Publications by authors named "J G Olveira"

25 Publications

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Role of rotifer () and () nauplii in the horizontal transmission of a natural nervous necrosis virus (NNV) reassortant strain to Senegalese sole () larvae.

Vet Q 2020 Dec;40(1):205-214

Instituto de Acuicultura, Departamento de Microbiología y Parasitología, Universidade de Santiago de Compostela, Santiago de Compostela, Spain.

Background: Marine invertebrates are provided as a first feed for marine fish larvae because of their strict nutritional requirements, despite also being a potential source of infectious agents.

Aim: To assess horizontal transmission of a nervous necrosis virus reassortant strain (NNV) to sole larvae via and rotifers.

Materials And Methods: Rotifer () and () nauplii cultures were bath infected with a reassortant (RGNNV/SJNNV) NNV strain isolated from gilthead sea bream and viral internalisation was confirmed by IFA. Senegalese sole () larvae were fed on infected and disease signs and mortality were recorded. In addition, NNV viability was checked in cultures of either unfed invertebrates or invertebrates fed on phytoplankton and in the supernatant of microalgae cultures. All samples were tested by RT-qPCR and inoculation in cell culture.

Results: Both rotifers and internalised NNV. Experimental transmission to sole larvae was achieved using infected and subsequently 60% mortality was recorded. At 24 h post-infection, orally infected individuals contained 9.34 × 10 copies of viral RNA, whereas the bath infection yielded 2.05 × 10 RNA copies larvae. Viral presence in both invertebrates was detected up to 8 days post infection but viral load decreased over time. Feeding with microalgae decreased viral detection even more and microalgae supernatants were demonstrated to significantly affect NNV viability.

Conclusions: Our results demonstrate that both invertebrates can bioaccumulate NNV and that Senegalese sole larvae fed on infected might develop viral encephalopathy and retinopathy and high mortality.
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http://dx.doi.org/10.1080/01652176.2020.1810357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7734120PMC
December 2020

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

Vet Res 2019 Jun 21;50(1):50. Epub 2019 Jun 21.

Instituto de Acuicultura, Departamento de Microbiología y Parasitología-Universidade de Santiago de Compostela, 15706, Santiago de Compostela, Spain.

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.
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http://dx.doi.org/10.1186/s13567-019-0669-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6588924PMC
June 2019

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

J Fish Dis 2019 Feb 3;42(2):221-227. Epub 2018 Dec 3.

Departamento de Microbiología y Parasitología, Instituto de Acuicultura, Universidade de Santiago de Compostela, Santiago de Compostela, Spain.

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.
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http://dx.doi.org/10.1111/jfd.12916DOI Listing
February 2019

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

Fish Shellfish Immunol 2019 Mar 11;86:14-24. Epub 2018 Nov 11.

Fish Innate Immune System Group, Department of Cell Biology and Histology, Faculty of Biology, Campus Regional de Excelencia Internacional "Campus Mare Nostrum", University of Murcia, 30100 Murcia, Spain. Electronic address:

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.
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http://dx.doi.org/10.1016/j.fsi.2018.11.024DOI Listing
March 2019

Betanodavirus infection in primary neuron cultures from sole.

Vet Res 2018 Sep 5;49(1):86. Epub 2018 Sep 5.

Departamento de Microbiología y Parasitología, Instituto de Acuicultura, Universidade de Santiago de Compostela, 15706, Santiago de Compostela, Spain.

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.
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http://dx.doi.org/10.1186/s13567-018-0580-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6125867PMC
September 2018

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

J Gen Virol 2018 09 24;99(9):1210-1220. Epub 2018 Jul 24.

1​Departamento de Microbiología y Parasitología, Instituto de Acuicultura, Universidade de Santiago de Compostela, 15706 Santiago de Compostela, Spain.

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.
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http://dx.doi.org/10.1099/jgv.0.001112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6230769PMC
September 2018

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

J Fish Dis 2018 Oct 20;41(10):1571-1578. Epub 2018 Jul 20.

Departamento de Microbiología y Parasitología, Instituto de Acuicultura, Universidade de Santiago de Compostela, Santiago de Compostela, Spain.

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.
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http://dx.doi.org/10.1111/jfd.12865DOI Listing
October 2018

The theoretical reliability of PCR-based fish viral diagnostic methods is critically affected when they are applied to fish populations with low prevalence and virus loads.

J Appl Microbiol 2018 Apr 22;124(4):977-989. Epub 2017 Oct 22.

Departamento de Microbiología, Facultad de Ciencias, Universidad de Malaga, Malaga, Spain.

Aims: The reliability of polymerase chain reaction (PCR) techniques is an important issue in viral diagnosis, and it is even crucial when they must be applied for detection of viruses in asymptomatic carriers. The problems will arise when the aim is to study wild fish populations, where the viral loads and prevalence values are extremely low. We have evaluated several PCR procedures employed by two laboratories for monitoring fish captured in several oceanographic campaigns performed in the Gulf of Cádiz.

Methods And Results: To evaluate the reliability of different diagnostics test used, we have re-analysed fish samples that had been previously subjected to diagnosis for a surveillance of viruses performed in 2010-2011 in wild fish populations. The following parameters were employed: the clinical sensitivity (Ss), the clinical specificity (Sp), the predictive positive value, the predictive negative value, and the positive and negative likelihood ratio (LR and LR ). For viral nervous necrosis virus, a RT-PCR procedure supplemented by nested PCR showed the highest values (100%) for all the parameters. For viral haemorrhagic septicaemia virus, the highest values were provided by RT-PCR supplemented by dot-blot hybridization. In the case of infectious pancreatic necrosis virus, none of the procedures yielded 100% for any parameter.

Conclusions: The results obtained for viral prevalence indicate: (i) that the conservation of the samples at -80°C did not affect to the capacity of detection of the virus in the tissues, and (ii) that the reproducibility of the diagnosis can be affected by factors including the staff experience and/or the materials employed. Finally, the use of a combination of procedures in advised to ensure the maximum reliability of the diagnosis when it is applied to asymptomatic fish populations.

Significance And Impact Of The Study: This paper describes a strategy of combining diagnostic tests for the surveillance and monitoring of wild fish populations to reduce underestimation of the prevalence of viruses this type of populations.
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http://dx.doi.org/10.1111/jam.13586DOI Listing
April 2018

Design and validation of a RT-qPCR procedure for diagnosis and quantification of most types of infectious pancreatic necrosis virus using a single pair of degenerated primers.

J Fish Dis 2017 Sep 27;40(9):1155-1167. Epub 2016 Dec 27.

Departamento de Microbiología y Parasitología, Instituto de Acuicultura, Universidade de Santiago de Compostela, Santiago de Compostela, Spain.

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 TCID  mL , 50 pfu mL or 66 RNA copies mL , depending on the standard. All the standard curves showed high reliability (R  > 0.95). The results support the high reliability of this new procedure for the diagnosis and quantification of IPNV.
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http://dx.doi.org/10.1111/jfd.12590DOI Listing
September 2017

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

Virology 2017 01 10;501:1-11. Epub 2016 Nov 10.

Departamento de Microbiología y Parasitología, Instituto de Acuicultura-Universidade de Santiago de Compostela, Santiago de Compostela 15706, Spain. Electronic address:

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.
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http://dx.doi.org/10.1016/j.virol.2016.11.003DOI Listing
January 2017

Reassortant betanodavirus infection in turbot (Scophthalmus maximus).

J Fish Dis 2016 Nov 2;39(11):1347-1356. Epub 2016 May 2.

Unidad de Ictiopatología-Patología Viral, Departamento de Microbiología y Parasitología, Instituto de Acuicultura, Universidad de Santiago de Compostela, Santiago de Compostela, Spain.

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 (rSs160.03 , rSs160.03 , rSs160.03 ) 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 rSs160.03 and rSs160.03 , 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.
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http://dx.doi.org/10.1111/jfd.12466DOI Listing
November 2016

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

PLoS One 2015 21;10(12):e0145131. Epub 2015 Dec 21.

Centro Oceanográfico de Murcia, Instituto Español de Oceanografía (IEO), Carretera de la Azohía s/n. Puerto de Mazarrón, Murcia, Spain.

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 17β-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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0145131PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4686992PMC
June 2016

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

Vet Microbiol 2015 Sep 8;179(3-4):162-7. Epub 2015 Jul 8.

Universidad de Santiago de Compostela, Departamento de Microbiología y Parasitología, Instituto de Acuicultura, Constantino Candeira, Santiago de Compostela, A Coruña CP-15705, Spain.

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.
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http://dx.doi.org/10.1016/j.vetmic.2015.07.004DOI Listing
September 2015

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

J Gen Virol 2015 Jun 27;96(Pt 6):1287-1296. Epub 2015 Jan 27.

Instituto de Acuicultura, Universidad de Santiago de Compostela, A Coruña, Spain.

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.
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http://dx.doi.org/10.1099/vir.0.000064DOI Listing
June 2015

Surveillance of viruses in wild fish populations in areas around the Gulf of Cadiz (South Atlantic Iberian Peninsula).

Appl Environ Microbiol 2014 Oct 15;80(20):6560-71. Epub 2014 Aug 15.

Unidad de Ictiopatología-Patologia Viral, Departamento de Microbiología y Parasitologia, Instituto de Acuicultura, Universidad de Santiago de Compostela, Santiago de Compostela, Spain

This report describes a viral epidemiological study of wild fish around the Gulf of Cadiz (southwestern Iberian Peninsula) and is focused on infectious pancreatic necrosis virus (IPNV), viral hemorrhagic septicemia virus (VHSV), and viral nervous necrosis virus (VNNV). One fish species (Chelon labrosus) was sampled inside the gulf, at the mouth of the San Pedro River. Another 29 were sampled, in three oceanographic campaigns, at sites around the Bay of Cadiz. The fish were processed individually and subjected to isolation in cell culture and molecular diagnosis. VHSV was not isolated from any species. Thirteen IPNV-type isolates were obtained from barracuda (Sphyraena sphyraena), axillary seabream (Pagellus acarne), common two-banded seabream (Diplodus vulgaris), common pandora (P. erythrinus), Senegal seabream (D. bellottii), and surmullet (Mullus surmuletus). Six VNNV isolates were obtained from axillary seabream, common pandora, black seabream (Spondyliosoma cantharus), red mullet (Mullet barbatus), Lusitanian toadfish (Halobatrachus didactylus), and tub gurnard (Chelidonichtys lucerna). In the river mouth, viruses were detected only after reamplification, obtaining prevalence percentages of IPNV and VNNV (44.4 and 63.0%, respectively) much higher than those observed in the oceanographic campaigns (25.7 and 19.6%, respectively). The opposite results were obtained in the case of VHSV after reamplification: 11.1% in the river mouth and 43.6% in the oceanic locations. Analyzing the results with respect to the proximity of the sampling sites to the coast, an anthropogenic influence on wild fish is suggested and discussed. The type of viruses and the presence of natural reassortants are also discussed.
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http://dx.doi.org/10.1128/AEM.02090-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4178629PMC
October 2014

Presence of viruses in wild eels Anguilla anguilla L, from the Albufera Lake (Spain).

J Fish Dis 2014 Jul 20;37(7):597-607. Epub 2014 May 20.

Unidad de Ictiopatología -Patología Viral, Departamento de Microbiología y Parasitología, Instituto de Acuicultura, Universidad de Santiago de Compostela, Santiago de Compostela, Spain.

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.
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http://dx.doi.org/10.1111/jfd.1392DOI Listing
July 2014

Effect of lipopolysaccharides from Vibrio alginolyticus on the Mx gene expression and virus recovery from gilthead sea bream (Sparus aurata L.) experimentally infected with Nodavirus.

Fish Shellfish Immunol 2013 Jan 22;34(1):383-6. Epub 2012 Oct 22.

Instituto Universitario de Sanidad Animal, Universidad de Las Palmas de Gran Canaria, Arucas, Spain.

Infections with nodavirus affect a wild and farmed fish species throughout the world, mostly from the marine environment. The aim of this work was to determine the immune status of gilthead sea bream that comes as a result of a Nodavirus infection, induced by activation of the interferon response pathway by lipopolysaccharides from Vibrio alginolyticus and the expression of interferoninduced Mx protein in liver samples. The enhancement of Mx protein gene expression was detected in liver samples of experimentally nodavirus infected fish and, furthermore, the immunostimulant LPS of V. alginolyticus decreased almost three times the virus titration with respect to no-immunized or infected with nodavirus group of fish.
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http://dx.doi.org/10.1016/j.fsi.2012.10.012DOI Listing
January 2013

Comparative analysis of both genomic segments of betanodaviruses isolated from epizootic outbreaks in farmed fish species provides evidence for genetic reassortment.

J Gen Virol 2009 Dec 26;90(Pt 12):2940-2951. Epub 2009 Aug 26.

Unidad de Ictiopatología-Patología Viral, Departamento de Microbiología y Parasitología, Instituto de Acuicultura, Universidad de Santiago de Compostela, Spain.

Sequencing of the full coding region of both genomic segments of seven betanodavirus strains isolated from different farmed species in Spain and Portugal revealed that six were reassortants, exhibiting a red-spotted grouper nervous necrosis virus (RGNNV)-type RNA1 and a striped jack nervous necrosis virus (SJNNV)-type RNA2. Analysis of sequences of reassortant strains at both the genomic and protein levels revealed the existence of differences compared with type strains of both genotypes. These differences were greater in the polymerase sequence, which is remarkable because viral structural proteins generally diverge more rapidly than non-structural proteins. Changes in two amino acids observed in the SJNNV capsid protein might be involved in the colonization of new host species by these reassortant strains. In addition, a more extensive phylogenetic analysis, including partial sequences of both RNA segments of 16 other Iberian nodaviruses, confirmed the existence of reassortment between RGNNV and SJNNV.
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http://dx.doi.org/10.1099/vir.0.013912-0DOI Listing
December 2009

Validation of real time RT-PCR applied to cell culture for diagnosis of any known genotype of viral haemorrhagic septicaemia virus.

J Virol Methods 2009 Dec 7;162(1-2):155-62. Epub 2009 Aug 7.

Unidad de Ictiopatología, Departamento de Microbiología y Parasitología, Instituto de Acuicultura, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain.

Viral haemorrhagic septicaemia virus (VHSV), a member of the Rhabdoviridae family, is a major viral pathogen of cultured salmonid fish, and also infects a wide range of marine fish species. In the present study, two real time PCR protocols (based on SYBR Green and TaqMan) were developed for the detection of strains belonging to all known genotypes of VHSV. Validation of the procedure, in terms of sensitivity, specificity and repeatability/reproducibility (R&R), was also performed. For this purpose, several pairs of primer amplifying regions corresponding to viral G and N genes were assayed. In the SYBR Green-based real time PCR, these primers failed to detect strains from some of the genotypes and/or showed low R&R. In order to improve the detection capacity, a multiplex procedure was designed, which enabled detection of all strains, with high R&R. The sensitivity of the procedure was measured, and a detection limit of 1 fg/microl of viral RNA or 10 copies of cloned plasmid was established. On the other hand, the TaqMan probe-based multiplex real time PCR detected all European strains, with similar levels of sensitivity and R&R, but failed to detect the American types.
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http://dx.doi.org/10.1016/j.jviromet.2009.07.033DOI Listing
December 2009

Antemortem versus postmortem methods for detection of betanodavirus in Senegalese sole (Solea senegalensis).

J Vet Diagn Invest 2008 Mar;20(2):215-9

Unidad de Ictiopatología-Patalogía Viral, Instituto de Acuicultura, Dpto. Microbiología y Parasitología, Universidad de Santiago de Compostela, Santiago de Compostela, Spain.

The suitability of nested reverse transcription polymerase chain reaction (nRT-PCR) to detect betanodavirus in blood samples from naturally infected Senegalese sole (Solea senegalensis) was evaluated in comparison with other diagnostic methods. Results indicated that histologic examination of brain lesions could be regarded as the most consistent indicator of nodavirus infection in this species. The nRT-PCR showed low to moderate levels of detection; the best values were obtained in brain samples followed by blood samples. Inoculation of SSN-1 and SAF-1 cells with fish samples did not cause cytopathic effect, although virus was detected by reverse transcription polymerase chain reaction in approximately 25% of the SSN-1 inoculated wells. The efficiency of detection of the viral genome was dramatically increased by the use of nRT-PCR, reaching 90.6% of positives in brain samples and 84.4% in blood samples. The sensitivity and the negative predictive value of nRT-PCR in blood samples were slightly lower than those obtained using brain samples. Nevertheless, it is suggested that the advantage of being able to perform diagnosis on live fish adequately counterbalances the slightly lower sensitivity of nRT-PCR on blood samples. This technique is proposed as a useful tool, not only for the selection of nodavirus-free breeders but also to check the fish status during ongrowing.
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http://dx.doi.org/10.1177/104063870802000212DOI Listing
March 2008

Emergence of pathogenic betanodaviruses belonging to the SJNNV genogroup in farmed fish species from the Iberian Peninsula.

J Fish Dis 2007 Apr;30(4):225-32

Unidad de Ictiopatología, Instituto de Acuicultura, Departamento de Microbiología y Parasitología, Universidad de Santiago de Compostela, Santiago de Compostela, Spain.

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.
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http://dx.doi.org/10.1111/j.1365-2761.2007.00803.xDOI Listing
April 2007

Susceptibility of the fish cell line SAF-1 to betanodavirus.

J Fish Dis 2006 Oct;29(10):633-6

Unidad de Ictiopatología, Instituto de Acuicultura, Departamento de Microbiología y Parasitología, Universidad de Santiago de Compostela, Santiago de Compostela, Spain.

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http://dx.doi.org/10.1111/j.1365-2761.2006.00757.xDOI Listing
October 2006

Isolation in cell culture and detection by PCR-based technology of IPNV-like virus from leucocytes of carrier turbot, Scophthalmus maximus (L.).

J Fish Dis 2005 Dec;28(12):713-22

Departamento de Microbiología y Parasitología, Unidad de Ictiopatología, Instituto de Acuicultura, Universidad de Santiago de Compostela, Santiago de Compostela, Spain.

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.
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http://dx.doi.org/10.1111/j.1365-2761.2005.00675.xDOI Listing
December 2005

Development of a rapid, sensitive and non-lethal diagnostic assay for the detection of viral haemorrhagic septicaemia virus.

J Virol Methods 2006 May 5;133(2):167-74. Epub 2005 Dec 5.

Unidad de Ictiopatología, Instituto de Acuicultura, Departamento de Microbiología y Parasitología, Universidad de Santiago de Compostela, Spain.

A non-lethal diagnostic procedure based on polymerase chain reaction (PCR) technology was developed to detect viral haemorrhagic septicaemia virus (VHSV). Sensitivity of the assay was tested using purified viral RNA and seeded tissues. Detection limits of the reverse transcriptase-polymerase chain reaction (RT-PCR) assay were estimated to be 10 fg of purified RNA and 0.97 x 10(3) or 10(0) TCID(50)/g of seeded tissue, depending on the experimental approach employed (viral adsorption allowed for 1 or 24h). Addition of nested PCR increased sensitivity up to 100-fold when cDNA excised from the agarose gel was used as template. Both, RT-PCR and nested RT-PCR, as well as Southern blot were applied to RNA extracted from blood of experimentally infected brown trout and the results were compared with those obtained by applying the same techniques to tissues and also with those of conventional viral isolation in cell culture. The superiority of the nested RT-PCR applied to blood samples has been clearly demonstrated in terms of sensitivity, obtaining positive results in 85% of fish tested, as against 40% obtained by RT-PCR and Southern blot, and only 5% viral isolations in cell culture. This procedure could turn into an important tool for screening of wild stocks as well as valuable individuals in commercial fish farms, since it makes to kill the fish unnecessary.
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http://dx.doi.org/10.1016/j.jviromet.2005.10.033DOI Listing
May 2006

Diversity of infectious pancreatic necrosis virus strains isolated from fish, shellfish, and other reservoirs in Northwestern Spain.

Appl Environ Microbiol 2000 Feb;66(2):839-43

Departamento de Microbioloxia e Parasitoloxia, Instituto de Acuicultura-Facultade de Bioloxia, Universidade de Santiago de Compostela, Santiago de Compostela 15706, Spain.

A comparison was done of 231 strains of birnavirus isolated from fish, shellfish, and other reservoirs in a survey study that began in 1986 in Galicia (northwestern Spain). Reference strains from all of the infectious pancreatic necrosis virus serotypes were included in the comparison, which was done by neutralization tests and agarose and polyacrylamide gel electrophoresis of the viral genome. The neutralization tests with antisera against the West Buxton, Spajarup (Sp), and Abild (Ab) strains showed that most of the Galician isolates were European types Sp and Ab; however, many isolates (30%) could not be typed. Results from agarose gels did not provided information for grouping of the strains, since all were found to have genomic segments of similar sizes. Analysis of polyacrylamide gels, however, allowed six electropherogroups (EGs) to be differentiated on the basis of genome mobility and separation among segments, and a certain relationship between EGs and serotypes was observed. A wide diversity of electropherotypes was observed among the Galician isolates, and as neutralization tests showed, most of the isolates were included in EGs corresponding to European types Ab and Sp. Only 6.5% of the isolates had the electropherotype characteristic of American strains.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC91907PMC
http://dx.doi.org/10.1128/aem.66.2.839-843.2000DOI Listing
February 2000