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. Betanodaviruses have been clustered into four genotypes, based on the RNA sequence of the T4 variable region of their capsid protein, and are named after the fish species from which they were first derived i.e. Striped Jack nervous necrosis virus (SJNNV), Tiger puffer nervous necrosis virus (TPNNV), Barfin flounder nervous necrosis virus (BFNNV) and Red-spotted grouper nervous necrosis virus (RGNNV), while an additional genotype turbot betanodavirus strain (TNV) has also been proposed. However, these genotypes tend to be associated with a particular water temperature range rather than being species-specific. Larvae and juvenile fish are especially susceptible to VER, with up to 100% mortality resulting in these age groups during disease episodes, with vertical transmission of the virus increasing the disease problem in smaller fish. A number of vaccine preparations have been tested in the laboratory and in the field e.g. inactivated virus, recombinant proteins, virus-like particles and DNA based vaccines, and their efficacy, based on relative percentage survival, has ranged from medium to high levels of protection to little or no protection. Ultimately a combination of effective prophylactic measures, including vaccination, is needed to control VER, and should also target larvae and broodstock stages of production to help the industry deal with the problem of vertical transmission. As yet there are no commercial vaccines for VER and the aquaculture industry eagerly awaits such a product. In this review we provide an overview on the current state of knowledge of the disease, the pathogen, and interactions between betanodavirus and its host, to provide a greater understanding of the multiple factors involved in the disease process. Such knowledge is needed to develop effective methods for controlling VER in the field, to protect the various aquaculture species farmed globally from the different Betanodavirus genotypes to which they are susceptible.

Tilapia tilapinevirus (also known as tilapia lake virus, TiLV) is considered to be a new threat to the global tilapia industry. The objective of this study was to develop simple cell culture-based heat-killed (HKV) and formalin-killed (FKV) vaccines for the prevention of disease caused by TiLV. The fish were immunized with 100 µl of either HKV or FKV by intraperitoneal injection with each vaccine containing 1.8 × 10 6 TCID 50-inactivated virus. A booster vaccination was carried out at 21-day postvaccination (dpv) using the same protocol. The fish were then challenged with a lethal dose of TiLV at 28 dpv. The expression of five immune genes (IgM, IgD, IgT, CD4 and CD8) in the head kidney and spleen of experimental fish was assessed at 14 and 21 dpv and again after the booster vaccination at 28 dpv. TiLV-specific IgM responses were measured by ELISA at the same time points. The results showed that both vaccines conferred significant protection, with relative percentage survival of 71.3% and 79.6% for HKV and FKV, respectively. Significant up-regulation of IgM and IgT was observed in the head kidney of fish vaccinated with HKV at 21 dpv, while IgM, IgD and CD4 expression increased in the head kidney of fish receiving FKV at the same time point. After booster vaccination, IgT and CD8 transcripts were significantlyThis is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

The pepscan procedure was used to identify betanodavirus B-cell epitopes recognized by neutralizing mouse monoclonal antibodies (MAbs) and serum samples obtained from sea bass, Dicentrarchus labrax, naturally infected with betanodavirus. Pepscan was performed with a panel of thirty-four 12-mer synthetic peptides that mimicked the entire betanodavirus capsid protein. Sea bass serum samples reacted strongly with three regions of the capsid protein comprising amino acid residues 1-32, 91-162 and 181-212. The latter region was also recognized by neutralizing MAbs and coincided with a region of high antigenic propensity identified by an antigen prediction algorithm. These data suggest that a region of the betanodavirus capsid protein spanning amino acid residues 181-212 may represent a neutralization domain that could potentially be used to inform the development of nodavirus vaccines and immunodiagnostic reagents.

Tilapia tilapinevirus (also known as tilapia lake virus, TiLV) is considered to be a new threat to the global tilapia industry. The objective of this study was to develop simple cell culture-based heat-killed (HKV) and formalin-killed (FKV) vaccines for the prevention of disease caused by TiLV. The fish were immunized with 100 μL of either HKV or FKV by intraperitoneal injection with each vaccine containing 1.8 × 106 TCID50 inactivated virus. A booster vaccination was carried out at 21-day post vaccination (dpv) using the same protocol. The fish were then challenged with a lethal dose of TiLV at 28 dpv. The expression of five immune genes (IgM, IgD, IgT, CD4 and CD8) in the head kidney and spleen of experimental fish was assessed at 14 and 21 dpv and again after the booster vaccination at 28 dpv. TiLV-specific IgM responses were measured by ELISA at the same time points. The results showed that both vaccines conferred significant protection, with relative percentage survival (RPS) of 71.3% and 79.6% for HKV and FKV, respectively. Significant up-regulation of IgM and IgT was observed in the head kidney of fish vaccinated with HKV at 21 dpv, while IgM, IgD and CD4 expression increased in the head kidney of fish receiving FKV at the same time point. After booster vaccination, IgT and CD8 transcripts were significantly increased in the spleen of fish vaccinated with the HKV, but not with FKV. Both vaccines induced a specific IgM response in both serum and mucus. In summary, this study showed that both HKV and FKV are promising injectable vaccines for the prevention of disease caused by TiLV in Nile tilapia.

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Immersion vaccination with a biomimetic mucoadhesive nanovaccine has been shown to induce a strong mucosal immune response against columnaris disease, a serious bacterial disease in farmed red tilapia caused by Flavobacterium columnare. However, the induction of a systemic immune response by the vaccine is yet to be investigated. Here, we examine if a specific humoral immune response is stimulated in tilapia by a biomimetic-mucoadhesive nanovaccine against Flavobacterium columnare using an indirect-enzyme-linked immunosorbent assay (ELISA), serum bactericidal activity (SBA) and the expression of immune-related genes within the head-kidney and spleen, together with assessing the relative percent survival of vaccinated fish after experimentally infecting them with F. columnare. The anti-IgM antibody titer of fish at 14 and 21 days post-vaccination was significantly higher in chitosan complex nanoemulsion (CS-NE) vaccinated fish compared to fish vaccinated with the formalin-killed vaccine or control fish, supporting the serum bactericidal activity results at these time points. The cumulative mortality of the unvaccinated control fish was 87% after challenging fish with the pathogen, while the cumulative mortality of the CS-NE vaccinated group was 24%, which was significantly lower than the formalin-killed vaccinated and control fish. There was a significant upregulation of IgM, IgT, TNF α, and IL1-β genes in the spleen and kidney of vaccinated fish. Significant upregulation of IgM and IgT genes was observed in the spleen of CS-NE vaccinated fish. The study confirmed the charged-chitosan-based mucoadhesive nanovaccine to be an effective platform for immersion vaccination of tilapia, with fish generating a humoral systemic immune response against columnaris disease in vaccinated fish.

One of the major disease threats affecting the Mediterranean aquaculture industry is viral encephalopathy and retinopathy (VER). The target organs for Betanodavirus detection are the brain and eyes, obtained through lethal sampling. This study aimed to evaluate the efficacy and suitability of non‐lethal samples for detecting Betanodavirus in European seabass (Dicentrarchus labrax). European seabass juveniles were infected with Betanodavirus, by either an intramuscular injection or immersion (107 TCID50/ml and 106 TCID50/ml, respectively), and samples collected 7, 15 and 30 days post‐infection (dpi). The brain was collected as a lethal sample, and gills, caudal fin and blood as non‐lethal tissues for detecting Betanodavirus by quantitative reverse transcription PCR (RT‐qPCR). The presence of virus in non‐lethal tissues was inconsistent, with lower viral loads than in the brain. For blood, higher viral loads were detected in intramuscular‐infected fish at 15 dpi until the end of the challenge. Serum antibodies against Betanodavirus were assessed using an enzyme‐linked immunosorbent assay (ELISA). Antibodies were detected as early as 7 dpi, with higher mean antibody titres at 15 and 30 dpi. The presence of Betanodavirus‐specific antibodies indicates that this is a suitable evaluation method for detecting early stages of the infection.

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