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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 L. Vázquez-Salgado would like to thank Interreg VA Spain–Portugal cooperation Program (POCTEP) 2014-2020, 0474_BLUEBIOLAB project, co-funded by FEDER for her research contract SI

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.

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.

Infectious pancreatic necrosis virus (IPNV) is an important virus which affects the salmonid aquaculture industry worldwide; therefore, it is important to develop rapid and reliable methods of diagnosis to detect the disease at early stages. Nowadays, RT-qPCR is replacing other methods because it provides additional information on the viral load, which is important to have a better understanding of the virus replication level and of the stage of the infection and its risk level. The main problem stems from the high diversity of this virus, which can compromise the reliability of the diagnosis. In this study, we have designed an RT-qPCR procedure for diagnosis and quantification of IPNV based on a single pair of primers targeted to segment B. The procedure has been validated, in vitro and in vivo, testing two different types of standards against seven reference strains and 23 field isolates from different types. The procedure is reliable for the detection of any type, with a detection limit of 31 TCID50 mL-1 , 50 pfu mL-1 or 66 RNA copies mL-1 , depending on the standard. All the standard curves showed high reliability (R2 > 0.95). The results support the high reliability of this new procedure for the diagnosis and quantification of IPNV.

In this study, the susceptibility of turbot juveniles to two betanodavirus strains was assessed, a RGNNV/SJNNV reassortant (Ss160.03) and a SJNNV strain. The reassortant isolate exhibits a slightly modified SJNNV CP, with two amino acid substitutions in the C-terminal domain (positions 247 and 270). To analyse the role of these residues as virulence and host determinants in turbot, three recombinant strains (rSs160.03247 , rSs160.03270 , rSs160.03247+270 ) harbouring site-specific mutations in the CP sequence were also tested in experimental trials. Moderate mortalities (up to 50%) were recorded at 18 °C in the fish challenged with the Ss160.03 strain, whereas low mortalities (17%) were observed in the group challenged with the SJNNV strain. A slight decrease (around 10%) was observed in the mortalities caused by the mutants rSs160.03247 and rSs160.03270 , whilst the mutation of both positions reduced mortality by more than half of that observed in fish challenged with the wild strain. These results are confirmed by the replication in brain tissues, because whereas the wild strain was detected from 5 to 30 dpi and reached the highest viral load, the recombinant virus harbouring both mutations was not detected in the brain until 20 dpi and with a moderate viral load.

A virological analysis was conducted on wild eels from the Albufera Lake (Spain). A total of 179 individuals at different growth stages were collected in two different surveys (2004 and 2008). Presence of anguillid herpesvirus (AngHV-1), aquabirnavirus and betanodavirus was confirmed by PCR procedures in both surveys, although the number of detections was clearly higher in 2008 (83% of the eels analysed resulted positive for virus presence). AngHV-1 was the viral agent most frequently detected, followed by aquabirnaviruses. Betanodaviruses were detected by the first time in wild eels, and although the detections were only made by nested PCR, high percentage of positives were achieved. In addition, in 2008, seven aquabirnaviruses were isolated. Phylogenetic analysis performed using partial sequences of both genomic segments of aquabirnaviruses indicated that the seven isolates could be typed as WB (genogroup I) on the basis of segment A sequences, but when segment B was used six of them clustered with C1 strain (genogroup V) and one was typed as Ab (genogroup II). These results indicate natural reassortment between different strains of aquabirnaviruses in the eels. Although betanodaviruses were not isolated in cell culture, the analysis of the sequence of the nested PCR product indicated that they clustered with SJNNV genotype. The diversity of viral agents and the high level of viral detections suggest that viral infections may play a more prominent role in the decline of the European eel than initially thought. © 2014 John Wiley & Sons Ltd.

Viral haemorrhagic septicaemia (VHS) is a notifiable fish disease, whose causative agent is a rhabdovirus isolated from a wide range of fish species, not only in fresh but also in marine and brackish waters. Phylogenetic studies have identified four major genotypes, with a strong geographical relationship. In this study, we have designed and validated a new procedure – named binary multiplex RT-qPCR (bmRT-qPCR) – for simultaneous detection and typing of all four genotypes of VHSV by real-time RT-PCR based on dual-labelled probes and composed by two multiplex systems designed for European and American/Asiatic isolates, respectively, using a combination of three different fluorophores. The specificity of the procedure was assessed by including a panel of 81 VHSV isolates covering all known genotypes and subtypes of the virus, and tissue material from experimentally infected rainbow trout, resulting in a correct detection and typing of all strains. The analytical sensitivity was evaluated in a comparative assay with titration in cell culture, observing that both methods provided similar limits of detection. The proposed method can be a powerful tool for epidemiological analysis of VHSV by genotyping unknown samples within a few hours.

A non-destructive procedure was utilized to determine the infectious pancreatic necrosis virus (IPNV) status of an apparently healthy turbot broodstock. Blood samples were used to detect IPNV by reverse transcriptase-polymerase chain reaction (RT-PCR), Southern blot hybridization and nested PCR. In addition, viral isolation from turbot leucocytes was performed. Around 22% of the fish were IPNV positive by RT-PCR, and this increased to close to 60% when nested PCR was performed. The present report supports the use of blood samples for the detection of IPNV-like viruses in brood fish. In addition, we demonstrate that it is possible to isolate the virus from the blood of carrier fish, as a non-lethal detection method, although it is much less sensitive than RT-PCR and nested PCR as a IPNV-like strain was isolated from only five of the 15 blood sample pools assayed. The viral isolate was identified as type Dry Mills (genogroup I) by means of restriction fragment length polymorphisms and DNA sequencing.

A dot-blot hybridization test has been developed for the detection of infectious pancreatic necrosis virus (IPNV) in infected fish. For this purpose, cloning of the dsRNA of the West Buxton strain of IPNV was carried out. Two cDNA clones (WB and A4) were characterized for use as diagnostic probes and corresponded to IPNV genome segments A and B. respectively. Clone WB1, with an insert of 812 base pairs, showed an 87 and 77% nuclcotidc sequence homology with the corresponding sequences of Jasper and N1 strains, respectively. Clone A4, with an insert size of 596bp, presented a nuclcotidc sequence homology of 90 and 80% with the corresponding sequences of the Jasper and Sp strains, respectively. Both probes were able to detect 15 ng of purified dsRNA, and were highly efficient in detecting the RNA of American IPNV strains. However, the A4 probe was less effective than WB1 in hybridizing to RNA from European and Spanish strains of IPNV. Both probes detected IPNV RNA in cells 4–8h post-infection with the homologous West Buxton strain, 8–12h post-infection with other American strains and 24h post-infection with the European strains of IPNV. The method was less sensitive in detecting IPNV RNA directly in infected fish tissues. However, the present authors obtained a 100% effectiveness to detect viral RNA in cells inoculated with fish tissues confirmed by conventional diagnostic methods as being infected with IPNV. Therefore, the hybridization test is appropriate if combined with conventional diagnostic procedures, e.g. applying the dot blot hybridization test on tissue cultures 12–24 h after inoculation with infected fish tissue homogenates.

An antigenic comparison of five rotaviruses isolated from fish in Spain, the USA and Canada was carried out using neutralization, immunodot and ELISA tests. The Spanish isolate (turbot rotavirus) was serologically different from the other four rotaviruses, by neutralization and immunodot. By these two techniques, among the four American strains the two isolates from Canada (smelt rolavirus and Atlantic salmon rotavirus) were closely related antigenically, and the two strains from USA (striped bass rotavirus and the Hetrick & Baya rotavirus, isolated from salmon) seemed to be the same scrotype. These results were supported by those obtained in the comparison of the RNA patterns of the five viruses. However, the antigenie relationships among these rotaviruses were very different when employing the ELISA technique.

In this study, we report the sequencing of the whole genome [including the 5' and 3' non-coding regions (NCR) of both segments A and B] of seven birnavirus strains isolated from wild fish from the Flemish Cap (FC) fishery at Newfoundland, Canada. From analysis and comparison of the sequences, most of the FC isolates clustered with the North American reference strains West Buxton (WB), Dry Mill and Jasper. One strain was included in the same genotype as the European strain Ab. In addition, at least in one case cohabitation of both type strains in an individual fish was demonstrated. These results clearly suggest the acquisition of the viruses from two different sources. The prevalence of the American type is easily explained by the close proximity of this fishing bank to the American coast whereas, although surprising, the presence of the European type strain could be because of migration of fish from European waters. In one strain, segment A and B sequences were typed differently (WB and Ab, respectively). These findings indicate natural reassortment between two strains of aquabirnaviruses in a host.

The susceptibility of turbot, Psetta maxima, to infection with two strains of viral haemorrhagic septicaemia virus (VHSV) obtained from wild Greenland halibut, Reinhardtius hippoglossoides, and from farmed turbot was examined. A marine VHSV strain known to be highly pathogenic for turbot was also utilized for comparative purposes. Fish were infected by intra-peritoneal (i.p.), immersion or cohabitation, and maintained at two different temperatures (8 and 15 degrees C). Infection trials showed that the three VHSV isolates were pathogenic for turbot fingerlings by i.p. injection at both temperatures, with high levels of mortality. Virus was recovered from most pools of dead fish i.p. challenged, but not from surviving fish. Although clinical signs were not induced following waterborne exposure, viral growth was obtained from some pools of surviving fish challenged by immersion with strain GH40 from Greenland halibut, which indicates that the virus can survive in sea water and infect other fish via horizontal transmission. Furthermore, although low, the clinical signs and mortality observed in fish cohabitating with turbot challenged with strain GH40 confirms horizontal transmission and indicates that the passage through fish increases the virulence of this strain for turbot. These findings indicate that Greenland halibut, as other wild fish, may play an important role in the epizootiology of VHSV and suggest a potential risk for the turbot farming industry.

Betanodaviruses are the causative agents of viral nervous necrosis (VNN) or viral encephalopathy and retinopathy (VER) in cultured marine fish. Based on the RNA2 gene fish nodaviruses have been traditionally classified into four different genotypes and recently a fifth genotype has been proposed. This study presents sequencing data of 24 new nodaviruses obtained from three different fish species: sea bass, Dicentrarchux labrax (L.), sea bream, Sparus aurata L., and Senegalese sole, Solea senegalensis Kaup, cultured in the Iberian Peninsula (Spain and Portugal). Sequence analysis was performed on the T4 region (388 nt) of the coat protein gene. In addition, phylogenetic analysis, according to maximum parsimony and neighbour-joining methods, was performed using these sequences and other nucleotide sequences available in the databases or in the literature. Results obtained indicate that all these new nodaviruses should be classified into the striped jack nervous necrosis virus (SJNNV) genotype. This finding suggests that SJNNV genotype is emerging in the Iberian Peninsula and could easily spread throughout the Mediterranean, representing a serious threat to the fish farming industry.