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.

Mass Production of Virus-Like Particles Using Chloroplast Genetic Engineering for Highly Immunogenic Oral Vaccine Against Fish Disease

Nervous necrosis virus (NNV) is the causative agent of viral nervous necrosis (VNN), which is one of the most serious fish diseases leading to mass mortality in a wide range of fish species worldwide. Although a few injectable inactivated vaccines are commercially available, there is a need for more labor-saving, cost-effective, and fish-friendly immunization methods. The use of transgenic plants expressing pathogen-derived recombinant antigens as edible vaccines is an ideal way to meet these requirements. In this study, chloroplast genetic engineering was successfully utilized to overexpress the red-spotted grouper NNV capsid protein (RGNNV-CP). The RGNNV-CP accumulated at high levels in all young, mature, and old senescent leaves of transplastomic tobacco plants (averaging approximately 3 mg/g leaf fresh weight). The RGNNV-CP efficiently self-assembled into virus-like particles (RGNNV-VLPs) in the chloroplast stroma of the transgenic lines, which could be readily observed by in situ transmission electron microscopy. Furthermore, intraperitoneal injection and oral administration of the crudely purified protein extract containing chloroplast-derived RGNNV-VLPs provided the sevenband grouper fish with sufficient protection against RGNNV challenge, and its immunogenicity was comparable to that of a commercial injectable vaccine. These findings indicate that chloroplast-derived VLP vaccines may play a promising role in the prevention of various diseases, not only in fish but also in other animals, including humans.

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.

Resistance to viral nervous necrosis in European sea bass (Dicentrarchus labrax L.): heritability and relationships with body weight, cortisol concentration, and antibody titer

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.

Prevalence of Betanodavirus in Sea Bass (Dicentrarchus Labrax) for All Production Stages in Turkey

Viral Nervous Necrosis (VNN) is one of the most important problems in sea bass aquaculture. Although there are many studies in the world on detection and molecular characterization of betanodaviruses causative agent of VNN, there are not enough studies related to prevalance to provide epidemiological maps. The purpose of this study was to investigate the betanodavirus prevalence in all bass hatcheries actively operating and in some farms in Turkey by RT-qPCR. A total of 2460 samples including fertilized egg, prelarva, postlarva, and fingerling were collected from 16 hatcheries as covering all production stages. A total of 600 sea bass were also collected from 20 farms. Betanodavirus was detected in a hatchery and a farm in fingerling sized sea bass and prevalances were found to be 6.25% and 5%, respectively. Virus isolation initially could not be achieved in E-11 but later SSN-1. Partial genome analysis of both RNA1 and RNA2 segments of the viruses, revealed that they were Redspotted grouper nervous necrosis virus genotype endemic in the Mediterranean basin. The absence of mortality related to VNN in the hatchery and farm, the healthy appearance of the sea bass, the low viral load detected and also output of retrospective epidemiological studies indicate that the infection is subclinical. Not detecting betanodavirus in other age groups where biosafety is implemented indicates that there is no active infection. In the light of these findings, it can be concluded that there are no circulating betanodavirus in hatcheries and the detected virus could be of sea-water origin.

Histopathological of Brain, Eye, Liver, Spleen Organs of Grouper Suspected VNN in Penyambuan Village, North Lombok

Cantang grouper is one of the leading NTB commodities that have high economic value. Cantang grouper (Epinephelus fuscoguttatus) has a wide distribution, one of which is in Penyambuan Village, North Lombok Regency. The disease that often attacks grouper fish is VNN (Viral Nervous Necrosis). One of the methods that can be used to detect VNN is the histopathological method. The purpose of this study was to determine the suspected cases and levels of infection with the VNN (Viral Nervous Necrosis) virus in grouper fish (Epinephelus fuscoguttatus) cultivated in Penyambuan Hamlet, Tanjung District, North Lombok Regency by using the histopathological method. The research method used is descriptive by explaining the results obtained from the histopathological test of 3 samples taken by looking at the presence or absence of necrosis in the eye and brain organs of the test sample. The results of observations using a microscope on the test sample after the histopathological process showed that the second sample (S2) of cantang grouper from the KJA in Pemambuan was suspected to be positive for VNN infection, as seen from the presence of necrosis in the form of vacuoles in the brain and eye organs in the level of chronic infection. From the results of this study, it is suggested that further research be carried out in the form of confirmation of the results using a more accurate method, namely real-time PCR. This study can serve as a detection guide for samples of suspected VNN.