Nervous Necrosis Virus-like Particle (VLP) Vaccine Stimulates European Sea Bass Innate and Adaptive Immune Responses and Induces Long-Term Protection against Disease.

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.

RT-qPCR assay for detection of mink astrovirus in outbreaks of diarrhea on Danish mink farms.

Diarrhea in mink kits is a major cause of disease and mortality in the mink production. The etiology remains unknown in most outbreaks due to a lack of diagnostic assays. In the current study we present an RT-qPCR method to detect mink astrovirus in fecal samples from mink kits with diarrhea. All sampled animals were classified based on age and patoanatomical evaluation as having pre-weaning diarrhea, diarrhea in the growth period or as having no macroscopic signs of diarrhea. Fecal samples were analyzed for MiAstV with RT-qPCR, next generation sequencing and electron microscopy in parallel. Mink astrovirus was detected with RT-qPCR in 92 out of 203 samples. This detection was confirmed by next generation sequencing in a high proportion of samples (22/27), and by visualization of astrovirus particles with EM in some of the samples. Mink astrovirus was highly prevalent (68%) among kits in the outbreaks of pre-weaning diarrhea, in particular outbreaks from May, while less prevalent in outbreaks in June. Mink astrovirus was detected in outbreaks of diarrhea in the growth period, though in a much lesser extent than in the pre-weaning period. The role of mink astrovirus in the diarrhea disease complex of mink remain to be investigated, and for that purpose this sensitive and robust RT-qPCR can be a valuable tool in the future.

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.