IDiBE https://hdl.handle.net/11000/5926 2026-04-03T22:21:37Z 2026-04-03T22:21:37Z Nombela, Ivan Carrion, Aurora Puente Marin, Sara CHICO GRAS, VERONICA Mercado, Luis Pérez, Luis coll, julio Ortega-Villaizan, Maria del Mar https://hdl.handle.net/11000/35039 2025-02-19T08:59:47Z 2025-01-20T13:49:30Z

Título : Infectious pancreatic necrosis virus triggers antiviral immune response in rainbow trout red blood cells, despite not being infective[version 2; referees: 2 approved] Autor : Nombela, Ivan; Carrion, Aurora; Puente Marin, Sara; CHICO GRAS, VERONICA; Mercado, Luis; Pérez, Luis; coll, julio; Ortega-Villaizan, Maria del Mar Resumen : Background Methods : Some fish viruses, such as piscine orthoreovirus and infectious salmon anemia virus, target red blood cells (RBCs), replicate inside them and induce an immune response. However, the roles of RBCs in the context of infectious pancreatic necrosis virus (IPNV) infection  have not been studied yet. : Ex vivo rainbow trout RBCs were obtained from peripheral blood, Ficoll purified and exposed to IPNV in order to analyze infectivity and immune response using RT-qPCR, immune fluorescence imaging, flow cytometry and western-blotting techniques. Results : IPNV could not infect RBCs; however, IPNV increased the expression of the INF1-related genes ifn-1 pkr ,  and mx genes. Moreover, conditioned media from IPNV-exposed RBCs conferred protection against IPNV infection in CHSE-214 fish cell line. Conclusions : Despite not being infected, rainbow trout RBCs could respond to IPNV with increased expression of antiviral genes. Fish RBCs could be considered as mediators of the antiviral response and therefore targets of new strategies against fish viral infections. Further research is ongoing to completely understand the molecular mechanism that triggers this antiviral response in rainbow trout RBCs.

2025-01-20T13:49:30Z Ortega-Villaizan, Maria del Mar Mercado, Luis CHICO GRAS, VERONICA https://hdl.handle.net/11000/34457 2025-02-19T08:55:35Z 2025-01-14T13:16:40Z

Título : Antiviral immune response in fish and shellfish Autor : Ortega-Villaizan, Maria del Mar; Mercado, Luis; CHICO GRAS, VERONICA Resumen : Innate immunity constitutes the main defense mechanism in fish and shellfish, which is the most antique and efficient system to protect organisms against microbial infections. In addition, lower vertebrates have less evolved adaptive immune responses compared to higher vertebrates, and therefore, rely on innate immune defenses for the control of pathogens (1). Nonetheless, it is worth noting that the contribution of both innate and adaptive immune responses leads to global protection against pathogens in the organism. The objective of this Research Topic is to reflect the significant advances in the study of the immune response against viral infections in fish. This topic contributes to identifying new mechanisms of response to infections and new effector molecules against viral pathogens. In addition, these studies provide a further understanding of the resistance of lower vertebrates to pathogens infection as well as to the prevention of exacerbated immune responses. The contributing articles collected in this Research Topic are grouped into the following proposed sections related to the antiviral defense in fish

2025-01-14T13:16:40Z Ortega-Villaizan, Maria del Mar CHICO GRAS, VERONICA Perez, Luis https://hdl.handle.net/11000/34456 2025-02-19T08:55:35Z 2025-01-14T13:09:17Z

Título : Fish Innate Immune Response to Viral Infection—An Overview of Five Major Antiviral Genes Autor : Ortega-Villaizan, Maria del Mar; CHICO GRAS, VERONICA; Perez, Luis Resumen : Fish viral diseases represent a constant threat to aquaculture production. Thus, a better understanding of the cellular mechanisms involved in establishing an antiviral state associated with protection against virus replication and pathogenesis is paramount for a sustainable aquaculture industry. This review summarizes the current state of knowledge on five selected host innate immunerelated genes in response to the most relevant viral pathogens in fish farming. Viruses have been classified as ssRNA, dsRNA, and dsDNA according to their genomes, in order to shed light on what those viruses may share in common and what response may be virus-specific, both in vitro (cell culture) as well as in vivo. Special emphasis has been put on trying to identify markers of resistance to viral pathogenesis. That is, those genes more often associated with protection against viral disease, a key issue bearing in mind potential applications into the aquaculture industry

2025-01-14T13:09:17Z Salvador Mira, Maria Elizabhet CHICO GRAS, VERONICA Arostica, Monica Guzmán Quimbayo, Fanny Roher, Nerea Perez, Luis Ortega-Villaizan, Maria del Mar https://hdl.handle.net/11000/34455 2025-02-19T08:55:35Z 2025-01-14T13:07:47Z

Título : Immunomodulatory Lectin-like Peptides for Fish Erythrocytes-Targeting as Potential Antiviral Drug Delivery Platforms Autor : Salvador Mira, Maria Elizabhet; CHICO GRAS, VERONICA; Arostica, Monica; Guzmán Quimbayo, Fanny; Roher, Nerea; Perez, Luis; Ortega-Villaizan, Maria del Mar Resumen : Download PDFsettingsOrder Article Reprints Open AccessCommunication Immunomodulatory Lectin-like Peptides for Fish Erythrocytes-Targeting as Potential Antiviral Drug Delivery Platforms by Maria Salvador-Mira 1,2,Veronica Chico 1,2ORCID,Monica Arostica 3,Fanny Guzmán 3ORCID,Nerea Roher 4,Luis Perez 1,2ORCID andMaria del Mar Ortega-Villaizan 1,2,*ORCID 1 Instituto de Biología Molecular y Celular (IBMC), Universidad Miguel Hernández (IBMC-UMH), 03202 Elche, Spain 2 Instituto de Investigación, Desarrollo e Innovación en Biotecnología Sanitaria de Elche (IDiBE), Universidad Miguel Hernández (IDiBE-UMH), 03202 Elche, Spain 3 Núcleo Biotecnológico de Curauma (NBC), Pontificia Universidad Católica de Valparaíso, Valparaíso 2373223, Chile 4 Department Biologia Cellular, Fisiologia Animal i Immunologia, Institut de Biotecnologia i de Biomedicina (IBB), Universitat Autònoma de Barcelona (UAB), 08193 Cerdanyola del Vallès, Spain * Author to whom correspondence should be addressed. Int. J. Mol. Sci. 2021, 22(21), 11821; https://doi.org/10.3390/ijms222111821 Submission received: 1 October 2021 / Revised: 24 October 2021 / Accepted: 26 October 2021 / Published: 30 October 2021 (This article belongs to the Section Biochemistry) Downloadkeyboard_arrow_down Browse Figures Versions Notes Abstract One of the challenges of science in disease prevention is optimizing drug and vaccine delivery. Until now, many strategies have been employed in this sector, but most are quite complex and labile. To overcome these limitations, great efforts are directed to coupling drugs to carriers, either of natural or synthetic origin. Among the most studied cell carriers are antigen-presenting cells (APCs), however, red blood cells (RBCs) are positioned as attractive carriers in drug delivery due to their abundance and availability in the body. Furthermore, fish RBCs have a nucleus and have been shown to have a strong involvement in modulating the immune response. In this study, we evaluated the binding of three peptides to rainbow trout RBCs, two lectin-like peptides and another derived from Plasmodium falciparum membrane protein, in order to take advantage of this peptide-RBCs binding to generate tools to improve the specificity, efficacy, immunostimulatory effect, and safety of the antiviral therapeutic or prophylactic administration systems currently used.

2025-01-14T13:07:47Z CHICO GRAS, VERONICA Salvador Mira, Maria Elizabhet Nombela, Ivan Puente Marin, Sara Perez, Luis Mercado, Luis Ortega-Villaizan, Maria del Mar https://hdl.handle.net/11000/34454 2025-02-19T08:55:35Z 2025-01-14T13:06:31Z

Título : Antiviral Function of NKEF against VHSV in Rainbow Trout Autor : CHICO GRAS, VERONICA; Salvador Mira, Maria Elizabhet; Nombela, Ivan; Puente Marin, Sara; Perez, Luis; Mercado, Luis; Ortega-Villaizan, Maria del Mar Resumen : Natural killer enhancing factor (NKEF) belongs to the peroxiredoxin family of proteins, a group of antioxidants that has been extensively studied in mammals. Recently, we identified NKEF in the immunoprecipitated proteome of rainbow trout red blood cells (RBCs) exposed to viral hemorrhagic septicemia virus (VHSV). In the present study, we evaluated the role of NKEF in the antiviral response of rainbow trout against VHSV by examining the expression profile of NKEFin VHSV-exposed RBCs and rainbow trout gonad-2 (RTG-2) cell line. We found an in vitro correlation between decreased VHSV replication and increased NKEF expression after RBCs were exposed to VHSV, however this was not found in RTG-2 cells where the infection highly increased and nkef transcripts remained almost unchanged. In addition, siRNA silencing of the nkef gene in rainbow trout RBCs and RTG-2 cells resulted in increased VHSV replication. We also found a correlation between nkef gene silencing and a decrease in the expression of genes related to type 1 interferon (IFN1) pathway. These findings indicated that NKEF is involved in the antiviral mechanisms of rainbow trout RBCs against VHSV and thus support its antiviral role and implication in the modulation of their immune response. Finally, overexpression of NKEF in an EPC cell line significantly reduced VHSV infectivity and was coupled to an increment in IFN1-related genes. In conclusion, NKEF may be a potential target for new therapeutic strategies against viral infections.

2025-01-14T13:06:31Z CHICO GRAS, VERONICA Salvador Mira, Maria Elizabhet Nombela, Ivan Puente Marin, Sara Ciordia, Sergio Mena, María Carmen Perez, Luis coll, julio Guzman, Fanny Encinar, José Antonio Mercado, Luis Ortega-Villaizan, Maria del Mar https://hdl.handle.net/11000/34453 2025-02-19T08:59:47Z 2025-01-14T13:05:33Z

Título : IFIT5 Participates in the Antiviral Mechanisms of Rainbow Trout Red Blood Cells Autor : CHICO GRAS, VERONICA; Salvador Mira, Maria Elizabhet; Nombela, Ivan; Puente Marin, Sara; Ciordia, Sergio; Mena, María Carmen; Perez, Luis; coll, julio; Guzman, Fanny; Encinar, José Antonio; Mercado, Luis; Ortega-Villaizan, Maria del Mar Resumen : Viral hemorrhagic septicemia virus (VHSV) infection appears to be halted in rainbow trout nucleated red blood cells (RBCs). Diverse mechanisms are thought to be related to the antiviral immune response of rainbow trout RBCs to VHSV. However, the specific rainbow trout RBC proteins that interact directly with VHSV are still unknown. In an attempt to identify VHSV-RBC protein interactions, we characterized the immunoprecipitated (IP) proteome of RBCs exposed to VHSV using an antibody against the N protein of VHSV. The IP proteomic characterization identified 31 proteins by mass spectrometry analysis. Among them, we identified interferon-induced protein with tetratricopeptide repeats 5 (IFIT5), a protein belonging to a family of proteins that are induced after the production of type I interferon. Importantly, IFIT5 has been implicated in the antiviral immune response. We confirmed the participation of IFIT5 in the rainbow trout RBC antiviral response by examining the expression profile of IFIT5 in RBCs after VHSV exposure at transcriptional and protein levels. We detected a correlation between the highest IFIT5 expression levels and the decline in VHSV replication at 6h post-exposure. In addition, silencing ifit5 resulted in a significant increase in VHSV replication in RBCs. Moreover, an increase in VHSV replication was observed in RBCs when the IFIT5 RNA-binding pocket cavity was modulated by using a natural compound from the SuperNatural II database. We performed a proximity ligation assay and detected a significant increase in positive cells among VHSV-exposed RBCs compared to unexposed RBCs, indicating protein-protein colocalization between IFIT5 and the glycoprotein G of VHSV. In summary, the

2025-01-14T13:05:33Z Jung, Myung-Hwa CHICO GRAS, VERONICA Ciordia, Sergio Mena, Maria Carmen Jung, Sung-Ju Ortega-Villaizan, Maria del Mar https://hdl.handle.net/11000/34452 2025-02-19T08:59:26Z 2025-01-14T13:00:59Z

Título : The Megalocytivirus RBIV Induces Apoptosis and MHC Class I Presentation in Rock Bream (Oplegnathus fasciatus) Red Blood Cells Autor : Jung, Myung-Hwa; CHICO GRAS, VERONICA; Ciordia, Sergio; Mena, Maria Carmen; Jung, Sung-Ju; Ortega-Villaizan, Maria del Mar Resumen : Rock bream iridovirus (RBIV) causes severe mass mortality in Korean rock bream (Oplegnathus fasciatus) populations. To date, immune defense mechanisms of rock bream against RBIV are unclear. While red blood cells (RBCs) are known to be involved in the immune response against viral infections, the participation of rock bream RBCs in the immune response against RBIV has not been studied yet. In this study, we examined induction of the immune response in rock bream RBCs after RBIV infection. Each fish was injected with RBIV, and virus copy number in RBCs gradually increased from 4 days post-infection (dpi), peaking at 10 dpi. A total of 318 proteins were significantly regulated in RBCs from RBIV-infected individuals, 183 proteins were upregulated and 135 proteins were downregulated. Differentially upregulated proteins included those involved in cellular amino acid metabolic processes, cellular detoxification, snRNP assembly, and the spliceosome. Remarkably, the MHC class I-related protein pathway was upregulated during RBIV infection. Simultaneously, the regulation of apoptosis-related proteins, including caspase-6 (CASP6), caspase-9 (CASP9), Fas cell surface death receptor (FAS), desmoplakin (DSP), and p21 (RAC1)activated kinase 2 (PAK2) changed with RBIV infection. Interestingly, the expression of genes within the ISG15 antiviral mechanism-related pathway, including filamin B (FLNB), interferon regulatory factor 3 (IRF3), nucleoporin 35 (NUP35), tripartite motif-containing 25 (TRIM25), and karyopherin subunit alpha 3 (KPNA3) were downregulated in RBCs from RBIV-infected individuals. Overall, these findings contribute to the understanding of RBIV pathogenesis and host interaction.

2025-01-14T13:00:59Z Puente Marin, Sara Nombela, Ivan CHICO GRAS, VERONICA Ciordia, Sergio Mena, Maria Carmen coll, julio Mercado, Luis Ortega-Villaizan, Maria del Mar https://hdl.handle.net/11000/34451 2025-02-19T08:59:26Z 2025-01-14T12:59:21Z

Título : Rainbow Trout Erythrocytes ex vivo Transfection With a DNA Vaccine Encoding VHSV Glycoprotein G Induces an Antiviral Immune Response Autor : Puente Marin, Sara; Nombela, Ivan; CHICO GRAS, VERONICA; Ciordia, Sergio; Mena, Maria Carmen; coll, julio; Mercado, Luis; Ortega-Villaizan, Maria del Mar Resumen : Fish red bloodcells (RBCs), areintegral in several biologic processes relevant to immunity, such as pathogen recognition, pathogen binding and clearance, and production of effector molecules and cytokines. So far, one of the best strategies to control and prevent viral diseases in aquaculture is DNA immunization. DNA vaccines (based on the rhabdoviral glycoprotein G [gpG] gene) have been shown to be effective against f ish rhabdoviruses. However, more knowledge about the immune response triggered by DNA immunization is necessary to develop novel and more effective strategies. In this study, we investigated the role of fish RBCs in immune responses induced by DNA vaccines. We show for the first time that rainbow trout RBCs express gpG of viral hemorrhagic septicaemia virus (VHSV) (GVHSV) when transfected with the DNA vaccine ex vivo and modulate the expression of immune genes and proteins. Functional network analysis of transcriptome profiling of RBCs expressing GVHSVrevealedchanges in gene expression related to G-protein coupled receptor (GPCR)-downstream signaling, complement activation, and RAR related orphan receptor α (RORA). Proteomic profile functional network analysis of GVHSV-transfected RBCs revealed proteins involved in the detoxification of reactive oxygen species, interferon-stimulated gene 15 (ISG15) antiviral mechanisms, antigen presentation of exogenous peptides, and the proteasome. Conditioned medium of GVHSV-transfected RBCs conferred antiviral protection and induced ifn1 and mx gene expression in RTG-2 cells infected with VHSV. In summary, rainbow trout nucleated RBCs could be actively participating in the regulation of the fish immune response to GVHSV DNA vaccine, and thus may represent a possible carrier cells for the development of new vaccine approaches.

2025-01-14T12:59:21Z Martinez-Lopez, Alicia Garcia Valtanen, Pablo Ortega-Villaizan, Maria del Mar CHICO GRAS, VERONICA Medina Gali, Regla María Perez, Luis coll, julio ESTEPA, AMPARO https://hdl.handle.net/11000/34450 2025-02-19T08:55:35Z 2025-01-14T12:58:29Z

Título : Increasing Versatility of the DNA Vaccines through Modification of the Subcellular Location of PlasmidEncoded Antigen Expression in the In Vivo Transfected Cells Autor : Martinez-Lopez, Alicia; Garcia Valtanen, Pablo; Ortega-Villaizan, Maria del Mar; CHICO GRAS, VERONICA; Medina Gali, Regla María; Perez, Luis; coll, julio; ESTEPA, AMPARO Resumen : 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 (gpG(LmPle20-462)) form of the VHSV-gpG. In vivo immunisation/challenge assays showed that only gpG(LmPle20-462) (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.

2025-01-14T12:58:29Z Puente Marin, Sara Nombela, Ivan Ciordia, Sergio CHICO GRAS, VERONICA coll, julio Ortega-Villaizan, Maria del Mar https://hdl.handle.net/11000/34449 2025-02-19T08:59:26Z 2025-01-14T12:57:26Z

Título : In Silico Functional Networks Identified in Fish Nucleated Red Blood Cells by Means of Transcriptomic and Proteomic Profiling Autor : Puente Marin, Sara; Nombela, Ivan; Ciordia, Sergio; CHICO GRAS, VERONICA; coll, julio; Ortega-Villaizan, Maria del Mar Resumen : Nucleated red blood cells (RBCs) of fish have, in the last decade, been implicated in several immune-related functions, such as antiviral response, phagocytosis or cytokine-mediated signaling. RNA-sequencing (RNA-seq) and label-free shotgun proteomic analyses were carried out for in silico functional pathway profiling of rainbow trout RBCs. For RNA-seq, a de novo assembly was conducted, in order to create a transcriptome database for RBCs. For proteome profiling, we developed a proteomic method that combined: (a) fractionation into cytosolic and membrane fractions, (b) hemoglobin removal of the cytosolic fraction, (c) protein digestion, and (d) a novel step with pH reversed-phase peptide fractionation and final Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC ESI-MS/MS) analysis of each fraction. Combined transcriptome- and proteome- sequencing data identified, in silico, novel and striking immune functional networks for rainbow trout nucleated RBCs, which are mainly linked to innate and adaptive immunity. Functional pathways related to regulation of hematopoietic cell differentiation, antigen presentation via major histocompatibility complex class II (MHCII), leukocyte differentiation and regulation of leukocyte activation were identified. These preliminary findings further implicate nucleated RBCs in immune function, such as antigen presentation and leukocyte activation.

2025-01-14T12:57:26Z