Poblaciones de peces salvajes: ¿responsables o sufridores de las patologías virales en acuicultura?

El desarrollo de la acuicultura en las últimas décadas ha sido el motor que ha favorecido el avance de la virología de peces. El número de grupos de investigación dedicados a esta disciplina ha ido incrementándose progresivamente, lo que ha implicado un crecimiento exponencial del conocimiento científico y el desarrollo tecnológico, y ha permitido mejoras en la prevención y el control de este tipo de patologías, que representan uno de los mayores temores del Sector. El estudio de los virus que afectan a peces en cultivo se beneficia de las especiales condiciones del mismo: área delimitada, poblaciones bien definidas y con un historial conocido, seguimiento de morbilidades y mortalidades –que aportan individuos en los que las altas cargas virales facilitan el diagnóstico y el estudio del agente causal– y disponibilidad de especímenes para experimentación científica. Estas condiciones, sin embargo, están ausentes –en la mayor parte de los casos– en ambientes naturales, sobre todo cuando se estudian entornos oceánicos. Éste es, con toda seguridad, el porqué de que, en las más de cinco décadas del marcado crecimiento de la investigación en esta área, el número de publicaciones científicas enfocadas a poblaciones salvajes no llegue a los 300. Sin embargo, en un número –creciente– de grupos se ha despertado el interés por conocer el estado sanitario de las poblaciones salvajes y silvestres de peces en todo el mundo. Fundamentalmente, pero no sólo, se pone la diana en aquellas poblaciones que pueden estar en la interfase de interacción con la acuicultura. Sin embargo, destaca también la lista creciente de campañas oceanográficas cuyo único fin inicial es el conocimiento del estado epidemiológico de poblaciones teóricamente muy alejadas, pero que han aportado interesantes datos que demuestran que la globalización ha existido en los océanos desde mucho antes que el Hombre inventara ese concepto. Los primeros trabajos de monitorización de poblaciones salvajes, que datan de mediados de los 70, estaban centrados en el estudio de la presencia del virus de la necrosis pancreática infecciosa (IPNV) en peces de lagos escoceses; hacia finales de esa década, se descubrió la presencia del virus de la septicemia hemorrágica viral (VHSV) en bacalao salvaje en la costa sur de Dinamarca. A lo largo de todos estos años, ambos virus han seguido siendo el objetivo de la mayor parte de las campañas de monitorización de este tipo de poblaciones de peces. Otros virus que han ido despertando el interés de los científicos son el virus de la necrosis nerviosa viral (VNNV) y el virus de la anemia infecciosa del salmón. Pero, hay una gran variedad de virus que han sido y son objeto de estudio en poblaciones naturales, y otros muchos –aún desconocidos– que, con toda seguridad, están esperando a ser descubiertos por los investigadores (como resultado de su estudio) o por los piscicultores en sus plantas de cultivo (como resultado de su efecto). No en vano todos estos virus representan una gran preocupación para los piscicultores, para los cuales –y también para algunos científicos– es tentador culpar a las poblaciones salvajes de las patologías emergentes y re-emergentes en acuicultura. Si bien es cierto que hay algunos estudios que demuestran –o pretenden demostrar–, en ocasiones con pruebas circunstanciales, que el efecto de dispersión de agentes virales desde ambientes naturales a la acuicultura no es recíproco, no se puede descartar el efecto amplificador que el cultivo intensivo de peces tiene sobre los agentes infecciosos, lo que hace que una gran carga viral pueda ser devuelta al medio. Por otro lado, sí está demostrado que el especial ecosistema del cultivo intensivo representa una presión adaptativa que acelera el reloj evolutivo de los virus en cuanto entran en ese entorno. Esto puede implicar que las poblaciones salvajes supuestamente responsables de una epizootia determinada se conviertan en poblaciones naif en cuanto reciban el virus devuelto por la población cultivada. La diferencia es que generalmente –aunque hay unos pocos casos demostrados– las mortalidades masivas en las poblaciones salvajes se nos pasan desapercibidas. No obstante, a pesar de los esfuerzos de muchos científicos, que hemos llegado a estar bajo el paraguas de una red internacional (DIPNET: Disease Interactions and Pathogens Exchange Network), sólo unos pocos casos de interacción salvaje-doméstico han podido ser confirmados. En este trabajo, aprovecho la oportunidad para dar unas pinceladas sobre el estado en que se encuentra el conocimiento del estatus epidemiológico de las poblaciones salvajes, el efecto de globalización de las corrientes, migraciones y la mano del hombre, y lo que puede ocurrir en la zona de interfase medio salvaje-acuicultura, así como qué medidas podemos tomar para reducir su efecto.Universidad de Málaga, Campus de Excelencia Internacional Andalucía Tec

Development and Validation of a SYBR Green Real Time PCR Protocol for Detection and Quantification of Nervous Necrosis Virus (NNV) Using Different Standards

The nervous necrosis virus (NNV) is a threat to fish aquaculture worldwide, especially in Mediterranean countries. Fast and accurate diagnosis is essential to control it, and viral quantification is required to predict the level of risk of new viral detections in field samples. For both, reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) is used by diagnostic laboratories. In the present study, we developed an RT-qPCR procedure for the diagnosis and simultaneous quantification of NNV isolates from any of the four genotypes. The method proved to be highly sensitive in terms of crude virus titer: 5.56–9.88 TCID50/mL (tissue culture infectious dose per mL), depending on the viral strain, and averaging 8.8 TCID50/mL or 0.08 TCID50/reaction. Other standards also yielded very low detection limits: 16.3 genome copies (cps) of purified virus per mL, 2.36 plasmid cps/mL, 7.86 in vitro synthetized RNA cps/mL, and 3.16 TCID50/mL of virus from infected tissues. The diagnostic parameters evaluated in fish samples were much higher in comparison to cell culture isolation and nested PCR. In addition, the high repeatability and reproducibility of the procedure, as well as the high coefficient of determination (R2) of all the calibration curves with any type of standard tested, ensure the high reliability of the quantification of NNV using this RT-qPCR procedure, regardless of the viral type detected and from the type of standard chosenThis research was partially funded by the Interreg VA Spain-Portugal cooperation program (POCTEP) 2014-2020, 0474_BLUEBIOLAB project, co-funded by FEDERS

Betanodavirus infection in primary neuron cultures from sole

Nervous necrosis virus (NNV), G. Betanodavirus, is the causative agent of viral encephalopathy and retinopathy, a disease that causes mass mortalities in a wide range of fish species. Betanodaviruses are neurotropic viruses and their replication in the susceptible fish species seems to be almost entirely restricted to nerve tissue. However, none of the cell lines used for NNV propagation has a nervous origin. In this study, first we established a protocol for the primary culture of neurons from Senegalese sole, which made it possible to further study virus-host cell interactions. Then, we compared the replication of three NNV strains with different genotypes (SJNNV, RGNNV and a RGNNV/SJNNV reassortant strain) in sole neuron primary cultures and E-11 cells. In addition, to study how two amino acid substitutions at the c-terminal of the capsid protein (positions 247 and 270) affect the binding to cell receptors, a recombinant strain was also tested. The results show that sole neural cells enabled replication of all the tested NNV strains. However, the recombinant strain shows a clearly delayed replication when compared with the wt strain. This delay was not observed in virus replicating in E-11 cells, suggesting a viral interaction with different cell receptors. The establishment of a sole primary neuronal culture protocol provides an important tool for research into betanodavirus infection in soleThis work was supported by Grant AGL2014-54532-C2-2-R from the Ministerio the Innovación y Competitividad (Spain), cofunded by FEDERS

Interspecies transmission between Solea senegalensis and Sparus aurata of reassortant Nervous Necrosis Virus (NNV) strains and effect of stress on the outcome of the infection

Viral Encephalopathy and Retinopathy (VER) episodes in Southern Europe have led to the isolation of several reassortant NNV strains from different fish species, including Senegalese sole and gilthead seabream. Polyculture of both species is being developed to optimize available resources. However, this farming technique can be threatened by NNV horizontal transmission between diseased fish or asymptomatic carriers and non-infected individuals from both fish species, which could lead to a VER outbreak in the facility and seriously affect fish production. Therefore, in this study we have assessed the susceptibility of gilthead seabream and Senegalese sole to two reassortant NNV strains isolated from each of the two fish species, and the possibility of interspecies transmission by cohabiting infected and naïve individuals. Our results showed that both NNV isolates caused moderate mortality rates and replicated in both fish species. In the cohabitation challenges, infective NNV particles were recovered from naïve cohabitants, demonstrating interspecies transmission from infected individuals that shed NNV into the water column. In addition, cumulative mortality in sole cohabitants was significantly higher, presumably due to the stress provoked by the aggressiveness of gilthead seabream. This is supported by the analysis of the hsp70 gene, a stress biomarker overexpressed in the sole cohabitants, especially in those that died on the first day of cohabitation. Therefore, despite the numerous advantages of polyculture, the risk of VER outbreaks represents a serious constraint for the implementation of this technique in Mediterranean aquaculturThis research was funded by the Ministerio de Ciencia, Innovación y Universidades (MCIUI), the Agencia Estatal de Investigación (AEI) and FEDER under Grant RTI2018-094687-B-C21 and also by the Interreg VA Spain-Portugal cooperation program (POCTEP) 2014-2020, 0474_BLUEBIOLAB project, co-funded by FEDER with a research contract granted to L. Vázquez- SalgadoS

Modification of betanodavirus virulence by substitutions in the 3’ terminal region of RNA2

Betanodaviruses have bi-segmented positive-sense RNA genomes, consisting of RNAs 1 and 2. For some members of the related genus alphanodavirus, the 3' terminal 50 nucleotides (nt) of RNA2, including a predicted stem-loop structure (3'SL), are essential for replication. We investigate the possible existence and role of a similar structure in a reassortant betanodavirus strain (RGNNV/SJNNV). In this study, we developed three recombinant strains containing nucleotide changes at positions 1408 and 1412. Predictive models showed stem-loop structures involving nt 1398–1421 of the natural reassortant whereas this structure is modified in the recombinant viruses harbouring point mutations r1408 and r1408– 1412, but not in r1412. Results obtained from infectivity assays showed differences between the reference strains and the mutants in both RNA1 and RNA2 synthesis. Moreover, an imbalance between the synthesis of both segments was demonstrated, mainly with the double mutant. All these results suggest an interaction between RNA1 and the 3' non-coding regions (3¢NCR) of RNA2. In addition, the significant attenuation of the virulence for Senegalese sole and the delayed replication of r1408–1412 in brain tissues may point to an interaction of RNA2 with host cellular proteinsThis work was supported by grant AGL2014-54532-C2-2-R from the Ministerio de Innovación y Competitividad (Spain), co-funded by FEDERS

Quantitative flow cytometry to measure viral production using infectious pancreatic necrosis virus as a model: a preliminary study

In recent decades, flow cytometry (FCM) has become an important tool in virology, due to its applications in viral replication and viral-cell interactions, as well as its capacity to quantify proteins (qFCM). In the present study, we have designed and evaluated a qFCM procedure for the in vitro analysis and quantification of fish viral proteins, using the infectious pancreatic necrosis virus (IPNV) as a model. We have also tested its use for viral titration and adapted the MARIS (method for analysing RNA following intracellular sorting) method for simultaneous quantification of viral RNA expression in infected cells. The procedure has proved to be repeatable and reproducible to an acceptable level, although to ensure reproducibility, the repetition of standard curves is inevitable. Regarding its use for viral quantification, a direct relationship (by a second-degree polynomial regression) between viral titres and Molecules of Equivalent Soluble Fluorochrome (MESF) was observed. Finally, the results support the use of this technology, not only for virus quantification, but also to study viral replication from a quantitative approachS

BEI Inactivated Vaccine Induces Innate and Adaptive Responses and Elicits Partial Protection upon Reassortant Betanodavirus Infection in Senegalese Sole

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 mechanismThis work was supported by grant RTI2018-094687-B-C21 from MICIU (Spain) co-funded by FEDERS

Steps of the Replication Cycle of the Viral Haemorrhagic Septicaemia Virus (VHSV) Affecting Its Virulence on Fish

The viral haemorrhagic septicaemia virus (VHSV), a single-stranded negative-sense RNA novirhabdovirus affecting a wide range of marine and freshwater fish species, is a main concern for European rainbow trout (Oncorhynchus mykiss) fish farmers. Its genome is constituted by six genes, codifying five structural and one nonstructural proteins. Many studies have been carried out to determine the participation of each gene in the VHSV virulence, most of them based on genome sequence analysis and/or reverse genetics to construct specific mutants and to evaluate their virulence phenotype. In the present study, we have used a different approach with a similar aim: hypothesizing that a failure in any step of the replication cycle can reduce the virulence in vivo, we studied in depth the in vitro replication of VHSV in different cell lines, using sets of strains from different origins, with high, low and moderate levels of virulence for fish. The results demonstrated that several steps in the viral replication cycle could affect VHSV virulence in fish, including adsorption, RNA synthesis and morphogenesis (including viral release). Notably, differences among strains in any step of the replication cycle were mostly strain-specific and reflected only in part the in vivo phenotype (high and low virulent). Our data, therefore, support the need for further studies aimed to construct completely avirulent VHSV recombinants targeting a combination of genes rather than a single one in order to study the mechanisms of genes interplay and their effect on viral phenotype in vitro and in vivoThe project has been funded under the ERANET. The content of this article reflects only the authors’ views, and the ERANET Consortium is not liable for any use that may be made of the information contained thereinS

Establecemento dos balances de materia no sector agroalimentario da provincia de Ourense a fin de optimizar os indicadores de circularidade material

O modelo económico actual é basicamente lineal, o que significa que se utiliza sempre materia prima virxe para fabricar o produto que remata no vertedoiro ao final da fase de uso. Por tratarse dunha situación insostible a longo prazo, estase a buscar o o paso cara a unha economía circular. É aquí onde toman importancia os indicadores de circularidade, ferramentas para transmitir información de interese. O indicador de circularidade material (ICM), desenvolvido pola Fundación Ellen MacArthur, avalía o rendemento da circularidade a nivel de produto. O obxectivo deste traballo foi adecuar o ICM para poder ser utilizado en empresas agroalimentarias da provincia de Ourense, e evitar así a penalización das actividades vinculadas a este sector, usando para iso datos de tres casos concretos (vitivinícola, cárnico e lácteo). Abárcase o cálculo do ICM desde dous puntos de vista. Primeiro, calcúlase o ICM tendo en conta só o proceso global do produto e logo calcúlase o ICM’, atendendo ás diferentes etapas que conforman o proceso. A modificación proposta consiste na inclusión dunha nova variable que modifica a cantidade final de residuos destinados a vertedoiro/enerxía/outros non recuperables. Estas novas variables son o coeficiente de dixestibilidade (CDixest) e o coeficiente de uso (CUso). A inclusión destes dous factores de corrección melloran significativamente o valor de ICM e semella repercutir no valor do ICM’, cando o produto se pode considerar materia prima dun subproceso posterio