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1.
J Appl Microbiol ; 124(4): 977-989, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28915317

RESUMO

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.


Assuntos
Doenças dos Peixes/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Viroses/veterinária , Vírus/isolamento & purificação , Animais , Doenças dos Peixes/diagnóstico , Peixes/virologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Viroses/diagnóstico , Viroses/virologia , Fenômenos Fisiológicos Virais , Vírus/classificação , Vírus/genética
2.
J Fish Dis ; 40(9): 1155-1167, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28026015

RESUMO

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


Assuntos
Infecções por Birnaviridae/veterinária , Doenças dos Peixes/diagnóstico , Vírus da Necrose Pancreática Infecciosa/isolamento & purificação , Perciformes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Salmão , Animais , Infecções por Birnaviridae/diagnóstico , Infecções por Birnaviridae/virologia , Linhagem Celular , Primers do DNA/genética , Doenças dos Peixes/virologia , Química Verde , Vírus da Necrose Pancreática Infecciosa/classificação , Vírus da Necrose Pancreática Infecciosa/genética , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos
3.
J Fish Dis ; 39(11): 1347-1356, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27135777

RESUMO

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


Assuntos
Suscetibilidade a Doenças/veterinária , Doenças dos Peixes/virologia , Linguados , Nodaviridae/fisiologia , Infecções por Vírus de RNA/veterinária , Vírus Reordenados/fisiologia , Animais , Suscetibilidade a Doenças/virologia , Infecções por Vírus de RNA/virologia , Carga Viral
4.
J Fish Dis ; 37(7): 597-607, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24846700

RESUMO

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.


Assuntos
Anguilla , Infecções por Vírus de DNA/veterinária , Doenças dos Peixes/epidemiologia , Infecções por Vírus de RNA/veterinária , Animais , Infecções por Vírus de DNA/epidemiologia , Infecções por Vírus de DNA/virologia , Vírus de DNA/classificação , Vírus de DNA/genética , Vírus de DNA/isolamento & purificação , Feminino , Doenças dos Peixes/virologia , Masculino , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase/veterinária , Infecções por Vírus de RNA/epidemiologia , Infecções por Vírus de RNA/virologia , Vírus de RNA/classificação , Vírus de RNA/genética , Vírus de RNA/isolamento & purificação , Estações do Ano , Análise de Sequência de DNA/veterinária , Espanha/epidemiologia , Proteínas Virais/genética
5.
Fish Shellfish Immunol ; 34(1): 383-6, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23092730

RESUMO

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.


Assuntos
Proteínas de Peixes/imunologia , Proteínas de Ligação ao GTP/imunologia , Nodaviridae/fisiologia , Dourada/imunologia , Vibrio alginolyticus/fisiologia , Animais , DNA Complementar/genética , Proteínas de Peixes/genética , Proteínas de Ligação ao GTP/genética , Expressão Gênica , Lipopolissacarídeos/administração & dosagem , Fígado/imunologia , Fígado/microbiologia , Fígado/virologia , Proteínas de Resistência a Myxovirus , Reação em Cadeia da Polimerase em Tempo Real , Dourada/genética , Dourada/microbiologia , Dourada/virologia
6.
Vet Q ; 40(1): 205-214, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32813983

RESUMO

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 Artemia and rotifers. MATERIALS AND METHODS: Rotifer (Brachionus plicatilis) and Artemia (Artemia salina) 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 (Solea senegalensis) larvae were fed on infected Artemia 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 Artemia internalised NNV. Experimental transmission to sole larvae was achieved using infected Artemia and subsequently 60% mortality was recorded. At 24 h post-infection, orally infected individuals contained 9.34 × 104 copies of viral RNA, whereas the bath infection yielded 2.05 × 106 RNA copies larvae-1. 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 Artemia might develop viral encephalopathy and retinopathy and high mortality.


Assuntos
Artemia/virologia , Doenças dos Peixes/virologia , Linguados/virologia , Vírus Reordenados/patogenicidade , Rotíferos/virologia , Viroses/veterinária , Animais , Doenças dos Peixes/mortalidade , Larva , Necrose/veterinária , Necrose/virologia , Vírus Reordenados/isolamento & purificação , Carga Viral , Viroses/transmissão
7.
J Gen Virol ; 90(Pt 12): 2940-2951, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19710256

RESUMO

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.


Assuntos
Doenças dos Peixes/virologia , Pesqueiros , Genoma Viral/genética , Nodaviridae , Infecções por Vírus de RNA/veterinária , Vírus Reordenados , Animais , Bass/virologia , Surtos de Doenças , Evolução Molecular , Nodaviridae/classificação , Nodaviridae/genética , Nodaviridae/isolamento & purificação , Filogenia , Infecções por Vírus de RNA/virologia , Recombinação Genética , Dourada/virologia , Análise de Sequência de DNA
8.
J Virol Methods ; 133(2): 167-74, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16332395

RESUMO

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.


Assuntos
Doenças dos Peixes/diagnóstico , Novirhabdovirus/isolamento & purificação , Reação em Cadeia da Polimerase/veterinária , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Infecções por Rhabdoviridae/veterinária , Animais , Southern Blotting , Células Cultivadas , Efeito Citopatogênico Viral , Células Epiteliais/citologia , Células Epiteliais/virologia , Estudos de Avaliação como Assunto , Doenças dos Peixes/virologia , Leucócitos/virologia , Novirhabdovirus/genética , RNA Viral/sangue , RNA Viral/genética , RNA Viral/isolamento & purificação , Infecções por Rhabdoviridae/diagnóstico , Sensibilidade e Especificidade , Truta/virologia
9.
J Virol Methods ; 162(1-2): 155-62, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19665481

RESUMO

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.


Assuntos
Doenças dos Peixes/diagnóstico , Peixes/virologia , Novirhabdovirus , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Infecções por Rhabdoviridae/veterinária , Animais , Benzotiazóis , Células Cultivadas , Primers do DNA , Diaminas , Doenças dos Peixes/virologia , Genótipo , Novirhabdovirus/classificação , Novirhabdovirus/genética , Novirhabdovirus/isolamento & purificação , Compostos Orgânicos , Quinolinas , RNA Viral/análise , RNA Viral/genética , RNA Viral/isolamento & purificação , Reprodutibilidade dos Testes , Infecções por Rhabdoviridae/diagnóstico , Infecções por Rhabdoviridae/virologia , Salmonidae/virologia , Sensibilidade e Especificidade , Taq Polimerase , Cultura de Vírus
10.
J Fish Dis ; 30(4): 225-32, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17394524

RESUMO

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.


Assuntos
Proteínas do Capsídeo/genética , Doenças dos Peixes/virologia , Nodaviridae/genética , Infecções por Vírus de RNA/veterinária , Animais , Sequência de Bases/genética , Pesqueiros , Genótipo , Dados de Sequência Molecular , Nodaviridae/isolamento & purificação , Nodaviridae/patogenicidade , Filogenia , Reação em Cadeia da Polimerase , Portugal , Infecções por Vírus de RNA/virologia , Homologia de Sequência do Ácido Nucleico , Espanha
11.
J Fish Dis ; 28(12): 713-22, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16336472

RESUMO

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.


Assuntos
Aquicultura/métodos , Infecções por Birnaviridae/veterinária , Doenças dos Peixes/virologia , Linguados , Vírus da Necrose Pancreática Infecciosa/genética , Filogenia , Viremia/veterinária , Animais , Sequência de Bases , Southern Blotting/veterinária , Análise por Conglomerados , Primers do DNA , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/veterinária , Polimorfismo de Fragmento de Restrição , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Análise de Sequência de DNA/veterinária , Espanha
12.
Appl Environ Microbiol ; 66(2): 839-43, 2000 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-10653762

RESUMO

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.


Assuntos
Infecções por Birnaviridae/veterinária , Doenças dos Peixes/virologia , Peixes/virologia , Vírus da Necrose Pancreática Infecciosa/classificação , Vírus da Necrose Pancreática Infecciosa/genética , Frutos do Mar/virologia , Animais , Infecções por Birnaviridae/virologia , Linhagem Celular , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Pesqueiros , Variação Genética , Vírus da Necrose Pancreática Infecciosa/isolamento & purificação , Testes de Neutralização , Espanha
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