Desarrollo de una vacuna DNA para el control de la enfermedad de linfocistis

Leiva, R., Borrego, J.J., Castro, D. y Labella, A.M. 2018. Desarrollo de una vacuna DNA para el control de la enfermedad de linfocistis. En: La Acuicultura en el Litoral Suratlántico. IX Jornadas de Acuicultura en el Litroral Suratlántico, p 96. Instituto de Investigación y Formación Agraria y Pesquera (IFAPA), Consejería de Agricultura, Pesca y Desarrollo Rural, Junta de Andalucía.El virus de la enfermedad de linfocistis (LCDV) es el agente etiológico de la enfermedad de linfocistis, que afecta tanto a peces marinos como dulceacuícolas. Esta enfermedad supone un grave problema para el sector de la acuicultura, provocando pérdidas económicas debido a su alta morbilidad. En el presente estudio se ha desarrollado un plásmido recombinante con objeto de ser utilizado como vacuna de DNA para limitar la incidencia de dicha enfermedad en el cultivo de dorada (Sparus aurata). El gen codificante de la proteína principal de la cápside (MCP) del LCDV de dorada (LCDV-Sa) se clonó en primer lugar en el vector de expresión pGEX-6P-3, con el fin de expresar la MCP viral como proteína de fusión con GST (glutathione S-transferase) en Escherichia coli BL-21. La expresión de la proteína de fusión MCP-GST en células bacterianas se comprobó mediante Western blot e inmunoensayo dot-blot con anticuerpos específicos dirigidos contra la MCP y la GST. Esta MCP recombinante se empleará como antígeno de captura para la detección mediante ELISA de anticuerpos anti-LCDV en peces vacunados. Por otra parte, el gen codificante de la MCP se subclonó en el vector de expresión pcDNA3.1/NT-GFP-TOPO, obteniéndose así el plásmido vacunal. Tras su amplificación en E. coli TOP 10, el plásmido se purificó y se utilizó para transfectar la línea celular de dorada SAF-1, evaluándose dos métodos de transfección: Nucleofector Kit y lipofectamina. La expresión de la MCP viral en células SAF-1 transfectadas con el plásmido vacunal se ha demostrado mediante detección de la GFP (green fluorescent protein) por microscopía de epifluorescencia, y por inmunodetección de la MCP viral utilizando anticuerpos específicos. El plásmido vacunal obtenido se inyectará intramuscularmente en ejemplares de dorada para determinar la distribución y expresión temporal de la vacuna, así como para detectar anticuerpos anti-LCDV en los peces vacunados.Proyecto de Excelencia de la Junta de Andalucía (P12-RNM-2261). Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Análisis de la expresión de inmunogenes en doradas vacunadas en respuesta a la infección por LCDV-Sa

El objetivo del presente estudio ha sido evaluar la respuesta inmune frente a la infección por Lymphocystis Disease Virus 3 (LCDV-Sa) en ejemplares de dorada (Sparus aurata) vacunados con un plásmido que codifica la proteína principal de la cápside del virus, con el fin de identificar inmunogenes relacionados con la protección inducida por la vacuna. Se utilizaron juveniles de dorada inyectados intramuscularmente con la vacuna (grupo vacunado) o el plásmido vacío (grupo placebo), así como con PBS (dos grupos). A los 30 d post-vacunación, los peces se inocularon con un aislado de LCDV-Sa (106 TCID50/pez), excepto uno de los grupos originalmente inyectado con PBS que se mantuvo como control no-infectado. Se tomaron muestras de riñón cefálico a los 1 y 3 d post-infección (dpi) (6 peces por tiempo) y se realizó un análisis de la expresión relativa de 49 inmunogenes de dorada utilizando la plataforma OpenArray®, basada en qPCR con sondas TaqMan, usando las muestras del control no-infectado como calibrador. Los resultados mostraron una mayor expresión diferencial de inmunogenes en los peces vacunados en comparación a los peces que recibieron el plásmido vacío o a los no vacunados. En todos los grupos experimentales se observó una sub-regulación génica a 1 dpi, que cambió a sobre-regulación a los 3 dpi. Además, en los peces vacunados se observó la estimulación temprana de la expresión del gen activador de recombinación (rag1) y una sobre-regulación tardía de mx1 y mx2.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Revisiting the genus Photobacterium: taxonomy, ecology and pathogenesis

The genus Photobacterium, one of the eight genera included in the family Vibrionaceae, contains 27 species with valid names and it has received attention because of the bioluminescence and pathogenesis mechanisms that some of its species exhibit. However, the taxonomy and phylogeny of this genus are not completely elucidated; for example, P. logei and P. fischeri are now considered members of the genus Aliivibrio, and previously were included in the genus Vibrio. In addition, P. damselae subsp. piscicida was formed as a new combination for former Vibrio damsela and Pasteurella piscicida. Moreover, P. damselae subsp. damselae is an earlier heterotypic synonym of P. histaminum. To avoid these incovenences draft and complete genomic sequences of members of Photobacterium are increasingly becoming available and their use is now routine for many research laboratories to address diverse goals: species delineation with overall genomic indexes, phylogenetic analyses, comparative genomics, and phenotypic inference. The habitats and isolation source of the Photobacterium species include seawater, sea sediments, saline lake waters, and a variety of marine organisms with which the photobacteria establish different relationships, from symbiosis to pathogenic interactions. Several species of this genus contain bioluminescent strains in symbiosis with marine fish and cephalopods; in addition, other species enhance its growth at pressures above 1 atmosphere, by means of several high-pressure adaptation mechanisms and for this, they may be considered as piezophilic (former barophilic) bacteria. Until now, only P. jeanii, P. rosenbergii, P. sanctipauli, and the two subspecies of P. damselae have been reported as responsible agents of several pathologies on animal hosts, such as corals, sponges, fish and homeothermic animals. In this review we have revised and updated the taxonomy, ecology and pathogenicity of several members of this genus. [Int Microbiol 20(1): 1-10 (2017)]Keywords: Photobacterium · taxonomy · symbiosis · pathogenesis · virulence factor

Nervous Necrosis Virus (NNV) Booster Vaccination Increases Senegalese Sole Survival and Enhances Immunoprotection

iral encephalopathy and retinopathy (VER), caused by nervous necrosis virus (NNV), is a serious threat to Senegalese sole farming. We have previously demonstrated that immunization with an inactivated vaccine confers partial protection against the infection. However, a vaccination program must be finely adjusted to achieve the best results in terms of immune system stimulation and protection. In this study we show that a booster injection 30 days after prime vaccination increases sole survival and reduces NNV replication in brain (viral target organ). The analysis of immune-related genes expression indicated that T CD4+ lymphocytes and the proteins Mx and HERC4 may play an important role in the protection. These findings increase our understanding of sole immune response against NNV and may contribute to the development of effective protection measures.This research was funded by Ministerio de Ciencia, Innovación y Universidades (MCIUI), the Agencia Estatal de Investigación (AEI) and FEDER, grant number RTI2018-094687-B-C21. Dr. Sandra Souto was funded with a postdoctoral grant from Consellería de Cultura, Educación e Universidade, Xunta de Galicia (grant number: ED481D-2022-024). Partial funding for open access charge: Universidad de Málag

Desarrollo de un chip de DNA para la detección de dianas génicas bacterianas de interés en el campo de la acuicultura

La adquisición bacteriana de multiresistencias frente a antimicrobianos es un fenómeno cada vez más preocupante, con gran repercusión para la salud pública, pero también con impacto en sectores de la producción animal, como es el caso particular de la acuicultura. Entre los años 2010-2012, como objetivo de un Proyecto de Investigación Italiano (Progetti d’interesse Nazionale, PRIN), se realizaron diferentes campañas de muestreo a lo largo de la costa del mar Adriático, en centros dedicados a la acuicultura y áreas costeras, estudiando la incidencia de bacterias multiresistentes, así como de los elementos genéticos móviles responsables de su adquisición, desarrollando además una herramienta biotecnológica que agiliza este tipo de estudios permitiendo detectar 164 dianas génicas en un único soporte. Se obtuvo una colección de 1274 aislados bacterianos, a partir de muestras de agua, sedimento y biofilm, resistentes a las distintas combinaciones de antibióticos utilizados según la legislación italiana. Del total, 105 (8,25%) aislados resultaron ser multiresistentes, detectándose en el 13,59% el integrón de clase I, en el 14,56% el plásmido pAb5s9, en el 5,82% el elemento SXT/R391, y en el 15,52% diferentes combinaciones de estos elementos genéticos. Basándonos en estos resultados, se ha desarrollado y validado un chip de DNA que permite detectar en un mismo soporte, genes que codifican para determinantes de resistencia a antimicrobianos, elementos genéticos móviles (MGE), genes para la detección de especies bacterianas patógenas con repercusión en acuicultura y para la salud del hombre, e indicadores de contaminación fecal.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Senegalese sole immune response against betanodavirus recombinants harboring modifications in the 3' terminal region of the RNA1

The nervous necrosis virus (NNV) is the etiological agent of the viral nervous necrosis (VNN), a disease affecting a high number of fish species worldwide. NNV genome is composed of two segments RNA1 and RNA2, encoding the RNA-dependent RNA polymerase and the capsid protein, respectively. NNV has been classified into four species: SJNNV, TPNNV, RGNNV and BFNNV. Furthermore, reassortants between RGNNV and SJNNV have been reported, such as wt160 isolated from Senegalese sole, which presents a RGNNV-RNA1 and a SJNNV-RNA2 type segments, and causes 100% mortality in this fish species. This isolate exhibited differences in the 3’ NCR of both genomic segments when compared to the reference strains of each genotype. In this study, the effect on virulence of the substitutions observed in the 3’NCR of the wt160-RNA1 has been evaluated, by the development of two recombinants harbouring mutations at position 3073 and 3093, which make the wt160-RNA1 similar to the reference RGNNV. Moreover, immune response of sole against the infection with these recombinants compared to the wild-type, has been evaluated using an OpenArray. The infection with the recombinants r3073 and r3093 decreased the mortality to 29.3% and 25.3%, respectively. Furthermore, the number of DEGs was higher at 3 days than at 2 days p.i., after the infection with the three viruses, being the number of DEG quite similar among viruses. Significant differences between DEG fold changes after infection with the mutants and the wt160 will be discussed. It should be highlighted that at 2 days p.i., the gene gig1 was not expressed after the infection with r3073 and r3093. However, at 3 days p.i. this gene was expressed at the highest level after the infection with the three viruses. Moreover, the infection with the wt160 induced the down-regulation of the genes gilt and magel2, which was not observed after the infections with r3073.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Co-occurrence of viral and bacterial pathogens in disease outbreaks affecting newly cultured sparid fish

Several microbial disease outbreaks in farm stocks of newly cultured sparid fish species, such as common seabream, redbanded seabream, and white seabream, were recorded from 2004 to 2006. This study describes the isolation and characterization of the potential causative agents, either bacteria or viruses, of these outbreaks. The isolated bacterial strains were characterized according to traditional taxonomical analyses and sequencing of a 16S rDNA fragment. Most bacteria were identified as Vibrio spp. and Photobacterium damselae subsp. damselae. The development of cytopathic effects (CPE) on different fish cell lines, the application of specific nested-PCR tests for infectious pancreatic necrosis virus (IPNV), viral nervous necrosis virus (VNNV) and viral hemorrhagic septicemia virus (VHSV), and subsequent sequence analyses were used for virus detection and identification. VNNV, related to the striped jack neural necrosis virus (SJNNV) genotype, and VHSV, related to the genotype Ia, were the only viruses detected. VNNV was isolated from the three fish species under study in five different outbreaks, whereas VHSV was isolated from common seabream and white seabream during two of these outbreaks. IPNV was not detected in any case. [Int Microbiol 2007; 10(3):193-199

Transcriptomic Profiles of Senegalese Sole Infected With Nervous Necrosis Virus Reassortants Presenting Different Degree of Virulence

Betanodaviruses [nervous necrosis virus (NNV)] are the causative agent of the viral encephalopathy and retinopathy, a disease that affects cultured Senegalese sole (Solea senegalensis). NNV reassortants, combining genomic segments from redspotted grouper nervous necrosis virus (RGNNV) and striped jack nervous necrosis virus (SJNNV) genotypes, have been previously isolated from several fish species. The wild-type reassortant wSs160.03, isolated from Senegalese sole, has been proven to be more virulent to sole than the parental genotypes (RGNNV and SJNNV), causing 100% mortality. Mutations at amino acids 247 (serine to alanine) and 270 (serine to asparagine) in the wSs160.03 capsid protein have allowed us to obtain a mutant reassortant (rSs160.03247+270), which provokes a 40% mortality decrease. In this study, the RNA-Seq technology has been used to comparatively analyze Senegalese sole transcriptomes in two organs (head kidney and eye/brain) after infection with wild-type and mutant strains. A total of 633 genes were differentially expressed (DEGs) in animals infected with the wild-type isolate (with higher virulence), whereas 393 genes were differentially expressed in animals infected with the mutant strain (37.9% decrease in the number of DEGs). To study the biological functions of detected DEGs involved in NNV infection, a gene ontology (GO) enrichment analysis was performed. Different GO profiles were obtained in the following subclasses: (i) biological process; (ii) cellular component; and (iii) molecular function, for each viral strain tested. Immune response and proteolysis have been the predominant biological process after the infection with the wild-type isolate, whereas the infection with the mutant strain induces proteolysis in head kidney and inhibition of vasculogenesis in nervous tissue. Regarding the immune response, genes coding for proteins acting as mediators of type I IFN expression (DHX58, IRF3, IRF7) and IFN-stimulated genes (ISG15, Mx, PKR, Gig1, ISG12, IFI44, IFIT-1, to name a few) were upregulated in animals infected with the wild-type isolate, whereas no-differential expression of these genes was observed in samples inoculated with the mutant strain. The different transcriptomic profiles obtained could help to better understand the NNV pathogenesis in Senegalese sole, setting up the importance as virulence determinants of amino acids at positions 247 and 270 within the RNA2 segment

Evaluación de la respuesta inmune en lenguado senegalés conferida por una vacuna inactivada frente a Betanodavirus

La necrosis nerviosa viral es una enfermedad que afecta a peces cultivados en todo el mundo. Su agente etiológico es el virus de la necrosis nerviosa, género Betanodavirus, familia Nodaviridae, que presenta un genoma compuesto por dos segmentos de RNA monocatenario. Los betanodavirus se clasifican en cuatro especies, Striped Jack-, Tiger puffer-, Redspotted grouper- y Barfin flonder nervous necrosis virus (SJNNV, TPNNV, RGNNV y BFNNV, respectivamente). En el sur de Europa se han descrito recombinantes de los segmentos genómicos de las especies SJNNV y RGNNV como agentes causantes de epizootías en lenguado senegalés y dorada. El control de esta enfermedad es de gran importancia para la acuicultura europea y la vacunación es una de las estrategias más prometedoras. Sin embargo, solo existen vacunas comercializadas contra la especie RGNNV las cuales no protegen frente a los aislados recombinantes, por lo que se ha desarrollado una vacuna inactivada utilizando el aislado recombinante SpSsIAusc160.03 que produce una moderada protección frente a la infección vírica en lenguado (Valero et al., 2021). El objetivo de este estudio es evaluar la capacidad de dicha vacuna de inducir una respuesta inmune eficaz en lenguado (Solea senegalensis). Se tomaron muestras de cerebro y riñón cefálico de lenguados vacunados y sin vacunar a 2, 3 y 7 días post-vacunación (dpv), analizándose la expresión de 106 inmunogenes mediante la plataforma OpenArray® (Gémez et al., 2020). Se detectó una respuesta inmune temprana en muestras de riñón, expresándose diferencialmente 39 y 29 genes a 2 y 3 dpv, respectivamente. Esta modulación fue significativamente menor a 7 dpv, con solo 3 genes expresados diferencialmente (DEG). En muestras de cerebro, tejido diana de la infección, se observó una menor modulación génica, detectándose expresión diferencial exclusivamente a 2 y 3 dpv (5 y 12 DEG, respectivamente). Financiación: Proyecto RTI2018-094687-B-C21/C22 del MICIU cofinanciado por FEDER.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Concurrence of iridovirus, polyomavirus, and a unique member of a new group of fish papillomaviruses in lymphocystis disease-affected gilthead sea bream

Lymphocystis disease is a geographically widespread disease affecting more than 150 different species of marine and freshwater fish. The disease, provoked by the iridovirus lymphocystis disease virus (LCDV), is characterized by the appearance of papillomalike lesions on the skin of affected animals that usually self-resolve over time. Development of the disease is usually associated with several environmental factors and, more frequently, with stress conditions provoked by the intensive culture conditions present in fish farms. In gilthead sea bream (Sparus aurata), an economically important cultured fish species in the Mediterranean area, a distinct LCDV has been identified but not yet completely characterized. We have used direct sequencing of the virome of lymphocystis lesions from affected S. aurata fish to obtain the complete genome of a new LCDV-Sa species that is the largest vertebrate iridovirus sequenced to date. Importantly, this approach allowed us to assemble the full-length circular genome sequence of two previously unknown viruses belonging to the papillomaviruses and polyomaviruses, termed Sparus aurata papillomavirus 1 (SaPV1) and Sparus aurata polyomavirus 1 (SaPyV1), respectively. Epidemiological surveys showed that lymphocystis disease was frequently associated with the concurrent appearance of one or both of the new viruses. SaPV1 has unique characteristics, such as an intron within the L1 gene, and as the first member of the Papillomaviridae family described in fish, provides evidence for a more ancient origin of this family than previously thought.Ministerio de Economía y Competitividad (RYC-2010-06300).Peer Reviewe