Fish HERC7: Phylogeny, Characterization, and Potential Implications for Antiviral Immunity in European Sea Bass.

E3 ubiquitin ligases, key components of the ubiquitin proteasome system, orchestrate protein degradation through ubiquitylation and profoundly impact cellular biology. Small HERC E3 ligases (HERC3-6) have diverse functions in mammals, including roles in spermatogenesis, protein degradation, and immunity. Until now, only mammals' HERC3, HERC5, and HERC6 are known to participate in immune responses, with major involvement in the antiviral response. Interestingly, an exclusive HERC7 has been characterized in fish showing great molecular conservation and antiviral roles. Thus, this study identifies and characterizes the herc7 gene in the European sea bass teleost. The European sea bass herc7 gene and the putative protein show good conservation of the promoter binding sites for interferons and the RCC1 and HECT domains characteristic of HERC proteins, respectively. The phylogenetic analysis shows a unique cluster with the fish-exclusive HERC7 orthologues. During ontogeny, the herc7 gene is expressed from 3 days post-fertilization onwards, being constitutively and widely distributed in adult tissues. In vitro, stimulated leucocytes up-regulate the herc7 gene in response to mitogens and viruses, pointing to a role in the immune response. Furthermore, sea bass herc7 expression is related to the interferon response intensity and viral load in different tissues upon in vivo infection with red-grouper betanodavirus (RGNNV), suggesting the potential involvement of fish HERC7 in ISGylation-based antiviral activity, similarly to mammalian HERC5. This study broadens the understanding of small HERC proteins in fish species and highlights HERC7 as a potential contributor to the immune response in European sea bass, with implications for antiviral defense mechanisms. Future research is needed to unravel the precise actions and functions of HERC7 in teleost fish immunity, providing insights into direct antiviral activity and viral evasion.

Synthetic antimicrobial Nkl and Dic peptides are immunomodulatory but only Dic peptide can be therapeutic against nodavirus infection.

Aquaculture is a prosperous economic sector threatened by viral infections. Among the viruses threatening fish culture, Betanodavirus (NNV) is extremely important in the Mediterranean Sea affecting to highly traded species as European sea bass. In this context, application of antimicrobial peptides (AMPs) has arisen as a potential biotechnological tool. The aim of this work was to evaluate the therapeutic application of two European sea bass-derived AMPs, NK-lysin (Nkl) and dicentracin (Dic), against NNV infections. Synthetic Dic peptide was able to significantly reduce NNV-induced mortalities while Nkl failed to do so. Although neither Dic nor Nkl peptides were able to alter the transcriptional levels of NNV and the number of infected cells, Nkl seemed to increase the viral load per cell. Interestingly, both Nkl and Dic peptides showed immunomodulatory roles. For instance, our data revealed an interplay among different AMPs, at both gene and protein levels. Otherwise, Nkl and Dic peptides provoked an anti-inflammatory balance upon NNV infection, as well as the recruitment of macrophages and B cells to the target site of the infection, the brain. In conclusion, Dic can be proposed as a therapeutic candidate to combat NNV.

A Potential Nervous Necrosis Virus (NNV) Live Vaccine for Sole Obtained by Genomic Modification.

Viral Encephalopathy and Retinopathy (VER) is a neurological infectious fish disease that causes vacuolization and necrosis in the central nervous system, which lead to swimming abnormalities and, generally, host death in the early stages of development. VER is caused by the Nervous Necrosis Virus (NNV), a non-enveloped virus with a bisegmented and positive-stranded (+) RNA genome. The largest segment (RNA1) codes for viral polymerase while capsid protein is encoded by RNA2. The aim of this study was to explore the potential of a reverse-engineered RGNNV/SJNNV strain that harbors mutations in both 3'NCRs (position 3073 of RNA1 and 1408 and 1412 of RNA2) as an attenuated live vaccine for sole. The attenuation of this strain was confirmed through experimental infections in sole at 22 °C. Vaccination trials were performed by bath, intramuscular, and intraperitoneal injection, at two temperatures (18 and 22 °C). Our results indicate the improved survival of vaccinated fish and delayed and poorer viral replication, as well as an overexpression of immune response genes linked to T cell markers (cd4 and cd8), to an early inflammatory response (tlr7 and tnfα), and to antiviral activity (rtp3 and mx). In conclusion, our study indicates that the attenuated strain is a good vaccine candidate as it favors sole survival upon infection with the wt strain while inducing a significant immune response.

Betanodavirus meningoencephalitis in an Atlantic blue marlin.

Viral nervous necrosis (viral encephalopathy and retinopathy) is caused by piscine nodavirus (Nodaviridae, Betanodavirus). Since 1986, this highly infectious virus has caused mass mortalities of up to 100% in farmed saltwater and freshwater fish around the world (with the exception of South America and Antarctica), affecting >60 species across 10 orders. The Atlantic blue marlin (Makaira nigricans Lacépède, 1802) is a top-level predator found throughout the tropical waters of the Atlantic and Indo-Pacific oceans. Despite their popularity as a sportfish, relatively little is known about the Atlantic blue marlin and other billfish. We describe here chronic betanodavirus infection in a juvenile Atlantic blue marlin, which is, to our knowledge, the first report of disease in M. nigricans.

Detection of different Betanodavirus genotypes in wild fish from Spanish Atlantic coastal waters (Galicia, northwestern Spain).

OBJECTIVE: The nervous necrosis virus (NNV; genus Betanodavirus) is an aquatic pathogen that is responsible for a neurological disease affecting marine fish. Despite its almost worldwide distribution, global warming could favor the spread of NNV to new areas, highlighting the importance of conducting epidemiological surveys on both wild and farmed marine fish species. In this study, we assessed NNV prevalence in wild fish caught along the Galician Atlantic coast. METHODS: In total, 1277 fish were analyzed by reverse transcription real-time polymerase chain reaction. RESULT: Twenty two (1.72%) of those fish tested positive for NNV, including two species in which the pathogen had not yet been reported. CONCLUSION: The reassortant RGNNV/SJNNV (red-spotted grouper NNV/striped jack NNV) was detected in 55% of NNV-positive individuals, while the remaining 45% harbored the SJNNV-type genome. Moreover, from European Pilchard Sardina pilchardus and Atlantic Mackerel Scomber scombrus, we isolated four reassortant strains that carried amino acid mutations at key sites related to NNV-host interaction.

Recombinant viral hemorrhagic septicemia virus with rearranged genomes as vaccine vectors to protect against lethal betanodavirus infection.

The outbreaks of viral hemorrhagic septicemia (VHS) and viral encephalopathy and retinopathy (VER) caused by the enveloped novirhabdovirus VHSV, and the non-enveloped betanodavirus nervous necrosis virus (NNV), respectively, represent two of the main viral infectious threats for aquaculture worldwide. Non-segmented negative-strand RNA viruses such as VHSV are subject to a transcription gradient dictated by the order of the genes in their genomes. With the goal of developing a bivalent vaccine against VHSV and NNV infection, the genome of VHSV has been engineered to modify the gene order and to introduce an expression cassette encoding the major protective antigen domain of NNV capsid protein. The NNV Linker-P specific domain was duplicated and fused to the signal peptide (SP) and the transmembrane domain (TM) derived from novirhabdovirus glycoprotein to obtain expression of antigen at the surface of infected cells and its incorporation into viral particles. By reverse genetics, eight recombinant VHSVs (rVHSV), termed NxGyCz according to the respective positions of the genes encoding the nucleoprotein (N) and glycoprotein (G) as well as the expression cassette (C) along the genome, have been successfully recovered. All rVHSVs have been fully characterized in vitro for NNV epitope expression in fish cells and incorporation into VHSV virions. Safety, immunogenicity and protective efficacy of rVHSVs has been tested in vivo in trout (Oncorhynchus mykiss) and sole (Solea senegalensis). Following bath immersion administration of the various rVHSVs to juvenile trout, some of the rVHSVs were attenuated and protective against a lethal VHSV challenge. Results indicate that rVHSV N2G1C4 is safe and protective against VHSV challenge in trout. In parallel, juvenile sole were injected with rVHSVs and challenged with NNV. The rVHSV N2G1C4 is also safe, immunogenic and efficiently protects sole against a lethal NNV challenge, thus presenting a promising starting point for the development of a bivalent live attenuated vaccine candidate for the protection of these two commercially valuable fish species against two major diseases in aquaculture.

Identification and characterization of immunoglobulin tau (IgT) in Asian Seabass (Lates calcarifer) and mucosal immune response to nervous necrosis virus.

Mucosal immunity plays a critical role in the protection of teleost fish against infection, but mucosal immunoglobulin of important aquaculture species unique to Southeast Asia remained greatly understudied. In this study, the sequence of immunoglobulin T (IgT) from Asian sea bass (ASB) is described for the first time. IgT of ASB possesses the characteristic structure of immunoglobulin with a variable heavy chain and four CH4 domains. The CH2-CH4 domains and full-length IgT were expressed and CH2-CH4 specific antibody was validated against full-length IgT expressed in Sf9 III cells. Subsequent use of the anti-CH2-CH4 antibody in immunofluorescence staining confirmed the presence of IgT-positive cells in the ASB gill and intestine. The constitutive expression of ASB IgT was characterized in different tissues and in response to red-spotted grouper nervous necrosis virus (RGNNV) infection. The highest basal expression of secretory IgT (sIgT) was observed in the mucosal and lymphoid tissues such as the gills, intestine and head kidney. Following NNV infection, IgT expression was upregulated in the head kidney and mucosal tissues. Moreover, a significant increase in localized IgT was found in gills and intestines of infected fish on day 14 post-infection. Interestingly, a significant increase in NNV-specific IgT secretion was only observed in the gills of the infected group. Our results suggest that ASB IgT may play an important role in the adaptive mucosal immune responses against viral infection and could potentially be adapted as a tool for the evaluation of prospective mucosal vaccines and adjuvants for the species.

Redspotted Grouper Nervous Necrosis Virus and the Reassortant RGNNV/SJNNV In Vitro Susceptibility against a Commercial Peroxy-Acid Biocide under Different Conditions of Use.

Aquaculture is a constantly growing sector. The intensification of fish production and the movement of aquatic animals could cause the spread of infectious diseases. Remarkably, the diffusion of viral agents represents the major bottleneck for finfish production, and viral encephalopathy and retinopathy (VER) is considered the most impacting disease for Mediterranean aquaculture. No effective therapies are available to contrast VER, and vaccination can be applied only in grow-out facilities. Hence, programs to minimize the sanitary risks in farms are paramount to implementing hygienic standards and biosecurity. This study aimed to evaluate the in vitro virucidal activity of a peroxy-acid disinfectant (Virkon® S, DuPont, Sudbury, UK) towards the two NNV strains most widespread in the Mediterranean Sea. Remarkably, two protocols were applied to assess the virucidal activity under different conditions of use: the suspension test and the net test. The latter has been applied to evaluate the efficacy of the biocide on instruments, simulating the in-field application. The obtained results demonstrated the suitability of the tested biocide for NNV inactivation, being effective under some of the tested conditions. However, the presence of organic matter, the concentration of the product, and the application conditions can significantly affect the result of the disinfection procedure.

The extensive transgenerational transcriptomic effects of ocean acidification on the olfactory epithelium of a marine fish are associated with a better viral resistance.

BACKGROUND: Progressive CO2-induced ocean acidification (OA) impacts marine life in ways that are difficult to predict but are likely to become exacerbated over generations. Although marine fishes can balance acid-base homeostasis efficiently, indirect ionic regulation that alter neurosensory systems can result in behavioural abnormalities. In marine invertebrates, OA can also affect immune system function, but whether this is the case in marine fishes is not fully understood. Farmed fish are highly susceptible to disease outbreak, yet strategies for overcoming such threats in the wake of OA are wanting. Here, we exposed two generations of the European sea bass (Dicentrarchus labrax) to end-of-century predicted pH levels (IPCC RCP8.5), with parents (F1) being exposed for four years and their offspring (F2) for 18 months. Our design included a transcriptomic analysis of the olfactory rosette (collected from the F2) and a viral challenge (exposing F2 to betanodavirus) where we assessed survival rates. RESULTS: We discovered transcriptomic trade-offs in both sensory and immune systems after long-term transgenerational exposure to OA. Specifically, RNA-Seq analysis of the olfactory rosette, the peripheral olfactory organ, from 18-months-old F2 revealed extensive regulation in genes involved in ion transport and neuronal signalling, including GABAergic signalling. We also detected OA-induced up-regulation of genes associated with odour transduction, synaptic plasticity, neuron excitability and wiring and down-regulation of genes involved in energy metabolism. Furthermore, OA-exposure induced up-regulation of genes involved in innate antiviral immunity (pathogen recognition receptors and interferon-stimulated genes) in combination with down-regulation of the protein biosynthetic machinery. Consistently, OA-exposed F2 challenged with betanodavirus, which causes damage to the nervous system of marine fish, had acquired improved resistance. CONCLUSION: F2 exposed to long-term transgenerational OA acclimation showed superior viral resistance, though as their metabolic and odour transduction programs were altered, odour-mediated behaviours might be consequently impacted. Although it is difficult to unveil how long-term OA impacts propagated between generations, our results reveal that, across generations, trade-offs in plastic responses is a core feature of the olfactory epithelium transcriptome in OA-exposed F2 offspring, and will have important consequences for how cultured and wild fish interacts with its environment.

Pathogenicity of Different Betanodavirus RGNNV/SJNNV Reassortant Strains in European Sea Bass.

European sea bass (Dicentrarchus labrax) is an important farmed marine species for Mediterranean aquaculture. Outbreaks of betanodavirus represent one of the main infectious threats for this species. The red-spotted grouper nervous necrosis virus genotype (RGNNV) is the most widely spread in Southern Europe, while the striped jack nervous necrosis virus genotype (SJNNV) has been rarely detected. The existence of natural reassortants between these genotypes has been demonstrated, the RGNNV/SJNNV strain being the most common. This study aimed to evaluate the pathogenicity of different RGNNV/SJNNV strains in European sea bass. A selection of nine European reassortants together with parental RGNNV and SJNNV strains were used to perform in vivo experimental challenges via intramuscular injection. Additional in vivo experimental challenges were performed by bath immersion in order to mimic the natural infection route of the virus. Overall, results on survival rates confirmed the susceptibility of European sea bass to reassortants and showed different levels of induced mortalities. Results obtained by RT-qPCR also highlighted high viral loads in asymptomatic survivors, suggesting a possible reservoir role of this species. Our findings on the comparison of complete genomic segments of all reassortants have shed light on different amino acid residues likely involved in the variable pathogenicity of RGNNV/SJNNV strains in European sea bass.

The first detection of betanodavirus reassortant genotype (RGNNV/SJNNV) isolated from gilthead sea bream (Sparus aurata) in the Turkish coastlines: The importance of screening and monitoring studies for identifying the source of the infection.

Viral nervous necrosis (VNN) is now endemic in the Mediterranean basin and the RGNNV genotype betanodavirus has caused frequent epidemics in European sea bass for a long time. Unexpected and increasing VNN epidemics have been reported in gilthead sea bream (GSB) farms in the last few years, from which the RGNNV/SJNNV genotype has been mostly isolated. The aim of this study was to perform a molecular characterization of the betanodavirus isolated from GSB (weighing 90-100 g) in a marine fish farm in the Aegean Sea and also, as an early warning exercise, to investigate the presence/absence of the virus in associated nearby farms (n:20) and in hatcheries (n:3). No virus was detected in any of the nearby farms or two hatcheries. However, in one hatchery, betanodavirus was detected in a 160-day-old GSB. The identified betanodavirus was genotyped as reassortant RGNNV/SJNNV and was phylogenetically related to the virus detected in the farm located in the Aegean sea. There have been multiple detections of the RGNNV genotype in Turkish coastal waters; however, the RGNNV/SJNNV genotype has been detected for the first time and it should be an early warning to focus attention on betanodaviruses in Turkish aquaculture.

Reservoirs of Red-Spotted Grouper Nervous Necrosis Virus (RGNNV) in Squid and Shrimp Species of Northern Alboran Sea.

The production of the aquaculture industry has increased to be equal to that of the world fisheries in recent years. However, aquaculture production faces threats such as infectious diseases. Betanodaviruses induce a neurological disease that affects fish species worldwide and is caused by nervous necrosis virus (NNV). NNV has a nude capsid protecting a bipartite RNA genome that consists of molecules RNA1 and RNA2. Four NNV strains distributed worldwide are discriminated according to sequence homology of the capsid protein encoded by RNA2. Since its first description over 30 years ago, the virus has expanded and reassortant strains have appeared. Preventive treatments prioritize the RGNNV (red-spotted grouper nervous necrosis virus) strain that has the highest optimum temperature for replication and the broadest range of susceptible species. There is strong concern about the spreading of NNV in the mariculture industry through contaminated diet. To surveil natural reservoirs of NNV in the western Mediterranean Sea, we collected invertebrate species in 2015 in the Alboran Sea. We report the detection of the RGNNV strain in two species of cephalopod mollusks (Alloteuthis media and Abralia veranyi), and in one decapod crustacean (Plesionika heterocarpus). According to RNA2 sequences obtained from invertebrate species and reported to date in the Mediterranean Sea, the strain RGNNV is predominant in this semienclosed sea. Neither an ecosystem- nor host-driven distribution of RGNNV were observed in the Mediterranean basin.

Mass mortalities associated with viral nervous necrosis in Murray cod in China.

In December 2019, a mass mortality among cultured Murray cod (Maccullochella peelii peelii) fry occurred on a freshwater farm located at Foshan city of Guangdong province, China. The cumulative mortality was up to 45% within 15 days. The diseased fish showed clinical signs, including abnormal swimming behaviour, loss of appetite and dark body colouration before mass mortality. Samples of brain and retina tissues were collected from affected fish and subjected to reverse transcriptase polymerase chain reaction detection and virus isolation in cell culture. Approximately 430 bp product was detected from the brain and retina tissues and culture supernatant of betanodavirus-infected SSN-1 cells. The typical cytopathic effect of betanodavirus infection, which is characterized by vacuolation, was observed in SSN-1 cells at three days after inoculating with the tissue filtrate of diseased Murry cod fry, and the TCID50 of the infected SSN-1 cell supernatant was 107.8 . Histopathological examinations revealed vacuolation and necrosis in the brain and retina of naturally and experimentally infected Murray cod fry. Electron microscopic observation also showed the aggregation of numerous spherical, non-enveloped viral particles measuring 22-28 nm in diameter in the cytoplasm of betanodavirus-infected SSN-1 cells. Sequence and phylogenetic analysis based on RdRp and Cp genes further indicated that the betanodavirus isolated from Murray cod belonged to the RGNNV genotype. Much higher mortality was obtained in challenged Murray cod fry compared with the controls through immersion challenge. This study is the first report of the natural infection of betanodavirus in freshwater fish in China.

Amantadine, a promising therapeutic agent against viral encephalopathy and retinopathy.

Outbreaks of viral encephalopathy and retinopathy (VER) in marine and freshwater species severely devastate the aquaculture worldwide. The causative agent of VER is nervous necrosis virus (NNV), which mainly infects the early developmental stages of fish, limiting the effectiveness of vaccines. To counter this case, the anti-NNV potentials of nine drugs with broad-spectrum antiviral activity were explored using ribavirin as a positive drug. Toxicity of the selected drugs to SSN-1 cells and grouper was firstly evaluated to determine the safety concentrations. For screening in vitro, amantadine and oseltamivir phosphate can relieve the cytopathic effects and inhibit NNV replication with the 90% inhibitory concentrations (IC90 ) of 38.74 and 106.75 mg/L, respectively. Amantadine has a stronger anti-NNV activity than ribavirin at the with- and post-NNV infection stages, indicating that it is a potential therapeutic agent against VER by acting directly on NNV. Similarly, amantadine also has a strong anti-NNV activity in vivo with the IC90 of 27.91 mg/L at the 7 days post-infection, while that was 73.25 mg/L for ribavirin. Following exposure to amantadine (40 mg/L) and ribavirin (100 mg/L) for 7 days, the survival rates of NNV-infected grouper were increased to 44% and 39%, respectively. The maximum amantadine content (11.88 mg/Kg) in grouper brain was reached following exposure for 24 hr, and amantadine can be quickly excreted from fish, reducing the risk of drug residue. Results so far indicated that amantadine is a promising therapeutic agent against NNV in aquaculture, providing an effective strategy for VER control at the early developmental stages of fish.

Update on the Inactivation Procedures for the Vaccine Development Prospects of a New Highly Virulent RGNNV Isolate.

Viral nervous necrosis (VNN) caused by the nervous necrosis virus (NNV) affects a broad range of primarily marine fish species, with mass mortality rates often seen among larvae and juveniles. Its genetic diversification may hinder the effective implementation of preventive measures such as vaccines. The present study describes different inactivation procedures for developing an inactivated vaccine against a new NNV isolate confirmed to possess deadly effects upon the European seabass (Dicentrarchus labrax), an important Mediterranean farmed fish species that is highly susceptible to this disease. First, an NNV isolate from seabass adults diagnosed with VNN was rescued and the sequences of its two genome segments (RNA1 and RNA2) were classified into the red-spotted grouper NNV (RGNNV) genotype, closely clustering to the highly pathogenic 283.2009 isolate. The testing of different inactivation procedures revealed that the virus particles of this isolate showed a marked resistance to heat (for at least 60 °C for 120 min with and without 1% BSA) but that they were fully inactivated by 3 mJ/cm2 UV-C irradiation and 24 h 0.2% formalin treatment, which stood out as promising NNV-inactivation procedures for potential vaccine candidates. Therefore, these procedures are feasible, effective, and rapid response strategies for VNN control in aquaculture.

Emergence of Reassortment between a New and Reported Types of Betanodavirus in Shellfish.

Recently, three types of betanodavirus including red spotted grouper nervous necrosis virus (RGNNV), barfin flounder nervous necrosis virus (BFNNV), and Korean shellfish nervous necrosis virus (KSNNV) (proposed as a new fifth type) have been detected in shellfish in the marine environment around Korea. To investigate the presence of reassortment between betanodavirus types, the type based on the RNA2 segment of betanodaviruses carried in 420 domestic shellfish (n = 306) and finfish (n = 35), as well as imported shellfish (n = 79), was compared with the type identified by reverse-transcriptase polymerase chain reaction (RT-PCR) for RNA1 segment. Only five samples carrying reassortant betanodaviruses were found, appearing as RG/KSNNV (n = 2), KS/RGNNV (n = 1), and SJ/RGNNV (n = 2) types. From these samples, we successfully isolated two reassortant strains from Korean and Chinese shellfish in E-11 cells and called them KG1-reKS/RG and CM1-reRG/KS, respectively. In the full genome sequences, each RNA segment of the reassortant strains exhibited the same gene length and high sequence homology (≥98%) with the reference strains corresponding to the type of each segment. Both these reassortant strains induced high mortality to sevenband grouper (Epinephelus septemfasciatus) larvae with high viral concentrations in the body (109 viral particles/mg) and severe vacuolation in the retina and brain. These are the first results showing the involvement of the KSNNV type in the reassortment of RNA segments in the reported types of betanodavirus, which could represent a new potential risk in fish.

Phylogeography of betanodavirus genotypes circulating in Tunisian aquaculture sites, 2012-2019.

The purpose of this study was to determine the phylogenetic relationships among the primary betanodavirus strains circulating in Tunisian coastal waters. A survey was conducted to investigate nodavirus infections at 15 European sea bass Dicentrarchus labrax and gilthead sea bream Sparus aurata farming sites located along the northern and eastern coasts of Tunisia. The primary objective of the study was to create epidemiological awareness of these infections by determining phylogenetic relationships between the main betanodavirus strains circulating during the period 2012-2019, using RNA1 and/or RNA2 genome segments. Approximately 40% (118 of 294) tissue pools tested were positive for betanodavirus. Positive pools were distributed across all of the sampling sites. While fish mortalities were always correlated with the presence of virus in sea bass, a severe outbreak was also identified in sea bream larvae in 2019. Phylogenetic analysis revealed that almost all Tunisian strains from both sea bass and sea bream irrespective of outbreaks clustered within the RGNNV genotype. It is noteworthy that samples collected during the 2019 outbreak from sea bream contained both RNA1 and RNA2 fragments belonging to the RGNNV and SJNNV genotype, respectively, an indication of viral genome reassortment. To our knowledge, this is the first report of reassortant betanodavirus in Tunisia.

Direct Amperometric Sensing of Fish Nodavirus RNA Using Gold Nanoparticle/DNA-Based Bioconjugates.

We describe the design of a simple and highly sensitive electrochemical bioanalytical method enabling the direct detection of a conserved RNA region within the capsid protein gene of a fish nodavirus, making use of nanostructured disposable electrodes. To achieve this goal, we select a conserved region within the nodavirus RNA2 segment to design a DNA probe that is tethered to the surface of nanostructured disposable screen-printed electrodes. In a proof-of-principle test, a synthetic RNA sequence is detected based on competitive hybridization between two oligonucleotides (biotinylated reporter DNA and target RNA) complimentary to a thiolated DNA capture probe. The method is further validated using extracted RNA samples obtained from healthy carrier Sparus aurata and clinically infected Dicentrarchus labrax fish specimens. In parallel, the sensitivity of the newly described biosensor is compared with a new real-time RT-PCR protocol. The current differences measured in the negative control and in presence of each concentration of target RNA are used to determine the dynamic range of the assay. We obtain a linear response (R2 = 0.995) over a range of RNA concentrations from 0.1 to 25 pM with a detection limit of 20 fM. The results are in good agreement with the results found by the RT-qPCR. This method provides a promising approach toward a more effective diagnosis and risk assessment of viral diseases in aquaculture.

Long-Term Protection and Serologic Response of European Sea Bass Vaccinated with a Betanodavirus Virus-Like Particle Produced in Pichia pastoris.

Viral Nervous Necrosis (VNN) causes high mortality and reduced growth in farmed European sea bass (Dicentrarchus labrax) in the Mediterranean. In the current studies, we tested a novel Pichia-produced virus-like particle (VLP) vaccine against VNN in European sea bass, caused by the betanodavirus "Red-Spotted Grouper Nervous Necrosis Virus" (RGNNV). European sea bass were immunized with a VLP-based vaccine formulated with different concentrations of antigen and with or without adjuvant. Antibody response was evaluated by ELISA and serum neutralization. The efficacy of these VLP-vaccine formulations was evaluated by an intramuscular challenge with RGNNV at different time points (1, 2 and 10 months post-vaccination) and both dead and surviving fish were sampled to evaluate the level of viable virus in the brain. The VLP-based vaccines induced an effective protective immunity against experimental infection at 2 months post-vaccination, and even to some degree at 10 months post-vaccination. Furthermore, the vaccine formulations triggered a dose-dependent response in neutralizing antibodies. Serologic response and clinical efficacy, measured as relative percent survival (RPS), seem to be correlated with the administered dose, although for the individual fish, a high titer of neutralizing antibodies prior to challenge was not always enough to protect against disease. The efficacy of the VLP vaccine could not be improved by formulation with a water-in-oil (W/O) adjuvant. The developed RGNNV-VLPs show a promising effect as a vaccine candidate, even without adjuvant, to protect sea bass against disease caused by RGNNV. However, detection of virus in vaccinated survivors means that it cannot be ruled out that survivors can transmit the virus.

Detection of RGNNV genotype betanodavirus in the Black Sea and monitoring studies.

Viral nervous necrosis (VNN), caused by betanodavirus, is a significant viral infection that threatens marine aquaculture. Freshwater and marine fish farms in Turkey are subjected to annual pathogen screenings. In 2016, during the Nervous Necrosis Virus screening program conducted in the Black Sea, betanodavirus was unexpectedly detected using real-time reverse transcription-polymerase chain reaction in apparently healthy sea bass. Phylogenetic analysis of both the RNA1 and RNA2 segments of the virus determined that the betanodavirus detected was red-spotted grouper nervous necrosis virus genotype (RGNNV). Following the initial discovery of betanodavirus in the Black Sea, monitoring studies performed over a 3 yr period have not indicated any additional presence of the virus. The absence of clinical symptoms related to VNN disease in the area's marine fish farms and the surrounding detection zone, and the fact that the virus has not been detected anew in monitoring programmes conducted following the initial detection, indicate that there is no virus circulation in the detection zone.