Diseño y evaluación de técnicas moleculares para diagnóstico de birnavirus acuáticos

De todos los problemas que afectan a la acuicultura, son las patologías uno de los más importantes. Al igual que otras especies animales en producción intensiva, los principales problemas sanitarios que afectan a los organismos acuáticos se atribuyen a una amplia variedad de agentes microbianos que engloban bacterias, virus, parásitos y hongos. De todas las enfermedades de origen microbiano, las infecciones virales son las de mayor trascendencia, debido a la ausencia de tratamientos efectivos y a que los peces supervivientes de una epizootia viral pueden convertirse en portadores asintomáticos de la enfermedad, favoreciendo así el mantenimiento y la diseminación de la enfermedad, así como la emergencia continua de nuevos agentes infecciosos. De hecho, siendo los agentes primarios de numerosas enfermedades, tienen la facultad de facilitar el desarrollo de agentes infecciosos secundarios

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

Amino acidic substitutions in the polymerase N-terminal region of a reassortant betanodavirus strain causing poor adaptation to temperature increase

Nervous necrosis virus (NNV), Genus Betanodavirus, is the causative agent of viral encephalopathy and retinopathy (VER), a neuropathological disease that causes fish mortalities worldwide. The NNV genome is composed of two single-stranded RNA molecules, RNA1 and RNA2, encoding the RNA polymerase and the coat protein, respectively. Betanodaviruses are classified into four genotypes: red-spotted grouper nervous necrosis virus (RGNNV), striped jack nervous necrosis virus (SJNNV), barfin flounder nervous necrosis virus (BFNNV) and tiger puffer nervous necrosis virus (TPNNV). In Southern Europe the presence of RGNNV, SJNNV and their natural reassortants (in both RNA1/RNA2 forms: RGNNV/SJNNV and SJNNV/RGNNV) has been reported. Pathology caused by these genotypes is closely linked to water temperature and the RNA1 segment encoding amino acids 1–445 has been postulated to regulate viral adaptation to temperature. Reassortants isolated from sole (RGNNV/SJNNV) show 6 substitutions in this region when compared with the RGNNV genotype (positions 41, 48, 218, 223, 238 and 289). We have demonstrated that change of these positions to those present in the RGNNV genotype cause low and delayed replication in vitro when compared with that of the wild type strain at 25 and 30 °C. The experimental infections confirmed the impact of the mutations on viral replication because at 25 °C the viral load and the mortality were significantly lower in fish infected with the mutant than in those challenged with the non-mutated virus. It was not possible to challenge fish at 30 °C because of the scarce tolerance of sole to this temperature.This work was supported by Grant AGL2014-54532-C2-2-R from the Ministerio the Innovación y Competitividad (Spain), cofunded by FEDER, and by Grant ED431C 2018/18 from Xunta de Galicia (Spain)S

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

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

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

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

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