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Vaccination and immune responses of European sea bass ( Dicentrarchus labrax L.) against betanodavirus
Abstract
This review summarizes the available knowledge on the immune defences of European sea bass against antigenic preparations derived from the viral encephalopathy and retinopathy virus (betanodavirus), which represents a major threat to the health of this fish species. The nodavirus is widely present and differentiates into several strains that infect invertebrates (in insects, alphanodavirus) and teleost fish, and thus may represent a great problem for farmed fish species. Many efforts have been directed to discovering new immunizations to induce protection in sea bass, especially at young stages, and these efforts have included employing diverse betanodavirus strains, antigen preparation, vaccination routes, and the addition of adjuvants and/or immunostimulants. The obtained results showed that inactivated preparations of betanodavirus that were administered intraperitoneally may induce both immune recognition and protection. Attempts at performing mucosal immunization by immersion and/or oral administration, which is a vaccination route that is highly preferred for sea bass, have shown intriguing results, and more studies are necessary for its improvement. Overall, the objective of identifying a reliable vaccine that also cross-protects against different genotypes or reassortant viruses for use in European sea bass against betanodavirus appears to be an attainable goal in the near future.
... The disease is preventable through vaccination, and several experimental VNN vaccines have been shown to induce neutralizing antibodies and protect against disease in experimental challenges [10]. Recently, two commercial vaccines were registered for use in selected Mediterranean countries [11,12]. ...
... The absorbance was measured at wavelengths 450 nm and 620 nm. The plot represents the mean absorbance (Abs450 nm -Abs620 nm) ± standard deviation (n = 5,9,5,6,6,10,9,5,6,6,9,9,5,6, 6 (order as in Figure)). ...
... Pathogens 2021,10, 1477 ...
The rapidly increasing Mediterranean aquaculture production of European sea bass is compromised by outbreaks of viral nervous necrosis, which can be recurrent and detrimental. In this study, we evaluated the duration of protection and immune response in sea bass given a single dose of a virus-like particle (VLP)-based vaccine. Examinations included experimental challenge with nervous necrosis virus (NNV), serological assays for NNV-specific antibody reactivity, and immune gene expression analysis. VLP-vaccinated fish showed high and superior survival in challenge both 3 and 7.5 months (1800 and 4500 dd) post-vaccination (RPS 87 and 88, OR (surviving) = 16.5 and 31.5, respectively, p < 0.01). Although not providing sterile immunity, VLP vaccination seemed to control the viral infection, as indicated by low prevalence of virus in the VLP-vaccinated survivors. High titers of neutralizing and specific antibodies were produced in VLP-vaccinated fish and persisted for at least ~9 months post-vaccination as well as after challenge. However, failure of immune sera to protect recipient fish in a passive immunization trial suggested that other immune mechanisms were important for protection. Accordingly, gene expression analysis revealed that VLP-vaccination induced a mechanistically broad immune response including upregulation of both innate and adaptive humoral and cellular components (mx, isg12, mhc I, mhc II, igm, and igt). No clinical side effects of the VLP vaccination at either tissue or performance levels were observed. The results altogether suggested the VLP-based vaccine to be suitable for clinical testing under farming conditions.
... European seabass, sea bream, meagre, Senegalese sole, turbot) resulting in elevated mortality rates of larval, juvenile and caged grown-out stages and consequently in large economic losses in the whole basin [22][23][24][25][26]. Much literature is available on D. labrax, the pathology and its serotypes [4,27,28] as well as on immune responses against the infection [29][30][31][32]. ...
... Others clearly showed that whole inactivated virus induced specific IgM titers and some degree of protection upon intraperitoneal (IP) administration [29]. An efficient uptake of the virus through the gills epithelium after immersion immunization was also demonstrated, but neither a systemic immune response nor an in vivo protection with respect to IP vaccination was induced [49]. ...
... Whole inactivated pathogens as vaccines represent a complex antigenic mixture and have a value in terms of cost/effectiveness; however, they may induce a limited protection time-wise as short as 5 weeks (Table 1). The virus that drawed the most attention for its severity in Mediterranean aquaculture is nodavirus, and many attempts have been made to investigate its effects on the immune system of target species, mainly seabass and turbot, with the aim of producing an efficient vaccine [29][30][31][32]53,54]. A licensed vaccine against nodavirus (RGNNV strain) has been recently released, thus closing the gap (Table 1). ...
This review describes and summarizes the knowledge on established and experimental vaccines developed against viral and bacterial pathologies affecting the most important farmed marine finfish species present in the Mediterranean area, namely European seabass Dicentrarchus labrax, sea bream Sparus aurata, turbot Psetta maxima and meagre Argyrosomus regius. The diseases that have been recorded in seabass, sea bream and meagre are caused by bacteria Vibrio anguillarum, Photobacterium damselae, Tenacibaculum maritimum as well as by viruses such as Viral Encephalopathy and Retinopathy/Viral Nervous Necrosis and Lymphocystic disease. The main pathologies of turbot are instead bacteriosis provoked by Tenacibaculum maritimum, Aeromonas sp. and Vibrio anguillarum, and virosis by viral hemorrhagic septicaemia virus. Some vaccines have been optimized and are now regularly available for the majority of the above-mentioned pathogens. A measurable immune protection has been conferred principally against Vibrio anguillarum, Photobacterium damselae sub. piscicida and VER/VNN.
... Four different genotype strains have been identified being red-spotted grouper nervous necrosis virus (RGNNV) the most common genotype found in the Mediterranean waters. Horizontal and vertical transmission of the NNV has been demonstrated, and the industry has applied different control strategies to avoid spreading of NNV, such as ozone sterilization, control of broodstocks and development of vaccines [1,[11][12][13]. In practice, the application of some of these procedures can be difficult, being the prevention by means of vaccination the most promising method to control virus disease [3]. ...
... In practice, the application of some of these procedures can be difficult, being the prevention by means of vaccination the most promising method to control virus disease [3]. Currently, there is only one commercial vaccine against NNV (PharmaQ) and high efforts to develop novel and more effective vaccines to prevent NNV outbreaks and dissemination are being made and can be found in the literature including attenuated, inactivated, recombinant and DNA vaccines [1,13]. ...
... Perhaps, this is due to high fish-to-fish variability and the fact that control groups also received whole or lysed E. coli, and it could be acting as a potent immunostimulant, regulating the expression of the studied genes and masking a possible regulation due to the rNNV protein in the vaccine. Previous studies have demonstrated early upregulation of some key immune genes encoding type-I IFN genes, Ig, Tcell markers or cytokines among others after vaccination with recombinant or inactivated NNV vaccines [13,21,33,37]. In sharp contrast to our data, although it is known the early induction and protective role of type-I IFN by NNV infection or vaccines [13,[38][39][40] few studies have documented increased expression of IFN-related genes 2-4 weeks after vaccination with NNV [31,41,42]. ...
Viral necrosis virus (NNV) or nodavirus causes fish viral encephalopathy and retinopathy worldwide. In some cases, mortalities in aquaculture industry can reach up to 100%, some species being especially sensitive as is the case of European sea bass (Dicentrarchus labrax), one of the main cultured species in the Mediterranean, with the consequent economical loses. Development of new vaccines against NNV is in the spotlight though few researches have focused in European sea bass. In this study we have generated a recombinant NNV (rNNV) vaccine produced in Escherichia coli expressing the capsid protein and administered it to European sea bass juveniles by two different routes (intraperitoneal and oral). The last being considered non-stressful and desired for fish farming of small fish, which in fact are the most affected by NNV. Oral vaccine was composed of feed pellets containing the recombinant whole bacteria, and injected vaccine was composed of recombinant bacteria previously lysed. Our results revealed production of specific anti-NNV IgM following the two vaccination procedures, levels that were further increased in orally-vaccinated group after challenge with NNV. Genes related to interferon (IFN), T-cell and immunoglobulin markers were scarcely regulated in head-kidney (HK), gut or brain. Vaccination by either route elicited a relative survival response of 100% after NNV challenge. To our knowledge, this is the first report of a recombinant vaccine followed by no purification steps which resulted in a complete protection in European sea bass when challenged with NNV.
... Several vaccination studies have been conducted for this disease, with very positive recent results [11] but these vaccines cannot yet be applied on larvae or early juveniles, for which this approach remains a challenge [12,13]. Genetic improvement is another potential approach to improve disease resistance in aquaculture, in general [14], and specifically VNN resistance [15]. ...
Background
Viral nervous necrosis (VNN) is a major disease that affects European sea bass, and understanding the biological mechanisms that underlie VNN resistance is important for the welfare of farmed fish and sustainability of production systems. The aim of this study was to identify genomic regions and genes that are associated with VNN resistance in sea bass.
Results
We generated a dataset of 838,451 single nucleotide polymorphisms (SNPs) identified from whole-genome sequencing (WGS) in the parental generation of two commercial populations (A: 2371 individuals and B: 3428 individuals) of European sea bass with phenotypic records for binary survival in a VNN challenge. For each population, three cohorts were submitted to a red-spotted grouper nervous necrosis virus (RGNNV) challenge by immersion and genotyped on a 57K SNP chip. After imputation of WGS SNPs from their parents, quantitative trait loci (QTL) were mapped using a Bayesian sparse linear mixed model (BSLMM). We found several QTL regions that were specific to one of the populations on different linkage groups (LG), and one 127-kb QTL region on LG12 that was shared by both populations and included the genes ZDHHC14, which encodes a palmitoyltransferase, and IFI6/IFI27-like , which encodes an interferon-alpha induced protein. The most significant SNP in this QTL region was only 1.9 kb downstream of the coding sequence of the IFI6/IFI27-like gene. An unrelated population of four large families was used to validate the effect of the QTL. Survival rates of susceptible genotypes were 40.6% and 45.4% in populations A and B, respectively, while that of the resistant genotype was 66.2% in population B and 78% in population A.
Conclusions
We have identified a genomic region that carries a major QTL for resistance to VNN and includes the ZDHHC14 and IFI6/IFI27-like genes. The potential involvement of the interferon pathway, a well-known anti-viral defense mechanism in several organisms (chicken, human, or fish), in survival to VNN infection is of particular interest. Our results can lead to major improvements for sea bass breeding programs through marker-assisted genomic selection to obtain more resistant fish.
... Nervous necrosis virus (NNV) is a highly important fish virus that has caused severe economic losses to the aquaculture industry around the world. The viral polymerase and capsid protein (CP) are encoded by two single-stranded RNA segments in the NNV genome (Buonocore et al., 2019). CP is the only structural protein on the NNV surface, and it plays a role in viral invasion and encapsidation. ...
Nervous necrosis virus (NNV) is a devastating infectious pathogen for fish species with 100% mortality. To date, no specific drugs or vaccines have been developed that can prevent infections in aquaculture caused by NNV. It has been found that the NNV utilizes capsid protein to enter into the host cell in Asian sea bass and cause disease. In this study, we evaluated the inhibitory potential of Allium sativum compounds that have been reported to show antiviral activity against various pathogens. The capsid protein was modeled and the binding affinity of all the compounds was calculated with the docking approach and top 2 (PubChem CID: 122130381 and CID 12303662) inhibitory compounds were selected for further ADMET properties and DFT analysis. Both the geometry optimization and redocking of the two inhibitory compounds (PubChem CID: 122130381 and CID 12303662) showed a strong binding affinity of-8.2 and-8.0 kcal/mol, respectively with the capsid protein. The molecular dynamic simulation approach further validated the capsid protein-CID: 122130381 and capsid protein-CID 12303662 complex stability. In conclusion, this study deduces that these Allium sativum phytochemicals might act as significant inhibitors of the NNV in sea bass, which can be further validated experimentally.
... The massive mortality of NNV-infected individuals, especially the almost 100% fatality rate in infected juveniles, has resulted in huge economic losses (Costa & Thompson, 2016). The NNV genome is split into two single-stranded RNA segments, encoding the viral polymerase and capsid protein (CP), respectively (Buonocore et al., 2019). As the only structural protein displayed on the NNV surface, CP is involved in the viral invasion and encapsidation processes. ...
As a highly important fish virus, nervous necrosis virus (NNV) has caused severe economic losses to the aquaculture industry worldwide. Autophagy, an evolutionarily conserved intracellular degradation process, is involved in the pathogenesis of several viruses. Although NNV can induce autophagy to facilitate infection in grouper fish spleen cells, how it initiates and mediates autophagy pathways during the initial stage of infection is still unclear. Here, we found that red-spotted grouper NNV (RGNNV) induced autophagosome formation in two fish cell lines at 1.5 and 3 h post infection, indicating that autophagy is activated upon entry of RGNNV. Moreover, autophagic detection showed that RGNNV entry induced incomplete autophagy by impairing the fusion of autophagosomes with lysosomes. Further investigation revealed that binding of the RGNNV capsid protein (CP) to the Lateolabrax japonicus heat shock protein HSP90ab1 (LjHSP90ab1), a cell surface receptor of RGNNV, contributed to RGNNV invasion-induced autophagy. Finally, we found that CP blocked the interaction of L. japonicus protein kinase B (AKT) with LjHSP90ab1 by competitively binding the NM domain of LjHSP90ab1 to inhibit the AKT-mechanistic target of the rapamycin (MTOR) pathway. This study provides novel insight into the relationship between NNV receptors and autophagy, which may help clarify the pathogenesis of NNV.
... This was made possible by the mAb DLT15, the first in fish immunology, which recognizes a thymocyte antigen and peripheral T cells. The antibody, together with nucleotide probes for T cell subpopulations, enabled a precise quantification of T cells in lymphoid and non-lymphoid tissues in unstimulated and stimulated conditions, including viral/bacterial infection and vaccination (Buonocore et al., 2019;Moreno et al., 2018), as well as in the modulation of intestinal microbiome (Picchietti, Fausto, et al., 2009), and also permitted immunopurification of T cells from lymphoid tissues . Importantly and as expected, a preponderance of T cells was confirmed in mucosal tissues by means of DLT15 (IIF) and through the identification of CD4/CD8 subpopulations by ISH, and interestingly, a density gradient was shown along the intestinal tract (Picchietti et al, 2011(Picchietti et al, , 2021b. ...
This review summarizes the current knowledge on immune defence activities of the European sea bass Dicentrarchus labrax by reporting the consistent amount of work done on this economically-important species. A draft genome sequence is available for this species, together with whole transcriptomes from lymphoid and non-lymphoid tissues. Available full-length coding sequences of many immunoregulatory and immune-related genes allow for targeted quantitative PCR analysis, nowadays needed for omics data verification, ex vivo and in vitro. The first anti-T cells monoclonal antibody teleost-wise was obtained in sea bass, followed by several monoclonal and polyclonal markers of lymphocyte populations, namely T cells (pan-T, CD3ε, TcRγ, CD45), and B cells (IgM, IgT, IgD). The combined use of molecular and biochemical tools enabled investigations on innate and acquired immune responses of sea bass in unstimulated/stimulated fish, along the development and under variable environmental conditions and food regimes. An overview of sea bass viral and bacterial pathogens and available vaccines against these microorganisms is also provided. The knowledge accumulated in the past 25 years validates the European sea bass as a reference marine model in the field of fish immunology.
... Interest in selective breeding as a tool to enhance genetic resistance to VNN in European sea bass has markedly increased in recent years, because of the major threat represented by NNV for sea bass hatcheries and aquaculture farms, combined with the lack of effective therapeutical remedies for this disease [1,43]. The presence of a genetic basis for resistance to various diseases in fish species, which is evidenced by moderate to high estimates of heritability, suggests that selective breeding can be an effective tool to reduce disease outbreaks or to limit negative consequences of such outbreaks [1,44]. ...
Background
Susceptibility of European sea bass ( Dicentrarchus labrax L.) to viral nervous necrosis (VNN) is well-known. Interest towards selective breeding as a tool to enhance genetic resistance in this species has increased sharply due to the major threat represented by VNN for farmed sea bass and limitations concerning specific therapeutical measures. A sea bass experimental population (N = 650) was challenged with nervous necrosis virus (NNV) to investigate genetic variation in VNN mortality. In addition, relationships of this trait with serum cortisol concentration after stress exposure, antibody titer against NNV antigens, and body weight at a fixed age were studied to identify potential indicator traits of VNN resistance.
Results
The estimate of heritability for VNN mortality was moderate and ranged from 0.15 (HPD95%, 95% highest posterior density interval: 0.02, 0.31) to 0.23 (HPD95%: 0.06, 0.47). Heritability estimates for cortisol concentration, antibody titer, and body weight were 0.19 (HPD95%: 0.07, 0.34), 0.36 (HPD95%: 0.16, 0.59) and 0.57 (HPD95%: 0.33, 0.84), respectively. Phenotypic relationships between traits were trivial and not statistically significant, except for the estimated correlation between antibody titer and body weight (0.24). Genetic correlations of mortality with body weight or antibody titer (− 0.39) exhibited a 0.89 probability of being negative. A negligible genetic correlation between mortality and cortisol concentration was detected. Antibody titer was estimated to be positively correlated with body weight (0.49).
Conclusions
Antibody titer against NNV offers the opportunity to use indirect selection to enhance resistance, while the use of cortisol concentration as an indicator trait in breeding programs for VNN resistance is questionable. The estimate of heritability for VNN mortality indicates the feasibility of selective breeding to enhance resistance to NNV and raises attention to the development of genomic prediction tools to simplify testing procedures for selection candidates.
... The knowledge of fish MALTs will help the researcher in the field of mucosal vaccine developments in aquaculture. Several advantages of the mucosal vaccine (Toffan et al., 2019;Somamoto and Nakanishi, 2020), have drawn the attention of fish immunologists. Earlier studies suggested that vaccination through mucosal tissues generates a significant immune response with long-term memory development in experimental fish (Salinas and Magadán, 2017;Somamoto and Nakanishi, 2020). ...
Polymeric immunoglobulin receptor (pIgR) is a protein that transports Immunoglobulins (Igs) from epithelial cells into the external secretion system of the animal. In the present study, we characterized the partial pIgR gene from Labeo rohita and analyzed its expression in response to the pDNA (pGPD-IFN) vaccine, and studied its correlation with the expression of IgM, in the mucosal-associated lymphoid tissues (MALT). The significant (p < 0.05) up-regulation of pIgR mRNA was observed in the mucosal tissues of vaccinated fish by qRT-PCR. The highest expression of the gene was detected in gill tissue at 15 days post-vaccination (dpv) followed by skin and gut at 30 dpv. The expression of IgM also showed a similar pattern and indicated a direct correlation of pIgR expression in all the tested tissues. The protective immune response of the DNA vaccine was measured as the relative percentage of survival (RPS) by challenging vaccinated fish with live Edwardsiella tarda (1 × 10⁵ CFU/Fish). The result showed a significantly high relative percentage survival (RPS) in the vaccinated group (47.05%) compared to the control group. Many factors contributing to the immune response of the vaccine. One of the most critical aspects is the rise in IgM level in local tissues. In other higher vertebrates, pIgR is reported to act as the transporter of IgM. The positive correlation in the expression of pIgR and IgM observed in the present study demonstrated the possible role of pIgR as the transporter of IgM in L rohita. The study suggests the possibility of the secretory nature of IgM in fish.
... This study showed that the immersion treatment induced low levels of specific IgM production and gave no protection compared to control fish [127]. While immersion vaccines against VER have not been proved to exhibit high efficacy, they have induced gene expression of relevant adaptive and antiviral genes [128]. ...
Immersion vaccines are used for a variety of aquacultured fish to protect against infectious diseases caused by bacteria and viruses. During immersion vaccination the antigens are taken up by the skin, gills or gut and processed by the immune system, where the resulting response may lead to protection. The lack of classical secondary responses following repeated immersion vaccination may partly be explained by the limited uptake of antigens by immersion compared to injection. Administration of vaccines depends on the size of the fish. In most cases, immersion vaccination is inferior to injection vaccination with regard to achieved protection. However, injection is problematic in small fish, and fry as small as 0.5 gram may be immersion vaccinated when they are considered adaptively immunocompetent. Inactivated vaccines are, in many cases, weakly immunogenic, resulting in low protection after immersion vaccination. Therefore, during recent years, several studies have focused on different ways to augment the efficacy of these vaccines. Examples are booster vaccination, administration of immunostimulants/adjuvants, pretreatment with low frequency ultrasound, use of live attenuated and DNA vaccines, preincubation in hyperosmotic solutions, percutaneous application of a multiple puncture instrument and application of more suitable inactivation chemicals. Electrostatic coating with positively charged chitosan to obtain mucoadhesive vaccines and a more efficient delivery of inactivated vaccines has also been successful.
... Alternative innovative vaccination immersion protocols have been described for sea bass [17], and specific antibodies were induced in grouper eggs by vertical transmission from broodfish injected with inactivated VNNV [18]. Vaccination methods against nodaviruses and their corresponding immune responses in European sea bass have been recently reviewed [19] including oral delivery alternatives such as those using inactivated bacteria encapsulating dsRNA from VNNV, and chitosan conjugated VNNV DNA [7]. Most recently, protection has been reported by using alive recombinant bacteria expressing the C protein sequence mixed with the feed [20]. ...
This work describes immunization of European sea bass (Dicentrarchus labrax) juveniles against viral nervous necrosis virus (VNNV), a betanodavirus causing worldwide mortalities in many fish species. Protection was obtained with the so-called spinycterin vehicles consisting of irreversibly DNA-damaged DNA-repair-less Escherichia coli displaying at their surface a downsized VNNV coat antigen. In this work we have i) maximized bacterial expression levels by downsizing the coat protein of VNNV to a fragment (frgC91-220) containing most of its previously determined antigenicity, ii) developed a scalable autoinduction culture media for E.coli based in soybean rather than in casein hydrolysates, iii) enriched surface expression by screening different anchors from several prokaryotic sources (anchor + frgC91-220 recombinant products), iv) preserved frgC91-220 antigenicity by inactivating bacteria by irreversible DNA-damage by means of Ciprofloxacin, and v) increased safety using a repair-less E.coli strain as chassis for the spinycterins. These spinycterins protected fish against VNNV challenge with partial (Nmistic + frgC91-220) or total (YBEL + frgC91-220) levels of protection, in contrast to fish immunized with frgC91-220 spinycterins. The proposed spinycterin platform has high levels of environmental safety and cost effectiveness and required no adjuvants, thus providing potential to further develop VNNV vaccines for sustainable aquaculture.
... Alternative innovative vaccination immersion protocols have been described for sea bass [17], and specific antibodies were induced in grouper eggs by vertical transmission from broodfish injected with inactivated VNNV [18]. Vaccination methods against nodaviruses and their corresponding immune responses in European sea bass have been recently reviewed [19] including oral delivery alternatives such as those using inactivated bacteria encapsulating dsRNA from VNNV, and chitosan conjugated VNNV DNA [7]. Most recently, protection has been reported by using alive recombinant bacteria expressing the C protein sequence mixed with the feed [20]. ...
This work describes practical immunization of European sea bass (Dicentrarchus labrax) juveniles against viral nervous necrosis virus (VNNV), a betanodavirus causing worldwide mortalities in many fish species. Protection was obtained with the so called spinycterin vehicles consisting in irreversibly DNA-damaged DNA-repair-less E.coli displaying at their surface a downsized antigen. In this work we, i) maximized bacterial expression levels by downsizing the C protein to a fragment (frgC91-220) containing most of its antigenicity, ii) developed an scalable autoinduction bacterial media based in soy-bean increasing membrane display and reproducibility, iii) enriched surface expression by screening different anchors from several prokaryotic origins (anchor+frgC91-220), iv) preserved frgC91-220 antigenicity by inactivating bacteria by irreversible DNA-damage by means of Ciprofloxacin, and v) increased safety using a repair-less E.coli strain as spinycterin chassis. These second generation of spinycterins protected fish against VNNV challenge with partial (Nmistic+frgC91-220) or 100 % (YBEL+frgC91-220 ) protection, in contrast to those fish immunized with frgC91-220 spinycterins. The proposed spinycterin platform has high levels of environmental safety and cost effectiveness, thus providing potential for small fish vaccines for sustainable aquaculture.
Diseases are a significant impediment to aquaculture’s sustainable and healthy growth. The aquaculture industry is suffering significant financial losses as a result of the worsening water quality and increasing frequency of aquatic disease outbreaks caused by the expansion of aquaculture. Drug control, immunoprophylaxis, ecologically integrated control, etc. are the principal control strategies for fish infections. For a long time, the prevention and control of aquatic diseases have mainly relied on the use of various antibiotics and chemical drugs. However, long-term use of chemical inputs not only increases pathogenic bacteria resistance but also damages the fish and aquaculture environments, resulting in drug residues in aquatic products, severely impeding the development of the aquaculture industry. The development and use of aquatic vaccines are the safest and most effective ways to prevent aquatic animal diseases and preserve the health and sustainability of aquaculture. To give references for the development and implementation of aquatic vaccines, this study reviews the development history, types, inoculation techniques, mechanisms of action, development prospects, and challenges encountered with aquatic vaccines.
Viral nervous necrosis (VNN) is a serious disease of marine and brackishwater fishes caused by nervous necrosis virus (NNV) resulting in up to 100% mortality in early larval and juvenile stages. Adult fish when infected are asymptomatic and spread the virus vertically to the offspring through milt and eggs. Prevention of vertical transmission of the disease is by using disease free broodstock and vaccinating the brooders. Estimation of antigen content and virus titre is essential to determine antigen/virus concentration in VNN vaccine. A monoclonal antibody based indirect sandwich ELISA was developed to quantify the NNV antigen and to estimate the virus titre by TCID50 coupled ELISA. Mouse hybridoma clones secreting monoclonal antibodies (MAb) against the capsid protein of NNV was developed and characterized. The antibodies reacted specifically with the recombinant capsid protein and purified virus in western blot. Polyclonal antibodies against NNV were used as capture antibodies and MAbs were used as detection antibodies to optimize an indirect sandwich ELISA to detect and quantify capsid protein of NNV. The developed assay had a sensitivity of 390 ng/ml and could detect the virus in clinical samples. The assay coupled with TCID50 could be used to estimate the virus titre rather than by observing the CPE which is laborious and subjective.
Nervous necrosis virus (NNV), causing almost 100% mortality among infected larvae and juvenile fish, is severely affecting the aquaculture industry. Although various vaccines against NNV were studied, they can hardly provoke strong enough immunity in fry with immature immune systems. To eliminate the infection possibility during fertilization, we applied an anti-NNV strategy by neutralization based on a single-chain variable fragment (ScFv) antibody, named Sc3F9, against orange-spotted grouper nervous necrosis virus (OGNNV). Sc3F9 binds to the shell domain of viral capsid protein (CP) with a dissociation constant (KD) of 48.6 nM by interacting with two discontinuous target sites on CP fragments of G122-L128 and L141-A148. The binding capacity was enhanced by prokaryotic expressing double copies of Sc3F9 (DSc3F9) in tandem, and the augmented avidity (KD = 29.5 nM) with irreversible binding to OGNNV was stable even in high salinity, indicating the wide application potential in seawater. DSc3F9 exhibited great recognition breadth within the Betanodavirus genus to bind all NNV serotypes in working concentration (40 μg/mL) and distinguished these three serotypes by different levels of binding capacity in low concentration (10 μg/mL). DSc3F9 blocked virus-like particle entry by destructing its virion as well as strongly neutralized OGNNV with an average half-maximal inhibitory concentration (IC50) value of 0.64 μM. Moreover, DSc3F9 efficiently eliminated the possible NNV infection during fertilization by washing grouper eggs before artificial insemination without reducing the fertility rate and hatchability. Thus, our results not only obtained a strong anti-NNV candidate but also provided an approach to NNV decontamination, especially in obtaining NNV-free grouper eggs.
Nervous necrosis virus (NNV) reassortant strains RGNNV/SJNNV have emerged as a potent threat to the Mediterranean marine aquaculture industry, causing viral encephalopathy and retinopathy (VER) in Senegalese sole (Solea senegalensis). In this study, a cheap and practical vaccine strategy using bacterial inclusion bodies made of the coat protein of a virulent reassortant strain of this betanodavirus was devised. The nanostructured recombinant protein nanoparticles, VNNV-CNP, were administered without adjuvant to two groups of juvenile sole, one by intraperitoneal injection and the other by oral intubation. Specific antibodies were raised in vivo against the NNV coat protein via both routes, with a substantial specific antibody expansion in the injected group 30 days post homologous prime boost. Expression levels of five adaptive immune-related genes, cd8a, cd4, igm, igt and arg2, were also quantified in intestine, spleen and head kidney. Results showed cd4 and igm were upregulated in the head kidney of injected fish, indicating activation of an adaptive systemic response, while intubated fish exhibited a mucosal response in the intestine. Neither route showed significant differential expression of cd8a. The specific antibody response elicited in vivo and the lack of any signs of toxicity over the 6-week study period in young fish (n = 100), evidences the potential of the nanoparticle as a vaccine candidate.
Fish mucosal organs are directly exposed to aquatic environment, which is suited to the colonization and growth of microorganisms, and thus are considered to play an important role in maintaining homeostasis and preventing entry of invading pathogens. Researches on fish mucosal immunity have shown that mucosal organs in fish such as gill, skin, intestine, olfactory organs harbour lymphoid cells including T and B cells as well as dendritic-like cells. The findings on immune responses following direct administration with antigens into the mucosal organs will help to shed light on developing fish mucosal vaccines. The present review highlighted vaccine deliveries via mucosal organs, especially focusing on methods other than typical mucosal vaccine platforms, such as oral and immersion vaccines. In addition, we proposed a hypothesis that mucosal tissues are important sites for generating cell-mediated immunity following vaccination with extracellular antigens.
Nodaviruses are small bipartite RNA viruses which belong to the family of Nodaviridae. They are categorized into alpha-nodavirus, which infects insects, and beta-nodavirus, which infects fishes. Another distinct group of nodavirus infects shrimps and prawns, which has been proposed to be categorized as gamma-nodavirus. Our current review focuses mainly on recent studies performed on nodaviruses. Nodavirus can be transmitted vertically and horizontally. Recent outbreaks have been reported in China, Indonesia, Singapore and India, affecting the aquaculture industry. It also decreased mullet stock in the Caspian Sea. Histopathology and transmission electron microscopy (TEM) are used to examine the presence of nodaviruses in infected fishes and prawns. For classification, virus isolation followed by nucleotide sequencing are required. In contrast to partial sequence identification, profiling the whole transcriptome using next generation sequencing (NGS) offers a more comprehensive comparison and characterization of the virus. For rapid diagnosis of nodavirus, assays targeting the viral RNA based on reverse-transcription PCR (RT-PCR) such as microfluidic chips, reverse-transcription loop-mediated isothermal amplification (RT-LAMP) and RT-LAMP coupled with lateral flow dipstick (RT-LAMP-LFD) have been developed. Besides viral RNA detections, diagnosis based on immunological assays such as enzyme-linked immunosorbent assay (ELISA), immunodot and Western blotting have also been reported. In addition, immune responses of fish and prawn are also discussed. Overall, in fish, innate immunity, cellular type I interferon immunity and humoral immunity cooperatively prevent nodavirus infections, whereas prawns and shrimps adopt different immune mechanisms against nodavirus infections, through upregulation of superoxide anion, prophenoloxidase, superoxide dismutase (SOD), crustin, peroxinectin, anti-lipopolysaccharides and heat shock proteins (HSP). Potential vaccines for fishes and prawns based on inactivated viruses, recombinant proteins or DNA, either delivered through injection, oral feeding or immersion, are also discussed in detail. Lastly, a comprehensive review on nodavirus virus-like particles (VLPs) is presented. In recent years, studies on prawn nodavirus are mainly focused on Macrobrachium rosenbergii nodavirus (MrNV). Recombinant MrNV VLPs have been produced in prokaryotic and eukaryotic expression systems. Their roles as a nucleic acid delivery vehicle, a platform for vaccine development, a molecular tool for mechanism study and in solving the structures of MrNV are intensively discussed.
Progressive research has been recently made in dissecting the molecular biology of Betanodavirus life cycle, the causative pathogen of viral encephalopathy and retinopathy in economic important marine fish species. Establishment of betanodavirus infectious clone allows the manipulation of virus genome for functional genomic study, which elucidates the biological event of the viral life cycle at molecular level. The betanodavirus strategizes its replication by expressing anti-apoptosis/antinecrotic proteins to maintain the cell viability during early infection. Subsequently utilizes and controls the biological machinery of the infected cells for viral genome replication. Towards the late phase of infection, mass production of capsid protein for virion assembly induces the activation of host apoptosis pathway. It eventually leads to the cell lysis and death, which the lysis of cell contributes to the accomplishment of viral shedding that completes a viral life cycle. The recent efforts to dissect the entire betanodavirus life cycle are currently reviewed.
Background
Immunoglobulins (Igs) are fundamental components of the adaptive immune system of vertebrates, with the IgT/IgZ isotype specific of Teleosts. In this paper we describe the identification of an IgT heavy chain from the European sea bass (Dicentrarchus labrax L.), its molecular characterization and tissue mRNA localization by in situ hybridization. ResultsSea bass IgT consists of 552 aa (Accession Number KM410929) and it contains a putative 19 amino acids long signal peptide and one potential N-glycosylation site. The C-region consists of four CH domains; each contains the cysteine and tryptophan residues required for their correct folding. Based on the recent sequencing of sea bass genome, we have identified five different genomic contigs bearing exons unequivocally pertaining to IgT (CH2, CH3 and CH4), but none corresponded to a complete IgH locus as IgT sequences were found in the highly fragmented assembled genomic regions which could not be assigned to any major scaffold. The 3D structure of sea bass IgT has been modelled using the crystal structure of a mouse Ig gamma as a template, thus showing that the amino acid sequence is suitable for the expected topology referred to an immunoglobulin-like architecture. The basal expression of sea bass IgT and IgM in different organs has been analysed: gut and gills, important mucosal organs, showed high IgT transcripts levels and this was the first indication of the possible involvement of sea bass IgT in mucosal immune responses. Moreover, sea bass IgT expression increased in gills and spleen after infection with nodavirus, highlighting the importance of IgT in sea bass immune responses. In situ hybridization confirmed the presence of IgT transcripts in the gut and it revealed a differential expression along the intestinal tract, with a major expression in the posterior intestine, suggesting the hindgut as a site for the recruitment of IgT+ cells in this species. IgT transcripts were also found in gill filaments and parallel lamellae and, for the first time, we identified scattered IgT positive cells in the liver, with a strong signal in the hepatic parenchyma. Conclusions
In conclusion, we performed a full molecular characterization of IgT in sea bass that points out its possible involvement in mucosal immune responses of this species.
Viral encephalopathy and retinopathy (VER), otherwise known as viral nervous necrosis (VNN), is a major devastating threat for aquatic animals. Betanodaviruses have been isolated in at least 70 aquatic animal species in marine and in freshwater environments throughout the world, with the notable exception of South America. In this review, the main features of betanodavirus, including its diversity, its distribution and its transmission modes in fish, are firstly presented. Then, the existing diagnosis and detection methods, as well as the different control procedures of this disease, are reviewed. Finally, the potential of selective breeding, including both conventional and genomic selection, as an opportunity to obtain resistant commercial populations, is examined.
The European sea bass (Dicentrarchus labrax) is an important farmed fish species in the Mediterranean area, very sensitive to the infection by encephalopathy and retinopathy virus (VERv), or Betanodavirus, which causes massive mortalities. Effective vaccines to fight the pathology are not yet available and in this work we describe a promising intraperitoneal immunization route against VERv of sea bass juveniles. We performed intraperitoneal and immersion immunization trials with a VERv (isolate 283.2009 RGNNV) inactivated by formalin, β-propiolactone and heat treatment. Interestingly, the intraperitoneal immunization with formalin-inactivated VERv induced a significant antigen-specific IgM production, differently from other inactivation protocols. However, the same formalin-inactivated antigen resulted in very low IgM antibodies when administered by immersion. Following the intraperitoneal injection with formalin-inactivated virus, the quantitative expression of the antiviral MxA gene showed a modulation of transcripts in the gut after 48 h and on head kidney after 24 h, whereas ISG12 gene was significantly up-regulated after 48 h on both tissues. In immersion immunization with formalin-inactivated VERv, a modulation of MxA and ISG12 genes after 24 h post-treatment was detected in the gills. An effective uptake of VERv particles in the gills was confirmed by immunohistochemistry using anti-VERv antibodies. Lastly, in challenge experiments using live VERv after intraperitoneal immunization with formalin-inactivated VERv, we observed a significant increase (81.9%) in relative survival percentage with respect to non-immunized fish, whereas immersion immunization resulted in no protection. Our results suggest that intraperitoneal immunization with formalin-inactivated VERv could be a safe and effective strategy to fight Betanodavirus infection in European sea bass.
Over the last three decades, the causative agent of viral encephalopathy and retinopathy (VER) disease has become a serious problem of marine finfish aquaculture, and more recently the disease has also been associated with farmed freshwater fish. The virus has been classified as a Betanodavirus within the family Nodaviridae, and the fact that Betanodaviruses are known to affect more than 120 different farmed and wild fish and invertebrate species, highlights the risk that Betanodaviruses pose to global aquaculture production.
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.
Viral nervous necrosis infections are causing severe problems on aquaculture industry due to ecological and economic impacts. Their causal agent is nervous necrosis virus or nodavirus, which has been classified into four genotypes. Different genotypes correlate with differences in viral pathogenicity. Therefore, rational development of effective vaccines and diagnostic reagents requires analysis of the genetic variation. The development and validation of a polymerase chain reaction amplification (PCR)-based methodology for nodavirus genotype assessment in a simple and robust format is described. Degenerate external primers and two genotype-specific internal primers were utilized for simultaneous amplification of nodavirus products in a single PCR. A first set of cycles produced a long PCR product, defined by the outer primers, and the internal primers amplified short DNA fragments specific for each genotype in lower annealing temperature. Detection was based on the size of the short products. Nodavirus infected and healthy samples were analyzed and none of the non-infected samples showed any bands, while all infected samples were positive. The proposed method can be performed within 4 h and consumes standard PCR and electrophoresis reagents, with costs lower than 2 per sample. Tetra-primer PCR is a suitable alternative for virus sequencing in medium scale research laboratories and farming facilities.
One of the most powerful innate immune responses against virus is mediated by the type I interferon (IFN). In teleost fish, it is known that virus infection triggers the expression of ifn and many IFN-stimulated genes but the viral RNA sensors and mediators leading to the IFN production are scarcely known. Thus, we have searched the presence of these genes in gilthead seabream (Sparus aurata) and European sea bass (Dicentrarchus labrax) and evaluated their expression after infection with viral nervous necrosis virus (VNNV) in the brain, the main viral target tissue, and the gonad, used to transmit the virus vertically. In seabream, a resistant fish species to the VNNV strain used, we found an up-regulation of the genes encoding MDA5, TBK1, IRF3, IFN, Mx and PKR proteins in the brain, which were unaltered in the gonad and could favour the dissemination by gonad fluids or gametes. Strikingly, in European sea bass, a very susceptible species, we identified, in addition, transcripts coding for LGP2, MAVS, TRAF3, TANK and IRF7 and found that all the genes analysed were up-regulated in the gonad but only mda5, lgp2, irf3, mx and pkr did in the brain. These findings support the notion that the European sea bass brain innate immune response is unable to clear the virus and points to the importance of the gonad immunity to control the dissemination of VNNV to the progenies, an aspect that is worth to investigate in aquatic animals.
This review describes the extant knowledge on the teleostean mucosal adaptive immune mechanisms, which is relevant for the development of oral or mucosal vaccines. In the last decade, a number of studies have shed light on the presence of new key components of mucosal immunity: a distinct immunoglobulin class (IgT or IgZ) and the polymeric Ig receptor (pIgR). In addition, intestinal T cells and their putative functions, antigen uptake mechanisms at mucosal surfaces and new mucosal vaccination strategies have been reported. New information on pIgR of Atlantic cod and common carp and comparison of natural and specific cell-mediated cytotoxicity in the gut of common carp and European seabass, is also included in this review. Based on the known facts about intestinal immunology and mucosal vaccination, suggestions are made for the advancement of fish vaccines.
Theefficacyof inactivatedbetanodavirusas a vaccineagainst viral nervousnecrosis (VNN)was evaluated using juvenilesevenband grouper Epinephelusseptemfasciatus. Fishwere intraperitoneallyinjectedonce withformalin-inactivatedredspotted grouper nervous necrosis virus (RGNNV). Virus-neutralizing antibodies were detected in the vaccinated fish from Day 10 to the end of the experimentalperiod (Day160), showing1: 2,000 or highermean antibodytiters fromDay 21 to Day 77. The vaccinated and unvaccinatedcontrol fish were challenged by intramuscular injectionwiththe homologousvirus at 14, 35 and 74 days post-vaccination. The vaccinated fish showed significantlylowermortalitiesat any challenges than the controlfish,withthe RPS (relative percentsurvival)values 67 or higher. Afieldtrial, inwhichfishwere exposed to natural infectionin net pens, also resulted in higher survivalrates in the vaccinated fish (RPS = 85) duringthe experi- mental period of 9 weeks. This high inductionof neutralizingantibodies and protectionindicates the potentialof the inactivatedvirus vaccine against VNN.
A total of 360 samples of trash fish/mollusk used for feeding cultured marine fish were collected from 4 stations of National Research Institute of Aquaculture, Fisheries Research Agency, Japan. The brain or eyes of the samples were examined by reverse transcription-polymerase chain reaction (RT-PCR) and nested PCR assays to detect the coat protein gene of betanodavirus. Two species of trash fish/mollusk samples: Japanese jack mackerel Trachrus japonicus and Japanese common squid Todarodes pacificus were positive for betanodavirus and the sequences of the nested PCR products (177 nucleotides) of these two species were closely related (98%) to the redspotted grouper nervous necrosis virus (RGNNV) genotype. Sevenband grouper Epinephelus septemfasciatus were intramuscularly injected (106 TCID50/fish) with betanodavirus isolates from trash fish/mollusk samples. The fish displayed abnormalities typical of viral nervous necrosis (VNN) infection, and mortalities ranged from 90 to 100%. These results suggest that trash fish/mollusk can be a source of betanodaviruses for cultured fish and that they pose a serious risk for outbreaks of VNN in susceptible cultured fish.
The effect of temperature, pH and electrolytes on the stability of two Mediterranean sea bass neuropathy nodavirus isolates (SBNN) and the inactivation of SBNN by heating, exposure to ultraviolet radiation and chemical disinfectants was investigated in vitro using a striped snakehead fish cell line (SSN-1) for virus propagation and assay. The two nodavirus isolates showed no significant differences in response to the procedures examined. Nodavirus held in cell culture medium containing 5% foetal bovine serum was effectively inactivated within 4 days at 37 degrees C and 3 months at 25 degrees C but showed no significant loss of titre over 6 months at 15 degrees C and still retained a measurable level of infectivity after storage for 1 year at this temperature. Virus suspensions in distilled water tolerated exposure to pH 2-11 with no significant loss of titre over 24 h. Thereafter, a steady diminution in infectivity was noted at pH 11 from day 3 and at pH 2 from day 15 onwards, but no loss of titre was
The distribution of red-spotted grouper nervous necrosis virus (RGNNV) antigens was examined by immunohistochemistry in the nervous and non-nervous organs of juvenile European seabass (Dicentrarchus labrax) during the course of an intramuscular infection. Histological changes resulting from the infection were evaluated from 3 days to 2 months post-infection. The specific antibody response was also studied 2 months post-challenge. Viral proteins were present throughout the experimental period in the retina (inner nuclear layer, ganglion layer, outer limiting membrane, and outer plexiform layer), brain(cerebellum and tectum opticum), and liver (hepatocytes and endothelial cells). These proteins were also observed in the renal tubular cells, white pulp of spleen, and in fibroblasts and cartilage of caudal fin. This is the first report of RGNNV proteins appearing in these organs, where the immunostaining was only detected at certain sampling times after the onset of mortality. Brain and retina of virus-exposed fish showed high levels of vacuolation, while accumulation of fat vacuoles was observed in the liver. RGNNV infection also induced a specific antibody response as measured by an ELISA. In summary, this is the first study demonstrating the presence of viral proteins in cells of caudal fin, kidney and spleen of European seabass.
Betanodaviruses are the causal agents of viral encephalo-retinopathy, an infectious disease affecting more than 40 marine fish species, characterized by high morbidity and mortality. Because of its severe impact, robust diagnostic tools are required. The aim of this work was to develop and validate a real-time TaqMan PCR assay to detect betanodaviruses in clinical specimens by amplifying a conserved region of the RNA2 strand. The method proved to be specific and sensitive, being capable of detecting as low as 10 TCID(50)/ml. For clinical validation, samples from 100 marine fish were collected during a natural outbreak of disease and tested by three distinct laboratory methods, namely real-time TaqMan PCR, RT-seminested PCR and virus isolation. The results indicated optimal agreement between tests. The assay that was developed is capable of detecting members of all of the betanodavirus genetic groups currently described and can be considered a valid alternative to the time-consuming and contamination-prone nested PCR.
Sea bass nervous necrosis virus is the causative agent of viral nervous necrosis, a disease responsible of high economic losses in larval and juvenile stages of cultured sea bass (Dicentrarchus labrax). To identify genes potentially involved in antiviral immune defense, gene expression profiles in response to nodavirus infection were investigated in sea bass head kidney using the suppression subtractive hybridization (SSH) technique. A total of 8.7% of the expressed sequence tags found in the SSH library showed significant similarities with immune genes, of which a prototype galectin (Sbgalectin-1), two C-type lectins (SbCLA and SbCLB) from groups II and VII, respectively, and a short pentraxin (Sbpentraxin) were selected for further characterization. Results of SSH were validated by in vivo up-regulation of expression of Sbgalectin-1, SbCLA, and SbCLB in response to nodavirus infection. To examine the potential role(s) of Sbgalectin-1 in response to nodavirus infection in further detail, the recombinant protein (rSbgalectin-1) was produced, and selected functional assays were conducted. A dose-dependent decrease of respiratory burst was observed in sea bass head kidney leukocytes after incubation with increasing concentrations of rSbgalectin-1. A decrease in IL-1beta, TNF-alpha, and Mx expression was observed in the brain of sea bass simultaneously injected with nodavirus and rSbgalectin-1 compared with those infected with nodavirus alone. Moreover, the protein was detected in the brain from infected fish, which is the main target of the virus. These results suggest a potential anti-inflammatory, protective role of Sbgalectin-1 during viral infection.
Viral encephalopathy and retinopathy (VER) is a severe infective disease characterized by neuropathological changes in several fish species associated with high mortality. The etiological agent is a virus belonging to the Nodaviridae family, genus Betanodavirus. To date, four different betanodavirus species have been officially recognized by International Committee on Taxonomy of Viruses (ICTV), namely the red-spotted grouper- (RGNNV), the striped jack- (SJNNV), the barfin flounder- (BFNNV) and the tiger puffer nervous necrosis virus (TPNNV). Moreover, two reassortants RGNNV/SJNNV and SJNNV/RGNNV have been described. Betanodaviruses can be classified into three different serotypes (A, B and C) that are antigenically different, so none (between serotype A and C) or partial (between serotype B and C) cross-immunoreactivity has been detected in vitro. In this study we investigated the in vivo cross-protection of the two main betanodavirus species (RGNNV and SJNNV), which belong to distinct serotype, by immunizing intraperitoneally (IP) juvenile sea bass with formalin inactivated RGNNV and SJNNV vaccines, followed by a challenge with RGNNV. Fish IP vaccinated with inactivated RGNNV showed a high protection value (85%). Serological analyses highlighted a great specific anti-NNV immunoglobulin M (IgM) production against the homologous virus, while a good seroconversion with low neutralization property was highlighted against the heterologous virus. In fish IP vaccinated with inactivated SJNNV the protection recorded was equal to 25%, significantly lower respect to the one provided by RGNNV IP vaccine. ELISA test detected good IgM production against the homologous virus, and a lower, but still detectable IgM production against the heterologous one. By contrast, serum neutralization test highlighted a poorly detectable antibody production unable to neutralize either the homologous or the heterologous virus. These results confirm that the two serotypes are not cross-protective in vivo. According to these findings, the production of multivalent formulation, or at least the provision of different types of vaccines based on both fish and virus species requirement, should be recommended in order to broaden the range of protection.
Viral Nervous Necrosis (VNN) disease is considered as one of the most serious threats for European sea bass cultured in Mediterranean Sea, with no simple and effective procedures to treat this disease. In this study, 1472 offspring resulting from artificial full factorial mating of western Mediterranean dams with sires from four different wild populations of European sea bass (Northern Atlantic, NAT; Western Mediterranean, WEM; Northern-East Mediterranean, NEM; and Southern-East Mediterranean, SEM) were challenged by experimental infection to W80 betanodavirus strain in order to evaluate genetic variations for VNN resistance among populations and genetic correlations between VNN resistance and production traits. The results showed a large variation of VNN resistance between the four populations tested as well as between sire families within strain. The survivals between pure wild populations SEM, NEM, WEM and NAT were estimated at 99%, 94%, 62%, and 44%, respectively. A moderate intra-population heritability of VNN resistance, calculated based on liability scale with sire model, was recorded for the first time in European sea bass (h²u = 0.26 ± 0.11). Finally, moderate negative genetic correlations between VNN resistance and daily growth coefficient (DGC) and body weight (BW) were also demonstrated (− 0.28 ± 0.20, − 0.35 ± 0.14, respectively) while the genetic correlation between resistance to VNN and fillet adiposity (FA) was weakly negative and not significant (− 0.13 ± 0.19). These results give good prospects of selective breeding of European sea bass for improved resistance to VNN disease.
Histones (H1 to H4) are the primary proteins which mediate the folding of DNA into chromatin; however, and in addition to this function, histones have been also related to antimicrobial peptides (AMPs) activity in vertebrates, in fact, mammalian H1 is mobilized as part as the anti-viral immune response. In fish, histones with AMP activity have been isolated and characterized mainly from skin and gonads. One of most threatening pathogens for wild and cultured fish species nowadays is nodavirus (NNV), which target tissues are the brain and retina, but it is also able to colonize the gonad and display vertical transmission. Taking all this into account we have identified the h1 and h2b coding sequences in European sea bass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata) fish species and studied their pattern of expression under naïve conditions and NNV in vivo infection. The data obtained prompted us to study their role on the immune response of gonad and head-kidney leucocytes upon viral (NNV), bacteria (Vibrio anguillarum or Photobacterium damselae), pathogen-associated molecular patterns (PAMPs) or mitogens stimulation. The h1 and h2b genes are expressed in a wide range of tissues and their expression is modify by infection or other immune stimuli, but further studies will be needed to determine the significance of these changes. These results suggest that h1 expression is related to the immune response against NNV in the brain, while h2b transcription seems to be more important in the head-kidney. Moreover, the potential role of histones as anti-viral agents is suggested and further characterization is in progress.
Vaccines for fish need to be improved for the aquaculture sector, with DNA vaccines and the oral administration route providing the most promising improvements. In this study, we have created an oral chitosan-encapsulated DNA vaccine (CP-pNNV) for the nodavirus (NNV) in order to protect the very susceptible European sea bass (Dicentrarchus labrax). Our data show that the oral CP-pNNV vaccine failed to induce serum circulating or neutralizing specific antibodies (immunoglobulin M) or to up-regulate their gene expression in the posterior gut. However, the vaccine up-regulated the expression of genes related to the cell-mediated cytotoxicity (CMC; tcrb and cd8a) and the interferon pathway (IFN; ifn, mx and ifng). In addition, 3 months after vaccination, challenged fish showed a retarded onset of fish death and lower cumulative mortality with a relative survival of 45%. Thus, we created a chitosan-encapsulated DNA vaccine against NNV that is partly protective to European sea bass juveniles and up-regulates the transcription of genes related to CMC and IFN. However, further studies are needed to improve the anti-NNV vaccine and to understand its mechanisms.
Betanodaviruses are the causative agents of a highly infectious disease of fish known as viral nervous necrosis (VNN). To date, 4 different nervous necrosis virus (NNV) genotypes have been described, but natural reassortant viruses have also been detected, which further increase viral variability. Water temperature plays an important role in determining the appearance and the severity of VNN disease. We assessed the effect of temperature (20°, 25° and 30°C) on mortality and virus load in the brain of European sea bass Dicentrarchus labrax experimentally infected with 4 genetically different betanodaviruses, namely red-spotted grouper NNV (RGNNV), striped jack NNV (SJNNV) and the reassortant strains RGNNV/SJNNV and SJNNV/RGNNV. The RGNNV/SJNNV virus possesses the polymerase gene of RGNNV and the coat protein gene of SJNNV, and vice versa for the SJNNV/RGNNV virus. The obtained results showed that the RGNNV strain is the most pathogenic for juvenile sea bass, but clinical disease and mortality ap peared only at higher temperatures. The SJNNV strain is weakly pathogenic for D. labrax regardless of the temperature used, while virus replication was detected in the brain of survivors only at 20°C. Finally, reassortant strains caused low mortality, independent of the temperature used, but the viral load in the brain was strongly influenced by water temperature and the genetic type of the polymerase gene. Taken together, these data show that nodavirus replication in vivo is a composite process regulated by both the genetic features of the viral strain and water temperatures.
Viral encephalopathy and retinopathy (VER) is one of the most devastating and economically relevant diseases for marine aquaculture. The presence of betanodavirus in freshwater fish is recorded, but very little is known about VER outbreaks in marine species reared in freshwater. Our study investigated the ability of betanodavirus to cause disease in European sea bass, Dicentrarchus labrax, reared at different salinity levels. Fish were challenged with RGNNV or mock infected by bath at different salinity levels (freshwater, 25‰ and 33‰). Fish were checked twice a day and the dead ones were examined by standard virological techniques, by rRT-PCR and by histochemical and immunohistochemical analyses. All the infected groups showed a significant higher mortality rate than the one of the mock-infected group. VERv presence was confirmed by rRT-PCR. Histochemical and immunohistochemical analyses highlighted the typical lesions associated with VER. Our results highlight that salinity does not affect the ability of betanodavirus to induce clinical signs and mortality in European sea bass infected under experimental conditions. These results underline the great adaptation potential of VERv, which in combination with its already known high environmental resistance and broad host range, may explain the diffusion of this disease and the threat posed to aquaculture worldwide.
Viral encephalopathy and retinopathy disease caused by betanodavirus, genus of the family Nodaviridae, affects marine, wild and farmed species including sea bass, one of the most important farmed species in Europe. This work describes a reliable and sensitive indirect ELISA assay to detect betanodavirus in biological samples using a polyclonal antiserum (pAb 283) against the 283/I09 virus strain, the most common red-spotted grouper nervous necrosis virus (RGNNV) genotype in the Mediterranean area, and a capture-based ELISA using a monoclonal antibody (mAb 4C3) specific to a common epitope present on the capsid protein. Using adsorbed, purified VERv preparation, the detection limit of indirect ELISA was 2 μg mL(-1) (3 × 10(5) TCID50 per mL), whereas for capture-based ELISA, the sensitivity for the antigen in solution was 17 μg mL(-1) (35 × 10(5) TCID50 per mL). The capture-based ELISA was employed to detect VERv in brain homogenates of in vivo infected sea bass and resulted positive in 22 of 32 samples, some of these with a high viral load estimates (about 1.1 × 10(8) TCID50 per mL). The ELISA system we propose may be helpful in investigations where coupling of viral content in fish tissues with the presence of circulating VERv-specific IgM is required, or for use in samples where PCR is difficult to perform.
The present paper describes the detection of striped jack nervous necrosis virus (SJNNV), the causative agent of viral nervous necrosis, in eggs, larvae, and brood stocks of hatchery-reared and captured striped jack Pseudocaranx dentex by indirect ELISA. An appropriate condition for the ELISA was determined using purified SJNNV and the detection limit of virus antigens was approximately 5 ng/well. All samples of larvae supposed to be affected with the disease were proved positive for the presence of SJNNV antigens in this ELISA test. The viral antigens were also detected in some samples of fertilized eggs and ovaries of spawners but not in the testis or brain. Thus, it was indicated that spawners were an inoculum source of this viral disease in striped jack.
We investigated the long-term kinetics of nervous necrosis virus (NNV) infectivity in sevenband grouper, Epinephelus septemfasciatus, injected with a live NNV vaccine at 17.3°C and reared at natural seawater temperature. We also evaluated horizontal infection of NNV from fish vaccinated with live NNV to naïve fish in a cohabitation experiment. Although 10.5% mortality was observed in the vaccinated fish, they were strongly protected from the challenge with homologous. The NNV infectivity titer was detected from day 5 after vaccination, peaked on day 10 at 107.43±0.35 TCID50/g, but NNV was under the detection limit (≤102.8 TCID50/g) between days 42 and 128. No mortalities or NNV were detected in any of the vaccinated and cohabitated naïve fish, suggesting that NNV spread from fish vaccinated with live NNV should be low if it is limited to fish in the late stage of vaccination (≥42 days from NNV inoculation). The present results demonstrate that a protective immune response to NNV was mounted in sevenband grouper by the live NNV vaccine without controlling fish rearing temperature.
Following a natural outbreak of viral encephalopa-thy and retinopathy (VER) at a commercial farm in Norway, surviving Atlantic halibut, Hippoglossus hippoglossus, were sequentially studied for distribu-tion of nodavirus, immune response and histopa-thology over 1 year. Typical clinical signs and histopathology of VER were observed during the acute stage of the disease. Most of the surviving fish became subclinical carriers of nodavirus with clus-ters of nodavirus-containing cells in the central nervous system. Four random samplings of pre-sumably healthy fish were performed from two fish groups, with low and high growth rates respectively, over a 7-month period. Immunohistochemical (IHC) examination revealed a higher number of nodavirus-positive cells in fish with a low growth rate than in fish with a high growth rate. All IHC positive fish were also reverse transcriptase polym-erase chain reaction (RT-PCR) positive for noda-virus and for nodavirus antibodies detected by enzyme-linked immunosorbent assay (ELISA) at all sampling points. The percentage of PCR-and ELISA-positive fish remained high throughout the year, while the number of IHC-positive fish decreased, especially in the group with a high growth rate. Several other histopathological changes were observed, including pericarditis, steatitis, changes in liver and kidney, and necrosis of the intestinal wall. None of these findings seemed to be related to the nodavirus infection. Nodavirus was reisolated in cell culture from subclinically infected fish one year after the acute VER outbreak, which indicates that the virus was still infectious.
This paper describes the protective immune responses of sevenband grouper, Epinephelus septemfasciatus Thunberg, immunized with live piscine nodavirus, the causative agent of viral nervous necrosis (VNN), or the Escherichia coli– expressed recombinant coat protein. Nodavirus-neutralizing antibodies were detected at titres ranging from 1:158 to 1:1257 in serum of sevenband grouper which survived intramuscular injection with the virus, by a cell culture assay system. The virus-neutralizing ability of immune serum was also confirmed by injecting virus previously treated with serum into fish. This indicates establishment of acquired immunity in survivors and thus explains why survivors from natural infection are resistant to recurrence of the disease. Young sevenband grouper were immunized twice by intramuscular injections with the recombinant coat protein. Immunized fish produced neutralizing antibodies at high titres for at least 110 days and showed significantly lower mortalities in virus challenge tests. These results suggest the potential for vaccination against VNN in sevenband grouper, which is susceptible to piscine nodavirus at all life-stages.
A polymerase chain reaction (PCR)-based assay to detect nervous necrosis virus (NNV) in fish was developed by using two sets of primers designed on a highly conserved region of the coat protein gene encoded by RNA2 of NNV. The first pair of primers amplified a fragment of 605 bp by one-step reverse-transcription (RT)-PCR, while the second pair amplified an internal segment of 255 bp by nested PCR. Addition of nested PCR increased the assay sensitivity 100-fold when carried out in a separate tube (two-step assay) and 10-fold when performed in the same tube (one-step assay). The sensitivity of the two-step assay was 104 times higher than that of virus cultivation. Nested PCR served also to confirm the specificity of the first amplification, as verified also by Southern hybridization analysis and direct sequencing. In species known to be susceptible to infection, such as European sea bass, Dicentrarchus labrax, and gilthead seabream, Sparus aurata, NNV was often detectable in brain tissue by RT-PCR alone but only by the two-step assay in blood, sperm, ovarian tissue or larvae. The same was true for sperm and ovarian tissue of shi drum, Umbrina cirrosa. NNV was also detected in the brains of Japanese red seabream, Pagrus major and brown meagre, Sciaena umbra, suggesting that these species can also be infected. No NNV was detected in samples of Artemia salina nauplii and rotifers obtained from a fish farm with an NNV outbreak. The inclusion of nested PCR in the assay appears to be necessary to screen out NNV-positive broodfish by blood sampling and testing of their larval progeny.
The use of an indirect elisa for the detection of the sea bass, Dicentrarchus labrax (L.), antibody to nodavirus is described. The sera of 110 adult sea bass (78 females and 32 males) maintained in captivity were analysed, and the females were individually classified in seropositive (16%) and seronegative (56%) groups, while some fish (28%) with low but detectable antibody levels were not classified. The proportion of seropositive males was smaller (3.1%) than the females. Repeated serological examination of 18 individually labelled females (spawners) revealed no changes over 5 months. The immunization of sea bass females with heat-killed nodavirus induced antibody titre as reflected by corresponding changes in elisa optical density readings. The antibody level increased 4 weeks post-immunization and was still detectable after 41 weeks. These preliminary results suggest the possibility of detecting virus-positive or virus-negative animals in attempts to reduce and prevent the vertical transmission of the virus in sea bass hatcheries.
In the last decade betanodavirus infections have emerged as major constraints on the culture of marine fish in all parts of the world with the exception of the African continent. The occurrence of these infections appears to be a function of the number of species cultured and the intensity of culture. This has been further complicated by the promiscuous translocation of stock within and between countries. Great strides have been made in defining these agents and producing diagnostic techniques but much more remains to be done. Lack of knowledge of the epidemiology of the diseases caused by nodaviruses, except for vertical transmission of the pathogen in some species, has impeded the development of control measures but, even so, the measures identified to date have not been adequately implemented by producers with the result that catastrophic losses still occur on a regular basis.
Mass mortalities of the European sea bass, Dicentrarchus labrax (L.), occurred in different ongrowing units in Greece. A presumptive diagnosis of viral nervous necrosis (VNN) was made on the basis of the light microscopic observations of a vacuolating encephalopathy and retinopathy. Positive peroxidase reaction with VNN antiserum confirmed nodavirus as the causative agent. Usually recorded in larvae and juvenile stages of different marine fish species, VNN is described in adult sea bass and sea bass reared in floating cages in the Mediterranean for the first time. The horizontal transmission of the disease has been strongly suspected. Histological analyses clearly demonstrate that cells other than nerve cells are also infected. Some epidemiological aspects of the disease are described and their implications for the establishment of prophylactic guidelines are discussed.
Adult broodstock sea bass, Dicentrarchus labrax (L.), were intramuscularly inoculated with live Sb-Atl nodavirus strain and maintained at two different water temperatures (13 and 20 °C) until spawning. Each spawn was then incubated separately in UV treated water for 3 days and newly hatched larvae reared for 22 days at 13–15 °C. The inoculated broodfish developed a specific immune response following inoculation with the live virus and specific antibodies were detected in the sera of all the inoculated fish whatever the temperature used (13 and 20 °C). Specific nodavirus antigen was also detected by enzyme-linked immunosorbent assay (ELISA) and nested reverse transcription-polymerase chain reaction (RT-PCR) in the brain and the liver of these fish. Vertical transmission of the nodavirus disease was demonstrated following inoculation of the broodfish with the live virus strain, as typical lesions associated with the nodavirus antigen were detected by immunohistochemistry in the brain of larvae at days 14–22 following hatching. The eggs from seven inoculated females that spawned during the experiment were tested for nodavirus by antigen ELISA, RT-PCR and nested RT-PCR. Fertilized and non-fertilized eggs, as well as developing larvae, were found nodavirus positive using these techniques. However, the nested RT-PCR was the only technique that could detect the nodavirus in ovarian biopsies before spawning. Therefore, the selection of nodavirus-free sea bass broodstock cannot be achieved by ELISA or RT-PCR testing of ovarian biopsies. Alternative methods such as serum nodavirus antibody detection of broodstock and/or nested RT-PCR testing of eggs before spawning should be used.
The CD83 cell surface marker is an important and intriguing component of immune system. It is considered the best marker for mature human dendritic cells, but it is also important for thymic development of T cells, and it also plays a role as a regulator of peripheral B-cell function and homeostasis. A CD83-like molecule was identified in sea bass (Dicentrarchus labrax) by EST sequencing of a thymus cDNA library; the CD83 cDNA is composed of 816 bp and the mature CD83 peptide consists of 195 amino acids, with a putative signal peptide of 18 amino acids and two possible N-glycosylation sites. The comparison of sea bass CD83 sequence with its homologues in other fish species and mammals shows some differences, with two cysteine residues conserved from fish to mammals and a high variability both in the total number of cysteines and in mature CD83 sequence polypeptide length. Basal transcripts levels of CD83 mRNA are highest in liver, followed by thymus. The in vitro treatment of head kidney leukocytes with LPS resulted in a down-regulation on CD83 mRNA leves both after 4 and 24 h, whereas with poly I:C an up-regulation after 4h followed by a down-regulation at 24 h was observed. An in vivo infection of sea bass juveniles with nodavirus induced an increase of CD83 expression on head kidney leukocytes both after 6 and 24 h and a decrease after 72 h. On the other hand, an in vivo infection with Photobacterium damselae bacteria induced a decrease of CD83 transcript levels after 6 and 24 h and an increase after 72 h. These findings suggest in sea bass CD83 expression could be modulated by viral and bacterial immune response.
Fish nodaviruses (betanodaviruses) are small, non-enveloped icosahedral single-stranded positive-sense RNA viruses that can cause viral encephalopathy and retinopathy (VER) in a number of cultured marine teleost species, including Atlantic halibut (Hippoglossus hippoglossus). A recombinant protein vaccine and a DNA vaccine were produced, based on the same capsid-encoding region of the Atlantic halibut nodavirus (AHNV) genome, and tested for protection in juvenile turbot (Scophthalmus maximus). Vaccine efficacy was demonstrated in the fish vaccinated with recombinant capsid protein but not in the DNA-vaccinated fish, despite the fact that in vivo expression of the DNA vaccine-encoded antigen was confirmed by RNA in situ hybridisation and immunohistochemistry. Combined DNA and recombinant vaccine administration did not improve the effect of the latter. Surprisingly, fish vaccinated with 50 μg recombinant protein demonstrated a threefold lower survival rate than the two groups that received 10 μg recombinant protein. Neither the recombinant protein vaccine nor the DNA vaccine induced anti-viral antibodies 9 weeks after immunisation, while antibodies reactive with the recombinant protein were detectable mainly in fish vaccinated with 50 μg recombinant protein. The study also demonstrates evidence of viral replication inside the myocytes of intramuscularly challenged fish.
Spherical virus particles were purified from larval striped jack (Pseudocaranx dentex with nervous necrosis. The virus consists of nonenveloped particles, about 25 nm in diameter, and contains two single-stranded, positive-sense RNA molecules with molecular weights of 1.01 × 106Da (RNA 1) and 0.49 × 106Da (RNA 2), respectively. The RNAs do not have poly(A) sequences at the 3′ terminus. Virus structural proteins consist of two proteins with molecular weights of 42 and 40 kDa. When translated into cell-free extracts of rabbit reticulocytes, RNA 1 directed the synthesis of the 1 a protein (100 kDa), whereas RNA 2 synthesized the 2a protein (42 kDa), which is probably the coat protein of the virus, and a polypeptide of 40 kDa which appears to be the processed form of the 42-kDa protein. Under electron microscopic observation, the virus particles were found in the tissues of the central nervous system of the affected larval striped jack. From morphorogical and biochemical properties of the virus, we identified this virus as a new member of the family of Nodaviridae and designated it striped jack nervous necrosis virus.
Nervous necrosis virus (NNV) is the causative agent of viral nervous necrosis (VNN), one of the most serious diseases in over 30 species of cultured marine fishes worldwide. Although several kinds of NNV vaccines have been developed, none of these vaccines have been yet marketed. Here, we demonstrate the potentiality of a live NNV vaccine for sevenband grouper Epinephelus septemfasciatus at a low rearing temperature (17 °C). Moreover, we investigated the kinetics of NNV infectivity titer in fish reared at low and optimum temperatures (17 °C and 26 °C) for VNN onset to determine why sevenband grouper reared at 17 °C survive NNV infection.
The distribution of viral genome in the tissues of juvenile European seabass (Dicentrarchus labrax) during the course of a Red Spotted Grouper Nervous Necrosis Virus (RGNNV) infection has not yet been described. The present study addresses this and indicates which target organs may be involved in viral replication. This information should enable more accurate detection of virus in asymptomatic carriers, and in turn help to control the spread of the disease. The aim of this study was to examine the pattern of expression of viral genomic segments RNA1 and RNA2, using two absolute real-time PCRs (RT-qPCR), over the course of a RGNNV infection after administering the virus by intramuscular injection. In situ hybridization was also used to locate the RNA2 viral segment in different organs throughout the infection. The experimental challenge provoked an acute form of viral nervous necrosis (VNN), with a resulting cumulative mortality of 37%. The RT-qPCRs designed allowed the detection of both genomic segments in all the organs tested (nervous and non-nervous tissues) at all sampling times examined. The highest viral RNA copy number was found in eyes, although viral replication appeared to begin in the brain. Viral replication was also recorded in pooled internal organs and in caudal fin. However, the increase in the viral RNA copy number in these organs did not result in an increased viral titre, which may indicate that a productive infection does not take place in non-nervous tissues, possibly due to a failure in a viral post-replication step.
Gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax) were subjected to either experimental infection with Photobacterium damselae subsp. piscicida or Nodavirus after a period of 2 weeks of crowding in which fish were subjected to a 5-fold increase in density (10-50 kg/m(3)). Samples were obtained before the crowding period (0 h or control) and at 24h and 72 h after crowding from both groups of infected fish. The Complement haemolytic activity and the expression of the C3 gene were evaluated in blood and liver samples respectively. The bacteriolytic and lysozyme activities were also assessed. The results showed that Complement haemolytic activity was reduced at 72 h with both bacteria and virus in high density Gilthead seabream, and a similar increase was observed at low density. Bacteriolytic activity under both bacterial and viral challenges for both species was increased at 24h, under low density. At high density, the bacterial challenge did not induce significant changes. C3 mRNA abundance was substantially increased after pathogen treatments in low density groups at 24h but no significant changes were detected at high densities. These results support the idea of the suppressor effect of stressors on the immune system since a reduction of Complement activity under virus and high density, or lack of response in C3 expression under high density were observed.
Naïve sea bass juveniles (38.4 + or - 4.5 g) were intramuscularly infected with a sublethal dose of betanodavirus isolate 378/I03, followed after 43 days by a similar boosting. This infection resulted in an overall mortality of 7.6%. At various intervals, sampling of fish tissues was performed to investigate: i) B and T lymphocyte content in organs and tissues; ii), proliferation of leucocytes re-stimulated in vitro with inactivated virus; iii) presence of serum antibody specific for betanodavirus; iv) expression of genes coding for the following immunoregulatory molecules involved in innate and acquired responses: type I IFN, Mx, IL-1, Cox-2; IL-10, TGF-beta, TCRbeta, CD4, CD8alpha, IgM, by using a quantitative PCR array system developed for sea bass. The obtained results showed a detectable increase of T cells and B cells in PBL during betanodavirus infection. Furthermore, leucocytes obtained from blood, head kidney, and gills showed a detectable "in vitro" increase in viability upon addition of inactivated viral particles, as determined by measuring intracellular ATP concentration. ELISA analysis of sera showed that exposure to nodavirus induced a low, but specific antibody titer measured 43 days after infection, despite the presence of measurable levels of natural antibody. Finally, a strong upregulation of genes coding for type I IFN, Mx, and IgM was identified after both infection and boosting. Interestingly, an upregulation of Cox-2 until boosting, and of TGF-beta and IL-10 after boosting was also observed, while the other tested genes did not show any significant variations with respect to mock-treated fish. Overall, our work represents a first comprehensive analysis of cellular and molecular immune parameters in a fish species exposed to a pathogenic virus.
A RT-qPCR assay that was developed and optimised for detection of betanodaviruses was validated for use as a diagnostic test for viral nervous necrosis disease of fish. Four betanodavirus genotypes were detected but the sensitivity was greatest for redspotted grouper nervous necrosis virus (RGNNV). The analytical sensitivity was 10-1000-fold greater than that of a nested RT-PCR assay and the limit of detection was <0.4 TCID(50) units per reaction. The assay was highly repeatable (standard deviation of estimated log(10)(viral copies) 0.10+/-0.08) and reproducible (standard deviation of estimated log(10)(viral copies) 0.08+/-0.06). Diagnostic accuracy was assessed in 2193 samples comprising tissue homogenates from Australian bass (Macquaria novemaculeata) and barramundi (Lates calcarifer), and also in SSN-1 tissue culture supernatants, using virus isolation in striped snake head (SSN-1) cell culture as the gold standard. Diagnostic sensitivity and specificity were 100% when the assay was applied to Australian bass tissue and SSN-1 tissue culture supernatants, but for barramundi tissue were 99.1% and 92.8%, respectively. The apparent imperfect specificity was shown by specific amplification of alternate regions of the betanodavirus genome to be due to the lower sensitivity of virus isolation. This is the first study to report the diagnostic performance of a RT-qPCR assay for detection of betanodavirus.