families, and Interferon-Related Genes

To investigate fish innate immunity, we have conducted organ and cell immune-related transcriptomic as well as immunohistologic analysis in mutant zebra fish (Danio rerio) lacking adaptive immunity (rag1−/−) at different developmental stages (egg, larvae, and adult), before and after infection with spring viremia carp virus (SVCV). The results revealed that, compared to immunocompetent zebra fish (rag1+/+), rag1−/− acquired increased resistance to SVCV with age, correlating with elevated transcript levels of immune genes in skin/fins and lymphoid organs (head kidney and spleen). Gene sets corresponding to apoptotic functions, immune-related multigene families, and interferon-related genes were constitutively upregulated in uninfected adult rag1−/− zebra fish. Overexpression of activated CASPASE-3 in different tissues before and after infection with SVCV further confirmed increased apoptotic function in rag1−/− zebra fish. Concurrently, staining of different tissue samples with a pan-leukocyte antibody marker showed abundant leukocyte infiltrations in SVCV-infected rag1−/− fish, coinciding with increased transcript expression of genes related to NK-cells and macrophages, suggesting that these genes played a key role in the enhanced immune response of rag1−/− zebra fish to SVCV lethal infection. Overall, we present evidence that indicates that rag1−/− zebra fish acquire an antiviral alert state while they reach adulthood in the absence of adaptive immunity. This antiviral state was characterized by (i) a more rapid response to viral infection, which resulted in increased survival, (ii) the involvement of NK-cell- and macrophage-mediated transcript responses rather than B- and/or T-cell dependent cells, and (iii) enhanced apoptosis, described here for the first time, as well as the similar modulation of multigene family/interferon-related genes previously associated to fish that survived lethal viral infections. From this and other studies, it might be concluded that some of the characteristics of mammalian trained immunity are present in lower vertebrates.

pH-Dependent Solution Structure and Activity of a Reduced Form of the Host-Defense Peptide Myticin C (Myt C) from the Mussel Mytilus galloprovincialis

Myticin C (Myt C) is a highly variable host-defense peptide (HDP) associated to the immune response in the mediterranean mussel (Mytilus galloprovincialis), which has shown to be active across species due to its strong antiviral activity against a fish rhabdovirus found in fish cells overexpressing this HDP. However, the potential antimicrobial properties of any synthetic analogue of Myt C has not yet been analysed. Thus, in this work we have synthesised the sequence of the mature peptide of Myt C variant c and analysed the structure activity relationships of its reduced (non-oxidized) form (red-MytCc). In contrast to results previously reported for oxidized isoforms of mussel myticins, red-MytCc was not active against bacteria at physiological pH and showed a moderate antiviral activity against the viral haemorrhagic septicaemia (VHS) rhabdovirus. However, its chemotactic properties remained active. Structure/function studies in neutral and acid environments by means of infrared spectroscopy indicated that the structure of red-MytCc is pH dependent, with acid media increasing its alpha-helical content. Furthermore, red-MytCc was able to efficiently aggregate artificial phospholipid membranes at low pH, as well as to inhibit the Escherichia coli growth, suggesting that this activity is attributable to its more structured form in an acidic environment. All together, these results highlight the dynamic and environmentally sensitive behavior of red-Myt C in solution, and provide important insights into Myt C structure/activity relationships and the requirements to exert its antimicrobial/immunomodulatory activities. On the other hand, the pH-dependent direct antimicrobial activity of Myt C suggests that this HDP may be a suitable template for the development of antimicrobial agents that would function selectively in specific pH environments, which are sorely needed in this “antibiotic-resistance era”.

Beta-glucan enhances the response to SVCV infection in zebrafish

&NA; The antiviral effects of beta‐glucan, an immunostimulatory agent were studied in zebrafish both in vitro and in vivo. Here we show that zebrafish ZF4 cells as well as whole fish primed with yeast &bgr;‐glucan zymosan exhibited increased cytokine expression and elevated response to spring viremia of carp virus (SVCV) infection. In vitro, previous treatment of &bgr;‐glucan enhanced ZF4 cell viability against SVCV infection which is associated to the activation of interferon signaling pathway and inflammatory cytokines gene expression. In vivo, the SVCV‐infected fish primed with &bgr;‐glucan had a higher survival rate (≈73%) than the control SVCV‐infected group (≈33%). Additionally, up‐regulation of the expression of a set of genes involved in innate immune response was detected in zebrafish intraperitoneally injected of &bgr;‐glucan: il1b, il6, il8, il10 and tnfa transcripts showed increased expression that appear to be rapid (2 days) but not long‐lived (less than 2 weeks). The present study is, to our knowledge, the first to combine cell culture and in vivo approaches to describe host response to &bgr;‐glucan stimulation and viral infection in zebrafish.

Increasing Versatility of the DNA Vaccines through Modification of the Subcellular Location of Plasmid-Encoded Antigen Expression in the In Vivo Transfected Cells

The route of administration of DNA vaccines can play a key role in the magnitude and quality of the immune response triggered after their administration. DNA vaccines containing the gene of the membrane-anchored glycoprotein (gpG) of the fish rhabdoviruses infectious haematopoietic necrosis virus (IHNV) or viral haematopoietic septicaemia virus (VHSV), perhaps the most effective DNA vaccines generated so far, confer maximum protection when injected intramuscularly in contrast to their low efficacy when injected intraperitoneally. In this work, taking as a model the DNA vaccine against VHSV, we focused on developing a more versatile DNA vaccine capable of inducing protective immunity regardless of the administration route used. For that, we designed two alternative constructs to gpG1-507 (the wild type membrane-anchored gpG of VHSV) encoding either a soluble (gpG1-462) or a secreted soluble (gpGLmPle20-462) form of the VHSV-gpG. In vivo immunisation/challenge assays showed that only gpGLmPle20-462 (the secreted soluble form) conferred protective immunity against VHSV lethal challenge via both intramuscular and intraperitoneal injection, being this the first description of a fish viral DNA vaccine that confers protection when administered intraperitoneally. Moreover, this new DNA vaccine construct also conferred protection when administered in the presence of an oil adjuvant suggesting that DNA vaccines against rhabdoviruses could be included in the formulation of current multicomponent-intaperitoneally injectable fish vaccines formulated with an oil adjuvant. On the other hand, a strong recruitment of membrane immunoglobulin expressing B cells, mainly membrane IgT, as well as t-bet expressing T cells, at early times post-immunisation, was specifically observed in the fish immunised with the secreted soluble form of the VHSV-gpG protein; this may indicate that the subcellular location of plasmid-encoded antigen expression in the in vivo transfected cells could be an important factor in determining the ways in which DNA vaccines prime the immune response.

Neutralization of viral infectivity by zebrafish c-reactive protein isoforms

HighlightsSeven C‐reactive protein isoforms were studied in zebrafish.Transcript (crp) and protein (CRP) levels changed heterogeneously after viral infections.Some of the crp1‐7/CRP1‐7 neutralized and protected against viral infections.Most anti‐viral effects were due to crp2/CRP2 and crp5/CRP5. &NA; This work explores the unexpected in vivo and in vitro anti‐viral functions of the seven c‐reactive protein (crp1‐7) genes of zebrafish (Danio rerio). First results showed heterogeneous crp1‐7 transcript levels in healthy wild‐type zebrafish tissues and organs and how those levels heterogeneously changed not only after bacterial but also after viral infections, including those in adaptive immunity‐deficient rag1−/− mutants. As shown by microarray hybridization and proteomic techniques, crp2/CRP2 and crp5/CRP5 transcripts/proteins were among the most modulated during in vivo viral infection situations including the highest responses in the absence of adaptive immunity. In contrast crp1/CRP1/and crp7/CRP7 very often remained unmodulated. All evidences suggested that zebrafish crp2‐6/CRP2‐6 may have in vivo anti‐viral activities in addition to their well known anti‐bacterial and/or physiological functions in mammalians. Confirming those expectations, in vitro neutralization and in vivo protection against spring viremia carp virus (SVCV) infections were demonstrated by crp2‐6/CRP2‐6 using crp1‐7 transfected and/or CRP1‐7‐enriched supernatant‐treated fish cells and crp2‐5‐injected one‐cell stage embryo eggs, respectively. All these findings discovered a crp1‐7/CRP1‐7 primitive anti‐viral functional diversity.These findings may help to study similar functions on the one‐gene‐coded human CRP, which is widely used as a clinical biomarker for bacterial infections, tissue inflammation and coronary heart diseases.

Turbot (Scophthalmus maximus) Nk-lysin induces protection against the pathogenic parasite Philasterides dicentrarchi via membrane disruption

Abstract P. dicentrarchi is one of the most threatening pathogens for turbot aquaculture. This protozoan ciliate is a causative agent of scuticociliatosis, which is a disease with important economic consequences for the sector. Neither vaccines nor therapeutic treatments are commercially available to combat this infection. Numerous antimicrobial peptides (AMPs) have demonstrated broad‐spectrum activity against bacteria, viruses, fungi, parasites and even tumor cells; an example is Nk‐lysin (Nkl), which is an AMP belonging to the saposin‐like protein (SAPLIP) family with an ability to interact with biological membranes. Following the recent characterization of turbot Nkl, an expression plasmid encoding Nkl was constructed and an anti‐Nkl polyclonal antibody was successfully tested. Using these tools, we demonstrated that although infection did not clearly affect nkl mRNA expression, it induced changes at the protein level. Turbot Nkl had the ability to inhibit proliferation of the P. dicentrarchi parasite both in vivo and in vitro. Moreover, a shortened peptide containing the active core of turbot Nkl (Nkl71‐100) was synthesized and showed high antiparasitic activity with a direct effect on parasite viability that probably occurred via membrane disruption. Therefore, the nkl gene may be a good candidate for genetic breeding selection of fish, and either the encoded peptide or its shortened analog is a promising antiparasitic treatment in aquaculture. HighlightsThe turbot nkl gene is not modulated during infection with P. dicentrarchi.Changes in the number of Nkl‐positive cells are observed after infection.Nkl is released into the peritoneal exudate during infection with P. dicentrarchi.Nkl and a synthetic shorter peptide have antiparasitic activity.Nkl reduces parasite viability via membrane disruption.

Chromatin immunoprecipitation and high throughput sequencing of SVCV-infected zebrafish reveals novel epigenetic histone methylation patterns involved in antiviral immune response

Abstract Chromatin immunoprecipitation (ChIP) and high throughput sequencing (ChIP‐seq) have been used to assess histone methylation (epigenetic modification) dynamics within the internal organs of zebrafish after spring viremia of carp virus (SVCV) infection. Our results show H3K4me3 up‐methylation in gene promoters associated with innate immune response during the first 5 days after SVCV infection. Gene Ontology (GO) enrichment analysis confirmed up‐methylation in 218 genes in the “immune system process” category. In particular, the promoters of interferon (ifn), interferon stimulated genes (isg), Toll‐like receptors (tlr) and c‐reactive protein (crp) multi gene sets were marked with the permissive H3K4 methylation. Higher histone 3 methylation was associated with higher transcription levels of the corresponding genes. Therefore, the evidence presented here suggests that transcriptional regulation at the promoter level of key immune genes of the interferon signaling pathway and c‐reactive proteins genes can be modulated by epigenetic modification of histones. This study emphasizes the importance of epigenetic control in the response of zebrafish to SVCV infection. Graphical abstract Figure. No Caption available. HighlightsFirst ChIPseq‐based study of SVCV‐infected zebrafish.Rapid increment of H3K4 methylation in promoters of immune‐related gene promoters after SVCV infection.Key inflammatory and antiviral genes showed transcriptional activation in correlation to gene promoter methylation.C‐reactive proteins genes exhibit the highest H3 methylation enrichment after 5 days of SVCV infection.

Restricted replication of viral hemorrhagic septicemia virus (VHSV) in a birnavirus-carrier cell culture

Viral hemorrhagic septicemia virus (VHSV) and infectious pancreatic necrosis virus (IPNV) are economically important pathogens of the salmonid aquaculture industry. In previous work we demonstrated that a cell line persistently infected with IPNV (EPCIPNV) exhibited antiviral activity against superinfection with the heterologous virus VHSV. This work extends our study by analyzing the replication of VHSV in the IPNV-persistently infected cells. At early and late stages of infection VHSV RNA synthesis, as well as VHSV-induced syncytia formation, were examined in EPCIPNV cultures. During the course of VHSV infection the accumulation of VHSV RNA is inhibited in EPCIPNV cells. Typical VHSV-induced membrane fusion at the late stages of infection is also absent in the IPNV carrier cultures. VHSV binding and fusion to EPCIPNV cells did not appear to be impaired, but a potent inhibitory effect on VHSV RNA synthesis is exerted at early times of infection in the IPNV carrier culture. In conclusion, the EPCIPNV cells are considered to be a useful system to study viral interference as well to analyze the mechanisms underlying the phenomenon of superinfection exclusion.