Publications by authors named "I Bandín"

52 Publications

Design and Evaluation of a Macroarray for Detection, Identification, and Typing of Viral Hemorrhagic Septicemia Virus (VHSV).

Animals (Basel) 2021 Mar 16;11(3). Epub 2021 Mar 16.

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

The viral hemorrhagic septicemia virus (VHSV) is the causative agent of an important disease in freshwater and marine fishes. Its diagnosis officially relies on the isolation of the virus in cell culture and its identification by serological or polymerase chain reaction (PCR) methodologies. Nowadays, reverse transcription real-time quantitative PCR (RT-qPCR) is the most widely employed technique for the detection of this virus and some studies have reported the validation of RT-qPCR procedures for the detection, typing, and quantification of VHSV isolates. However, although the efficacy of this technique is not in doubt, it can be cumbersome and even impractical when it comes to processing large numbers of samples, a situation in which cross-contamination problems cannot be ruled out. In the present study, we have designed and validated a macroarray for the simultaneous detection, typing, and quantification of VHSV strains. Its analytical sensitivity (5-50 TCID/), analytical specificity (intra and intergroup), efficiency (E = 100.0-101.1) and reliability (repeatability and reproducibility with CV < 5%, and standard curves with R < 0.95) with strains from any VHSV genotype have been widely demonstrated. The procedure is based on the 'binary multiplex RT-qPCR system (bmRT-qPCR)' previously reported by the same team, applied to arrays of 96-well PCR strip tubes plates, which can be stored at -25 °C for three months and up to one year before their use, without significant loss of efficiency.
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http://dx.doi.org/10.3390/ani11030841DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8002285PMC
March 2021

Immunogene expression analysis in betanodavirus infected-Senegalese sole using an OpenArray® platform.

Gene 2021 Mar 11;774:145430. Epub 2021 Jan 11.

Universidad de Málaga, Instituto de Biotecnología y Desarrollo Azul, IBYDA, Departamento de Microbiología, Facultad de Ciencias, Málaga, Spain. Electronic address:

The transcriptomic response of Senegalese sole (Solea senegalensis) triggered by two betanodaviruses with different virulence to that fish species has been assessed using an OpenArray® platform based on TaqMan™ quantitative PCR. The transcription of 112 genes per sample has been evaluated at two sampling times in two organs (head kidney and eye/brain-pooled samples). Those genes were involved in several roles or pathways, such as viral recognition, regulation of type I (IFN-1)-dependent immune responses, JAK-STAT cascade, interferon stimulated genes, protein ubiquitination, virus responsive genes, complement system, inflammatory response, other immune system effectors, regulation of T-cell proliferation, and proteolysis and apoptosis. The highly virulent isolate, wSs160.3, a wild type reassortant containing a RGNNV-type RNA1 and a SJNNV-type RNA2 segments, induced the expression of a higher number of genes in both tested organs than the moderately virulent strain, a recombinant harbouring mutations in the protruding domain of the capsid protein. The number of differentially expressed genes was higher 2 days after the infection with the wild type isolate than at 3 days post-inoculation. The wild type isolate also elicited an exacerbated interferon 1 response, which, instead of protecting sole against the infection, increases the disease severity by the induction of apoptosis and inflammation-derived immunopathology, although inflammation seems to be modulated by the complement system. Furthermore, results derived from this study suggest a potential important role for some genes with high expression after infection with the highly virulent virus, such as rtp3, sacs and isg15. On the other hand, the infection with the mutant does not induce immune response, probably due to an altered recognition by the host, which is supported by a different viral recognition pathway, involving myd88 and tbkbp1.
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http://dx.doi.org/10.1016/j.gene.2021.145430DOI Listing
March 2021

Steps of the Replication Cycle of the Viral Haemorrhagic Septicaemia Virus (VHSV) Affecting Its Virulence on Fish.

Animals (Basel) 2020 Dec 1;10(12). Epub 2020 Dec 1.

Instituto de Acuicultura-Dpt Microbiología, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain.

The viral haemorrhagic septicaemia virus (VHSV), a single-stranded negative-sense RNA novirhabdovirus affecting a wide range of marine and freshwater fish species, is a main concern for European rainbow trout () fish farmers. Its genome is constituted by six genes, codifying five structural and one nonstructural proteins. Many studies have been carried out to determine the participation of each gene in the VHSV virulence, most of them based on genome sequence analysis and/or reverse genetics to construct specific mutants and to evaluate their virulence phenotype. In the present study, we have used a different approach with a similar aim: hypothesizing that a failure in any step of the replication cycle can reduce the virulence in vivo, we studied in depth the in vitro replication of VHSV in different cell lines, using sets of strains from different origins, with high, low and moderate levels of virulence for fish. The results demonstrated that several steps in the viral replication cycle could affect VHSV virulence in fish, including adsorption, RNA synthesis and morphogenesis (including viral release). Notably, differences among strains in any step of the replication cycle were mostly strain-specific and reflected only in part the in vivo phenotype (high and low virulent). Our data, therefore, support the need for further studies aimed to construct completely avirulent VHSV recombinants targeting a combination of genes rather than a single one in order to study the mechanisms of genes interplay and their effect on viral phenotype in vitro and in vivo.
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http://dx.doi.org/10.3390/ani10122264DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7761041PMC
December 2020

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

Betanodavirus and VER Disease: A 30-year Research Review.

Pathogens 2020 Feb 9;9(2). Epub 2020 Feb 9.

Departamento de Microbioloxía e Parasitoloxía-Instituto de Acuicultura. Universidade de Santiago de Compostela. 15782 Santiago de Compostela, Spain.

The outbreaks of viral encephalopathy and retinopathy (VER), caused by nervous necrosis virus (NNV), represent one of the main infectious threats for marine aquaculture worldwide. Since the first description of the disease at the end of the 1980s, a considerable amount of research has gone into understanding the mechanisms involved in fish infection, developing reliable diagnostic methods, and control measures, and several comprehensive reviews have been published to date. This review focuses on host-virus interaction and epidemiological aspects, comprising viral distribution and transmission as well as the continuously increasing host range (177 susceptible marine species and epizootic outbreaks reported in 62 of them), with special emphasis on genotypes and the effect of global warming on NNV infection, but also including the latest findings in the NNV life cycle and virulence as well as diagnostic methods and VER disease control.
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http://dx.doi.org/10.3390/pathogens9020106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7168202PMC
February 2020

Capsid amino acids at positions 247 and 270 are involved in the virulence of betanodaviruses to European sea bass.

Sci Rep 2019 Oct 1;9(1):14068. Epub 2019 Oct 1.

Universidad de Málaga, Departamento de Microbiología, Málaga, Spain.

European sea bass (Dicentrarchus labrax) is severely affected by nervous necrosis disease, caused by nervous necrosis virus (NNV). Two out of the four genotypes of this virus (red-spotted grouper nervous necrosis virus, RGNNV; and striped jack nervous necrosis virus, SJNNV) have been detected in sea bass, although showing different levels of virulence to this fish species. Thus, sea bass is highly susceptible to RGNNV, whereas outbreaks caused by SJNNV have not been reported in this fish species. The role of the capsid protein (Cp) amino acids 247 and 270 in the virulence of a RGNNV isolate to sea bass has been evaluated by the generation of recombinant RGNNV viruses harbouring SJNNV-type amino acids in the above mentioned positions (Mut247Dl965, Mut270Dl965 and Mut247 + 270Dl965). Viral in vitro and in vivo replication, virus virulence and fish immune response triggered by these viruses have been analysed. Mutated viruses replicated on E-11 cells, although showing some differences compared to the wild type virus, suggesting that the mutations can affect the viral cell recognition and entry. In vivo, fish mortality caused by mutated viruses was 75% lower, and viral replication in sea bass brain was altered compared to non-mutated virus. Regarding sea bass immune response, mutated viruses triggered a lower induction of IFN I system and inflammatory response-related genes. Furthermore, mutations caused changes in viral serological properties (especially the mutation in amino acid 270), inducing higher seroconversion and changing antigen recognition.
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http://dx.doi.org/10.1038/s41598-019-50622-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6773868PMC
October 2019

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

Transcriptomic Profiles of Senegalese Sole Infected With Nervous Necrosis Virus Reassortants Presenting Different Degree of Virulence.

Front Immunol 2018 17;9:1626. Epub 2018 Jul 17.

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

Betanodaviruses [nervous necrosis virus (NNV)] are the causative agent of the viral encephalopathy and retinopathy, a disease that affects cultured Senegalese sole (). NNV reassortants, combining genomic segments from redspotted grouper nervous necrosis virus (RGNNV) and striped jack nervous necrosis virus (SJNNV) genotypes, have been previously isolated from several fish species. The wild-type reassortant wSs160.03, isolated from Senegalese sole, has been proven to be more virulent to sole than the parental genotypes (RGNNV and SJNNV), causing 100% mortality. Mutations at amino acids 247 (serine to alanine) and 270 (serine to asparagine) in the wSs160.03 capsid protein have allowed us to obtain a mutant reassortant (rSs160.03), which provokes a 40% mortality decrease. In this study, the RNA-Seq technology has been used to comparatively analyze Senegalese sole transcriptomes in two organs (head kidney and eye/brain) after infection with wild-type and mutant strains. A total of 633 genes were differentially expressed (DEGs) in animals infected with the wild-type isolate (with higher virulence), whereas 393 genes were differentially expressed in animals infected with the mutant strain (37.9% decrease in the number of DEGs). To study the biological functions of detected DEGs involved in NNV infection, a gene ontology (GO) enrichment analysis was performed. Different GO profiles were obtained in the following subclasses: (i) biological process; (ii) cellular component; and (iii) molecular function, for each viral strain tested. Immune response and proteolysis have been the predominant biological process after the infection with the wild-type isolate, whereas the infection with the mutant strain induces proteolysis in head kidney and inhibition of vasculogenesis in nervous tissue. Regarding the immune response, genes coding for proteins acting as mediators of type I IFN expression () and IFN-stimulated genes (, to name a few) were upregulated in animals infected with the wild-type isolate, whereas no-differential expression of these genes was observed in samples inoculated with the mutant strain. The different transcriptomic profiles obtained could help to better understand the NNV pathogenesis in Senegalese sole, setting up the importance as virulence determinants of amino acids at positions 247 and 270 within the RNA2 segment.
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http://dx.doi.org/10.3389/fimmu.2018.01626DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6056728PMC
July 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

Molecular characterization and expression analyses of the Solea senegalensis interferon-stimulated gene 15 (isg15) following NNV infections.

Fish Shellfish Immunol 2017 Jul 17;66:423-432. Epub 2017 May 17.

Universidad de Málaga, Departamento de Microbiología, Facultad de Ciencias, Campus de Teatinos s/n, 29071 Málaga, Spain. Electronic address:

Interferons are essential in fish resistance to viral infections. They induce interferon-stimulated genes, such as isg15. In this study, the Senegalese sole isg15 gene (ssisg15) has been characterized. As other isg15, ssisg15 contains a 402-bp intron sited in the 5'-UTR, and the full length cDNA is 1492-bp, including a 480-bp ORF. The expression analyses revealed basal levels of isg15 transcripts, and a clear induction after poly I:C injection, that reached maximum values in brain, head kidney and gills. The ssisg15 induction patterns were similar in RGNNV- and SJNNV-inoculated fish, whereas the reassortant (RG/SJ) isolate, which has higher replication fitness, triggered delayed but higher transcript levels. Furthermore, RG/SJ infection after poly I:C treatment reduced the induction of ssisg15 transcripts, suggesting an antagonistic mechanism against interferon type I system, that might allow an efficient viral replication at the initial steps of the infective process.
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http://dx.doi.org/10.1016/j.fsi.2017.05.040DOI Listing
July 2017

In vivo study of viral haemorrhagic septicaemia virus and infectious pancreatic necrosis virus coexistence in Senegalese sole (Solea senegalensis).

J Fish Dis 2017 Sep 29;40(9):1129-1139. Epub 2016 Dec 29.

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

The effect of IPNV-VHSV coinfection and superinfection on the mortality caused by both viruses in Senegalese sole has been analysed. No effect was observed after coinfection. However, a clear viral interference was recorded between a primary IPNV and a subsequent VHSV infection, which led to a survival increase in the infected sole of 50% points when compared with fish infected only with VHSV. The significantly higher Mx transcriptional values in the fish pre-exposed to IPNV (at least at first days after superinfection) and the increased daily mortality when low Mx transcriptional levels were recorded suggest that Mx may be involved in the protective effect against VHSV infection. However, in fish subjected to VHSV primary/IPNV secondary infection, no interference was observed.
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http://dx.doi.org/10.1111/jfd.12585DOI 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

Aquabirnavirus polyploidy: a new strategy to modulate virulence?

J Gen Virol 2016 May;97(5):1168-1177

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

One of the main research issues regarding infectious pancreatic necrosis virus (IPNV) is its virulence mechanisms. The basis for understanding the molecular virulence determinants of this virus was established over the last decade when it was demonstrated that certain amino acid domains in the VP2 and VP2-NS inter-region determined the level of virulence of IPNV. However, certain variability was still inexplicable and therefore other factors may also be involved. To this end, it was demonstrated recently that infectious bursal disease virus (IBDV), a virus in a different genus of the same family as IPNV, can package more than two dsRNA segments, and that polyploidy may be associated with virulence. In the present report, we analysed the viral fractions obtained after gradient centrifugation to demonstrate that IPNV virions can also package more than two segments, thus indicating that polyploidy is a common birnavirus trait. The differential replication and virulence additionally suggested that such a characteristic is involved in the modulation of virus infectivity. However, although the results clearly demonstrated that the replication capacity was enhanced as the viral ploidy increased, the results could not strongly support a direct relationship between ploidy and virulence to the host, thus suggesting that other virulence determinants are also involved.
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http://dx.doi.org/10.1099/jgv.0.000434DOI Listing
May 2016

Role of the IFN I system against the VHSV infection in juvenile Senegalese sole (Solea senegalensis).

Vet Res 2016 Jan 8;47. Epub 2016 Jan 8.

Departamento de Microbiología, Facultad de Ciencias, Universidad de Málaga, 29071, Málaga, Spain.

Senegalese sole is susceptible to marine VHSV isolates but is not affected by freshwater isolates, which may indicate differences regarding virus-host immune system interaction. IFN I induces an antiviral state in fish, stimulating the expression of genes encoding antiviral proteins (ISG). In this study, the stimulation of the Senegalese sole IFN I by VHSV infections has been evaluated by the relative quantification of the transcription of several ISG (Mx, Isg15 and Pkr) after inoculation with marine (pathogenic) and freshwater (non-pathogenic) VHSV isolates. Compared to marine VHSV, lower levels of RNA of the freshwater VHSV induced transcription of ISG to similar levels, with the Isg15 showing the highest fold induction. The protective role of the IFN I system was evaluated in poly I:C-inoculated animals subsequently challenged with VHSV isolates. The cumulative mortality caused by the marine isolate in the control group was 68%, whereas in the poly I:C-stimulated group was 5%. The freshwater VHSV isolate did not cause any mortality. Furthermore, viral RNA fold change and viral titers were lower in animals from the poly I:C + VHSV groups than in the controls. The implication of the IFN I system in the protection observed was confirmed by the transcription of the ISG in animals from the poly I:C + VHSV groups. However, the marine VHSV isolate exerts a negative effect on the ISG transcription at 3 and 6 h post-inoculation (hpi), which is not observed for the freshwater isolate. This difference might be partly responsible for the virulence shown by the marine isolate.
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http://dx.doi.org/10.1186/s13567-015-0299-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4705576PMC
January 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

Real-time RT-PCR for detection, identification and absolute quantification of viral haemorrhagic septicaemia virus using different types of standards.

Dis Aquat Organ 2015 May;114(2):99-116

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

In the present study, 2 systems of real-time RT-PCR-one based on SYBR Green and the other on TaqMan-were designed to detect strains from any genotype of viral haemorrhagic septicaemia virus (VHSV), with high sensitivity and repeatability/reproducibility. In addition, the method was optimized for quantitative purposes (qRT-PCR), and standard curves with different types of reference templates were constructed and compared. Specificity was tested against 26 isolates from 4 genotypes. The sensitivity of the procedures was first tested against cell culture isolation, obtaining a limit of detection (LD) of 100 TCID50 ml-1 (100-fold below the LD using cell culture), at a threshold cycle value (Ct) of 36. Sensitivity was also evaluated using RNA from crude (LD = 1 fg; 160 genome copies) and purified virus (100 ag; 16 copies), plasmid DNA (2 copies) and RNA transcript (15 copies). No differences between both chemistries were observed in sensitivity and dynamic range. To evaluate repeatability and reproducibility, all experiments were performed in triplicate and on 3 different days, by workers with different levels of experience, obtaining Ct values with coefficients of variation always <5. This fact, together with the high efficiency and R2 values of the standard curves, encouraged us to analyse the reliability of the method for viral quantification. The results not only demonstrated that the procedure can be used for detection, identification and quantification of this virus, but also demonstrated a clear correlation between the regression lines obtained with different standards, which will help scientists to compare sensitivity results between different studies.
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http://dx.doi.org/10.3354/dao02840DOI Listing
May 2015

Experimental susceptibility of European sea bass and Senegalese sole to different betanodavirus isolates.

Vet Microbiol 2015 May 4;177(1-2):53-61. Epub 2015 Mar 4.

Universidad de Santiago de Compostela, Instituto de Acuicultura, Constantino Candeira C.P.: 15705, Santiago de Compostela, A Coruña, Spain.

The susceptibility of juvenile European sea bass and Senegalese sole to three VNNV isolates (a reassortant RGNNV/SJNNV, as well as the parental RGNNV and SJNNV genotypes) has been evaluated by challenges using two inoculation ways (bath and intramuscular injection). The results demonstrate that these two fish species are susceptible to all the VNNV isolates tested. In European sea bass, RGNNV caused the highest cumulative mortality, reaching maximum values of viral RNA and titres. Although the SJNNV isolate did not provoke mortality or clinical signs of disease in this fish species, viral production in survivor fish was determined; on the other hand the reassortant isolate did cause mortality and clinical signs of disease, although less evident than those recorded after RGNNV infection. These results suggest that the changes suffered by the SJNNV RNA2 segment of the reassortant isolate, compared to the parental SJNNV, may have involved host-specificity and/or virulence determinants for European sea bass. Regarding Senegalese sole, although the three isolates caused 100% mortality, the reassortant strain provoked the most acute symptoms, and more quickly, especially in the bath challenge. This was also the isolate showing less difference between the number of RNA copies and viral titre, reaching the highest titres of infective viral particles in nervous tissue of infected animals. The RGNNV isolate produced the lowest values of infective viral particles. All these results suggest that the RGNNV and the reassortant isolates are the most suited for infecting European sea bass and Senegalese sole, respectively.
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http://dx.doi.org/10.1016/j.vetmic.2015.02.030DOI Listing
May 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

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

Use of reverse transcription-real time polymerase chain reaction (real time RT-PCR) assays with Universal Probe Library (UPL) probes for the detection and genotyping of infectious pancreatic necrosis virus strains isolated in Chile.

J Virol Methods 2012 Jul 30;183(1):80-5. Epub 2012 Mar 30.

Laboratorio de Patología de Organismos Acuáticos y Biotecnología Acuícola, Facultad de Ciencias Biológicas, Universidad Andrés Bello, Viña del Mar, Chile.

Reverse transcription-real time polymerase chain reaction (real time RT-PCR) assay with Universal Probe Library (UPL) probes has been developed for the detection and genotyping of Chilean infectious pancreatic necrosis virus (IPNV) isolates from infected cell culture. Partial nucleotide sequences (1175 bp) of the VP2 coding region from a selection of 7 Chilean IPNV isolates showed that they clustered into two main groups strongly correlated with Genogroups 1 and 5 proposed by Blake et al. (2001), corresponding to types West Buxton (WB) and Spajarup (Sp), respectively. Based on the VP2 gene sequences of those 7 Chilean isolates and different reference IPNV strains, 2 sets of candidate primer/UPL probes (# 8 and # 117) were designed and evaluated with a total of 32 field isolates isolated from Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss) and Pacific salmon (Oncorhynchus kisutch) farms from 2006 to 2010 in Chile. The UPL probes clearly differentiated the same two major Genogroups that those recognized by sequencing analysis. Among the Chilean isolates examined, 18 yielded amplification with UPL probe # 8, and 14 with probe # 117, respectively corresponding to types Sp and WB, as demonstrated by typing by sequencing. Based on the findings reported below, it has been demonstrated that the combined real time RT-PCR protocol with UPLs approach was efficient in discriminating distinct Genogroups of IPNV cultured in fish cell lines and, therefore, recommended its use for detection and typing of IPN viruses. The study also confirmed the existence of two IPNV type strains in Chilean salmonid aquaculture.
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http://dx.doi.org/10.1016/j.jviromet.2012.03.022DOI Listing
July 2012

Antiviral properties of polymeric aziridine- and biguanide-modified core-shell magnetic nanoparticles.

Langmuir 2012 Mar 22;28(9):4548-58. Epub 2012 Feb 22.

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.

Polycationic superparamagnetic nanoparticles (∼150-250 nm) were evaluated as virucidal agents. The particles possess a core-shell structure, with cores consisting of magnetite clusters and shells of functional silica covalently bound to poly(hexamethylene biguanide) (PHMBG), polyethyleneimine (PEI), or PEI terminated with aziridine moieties. Aziridine was conjugated to the PEI shell through cationic ring-opening polymerization. The nanometric core-shell particles functionalized with biguanide or aziridine moieties are able to bind and inactivate bacteriophage MS2, herpes simplex virus HSV-1, nonenveloped infectious pancreatic necrosis virus (IPNV), and enveloped viral hemorrhagic septicaemia virus (VHSV). The virus-particle complexes can be efficiently removed from the aqueous milieu by simple magnetocollection.
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http://dx.doi.org/10.1021/la205127xDOI Listing
March 2012

Host range, host specificity and hypothesized host shift events among viruses of lower vertebrates.

Vet Res 2011 May 18;42:67. Epub 2011 May 18.

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

The successful replication of a viral agent in a host is a complex process that often leads to a species specificity of the virus and can make interspecies transmission difficult. Despite this difficulty, natural host switch seems to have been frequent among viruses of lower vertebrates, especially fish viruses, since there are several viruses known to be able to infect a wide range of species. In the present review we will focus on well documented reports of broad host range, variations in host specificity, and host shift events hypothesized for viruses within the genera Ranavirus, Novirhabdovirus, Betanodavirus, Isavirus, and some herpesvirus.
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http://dx.doi.org/10.1186/1297-9716-42-67DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3125225PMC
May 2011

Susceptibility of juvenile sole Solea senegalensis to marine isolates of viral haemorrhagic septicaemia virus from wild and farmed fish.

Dis Aquat Organ 2011 Jan;93(2):111-6

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

The susceptibility of sole Solea senegalensis to infection with 3 viral haemorrhagic septicaemia virus (VHSV) strains obtained from wild Greenland halibut Reinhardtius hippoglossoides and farmed turbot Psetta maxima was demonstrated. Fish were infected by an intraperitoneal (i.p.), immersion or cohabitational route, and maintained at 16 degrees C. Infection trials showed that VHSV isolates were pathogenic for sole fingerlings by i.p. injection and waterborne exposure causing moderate levels of mortality (10 to 55%). In addition, the mortality observed in fish cohabitating with i.p.-infected sole confirms horizontal transmission of the virus. However, the low rates of mortality registered in this challenge suggest that there is a low dissemination of virus by the i.p.-infected sole, which results in lower secondary challenge of the cohabitating fish. External signs of disease included haemorrhaging of the ventral area and ascitic fluid in the body cavity. Dead fish were tested for VHSV by both cell culture and RT-PCR assay, using pools of kidney and spleen from 10 individuals. Virus was recovered from most of the pools composed of dead fish. The results obtained in this study not only demonstrate the susceptibility of sole to the VHSV strains employed but also indicate that wild VHSV marine isolates represent a potential risk for sole aquaculture.
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http://dx.doi.org/10.3354/dao02299DOI Listing
January 2011

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