Water-in-oil adjuvant challenges in fish vaccination: An experimental inactivated adjuvanted vaccine against betanodavirus infection in Senegalese sole.

The extensive growth of intensive fish farming has led to a massive spread of infectious diseases. Nervous necrosis virus (NNV) is the causative agent of the viral encephalo- and retinopathy disease which has become a major threat for fish farming all over the globe. The devastating mortality rates recorded in disease outbreaks, especially when infected specimens are at early stages of development, have a high economic impact on the sector. Currently, vaccines are the most cost-effective preventing tool in the fight against viruses. Inactivated vaccines have the advantage of simplicity in their development at the same time as present the antigen in a similar manner than the natural infection in the host. Nevertheless, they usually trigger weaker immune responses needing adjuvants to boost their effectiveness. In this work, we have intraperitoneally vaccinated Senegalese sole juveniles (Solea senegalensis) with a previously designed inactivated vaccine against NNV based on binary ethylenimine (BEI), mixed or not with an oil-adjuvant. Our results demonstrated the potential activation of different immune pathways when the vaccine was administered alone compared to the oil-adjuvanted vaccine, both resulting in an equivalent partial improvement in survival following a NNV challenge. However, whilst the vaccine alone led to a significant increase in specific antibodies, in the adjuvanted version those antibodies were kept basal although with a slight improvement in their neutralization capacity. At transcriptional level, neither vaccine (adjuvanted or not) triggered the immune system activation during the vaccination period. However, after NNV infection, the BEI-inactivated vaccines alone and oil-adjuvanted both elicited the stimulation of antiviral responsive genes (rtp3, herc4), antigen presentation molecules (mhcii) and T-cell markers (cd8a) in the head-kidney. Additionally, the oil-adjuvanted vaccine appears to stimulate mediator cytokines (il6) and B-cell markers (ight and ighm). Surprisingly, when the adjuvant was administered alone, fish showed the highest survival rates concomitantly with a lack of NNV-IgM production, pointing to the possible induction of different immune pathways than the B-cell responses via antibodies by the adjuvant. Since this combined vaccine did not succeed in the full extension of protection against the pathogen, further studies should be performed focusing on unravelling the molecular mechanisms through which adjuvants trigger the immune response, both independently and when added to a vaccine antigen.

A Potential Nervous Necrosis Virus (NNV) Live Vaccine for Sole Obtained by Genomic Modification.

Viral Encephalopathy and Retinopathy (VER) is a neurological infectious fish disease that causes vacuolization and necrosis in the central nervous system, which lead to swimming abnormalities and, generally, host death in the early stages of development. VER is caused by the Nervous Necrosis Virus (NNV), a non-enveloped virus with a bisegmented and positive-stranded (+) RNA genome. The largest segment (RNA1) codes for viral polymerase while capsid protein is encoded by RNA2. The aim of this study was to explore the potential of a reverse-engineered RGNNV/SJNNV strain that harbors mutations in both 3'NCRs (position 3073 of RNA1 and 1408 and 1412 of RNA2) as an attenuated live vaccine for sole. The attenuation of this strain was confirmed through experimental infections in sole at 22 °C. Vaccination trials were performed by bath, intramuscular, and intraperitoneal injection, at two temperatures (18 and 22 °C). Our results indicate the improved survival of vaccinated fish and delayed and poorer viral replication, as well as an overexpression of immune response genes linked to T cell markers (cd4 and cd8), to an early inflammatory response (tlr7 and tnfα), and to antiviral activity (rtp3 and mx). In conclusion, our study indicates that the attenuated strain is a good vaccine candidate as it favors sole survival upon infection with the wt strain while inducing a significant immune response.

Detection of different Betanodavirus genotypes in wild fish from Spanish Atlantic coastal waters (Galicia, northwestern Spain).

OBJECTIVE: The nervous necrosis virus (NNV; genus Betanodavirus) is an aquatic pathogen that is responsible for a neurological disease affecting marine fish. Despite its almost worldwide distribution, global warming could favor the spread of NNV to new areas, highlighting the importance of conducting epidemiological surveys on both wild and farmed marine fish species. In this study, we assessed NNV prevalence in wild fish caught along the Galician Atlantic coast. METHODS: In total, 1277 fish were analyzed by reverse transcription real-time polymerase chain reaction. RESULT: Twenty two (1.72%) of those fish tested positive for NNV, including two species in which the pathogen had not yet been reported. CONCLUSION: The reassortant RGNNV/SJNNV (red-spotted grouper NNV/striped jack NNV) was detected in 55% of NNV-positive individuals, while the remaining 45% harbored the SJNNV-type genome. Moreover, from European Pilchard Sardina pilchardus and Atlantic Mackerel Scomber scombrus, we isolated four reassortant strains that carried amino acid mutations at key sites related to NNV-host interaction.

Designing and Validation of a Droplet Digital PCR Procedure for Diagnosis and Accurate Quantification of Nervous Necrosis Virus in the Mediterranean Area.

The viral nervous necrosis virus (VNNV) is the causative agent of an important disease affecting fish species cultured worldwide. Early and accurate diagnosis is, at present, the most effective control and prevention tool, and molecular techniques have been strongly introduced and accepted by official organizations. Among those, real-time quantitative polymerase chain reaction (rt-qPCR) is nowadays displacing other molecular techniques. However, another PCR-based technology, droplet digital PCR (ddPCR), is on the increase. It has many advantages over qPCR, such as higher sensitivity and more reliability of the quantification. Therefore, we decided to design and validate a protocol for the diagnosis and quantification of SJ and RG type VNNV using reverse transcription-ddPCR (RT-ddPCR). We obtained an extremely low limit of detection, 10- to 100-fold lower than with RT-qPCR. Quantification by RT-ddPCR, with a dynamic range of 6.8-6.8 × 104 (SJ type) or 1.04 × 101-1.04 × 105 (RG type) cps/rctn, was more reliable than with RT-qPCR. The procedure was tested and validated in field samples, providing high clinical sensitivity and negative predictive values. In conclusion, we propose this method to substitute RT-qPCR protocols because it exceeds the expectations of qPCR in the diagnosis and quantification of VNNV.

Nervous necrosis virus viability modulation by water salinity and temperature.

Nervous necrosis virus (NNV) is a hazardous aquatic pathogen, distributed worldwide and in a wide range of temperatures. Viral persistence in water has been demonstrated to be affected by different factors, such as temperature, UV, or biological load. In this study, we have investigated the viability of NNV strains in low- and high-salinity seawater (LS and HS, respectively) both in laboratory and aquarium conditions, at different storage temperatures, and for comparative purposes, in culture medium. Our results showed the highest NNV viability in seawater at 15°C and as temperature increased, a drop in viral persistence was observed. Additionally, survival at 15 and 30°C was strongly affected by increasing salt content, while no differences were observed between LS and HS groups at 20 and 25°C. The results of the incubation under aquarium conditions indicated that the effect of UV light and oxygen exposure accelerate the inactivation of infective particles. According to previous studies, NNV persistence in cell culture medium was higher than in seawater, and as observed in the latter, increasing incubation temperatures led to a decrease in viral survival.

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

A re-immunization programme has been tested to improve the protective response elicited in sole by a previously developed BEI-inactivated betanodavirus vaccine. The vaccine was prepared using a reassortant RGNNV/SJNNV strain which is highly pathogenic for sole, and vaccination assays were performed by intraperitoneal injection. Experimental design included a prime- and a booster-vaccination group, which consisted of individuals that received a second vaccine injection at 30 days post vaccination), and their respective controls. A month after prime/booster vaccination, fish were challenged by intramuscular injection with the homologous NNV strain. Samples were collected at different times post vaccination and post challenge to assess the immune response and viral replication. Booster dose enhanced the protection against NNV infection because a significant increase in survival was recorded when compared with prime-vaccinated individuals (relative percent survival 77 vs. 55). In addition, a clear decrease in viral replication in the brain of challenged sole was observed. During the immune induction period, no differences in IgM production were observed between prime- and booster-vaccinated fish, and the expression of the antigen presenting cells (APC)-related molecule MHC class II antigen was the only differential stimulation recorded in the re-immunized individuals. However, a significant upregulation of mhcII and the lymphocytes T helper (Th) marker cd4 was observed after the challenge in the booster-vaccinated group, suggesting these cells play a role in the protection conferred by the booster injection. In addition, after viral infection, re-immunized fish showed specific and neutralizing antibody production and overexpression of other immune-related genes putatively involved in the control of NNV replication.

Differential Nervous Necrosis Virus (NNV) Replication in Five Putative Susceptible Cell Lines.

Viral encephalopathy and retinopathy caused by nervous necrosis virus (NNV), is one of the most threatening viral diseases affecting marine fish worldwide. In vitro propagation of NNV strains is essential for the design of effective control measures. In the present study we analysed both the susceptibility and the permissiveness of five fish cell lines (E-11, GF-1, SAF-1, DLB-1, and SaB-1) to three NNV strains (one RGNNV, one SJNNV, and one reassortant RGNNV/SJNNV). E-11 and DLB-1 were demonstrated to be highly susceptible to NNV strains, with average adsorption efficiency (AE) values higher than 90%. SAF-1 also showed high susceptibility (AE 88%), whereas GF-1 can be regarded as moderately susceptible (AE around 50%). On the contrary, SaB-1 can be considered a poorly susceptible cell line (AE values below 20%). E-11 and GF-1 cell lines provided the highest production rates for RGNNV and RG/SJ (around 103) and both cell lines can be regarded as fully permissive for these viral types. However, the SJNNV production rate in GF-1 was only 17.8 and therefore this cell line should be considered semi-permissive for this genotype. In SAF-1 cells, moderate viral replication was recorded but differences in intracellular and extracellular production suggest that viral progeny was not efficiently released. In DLB-1 and SaB-1 the final viral titres obtained in E-11 were lower than those of the inoculum. However, RNA1 synthesis values seem to indicate that RGNNV replication in DLB-1 and SAF-1 could have been underestimated, probably due to a poor adaptation of the virus grown in these cell lines to E-11. Based on all these results, E-11 seems to be the most appropriate cell for in vitro culture of RGNNV, SJNNV, and reassortant strains.

Effect of rearing density on nervous necrosis virus infection in Senegalese sole (Solea senegalensis).

Intensive fish farming at high densities results in a wide range of adverse consequences on fish welfare, including pathogen spreading, stress and increased mortality rates. In this work, we have assessed whether the survival of Senegalese sole infected with the nervous necrosis virus (NNV), a pathogen responsible for severe disease outbreaks, is affected by rearing density. Based on the different fish ratios per surface area (g cm-2 ) and water volume (g L-1 ), our research showed an earlier mortality onset in the tanks containing NNV-infected fish reared at medium density (MD: 0.071 g cm-2 /5 g L-1 ) and high density (HD: 0.142 g cm-2 /10 g L-1 ), as well as higher cumulative mortality values. However, transcription analysis of hsp70, gr1 and pepck genes, well-known stress biomarkers, seems to indicate that none of the challenged fish were under high stress conditions. NNV load was slightly higher both in dead and in sampled fish from MD and HD groups, and especially in the rearing water from these groups, where peaks in mortality seemed to correlate with increasing NNV load in the water. In conclusion, our results suggest that rearing NNV-infected Senegalese sole at high densities resulted in an earlier mortality onset and higher cumulative values and viral load.

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 mechanism.

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 chosen.

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.

European sea bass brain DLB-1 cell line is susceptible to nodavirus: A transcriptomic study.

Viral diseases are responsible for high rates of mortality and subsequent economic losses in modern aquaculture. The nervous necrosis virus (NNV) produces viral encephalopathy and retinopathy (VER), which affects the fish central nervous system. It is considered one of the most serious viral diseases in marine aquaculture, the European sea bass (Dicentrarchus labrax) being amongst the most susceptible. We have evaluated the European sea bass brain derived cell line (DLB-1) susceptibility to NNV genotypes and evaluated its transcriptomic profile. DLB-1 cells supported NNV gene transcription and replication since strains belonging to the four NNV genotypes produce cytopathic effects. Afterwards, DLB-1 cells were infected with an RGNNV strain, the one which showed the highest replication, for 12 and 72 h and an RNA-seq analysis was performed to identify potential genes involved in the host-NNV interactions. Differential expression analysis showed the up-regulation of many genes related to immunity, heat-shock proteins or apoptosis but not to proteasome or autophagy processes. These data suggest that the immune response, mainly the interferon (IFN) pathway, is not powerful enough to abrogate the infection, and cells finally suffer stress and die by apoptosis liberating infective particles. GO enrichment also revealed, for the first time, the down-regulation of terms related to brain/neuron biology indicating molecular mechanisms causing the pathogenic effect of NNV. This study opens the way to understand key elements in sea bass brain and NNV interactions.

Amino acid changes in the capsid protein of a reassortant betanodavirus strain: Effect on viral replication in vitro and in vivo.

Betanodavirus reassortant strains (RGNNV/SJNNV) isolated from Senegalese sole harbour an SJNNV capsid featuring several changes with respect to the SJNNV-type strain, sharing three hallmark substitutions. Here, we have employed recombinant strains harbouring mutations in these positions (r20 and r20 + 247 + 270) and have demonstrated that the three substitutions affect different steps of the viral replication process. Adsorption ability and efficiency of viral attachment were only affected by substitutions in the C-terminal side of the capsid. However, the concurrent mutation in the N-terminal side seems to slightly decrease these properties, suggesting that this region could also be involved in viral binding. Differences in the intracellular and extracellular production of the mutant strains suggest that both the C-terminal and N-terminal regions of the capsid protein may be involved in the particle budding. Furthermore, viral replication in sole brain tissue of the mutant strains, and especially double- and triple-mutant strains, is clearly delayed with respect to the wt strain. These data support previous findings indicating that the C-terminal side plays a role in virulence because of a slower spread in the fish host brain and suggest that the concurrent participation of the N-terminal side is also important for viral replication in vivo.

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 sole.

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 proteins.

Betanodavirus infection in bath-challenged Solea senegalensis juveniles: A comparative analysis of RGNNV, SJNNV and reassortant strains.

Senegalese sole has been shown to be highly susceptible to betanodavirus infection, although virulence differences were observed between strains. To study the mechanisms involved in these differences, we have analysed the replication in brain tissue of three strains with different genotypes during 15 days after bath infection. In addition, possible portals of entry for betanodavirus into sole were investigated. The reassortant RGNNV/SJNNV and the SJNNV strain reached the brain after 1 and 2 days postinfection, respectively. Although no RGNNV replication was detected until day 3-4 postinfection, at the end of the experiment this strain yielded the highest viral load; this is in accordance with previous studies in which sole infected with the reassortant showed more acute signs and earlier mortality than the RGNNV and SJNNV strains. Differences between strains were also observed in the possible portals of entry. Thus, whereas the reassortant strain could infect sole mainly through the skin or the oral route, and, to a minor extent, through the gills, the SJNNV strain seems to enter fish only through the gills and the RGNNV strain could use all tissues indistinctly. Taken together, all these results support the hypothesis that reassortment has improved betanodavirus infectivity for sole.

In vitro reassortment between Infectious Pancreatic Necrosis Virus (IPNV) strains: The mechanisms involved and its effect on virulence.

Reassortment is one of the main mechanisms of evolution in dsRNA viruses with segmented genomes. It contributes to generate genetic diversity and plays an important role in the emergence and spread of new strains with altered virulence. Natural reassorment has been demonstrated among infectious pancreatic necrosis-like viruses (genus Aquabirnavirus, Birnaviridae). In the present study, coinfections between different viral strains, and genome sequencing by the Sanger and Illumina methods were applied to analyze the frequency of reassortment of this virus in vitro, the possible mechanisms involved, and its effect on virulence. Results have demonstrated that reassortment is a cell-dependent and non-random process, probably through differential expression of the different mRNA classes in the ribosomes of a specific cell, and by specific associations between the components to construct the ribonucleoprotein (RNP) complexes and/or RNP cross-inhibition. However, the precise mechanisms involved, known in other viruses, still remain to be demonstrated in birnaviruses.

Nodavirus Colonizes and Replicates in the Testis of Gilthead Seabream and European Sea Bass Modulating Its Immune and Reproductive Functions.

Viruses are threatening pathogens for fish aquaculture. Some of them are transmitted through gonad fluids or gametes as occurs with nervous necrosis virus (NNV). In order to be transmitted through the gonad, the virus should colonize and replicate inside some cell types of this tissue and avoid the subsequent immune response locally. However, whether NNV colonizes the gonad, the cell types that are infected, and how the immune response in the gonad is regulated has never been studied. We have demonstrated for the first time the presence and localization of NNV into the testis after an experimental infection in the European sea bass (Dicentrarchus labrax), and in the gilthead seabream (Sparus aurata), a very susceptible and an asymptomatic host fish species, respectively. Thus, we localized in the testis viral RNA in both species using in situ PCR and viral proteins in gilthead seabream by immunohistochemistry, suggesting that males might also transmit the virus. In addition, we were able to isolate infective particles from the testis of both species demonstrating that NNV colonizes and replicates into the testis of both species. Blood contamination of the tissues sampled was discarded by completely fish bleeding, furthermore the in situ PCR and immunocytochemistry techniques never showed staining in blood vessels or cells. Moreover, we also determined how the immune and reproductive functions are affected comparing the effects in the testis with those found in the brain, the main target tissue of the virus. Interestingly, NNV triggered the immune response in the European sea bass but not in the gilthead seabream testis. Regarding reproductive functions, NNV infection alters 17ß-estradiol and 11-ketotestosterone production and the potential sensitivity of brain and testis to these hormones, whereas there is no disruption of testicular functions according to several reproductive parameters. Moreover, we have also studied the NNV infection of the testis in vitro to assess local responses. Our in vitro results show that the changes observed on the expression of immune and reproductive genes in the testis of both species are different to those observed upon in vivo infections in most of the cases.

Influence of temperature on Betanodavirus infection in Senegalese sole (Solea senegalensis).

In this study Senegalese sole juveniles were experimentally infected with a reassortant Betanodavirus strain at three different temperatures: 22 °C, 18 °C and 16 °C by bath challenge and cohabitation. The results obtained showed that virus virulence decreased by reducing the water temperature. At 22 °C mortalities reached 100%, at 18 °C they ranged from 75 to 80% and at 16 °C only 8% of the fish died. In addition, horizontal transmission was demonstrated regardless of the rearing temperature. At 16 °C active viral replication was detected up to 66 days post-infection, but no signs of the disease were observed and only a very low level of mortality was recorded. The increase in water temperature from 16 to 22 °C caused a quick rise in the viral load and a subsequent outbreak of mortalities. These findings demonstrate that this reassortant Betanodavirus strain can cause a persistent infection in Senegalese sole at low temperatures (16 °C) for long periods of time, and when temperature increases the virus is able to trigger an acute infection and provoke high mortalities.

In vitro and in vivo characterization of molecular determinants of virulence in reassortant betanodavirus.

We previously reported that betanodavirus reassortant strains [redspotted grouper nervous necrosis virus/striped jack nervous necrosis virus (SJNNV)] isolated from Senegalese sole (Solea senegalensis) exhibited a modified SJNNV capsid amino acid sequence, with changes at aa 247 and 270. In the current study, we investigated the possible role of both residues as putative virulence determinants. Three recombinant viruses harbouring site-specific mutations in the capsid protein sequence, rSs160.03247 (S247A), rSs160.03270 (S270N) and rSs160.03247+270 (S247A/S270N), were generated using a reverse genetics system. These recombinant viruses were studied in cell culture and in vivo in the natural fish host. The three mutant viruses were shown to be infectious and able to replicate in E-11 cells, reaching final titres similar to the WT virus, although with a somewhat slower kinetics of replication. When the effect of the amino acid substitutions on virus pathogenicity was evaluated in Senegalese sole, typical clinical signs of betanodavirus infection were observed in all groups. However, fish mortality induced by all three mutant viruses was clearly affected. Roughly 40 % of the fish survived in these three groups in contrast with the WT virus which killed 100 % of the fish. These data demonstrated that aa 247 and 270 play a major role in betanodavirus virulence although when both mutated aa 247 and 270 are present, corresponding recombinant virus was not further attenuated.