Publications by authors named "José G Olveira"

11 Publications

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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

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

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