Gamma-Ray Inactivation of Betanodavirus: Antigen Preservation and Vaccine Potential

Gamma-Ray Inactivation of Betanodavirus: Antigen Preservation and Vaccine Potential | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Gamma-Ray Inactivation of Betanodavirus: Antigen Preservation and Vaccine Potential Houda Agrebi, Alessandra Buratin, Paola Berto, Semah Najahi, Richard Thiga Kangethe, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8549652/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Viral nervous necrosis (VNN), caused by Betanodavirus , poses a significant challenge to Mediterranean aquaculture, especially for European sea bass ( Dicentrarchus labrax ). Conventional chemical inactivation methods often compromise viral immunogenicity or introduce cytotoxic effects, highlighting the need for safer and more effective alternatives for virus inactivation. This study explores gamma irradiation as an alternative approach to inactivate reassortant Betanodavirus (RGNNV/SJNNV) while preserving its antigenic properties for vaccine development. Virus samples were exposed to gamma radiation doses ranging from 5 to 60 kGy. The D₁₀ value (7.55 kGy), the inactivation dose (55.35 kGy), and the gamma radiation dose (70.45 kGy) ensuring a Sterility Assurance Level (SAL) of the Tunisian Betanodavirus were established based on viral infectivity reduction. In vitro immunological assays, including enzyme-linked immunosorbent assay (ELISA) demonstrated that the structural proteins of the virus retained antigenicity post-irradiation, as evidenced by consistent IgM antibody recognition titers. Transmission electron microscopy confirmed that irradiation did not alter the viral shape or structural integrity. The addition of trehalose as a radioprotectant prior to irradiation did not enhance the immunogenic response, as measured by ELISA. Notably, the highest immune response characterized by 92% IgM-positive individuals, was observed in fish vaccinated with irradiated virus in absence of trehalose. These results highlight gamma irradiation as a promising, non-chemical method for the development of safe and immunogenic inactivated vaccines against Betanodavirus in aquaculture. Antigenicity Aquaculture Betanodavirus Dicentrarchus labrax Gamma irradiation Inactivated vaccine RGNNV/SJNNV Sterility Assurance Level Trehalose Viral nervous necrosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Aquaculture is one of the fastest growing sectors of the global food industry, currently surpassing traditional marine fisheries in the production of fish protein for human consumption (FAO 2012 ). This rapid expansion has been associated with an increased incidence of infectious diseases, facilitated by the continuous movement of eggs, larvae and adult fish between aquaculture facilities worldwide. In addition, survivors of disease outbreaks can act as asymptomatic carriers, complicating disease management and control strategies. One of the most widespread and devastating pathogens in marine aquaculture is nervous necrosis virus (NNV), a member of the genus Betanodavirus and family Nodaviridae according to the International Committee on Taxonomy of Viruses(The International Committee on Taxonomy of Viruses (ICTV)). NNV is the causative agent of viral encephalopathy and retinopathy (VER), a severe neuropathological disease that results in mortality rates of up to 100%, particularly in larval and juvenile fish (Bandín and Souto 2020 ). NNV is a non-enveloped, icosahedral virus with a bi-segmented, positive-sense single-stranded RNA genome: RNA1 (3.1 Kb) and RNA2 (1.4 Kb), which encode the RNA-dependent RNA polymerase (RdRp) and the capsid protein (CP), respectively (Mori et al. 1992 ). Based on the small variable region of RNA2, four genotypes have been identified: Betanodavirus pseudocarangis (SJNNV), Betanodavirus takifugui (TPNNV), Betanodavirus verasperi (BFNNV), and Betanodavirus epinepheli (RGNNV) (Nishizawa et al. 1997 ; Bandín and Souto 2020 ). Additionally, reassortant strains, such as RGNNV/SJNNV and SJNNV/RGNNV, have emerged, posing further challenges to fish health (Toffolo et al. 2007 ; Olveira et al. 2009 ; Panzarin et al. 2012 ; Toffan et al. 2017 ; Chérif et al. 2021 ). These NNV genotypes have distinct host ranges, geographic distributions, serotypes, and in vitro growth temperature preferences. The BFNNV infects species such as barfin flounder ( Verasper moseri ) and Atlantic cod ( Gadus morhua ); it is predominantly found in the North Sea, Norwegian Sea, Baltic Sea, and Northern Atlantic Ocean, belongs to serotype B, and grows optimally at 15–20°C. The RGNNV, the most widely distributed genotype, infects various groupers ( Epinephelus spp.) , Japanese flounder ( Paralichthys olivaceus ), and European sea bass ( Dicentrarchus labrax ); it is prevalent in the Mediterranean and in the surrounding seas of Australia and Asia, corresponds to serotype C, and replicates efficiently at 25–30°C. The SJNNV affects hosts like striped jack ( Pseudocaranx dentex ) and Senegalese sole ( Solea senegalensis ); it occurs mainly in Japan and the Atlantic ocean in front of the Iberian Peninsula, is classified under serotype A, and grows best at 20–25°C. The TPNNV has been isolated only from tiger puffer ( Takifugu rubripes ); it is restricted to Japan, belongs to serotype B, and has an optimal growth temperature of 20°C(Barsøe 2021 ). European seabass ( Dicentrarchus labrax ), an important species in Mediterranean aquaculture (FEAP 2022 ), is highly susceptible to RGNNV, with mortality rates up to 100% in infected populations (Chérif et al. 2009 ; Shetty et al. 2012 ). However, reassortant viruses RGNNV/SJNNV can cause infection in this species, and natural outbreaks caused by reassortant RGNNV/SJNNV has been described in European sea bass larvae and juveniles with variable cumulative mortality (Volpe et al. 2020 ; Biasini et al. 2022 ). Recently, RGNNV/SJNNV has also been isolated from seabass in Tunisia, suffering significant mortality (Agrebi et al. 2025 ). Additionally, the increasing frequency of Betanodavirus outbreaks caused by RGNNV/SJNNV strains in Sparus aurata farms (NaveenKumar et al. 2017 ; Savoca et al. 2021 ) highlight the urgent need for prophylactic measures such as vaccines. Currently, two commercial vaccines are available for the prevention of VNN in aquaculture: Alphaject micro-1-noda (Pharmaq AS)(Pharmaq 2021 ), which contains inactivated RGNNV strain ALV1107 with a mineral oil adjuvant, administered at 0.05 mL intraperitoneally (IP) to fish ≥ 12 g, providing immunity after 466 degree days and lasting for one year; and Icthiovac VNN (Laboratorios HIPRA)(HipraIcthiovac. 2021), which uses inactivated Betanodavirus strain 1103 with a non-mineral oil adjuvant, administered at 0.1 mL IP to fish ≥ 15 g, with an onset of immunity at 924 degree days, though its duration is not established. Both vaccines are designed for conferring protection against RGNNV virus, while for the RGNNV/SJNNV viruses, which possess different antigenic properties, no vaccine are available. These vaccines are inactivated by formalin, which, while effective, can compromise the immunogenicity of the virus, reducing the efficacy of the resulting vaccine (Valero et al. 2018 ; Barsøe 2021 ). Other chemical inactivation techniques, such as binary ethylenimine (BEI), have shown promising but still face limitations (Valero et al. 2021 , p. 20). As a result, there has been a growing interest in alternative inactivation methods, with gamma irradiation emerging as a promising approach for inactivating viruses while preserving their antigenicity (Unger et al. 2022 ). Compared to chemical inactivation methods, gamma irradiation offers a safer and more effective alternative by avoiding the risk of chemical residues while efficiently inactivating pathogens through targeted damage to nucleic acids (DNA or RNA). Importantly, this approach can preserve surface antigens and overall viral structure, particularly when radioprotectants such as trehalose are employed, thereby maintaining immunogenic properties essential for vaccine development (Tobin et al. 2020 ). Gamma irradiation has already been successfully used in the development of vaccines for human and animal diseases such as malaria, influenza and many others (Bhatia and Pillai 2022 ). This study investigates the use of gamma irradiation as a method to inactivate reassortant Betanodavirus (RGNNV/SJNNV) while preserving its antigenicity for vaccine development. The aim is to determine the optimal irradiation dose in combination with different percentual of trehalose that ensures complete viral inactivation without compromising the ability of the virus to stimulate a satisfactory immune response. This research will contribute to the development of safer and more effective vaccines for Mediterranean aquaculture, potentially providing a novel solution to the ongoing challenge of Betanodavirus outbreaks in European seabass farming. 2. Materials and Methods 2.1 Virus Stock Preparation and Cell Cultures The Tunisian reassortant RGNNV/SJNNV Betanodavirus strain, VNNV/D.labrax/TUN/148-8/Dec23, was isolated from D. labrax in 2023(Agrebi et al. 2025 ) and propagated in striped snakehead cells (SNN-1) (Frerichs et al. 1991 ). The SNN-1 cells were inoculated with the isolate and incubated at 25°C until the cytopathic effect (CPE) was extensive. The supernatant was then harvested, centrifuged at 3000 × g for 10 minutes to remove cell debris and stored at − 80°C until further use. Viral titers were determined on SNN-1 cell culture and calculated with the TCID 50 method as described by Reed and Muench ( 1938 ) (Reed and Muench 1938 ). 2.2 Betanodavirus Inactivation by Gamma Irradiation Virus inactivation was carried out at the International Atomic Energy Agency (IAEA) laboratories in Seibersdorf, Austria, following established protocols, using a Co-60 irradiator Model 812 (Foss Therapy services, Inc., California, and USA). Gamma ray doses of 5, 10, 15, 20, 30, 40, 50 and 60 kGy were applied to triplicate aliquots of 3 mL each of Betanodavirus suspensions with 12% trehalose (Sigma) (w/v), under frozen conditions. The irradiated samples were titrated as reported before and a dose-response curve was constructed to determine the D 10 value and the optimal dose for complete virus inactivation also known as sterility assurance level (SAL) dose. After determination of the gamma radiation dose (70.45 kGy) ensuring the SAL of the Tunisian Betanodavirus , vials containing 10 ml each of Betanodavirus suspensions with different radioprotector (trehalose) concentrations (0, 6 and 12%) were irradiated in triplicate at the determined SAL dose. These samples were evaluated to assess the preservation of viral antigens by ELISA assay, morphology post-irradiation by transmission electron microscopy (TEM) observation and immunogenicity evaluation by western blot analysis and fish immunization. 2.3 Characterization of Inactivated Betanodavirus 2.3.1. ELISA assay Recognition of antigens in irradiated Betanodavirus compared to the unirradiated control virus was determined by indirect ELISA. Briefly, for antigen coating, 100 µL of ultracentrifuged Betanodavirus particles (diluted 1:10 in carbonate-bicarbonate buffer, pH 8.9) were added to MaxiSorb ELISA plates (In Vitro , Denmark) and incubated overnight at 4°C. After washing the plates and blocking step them with PBS containing 3% bovine serum albumin (Merck Products) for 1 hour at 37°C, the coated wells were incubated with primary antibodies specific for NNV A serotype. Rabbit anti-RGNNV/SJNNV (Anti-378) and Rabbit anti-SJNNV (Anti-484) (IZSVe, Padua, Italy) were diluted 1:1000 and 1:10.000, respectively and tested separately against treated and untreated virus. After incubation with a secondary anti-rabbit IgG peroxidase-conjugated antibody (Merck Products) at a 1:4000 dilution, color development was performed using TMB ELISA substrate (Prodotti Gianni) and stopped with H₂SO₄ (2N). Specific absorbances (OD) were recorded at 450 nm. 2.3.2 Western Blot Analysis Western blot analysis was performed to confirm the presence of viral antigens in irradiated Betanodavirus . Viral samples (20µl) were prepared with Laemmli loading buffer and run on a 4–15% gradient SDS-PAGE gel (Mini Protean TGX Stain Free Precast gel_Bio-Rad). After electrophoresis, the protein profile in the gel was transferred to PVDF membrane (Immobilon). For immunoblotting analysis, the membrane was saturated with blocking buffer (5% in PBS) for 1h and incubated for 2h with the primary antibody: rabbit anti-RGNNV/SJNNV diluted 1:500 (Anti-378,) or rabbit anti-SJNNV diluted 1:1000 (Anti-484) (IZSVe, Padua, Italy). A second incubation of 1h with HRP-conjugated goat anti-rabbit IgG secondary antibody (diluted 1:1000) was performed. Labeled proteins were visualized with an Opti-2CN substrate Kit (Bio-Rad). 2.3.3 Transmission Electron Microscopy (TEM) Transmission electron microscopy (TEM) was used to assess the preservation of morphology in irradiated Betanodavirus . Both ultracentrifuged unirradiated and irradiated Betanodavirus samples were analyzed by negative staining. A Formvar carbon-supported copper grid (Electron Microscopy Sciences, Formvar/Carbon Copper Grid, 200 mesh) was placed flat at the bottom of a tube, and 100 µL of the sample was dispensed onto the grid. The grid was then ultracentrifuged for 15 minutes at 28–30 psi using a Beckman air-driven ultracentrifuge (Airfuge). After blotting the liquid in excess, the grid was subjected to negative staining for 1 min with 50 µL of 2% phosphotungstic acid (PTA) at pH 7.0. The grid was subsequently dried for 5 min and exanimated with Philips EM 208 instrument operating at 80KV. 2.4 Fish immunization with irradiated Betanodavirus European sea bass specimens (weight: 16 ± 2 g) were divided into three tanks (350 L each) with 15 fish each forming three experimental groups. The fish were first anesthetized with 100 ppm of tricaine (Pharmaq), blood samples (about 100 µL) were taken from the caudal peduncles just before vaccination (T0), and they were then intracoelomically immunized as follows: 100 µL/fish of Betanodavirus treated with gamma radiation dose of 70.45 kGy (ensuring the SAL) without trehalose, with 6% trehalose, and with 12% trehalose respectively. Blood samples were collected at 21- and 42-days post-vaccination (dpv), as described above. Serum samples were separated from blood cloth by centrifugation at 3.500 g for 10 minutes at 4°C and immediately stored at − 80°C until use. The collected sera were tested for fish IgM antibody presence by indirect ELISA after being heat inactive with a treatment at 56°C for 30 minutes. Briefly, the MaxiSorb ELISA plates were coated with ultracentrifuged SJNNV Betanodavirus antigen (484) (IZSVe, Padua, Italy) diluted 1:100 in carbonate-bicarbonate buffer (pH 8.9) and incubated overnight at 4°C. The plates were then blocked with PBS added with 3% BSA. The sera were analyzed in 1:100 dilution with PBS in duplicates using rabbit anti-sea bass IgM (1:4000) (IZSVe, Padua, Italy) and goat anti-rabbit-HRP conjugated (1:4000) (Sigma-Aldrich, St. Louis, MO, USA) as secondary and tertiary antibodies, respectively. Standard washing (3 times with PBS-tween) was performed in between passages. TMB was added to activate the HRP, and the reaction was stopped with H₂SO₄ 2N after 5 minutes when a clear color change was evident. The OD was measured at 450 nm in duplicate wells, and the average OD was calculated. Three positive and three negative control samples were included on each plate and used to calculate the sample/positive ratio (S/P) as follow: S/P = (mean OD (sample) − mean OD (neg control)) / (mean OD (pos control) − mean OD (neg control)) × 100. An S/P 56% was considered positive according to internal validation. 2.6 Statistical Analysis Statistical analyses were performed using one-way analysis of variance (ANOVA) in Excel 2019 to compare the means of direct ELISA results. Data are expressed as mean ± standard deviation, and a p-value ≤ 0.05 was considered statistically significant (Motamedi-sedeh et al. 2022 ). 3. Results 3.1 Virus Titration and Gamma Radiation Inactivation of Betanodavirus To determine the dose of gamma radiation required for complete inactivation of Betanodavirus , virus suspensions with an initial titer of \(\:{10}^{7.43}\) TCID50/ml were irradiated with increasing doses from 5 to 60 kGy using a cobalt-60 gamma irradiator. The experiment was performed in triplicate. After irradiation, viral infectivity was quantified by the calculation of TCID 50 on SSN-1 cells. A dose-response curve (Fig. 1 ) was then generated (Table 1 ). The D10 value, which indicates the gamma radiation dose required to achieve a one log10 reduction in viral infectivity (Whitby JL and Gelda AK 1979; Silva Aquino 2012 ) was calculated using log-linear regression of the dose-survival curve (y = − 0.1324x + 7.328), yielding a D 10 value of 7.55 kGy. To achieve a SAL, the gamma-ray dose was calculated by adding double the D₁₀ value to the inactivation dose of 55.35 kGy, yielding a final SAL-related dose of 70.45 kGy. This SAL dose ensures complete and reliable inactivation of the Betanodavirus, an essential step for its potential use in vaccine development. No cytopathic effects (CPE) were observed at doses ≥ 50 kGy, as confirmed by serial passages of irradiated samples in SSN-1 cell cultures, further supporting the efficacy of the gamma radiation dose (70.45 kGy) ensuring a SAL in achieving virus inactivation (Wang et al. 2010 ; Hume et al. 2016 ). Table 1 Viral titers of different kGy irradiated Betanodavirus expressed as log10 of the TCID 50 /ml value. SD = standard deviation Dose kGy 0 5 10 15 20 30 40 50 60 Log10 of TCID50/ml ± SD 7,43 ± 0,21 6,61 ± 0,19 6,07 ± 0,26 5,41 ± 0,3 4,52 ± 0,25 3,67 ± 0,54 1,8 < 1.8 < 1.8 3.2 Characterization of Inactivated Betanodavirus ELISA and Western blot Betanodavirus samples irradiated at the calculated SAL-related dose in the presence of different concentrations (0-6-12%) of trehalose were used to assess the efficacy of this radioprotecting agent. The OD values obtained by ELISA assay of all samples are shown in Fig. 2 , and the SDS-PAGE gel results are presented in Fig. 3 . a: with RGNNV/SJNNV antibody (Anti-378); b: with SJNNV antibody (Anti-484). A: with anti –RGNNV/SJNNV B: with anti SJNNV. Lane 1 : size marker (KDa); lane 2 : Irradiated Betanodavirus without trehalose; l ane 3 : Irradiated Betanodavirus + 6% trehalose; lane 4 : Irradiated Betanodavirus + 12% trehalose; lane 5: (wt) non-irradiated Betanodavirus The results of the ELISA(Fig. 2 ) assay showed that antigenic recognition of the capsid protein remained intact in the gamma-irradiated samples, with no significant differences in OD values observed compared to the unirradiated control virus (P > 0.05). The presence of trehalose appeared to reduce the binding efficiency of antigen-antibody complexes, suggesting that may limit access to antigenic sites on the virus. These results suggested further investigation into the protective role of trehalose as radio protector for Betanodavirus . Conversely, Western blot analysis (Fig. 3 ) showed the presence of viral capsid protein (37KDa) in both irradiated and non-irradiated samples without any differences linked to the presence of trehalose. 3.2.1 Transmission Electron Microscopy (TEM) The structural integrity of the Betanodavirus irradiated at the SAL dose was confirmed using transmission electron microscopy (TEM), as shown in Fig. 4 . (a, a’) Unirradiated Betanodavirus (56,000x, 71,000x); (b, b’) Irradiated Betanodavirus without trehalose (56,000x, 71,000x); (c) Irradiated Betanodavirus + 6% trehalose (44,000x); (d) Irradiated Betanodavirus + 12% trehalose (44,000x). The TEM images revealed that gamma irradiation effectively preserved the morphological integrity of the Betanodavirus particles. The inactivated virus retained its characteristical size, shape and surface appearance, similar to that of the unirradiated control. These results suggest that gamma irradiation did not cause significant morphological changes or structural damage to the virus particles. This preservation of the viral structure is crucial for maintaining the immunogenic potential of the virus and ensuring its ability to stimulate an effective immune response. 3.2.2 Fish immunization with irradiated Betanodavirus Fish recover well from the injection procedure. During the whole experiment, only three fish died in two different tanks for unidentified causes. European sea bass immunized with irradiated Betanodavirus at SAL dose without trehalose increased from 21% (at day 21) to 92% (at day 42) as proved by the S/P results of the sera collected form this group (Fig. 5 ). In contrast, the percentage of IgM-positive fish immunized with irradiated Betanodavirus with 6% trehalose increased from 29 to 71%, and from 50 to 66% for the 12% trehalose group. 4. Discussion Betanodavirus , the causative agent of VNN, continues to present a major challenge to Mediterranean aquaculture, particularly affecting the early life stages of European seabass ( D. labrax ). The high mortality rates associated with VNN result in significant economic losses, underscoring the urgent need for effective preventive strategies, such as vaccines. While several vaccine approaches targeting Betanodavirus have been explored (Nishizawa et al. 1997 ; Sommerset et al. 2003 ; Kai and Chi 2008 ; Vimal et al. 2014 ; Kai et al. 2014 ; Lin et al. 2016 ; Nuñez-Ortiz et al. 2016 ; Valero et al. 2016 ; Luu et al. 2017 ), concerns remain regarding the safety and public perception of chemical and genetic modifications commonly employed in vaccine production. As consumer preferences shift toward more natural, chemical-free food sources, there is a growing imperative to develop safer and more efficient methods for virus inactivation in vaccine production, particularly given that aquaculture accounts for over 50% of global fish supply (FAO 2016). Furthermore, no vaccine is available against the reassortant RGNNV/SJNNV nodavirus strain in the Mediterranean Sea. This virus in the recent years is causing severe mortalities in some of the most important species for the aquaculture: gilthead sea bream, European sea bass, and Senegale sole (Toffan et al. 2017 ; Agrebi et al. 2025 ). To address these challenges, our study aimed to investigate gamma irradiation as a novel and promising method for inactivating Betanodavirus while preserving its structural and antigenic integrity, two essential factors for generating an effective immune response. The selected Betanodavirus strain was recently isolated of RGNNV/SJNNV, a strain which lacks a commercially available vaccine. Betanodavirus are renowned for their extreme resistance to inactivation (Arimoto et al. 1996 ; Frerichs et al. 2000 ; Nuñez-Ortiz et al. 2016 ; Falco et al. 2021 ). Traditional virus inactivation methods, such as heat or formalin treatment, are commonly used in VNN vaccine production but are fraught with limitations, including the potential for residual toxicity and the degradation of viral epitopes, which can undermine vaccine efficacy(Falco et al. 2021 ). In contrast, gamma irradiation provides an effective alternative by inactivating the virus while preserving its antigenic properties, often through the use of radioprotectants such as trehalose, which are considered essential for inducing long-term protective immunity (Seo 2015 ; Turan et al. 2021 ). Gamma irradiation, particularly with cobalt-60 as the radiation source, has been shown to be effective in ionizing radiation inactivation of a wide range of pathogens. One of its key advantages is the ability to minimally disrupt protein structures and antigenic epitopes, making it an ideal candidate for virus sterilization without compromising vaccine quality. The high penetration of gamma radiation, coupled with its efficacy in frozen conditions, reduces the damage caused by free radical generation during water radiolysis (Stauffer et al. 2006 ; David et al. 2017 ). Indeed, our results show that gamma irradiation effectively inactivated Betanodavirus too without altering its antigenicity, as confirmed by ELISA, TEM and WB, proving that capsid proteins of the irradiated virus retained their immunoreactivity. These results are consistent with previous studies on other viruses, such as foot-and-mouth disease virus (FMDV), herpes simplex virus (HSV) and white spot syndrome virus (WSSV), where gamma irradiation preserved viral antigenicity, further supporting its potential for vaccine development (Motamedi-Sedeh et al. 2015 , 2016 , 2017 ). It has to be recall that the dose of radiation used to inactivate the virus is a critical factor. Determining the optimal dose of gamma irradiation that effectively inactivates the virus without compromising its antigenicity is a primary objective in many irradiated vaccine studies (Sedeh et al. 2008 ; Syaifudin et al. 2011 ). The optimal dose depends on several factors, including irradiation temperature, size and structural configuration of the viral genome, presence of oxygen during irradiation, water content and post-irradiation conditions (Whitby JL and Gelda AK 1979; Silva Aquino 2012 ). The sterilizing dose for γ-irradiated materials is determined using the SAL, which quantifies the probability of a single viable pathogen surviving the sterilization process. For products intended to contact compromised tissues, such as vaccines, the International Atomic Energy Agency (IAEA) recommends a SAL of 10⁻⁶, corresponding to a one-in-a-million chance of pathogen survival. The irradiation dose required to meet this standard, DS SAL , can be calculated using established mathematical models (Singleton et al. 2020 ). However, previous research suggested that practical validation may take precedence over theoretical dose estimations to confirm the reliability and safety of the inactivation process (Leung et al. 2020 ; Singleton et al. 2020 ). To support this point, Fig. 6 illustrates the distribution of estimated minimum and maximum D₁₀ values across selected viral families within a genome-based phylogenetic framework. Herein, the SAL-related gamma irradiation dose was calculated by adding twice the D₁₀ value (7.55 kGy) to the inactivation dose of 55.35 kGy, resulting in a final SAL-related dose of 70.45 kGy. Based on this, a SAL dose of 70.45 kGy was sufficient to ensure complete virus inactivation, which was further confirmed by the absence of cytopathic effects (CPE) in SSN-1 cells after three passages. These results are consistent with established standards for microbial inactivation and confirm gamma irradiation as a reliable and safe method for viral inactivation (Wang et al. 2010 ; Hume et al. 2016 ). Trehalose is a disaccharide sugar that acts as a cryoprotectant and free radical quencher, stabilizing proteins and preserving vital biological processes (Richards et al. 2002 ; Lee et al. 2018 ; Martinon et al. 2020 ; Motamedi-sedeh et al. 2022 ). This sugar however, presents some limitations: it increases the viscosity of the solution in which it is added and it has a high commercial price. For this reason, our study also evaluated the advantage of adding different concentration of trehalose to preserve Betanodavirus integrity during freezing and irradiation. Trehalose did not significantly affect the shape and the WB results while a slight reduction in antibody binding efficiency was observed as proved by ELISA and by IgM production in fish. This effect may be explained by the fact that trehalose forms a rigid hydrogen-bonded matrix around proteins, inducing steric hindrance that masks hydrophobic regions and consequently reduces the accessibility of immunogenic epitopes as already reported (Jonsson et al. 2024 ). Structural preservation is critical for the immune system to recognize the virus and mount an appropriate immune response. Transmission electron microscopy (TEM) supported our findings, showing that the irradiated Betanodavirus retained its characteristic morphology, with no significant changes in size, shape, or surface features. The combination of intact viral structure and preserved antigenicity support gamma irradiation as an ideal method for the inactivation of Betanodavirus without compromising vaccine quality. The preservation of antigenicity in irradiated Betanodavirus resulted in a high immune response in the European sea bass group immunised with irradiated Betanodavirus without trehalose, with 92% of the fish-testing positive for IgM at 42 days post-vaccination. In contrast, the addition of trehalose to the vaccine formulation did not significantly enhance the immune response compared to irradiated Betanodavirus alone. Although a gradual increase in the number of IgM-positive fish was observed over time, the addition of either 6 or 12% trehalose did not result in a significant improvement in antibody production. These results suggest that trehalose may not significantly affect the immunogenicity of the irradiated Betanodavirus vaccine in European seabass. This is in contrast to other studies where trehalose has been reported to improve immunogenicity, suggesting that its effects may depend on specific vaccine formulations or experimental conditions (Motamedi-Sedeh et al. 2023 ). Genome-based phylogenetic trees—including accession numbers, viral family names, and D₁₀ values (kGy)—were downloaded from the Virus Variation Resource ( Hatcher et al. 2017 ). The trees were visualized and annotated with corresponding datasets (minimum and maximum D₁₀ values) using EvolView ( Zhang et al. 2012 ). D₁₀ values were retrieved from published data sources ( Mietanko et al. 2022 ; Al-Hadyan et al. 2021 )). 5. Conclusion Betanodavirus remains a critical threat to Mediterranean aquaculture, particularly affecting the early developmental stages of European seabass ( D. labrax) and causing substantial economic losses to fish farms. Emergence of reassortant strains for which no commercial vaccines are available is of particular concern. Given the limitations and safety concerns associated with conventional methods, there is an urgent need for alternative virus inactivation strategies that ensure both safety and efficacy. Our study demonstrates that gamma irradiation, specifically using cobalt-60, effectively inactivates Betanodavirus while preserving its structural integrity and antigenicity. The absence of cytopathic effects in SSN-1 cells following irradiation at a SAL-related dose of 70.45 kGy confirms the reliability and safety of this virus inactivation method. Transmission electron microscopy further corroborated the preservation of viral morphology post-irradiation. The use of trehalose as a radioprotectant, although potentially beneficial in stabilizing viral proteins during freezing and irradiation, in our study resulted in a slight but non-significant reduction in antibody binding efficiency and did not enhance the immunogenicity of the irradiated virus in European seabass. Therefore, we conclude that trehalose is not an essential ingredient of a future irradiate vaccine against VNN. Overall, gamma irradiation emerges as a promising, non-chemical approach for producing Betanodavirus vaccines that maintain immunogenic properties and induce robust humoral immune responses without the drawbacks of traditional inactivation techniques. This technology holds substantial potential for advancing vaccine development against Betanodavirus and other aquaculture pathogens, thereby supporting sustainable and safe fish farming practices globally. Declarations Conflicts of Interest: The authors declare the absence of any conflicts of interest, whether financial or non-financial, that could influence the research. Ethic statement: All animal experimental trials were carried out in compliance with European Directive 2010/63/EU, 2010. The experimental protocol was authorized by the Italian Ministry of Health (Law decree 529/2020-PR on 26/5/2022). Author Contribution Conceptualization, experimental design, C.N, S.H. and T.A .; *in vivo* and *in vitro* experiments, A.H, B.A. and B.P .; Irradiation experiments ,T.K.R. and W.V .; Data analysis and interpretation , A.H, B.A. and N.S.; Writing original draft preparation, A.H.; Writing, review and editing of the manuscript, C.N, S.H. T.A, B.Z.B., coordination of financial and technical support, H.S., R.T.K., and V.W. All authors read and approved the final manuscript. Acknowledgements We sincerely acknowledge the financial support provided by the Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture through the International Atomic Energy Agency (IAEA) Technical Cooperation (TC) Project TUN5032 (AquaVac-ir, http://www.aquavac-ir.tn/ ) and the IAEA CRP 2296 (Project Code: D3.20.37). This research was conducted under Contract Number 26187 (Immune2AquaVac-ir). 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Cherif","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBElEQVRIiWNgGAWjYHACgwNAIoGBgbHhwwcDErU0zpwB1MIDFYUxsGphgGhhYJwNUkVQizn74Y0HfjDY5fFLH25stim4Y28vdoDtc+GePwz20g1YtVj2pBUc7GFILpbsS2xszjF4ltgjncA8e8YzoC0yB3B4JMfgAA/DgcQNZxjbH+cYHE7gAWph5jkA1CKRgF3L+TcGB/8Atew/w9jYbGFw2J6wlhtAk8G28AC1MBgcZuwhrOVZwWEZg+TEGUBbGnsMDif23E5sBmox5uG5gcthyZs/vqmwS+zvYX/Y8OPPYXv22cmHgVrk5NhnYNcC1YjCY2wAkXhichSMglEwCkYBIQAAbBJbzNMKNd0AAAAASUVORK5CYII=","orcid":"","institution":"National Institute for Marine Sciences and Technologies","correspondingAuthor":true,"prefix":"","firstName":"Nadia","middleName":"","lastName":"Cherif","suffix":""},{"id":573141527,"identity":"371b5d65-8ca9-4039-bc40-6f5fd5f12681","order_by":10,"name":"Haitham Sghaier","email":"","orcid":"","institution":"Centre National des Sciences et Technologies Nucléaires","correspondingAuthor":false,"prefix":"","firstName":"Haitham","middleName":"","lastName":"Sghaier","suffix":""}],"badges":[],"createdAt":"2026-01-08 09:38:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8549652/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8549652/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":100850967,"identity":"a6726bd9-763c-4cc5-9f6d-19d1f79fcc8e","added_by":"auto","created_at":"2026-01-22 06:00:37","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":66297,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDose/response curve and regression line for irradiated Betanodavirus ranging from 5 to 60 kGy.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8549652/v1/d1c7bb954486995ebadabadf.jpg"},{"id":101203050,"identity":"ac25aef8-c15f-4309-9314-d05033d7d3a5","added_by":"auto","created_at":"2026-01-27 09:38:39","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":96618,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMean OD values obtained by direct ELISA assay on irradiated and unirradiated Betanodavirus\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea: with RGNNV/SJNNV antibody (Anti-378); b: with SJNNV antibody (Anti-484).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8549652/v1/64a6e89e6e2588cb1d85a684.jpg"},{"id":100850969,"identity":"dde314e4-8629-4f25-8f5a-b19635b1c629","added_by":"auto","created_at":"2026-01-22 06:00:37","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":74653,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWestern blot labelled with rabbit serum anti-Betanodavirus.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA: with anti –RGNNV/SJNNV B: with anti SJNNV.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLane 1:\u003c/strong\u003e size marker (KDa); \u003cstrong\u003elane 2:\u003c/strong\u003e Irradiated \u003cem\u003eBetanodavirus\u003c/em\u003e without trehalose; l\u003cstrong\u003eane 3:\u003c/strong\u003eIrradiated \u003cem\u003eBetanodavirus\u003c/em\u003e + 6% trehalose; \u003cstrong\u003elane\u003c/strong\u003e \u003cstrong\u003e4:\u003c/strong\u003e Irradiated \u003cem\u003eBetanodavirus\u003c/em\u003e + 12% trehalose; \u003cstrong\u003elane 5: (wt)\u003c/strong\u003e non-irradiated \u003cem\u003eBetanodavirus\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8549652/v1/3ea8f15107ad2dce4c38325a.jpg"},{"id":100850972,"identity":"b12d0d28-b1ba-46c5-a95c-bb34cc50bc33","added_by":"auto","created_at":"2026-01-22 06:00:37","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":285468,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eElectron micrograph of Betanodavirus.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a, a’) Unirradiated \u003cem\u003eBetanodavirus\u003c/em\u003e (56,000x, 71,000x)\u003cem\u003e;\u003c/em\u003e (b, b’) Irradiated \u003cem\u003eBetanodavirus\u003c/em\u003e without trehalose (56,000x, 71,000x); (c) Irradiated \u003cem\u003eBetanodavirus\u003c/em\u003e +6% trehalose (44,000x); (d) Irradiated \u003cem\u003eBetanodavirus\u003c/em\u003e+12% trehalose (44,000x).\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8549652/v1/28e6f48301a7b7827b0a9f78.jpg"},{"id":100850970,"identity":"d37a2bcb-d200-4989-8b09-ec8bc87299de","added_by":"auto","created_at":"2026-01-22 06:00:37","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":166750,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePrevalence of IgM detected by ELISA in fish vaccinated with irradiated Betanodavirus at SAL dose and formulated with different trehalose concentrations. Serum from vaccinated fish was sampled on 21 days post vaccination (21 dpv) and on 42 days post vaccination (42dpv).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8549652/v1/89f52dca0e3b0f3ea6121114.jpg"},{"id":100949807,"identity":"36773c79-175c-4c5d-a5f8-860bdcab75f0","added_by":"auto","created_at":"2026-01-23 07:05:49","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":64577,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEstimated Minimum and Maximum Gamma Irradiation D₁₀ Values for Viral Families: Adenoviridae, Coronaviridae, Nodaviridae, Parvoviridae, and Togaviridae\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenome-based phylogenetic trees—including accession numbers, viral family names, and D₁₀ values (kGy)—were downloaded from the Virus Variation Resource (Hatcher et al. 2017). The trees were visualized and annotated with corresponding datasets (minimum and maximum D₁₀ values) using EvolView (Zhang et al. 2012). D₁₀ values were retrieved from published data sources (Al-Hadyan et al. 2021; Mietanko et al. 2020)).\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8549652/v1/632c629347b3f6641a1df8c0.jpg"},{"id":101751745,"identity":"0c2b14a5-9805-4ca4-8a0b-c2929073f068","added_by":"auto","created_at":"2026-02-03 10:23:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1909738,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8549652/v1/3febd2af-ce7c-494d-a74f-9f80d806c7d1.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Gamma-Ray Inactivation of Betanodavirus: Antigen Preservation and Vaccine Potential","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAquaculture is one of the fastest growing sectors of the global food industry, currently surpassing traditional marine fisheries in the production of fish protein for human consumption (FAO \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). This rapid expansion has been associated with an increased incidence of infectious diseases, facilitated by the continuous movement of eggs, larvae and adult fish between aquaculture facilities worldwide. In addition, survivors of disease outbreaks can act as asymptomatic carriers, complicating disease management and control strategies. One of the most widespread and devastating pathogens in marine aquaculture is nervous necrosis virus (NNV), a member of the genus \u003cem\u003eBetanodavirus\u003c/em\u003e and family \u003cem\u003eNodaviridae\u003c/em\u003e according to the International Committee on Taxonomy of Viruses(The International Committee on Taxonomy of Viruses (ICTV)). NNV is the causative agent of viral encephalopathy and retinopathy (VER), a severe neuropathological disease that results in mortality rates of up to 100%, particularly in larval and juvenile fish (Band\u0026iacute;n and Souto \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNNV is a non-enveloped, icosahedral virus with a bi-segmented, positive-sense single-stranded RNA genome: RNA1 (3.1 Kb) and RNA2 (1.4 Kb), which encode the RNA-dependent RNA polymerase (RdRp) and the capsid protein (CP), respectively (Mori et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). Based on the small variable region of RNA2, four genotypes have been identified: \u003cem\u003eBetanodavirus pseudocarangis (SJNNV), Betanodavirus takifugui (TPNNV), Betanodavirus verasperi (BFNNV), and Betanodavirus epinepheli (RGNNV)\u003c/em\u003e (Nishizawa et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Band\u0026iacute;n and Souto \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Additionally, reassortant strains, such as RGNNV/SJNNV and SJNNV/RGNNV, have emerged, posing further challenges to fish health (Toffolo et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Olveira et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Panzarin et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Toffan et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Ch\u0026eacute;rif et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These NNV genotypes have distinct host ranges, geographic distributions, serotypes, and \u003cem\u003ein vitro\u003c/em\u003e growth temperature preferences. The BFNNV infects species such as barfin flounder (\u003cem\u003eVerasper moseri\u003c/em\u003e) and Atlantic cod (\u003cem\u003eGadus morhua\u003c/em\u003e); it is predominantly found in the North Sea, Norwegian Sea, Baltic Sea, and Northern Atlantic Ocean, belongs to serotype B, and grows optimally at 15\u0026ndash;20\u0026deg;C. The RGNNV, the most widely distributed genotype, infects various groupers (\u003cem\u003eEpinephelus spp.)\u003c/em\u003e, Japanese flounder (\u003cem\u003eParalichthys olivaceus\u003c/em\u003e), and European sea bass (\u003cem\u003eDicentrarchus labrax\u003c/em\u003e); it is prevalent in the Mediterranean and in the surrounding seas of Australia and Asia, corresponds to serotype C, and replicates efficiently at 25\u0026ndash;30\u0026deg;C. The SJNNV affects hosts like striped jack (\u003cem\u003ePseudocaranx dentex\u003c/em\u003e) and Senegalese sole (\u003cem\u003eSolea senegalensis\u003c/em\u003e); it occurs mainly in Japan and the Atlantic ocean in front of the Iberian Peninsula, is classified under serotype A, and grows best at 20\u0026ndash;25\u0026deg;C. The TPNNV has been isolated only from tiger puffer (\u003cem\u003eTakifugu rubripes\u003c/em\u003e); it is restricted to Japan, belongs to serotype B, and has an optimal growth temperature of 20\u0026deg;C(Bars\u0026oslash;e \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEuropean seabass (\u003cem\u003eDicentrarchus labrax\u003c/em\u003e), an important species in Mediterranean aquaculture (FEAP \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), is highly susceptible to RGNNV, with mortality rates up to 100% in infected populations (Ch\u0026eacute;rif et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Shetty et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). However, reassortant viruses RGNNV/SJNNV can cause infection in this species, and natural outbreaks caused by reassortant RGNNV/SJNNV has been described in European sea bass larvae and juveniles with variable cumulative mortality (Volpe et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Biasini et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Recently, RGNNV/SJNNV has also been isolated from seabass in Tunisia, suffering significant mortality (Agrebi et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Additionally, the increasing frequency of \u003cem\u003eBetanodavirus\u003c/em\u003e outbreaks caused by RGNNV/SJNNV strains in \u003cem\u003eSparus aurata\u003c/em\u003e farms (NaveenKumar et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Savoca et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) highlight the urgent need for prophylactic measures such as vaccines.\u003c/p\u003e \u003cp\u003eCurrently, two commercial vaccines are available for the prevention of VNN in aquaculture: Alphaject micro-1-noda (Pharmaq AS)(Pharmaq \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), which contains inactivated RGNNV strain ALV1107 with a mineral oil adjuvant, administered at 0.05 mL intraperitoneally (IP) to fish\u0026thinsp;\u0026ge;\u0026thinsp;12 g, providing immunity after 466 degree days and lasting for one year; and Icthiovac VNN (Laboratorios HIPRA)(HipraIcthiovac. 2021), which uses inactivated \u003cem\u003eBetanodavirus\u003c/em\u003e strain 1103 with a non-mineral oil adjuvant, administered at 0.1 mL IP to fish\u0026thinsp;\u0026ge;\u0026thinsp;15 g, with an onset of immunity at 924 degree days, though its duration is not established. Both vaccines are designed for conferring protection against RGNNV virus, while for the RGNNV/SJNNV viruses, which possess different antigenic properties, no vaccine are available. These vaccines are inactivated by formalin, which, while effective, can compromise the immunogenicity of the virus, reducing the efficacy of the resulting vaccine (Valero et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Bars\u0026oslash;e \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Other chemical inactivation techniques, such as binary ethylenimine (BEI), have shown promising but still face limitations (Valero et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, p. 20). As a result, there has been a growing interest in alternative inactivation methods, with gamma irradiation emerging as a promising approach for inactivating viruses while preserving their antigenicity (Unger et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Compared to chemical inactivation methods, gamma irradiation offers a safer and more effective alternative by avoiding the risk of chemical residues while efficiently inactivating pathogens through targeted damage to nucleic acids (DNA or RNA). Importantly, this approach can preserve surface antigens and overall viral structure, particularly when radioprotectants such as trehalose are employed, thereby maintaining immunogenic properties essential for vaccine development (Tobin et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Gamma irradiation has already been successfully used in the development of vaccines for human and animal diseases such as malaria, influenza and many others (Bhatia and Pillai \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This study investigates the use of gamma irradiation as a method to inactivate reassortant \u003cem\u003eBetanodavirus\u003c/em\u003e (RGNNV/SJNNV) while preserving its antigenicity for vaccine development. The aim is to determine the optimal irradiation dose in combination with different percentual of trehalose that ensures complete viral inactivation without compromising the ability of the virus to stimulate a satisfactory immune response. This research will contribute to the development of safer and more effective vaccines for Mediterranean aquaculture, potentially providing a novel solution to the ongoing challenge of \u003cem\u003eBetanodavirus\u003c/em\u003e outbreaks in European seabass farming.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Virus Stock Preparation and Cell Cultures\u003c/h2\u003e \u003cp\u003eThe Tunisian reassortant RGNNV/SJNNV \u003cem\u003eBetanodavirus\u003c/em\u003e strain, VNNV/D.labrax/TUN/148-8/Dec23, was isolated from \u003cem\u003eD. labrax\u003c/em\u003e in 2023(Agrebi et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) and propagated in \u003cem\u003estriped snakehead\u003c/em\u003e cells (SNN-1) (Frerichs et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1991\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe SNN-1 cells were inoculated with the isolate and incubated at 25\u0026deg;C until the cytopathic effect (CPE) was extensive. The supernatant was then harvested, centrifuged at 3000 \u0026times; g for 10 minutes to remove cell debris and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until further use. Viral titers were determined on SNN-1 cell culture and calculated with the TCID\u003csub\u003e50\u003c/sub\u003e method as described by Reed and Muench (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1938\u003c/span\u003e) (Reed and Muench \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1938\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 \u003cem\u003eBetanodavirus\u003c/em\u003e Inactivation by Gamma Irradiation\u003c/h2\u003e \u003cp\u003eVirus inactivation was carried out at the International Atomic Energy Agency (IAEA) laboratories in Seibersdorf, Austria, following established protocols, using a Co-60 irradiator Model 812 (Foss Therapy services, Inc., California, and USA). Gamma ray doses of 5, 10, 15, 20, 30, 40, 50 and 60 kGy were applied to triplicate aliquots of 3 mL each of \u003cem\u003eBetanodavirus\u003c/em\u003e suspensions with 12% trehalose (Sigma) (w/v), under frozen conditions. The irradiated samples were titrated as reported before and a dose-response curve was constructed to determine the D\u003csub\u003e10\u003c/sub\u003e value and the optimal dose for complete virus inactivation also known as sterility assurance level (SAL) dose.\u003c/p\u003e \u003cp\u003eAfter determination of the gamma radiation dose (70.45 kGy) ensuring the SAL of the Tunisian \u003cem\u003eBetanodavirus\u003c/em\u003e, vials containing 10 ml each of \u003cem\u003eBetanodavirus\u003c/em\u003e suspensions with different radioprotector (trehalose) concentrations (0, 6 and 12%) were irradiated in triplicate at the determined SAL dose. These samples were evaluated to assess the preservation of viral antigens by ELISA assay, morphology post-irradiation by transmission electron microscopy (TEM) observation and immunogenicity evaluation by western blot analysis and fish immunization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Characterization of Inactivated \u003cem\u003eBetanodavirus\u003c/em\u003e\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1. ELISA assay\u003c/h2\u003e \u003cp\u003eRecognition of antigens in irradiated \u003cem\u003eBetanodavirus\u003c/em\u003e compared to the unirradiated control virus was determined by indirect ELISA. Briefly, for antigen coating, 100 \u0026micro;L of ultracentrifuged \u003cem\u003eBetanodavirus\u003c/em\u003e particles (diluted 1:10 in carbonate-bicarbonate buffer, pH 8.9) were added to MaxiSorb ELISA plates (In \u003cem\u003eVitro\u003c/em\u003e, Denmark) and incubated overnight at 4\u0026deg;C. After washing the plates and blocking step them with PBS containing 3% bovine serum albumin (Merck Products) for 1 hour at 37\u0026deg;C, the coated wells were incubated with primary antibodies specific for NNV A serotype. Rabbit anti-RGNNV/SJNNV (Anti-378) and Rabbit anti-SJNNV (Anti-484) (IZSVe, Padua, Italy) were diluted 1:1000 and 1:10.000, respectively and tested separately against treated and untreated virus. After incubation with a secondary anti-rabbit IgG peroxidase-conjugated antibody (Merck Products) at a 1:4000 dilution, color development was performed using TMB ELISA substrate (Prodotti Gianni) and stopped with H₂SO₄ (2N). Specific absorbances (OD) were recorded at 450 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2 Western Blot Analysis\u003c/h2\u003e \u003cp\u003eWestern blot analysis was performed to confirm the presence of viral antigens in irradiated \u003cem\u003eBetanodavirus\u003c/em\u003e. Viral samples (20\u0026micro;l) were prepared with Laemmli loading buffer and run on a 4\u0026ndash;15% gradient SDS-PAGE gel (Mini Protean TGX Stain Free Precast gel_Bio-Rad). After electrophoresis, the protein profile in the gel was transferred to PVDF membrane (Immobilon). For immunoblotting analysis, the membrane was saturated with blocking buffer (5% in PBS) for 1h and incubated for 2h with the primary antibody: rabbit anti-RGNNV/SJNNV diluted 1:500 (Anti-378,) or rabbit anti-SJNNV diluted 1:1000 (Anti-484) (IZSVe, Padua, Italy). A second incubation of 1h with HRP-conjugated goat anti-rabbit IgG secondary antibody (diluted 1:1000) was performed. Labeled proteins were visualized with an Opti-2CN substrate Kit (Bio-Rad).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3 Transmission Electron Microscopy (TEM)\u003c/h2\u003e \u003cp\u003eTransmission electron microscopy (TEM) was used to assess the preservation of morphology in irradiated \u003cem\u003eBetanodavirus\u003c/em\u003e. Both ultracentrifuged unirradiated and irradiated \u003cem\u003eBetanodavirus\u003c/em\u003e samples were analyzed by negative staining. A Formvar carbon-supported copper grid (Electron Microscopy Sciences, Formvar/Carbon Copper Grid, 200 mesh) was placed flat at the bottom of a tube, and 100 \u0026micro;L of the sample was dispensed onto the grid. The grid was then ultracentrifuged for 15 minutes at 28\u0026ndash;30 psi using a Beckman air-driven ultracentrifuge (Airfuge). After blotting the liquid in excess, the grid was subjected to negative staining for 1 min with 50 \u0026micro;L of 2% phosphotungstic acid (PTA) at pH 7.0. The grid was subsequently dried for 5 min and exanimated with Philips EM 208 instrument operating at 80KV.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Fish immunization with irradiated Betanodavirus\u003c/h2\u003e \u003cp\u003eEuropean sea bass specimens (weight: 16\u0026thinsp;\u0026plusmn;\u0026thinsp;2 g) were divided into three tanks (350 L each) with 15 fish each forming three experimental groups. The fish were first anesthetized with 100 ppm of tricaine (Pharmaq), blood samples (about 100 \u0026micro;L) were taken from the caudal peduncles just before vaccination (T0), and they were then intracoelomically immunized as follows: 100 \u0026micro;L/fish of \u003cem\u003eBetanodavirus\u003c/em\u003e treated with gamma radiation dose of 70.45 kGy (ensuring the SAL) without trehalose, with 6% trehalose, and with 12% trehalose respectively. Blood samples were collected at 21- and 42-days post-vaccination (dpv), as described above. Serum samples were separated from blood cloth by centrifugation at 3.500 g for 10 minutes at 4\u0026deg;C and immediately stored at \u0026minus;\u0026thinsp;80\u0026deg;C until use.\u003c/p\u003e \u003cp\u003eThe collected sera were tested for fish IgM antibody presence by indirect ELISA after being heat inactive with a treatment at 56\u0026deg;C for 30 minutes. Briefly, the MaxiSorb ELISA plates were coated with ultracentrifuged SJNNV \u003cem\u003eBetanodavirus\u003c/em\u003e antigen (484) (IZSVe, Padua, Italy) diluted 1:100 in carbonate-bicarbonate buffer (pH 8.9) and incubated overnight at 4\u0026deg;C. The plates were then blocked with PBS added with 3% BSA. The sera were analyzed in 1:100 dilution with PBS in duplicates using rabbit anti-sea bass IgM (1:4000) (IZSVe, Padua, Italy) and goat anti-rabbit-HRP conjugated (1:4000) (Sigma-Aldrich, St. Louis, MO, USA) as secondary and tertiary antibodies, respectively. Standard washing (3 times with PBS-tween) was performed in between passages. TMB was added to activate the HRP, and the reaction was stopped with H₂SO₄ 2N after 5 minutes when a clear color change was evident. The OD was measured at 450 nm in duplicate wells, and the average OD was calculated. Three positive and three negative control samples were included on each plate and used to calculate the sample/positive ratio (S/P) as follow:\u003c/p\u003e \u003cp\u003eS/P = (mean OD (sample)\u0026thinsp;\u0026minus;\u0026thinsp;mean OD (neg control)) / (mean OD (pos control)\u0026thinsp;\u0026minus;\u0026thinsp;mean OD (neg control)) \u0026times; 100.\u003c/p\u003e \u003cp\u003eAn S/P\u0026thinsp;\u0026lt;\u0026thinsp;42% was considered negative, 42\u0026ndash;56% was considered doubtful, and \u0026gt;\u0026thinsp;56% was considered positive according to internal validation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using one-way analysis of variance (ANOVA) in Excel 2019 to compare the means of direct ELISA results. Data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, and a p-value\u0026thinsp;\u0026le;\u0026thinsp;0.05 was considered statistically significant (Motamedi-sedeh et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Virus Titration and Gamma Radiation Inactivation of \u003cem\u003eBetanodavirus\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eTo determine the dose of gamma radiation required for complete inactivation of \u003cem\u003eBetanodavirus\u003c/em\u003e, virus suspensions with an initial titer of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{10}^{7.43}\\)\u003c/span\u003e\u003c/span\u003eTCID50/ml were irradiated with increasing doses from 5 to 60 kGy using a cobalt-60 gamma irradiator. The experiment was performed in triplicate. After irradiation, viral infectivity was quantified by the calculation of TCID\u003csub\u003e50\u003c/sub\u003e on SSN-1 cells. A dose-response curve (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) was then generated (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The D10 value, which indicates the gamma radiation dose required to achieve a one log10 reduction in viral infectivity (Whitby JL and Gelda AK 1979; Silva Aquino \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) was calculated using log-linear regression of the dose-survival curve (y = \u0026minus;\u0026thinsp;0.1324x\u0026thinsp;+\u0026thinsp;7.328), yielding a D\u003csub\u003e10\u003c/sub\u003e value of 7.55 kGy. To achieve a SAL, the gamma-ray dose was calculated by adding double the D₁₀ value to the inactivation dose of 55.35 kGy, yielding a final SAL-related dose of 70.45 kGy. This SAL dose ensures complete and reliable inactivation of the Betanodavirus, an essential step for its potential use in vaccine development. No cytopathic effects (CPE) were observed at doses\u0026thinsp;\u0026ge;\u0026thinsp;50 kGy, as confirmed by serial passages of irradiated samples in SSN-1 cell cultures, further supporting the efficacy of the gamma radiation dose (70.45 kGy) ensuring a SAL in achieving virus inactivation (Wang et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Hume et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eViral titers of different kGy irradiated Betanodavirus expressed as log10 of the TCID\u003csub\u003e50\u003c/sub\u003e/ml value. SD\u0026thinsp;=\u0026thinsp;standard deviation\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDose kGy\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLog10 of TCID50/ml\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7,43\u003c/p\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0,21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6,61\u0026thinsp;\u0026plusmn;\u0026thinsp;0,19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6,07\u0026thinsp;\u0026plusmn;\u0026thinsp;0,26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5,41\u0026thinsp;\u0026plusmn;\u0026thinsp;0,3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4,52\u0026thinsp;\u0026plusmn;\u0026thinsp;0,25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3,67\u0026thinsp;\u0026plusmn;\u0026thinsp;0,54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1,8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;1.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Characterization of Inactivated \u003cem\u003eBetanodavirus\u003c/em\u003e ELISA and Western blot\u003c/h2\u003e \u003cp\u003e \u003cem\u003eBetanodavirus\u003c/em\u003e samples irradiated at the calculated SAL-related dose in the presence of different concentrations (0-6-12%) of trehalose were used to assess the efficacy of this radioprotecting agent. The OD values obtained by ELISA assay of all samples are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, and the SDS-PAGE gel results are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ea: with RGNNV/SJNNV antibody (Anti-378); b: with SJNNV antibody (Anti-484).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003e\u003c/div\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eA: with anti \u0026ndash;RGNNV/SJNNV B: with anti SJNNV.\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eLane 1\u003c/b\u003e: size marker (KDa); \u003cb\u003elane 2\u003c/b\u003e: Irradiated \u003cem\u003eBetanodavirus\u003c/em\u003e without trehalose; l\u003cb\u003eane 3\u003c/b\u003e: Irradiated \u003cem\u003eBetanodavirus\u003c/em\u003e\u0026thinsp;+\u0026thinsp;6% trehalose; \u003cb\u003elane 4\u003c/b\u003e: Irradiated \u003cem\u003eBetanodavirus\u003c/em\u003e\u0026thinsp;+\u0026thinsp;12% trehalose; \u003cb\u003elane 5: (wt)\u003c/b\u003e non-irradiated \u003cem\u003eBetanodavirus\u003c/em\u003e\u003c/p\u003e \u003cp\u003eThe results of the ELISA(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) assay showed that antigenic recognition of the capsid protein remained intact in the gamma-irradiated samples, with no significant differences in OD values observed compared to the unirradiated control virus (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The presence of trehalose appeared to reduce the binding efficiency of antigen-antibody complexes, suggesting that may limit access to antigenic sites on the virus. These results suggested further investigation into the protective role of trehalose as radio protector for \u003cem\u003eBetanodavirus\u003c/em\u003e. Conversely, Western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) showed the presence of viral capsid protein (37KDa) in both irradiated and non-irradiated samples without any differences linked to the presence of trehalose.\u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003cdiv class=\"Heading\"\u003e3.2.1 Transmission Electron Microscopy (TEM)\u003c/div\u003e \u003cp\u003eThe structural integrity of the \u003cem\u003eBetanodavirus\u003c/em\u003e irradiated at the SAL dose was confirmed using transmission electron microscopy (TEM), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(a, a\u0026rsquo;) Unirradiated \u003cem\u003eBetanodavirus\u003c/em\u003e (56,000x, 71,000x); (b, b\u0026rsquo;) Irradiated \u003cem\u003eBetanodavirus\u003c/em\u003e without trehalose (56,000x, 71,000x); (c) Irradiated \u003cem\u003eBetanodavirus\u003c/em\u003e\u0026thinsp;+\u0026thinsp;6% trehalose (44,000x); (d) Irradiated \u003cem\u003eBetanodavirus\u003c/em\u003e\u0026thinsp;+\u0026thinsp;12% trehalose (44,000x).\u003c/p\u003e \u003cp\u003eThe TEM images revealed that gamma irradiation effectively preserved the morphological integrity of the \u003cem\u003eBetanodavirus\u003c/em\u003e particles. The inactivated virus retained its characteristical size, shape and surface appearance, similar to that of the unirradiated control. These results suggest that gamma irradiation did not cause significant morphological changes or structural damage to the virus particles. This preservation of the viral structure is crucial for maintaining the immunogenic potential of the virus and ensuring its ability to stimulate an effective immune response.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003cdiv class=\"Heading\"\u003e3.2.2 Fish immunization with irradiated \u003cem\u003eBetanodavirus\u003c/em\u003e\u003c/div\u003e \u003cp\u003eFish recover well from the injection procedure. During the whole experiment, only three fish died in two different tanks for unidentified causes.\u003c/p\u003e \u003cp\u003eEuropean sea bass immunized with irradiated \u003cem\u003eBetanodavirus\u003c/em\u003e at SAL dose without trehalose increased from 21% (at day 21) to 92% (at day 42) as proved by the S/P results of the sera collected form this group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In contrast, the percentage of IgM-positive fish immunized with irradiated \u003cem\u003eBetanodavirus\u003c/em\u003e with 6% trehalose increased from 29 to 71%, and from 50 to 66% for the 12% trehalose group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e \u003cem\u003eBetanodavirus\u003c/em\u003e, the causative agent of VNN, continues to present a major challenge to Mediterranean aquaculture, particularly affecting the early life stages of European seabass (\u003cem\u003eD. labrax\u003c/em\u003e). The high mortality rates associated with VNN result in significant economic losses, underscoring the urgent need for effective preventive strategies, such as vaccines. While several vaccine approaches targeting \u003cem\u003eBetanodavirus\u003c/em\u003e have been explored (Nishizawa et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Sommerset et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Kai and Chi \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Vimal et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Kai et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Lin et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Nu\u0026ntilde;ez-Ortiz et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Valero et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Luu et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), concerns remain regarding the safety and public perception of chemical and genetic modifications commonly employed in vaccine production. As consumer preferences shift toward more natural, chemical-free food sources, there is a growing imperative to develop safer and more efficient methods for virus inactivation in vaccine production, particularly given that aquaculture accounts for over 50% of global fish supply (FAO 2016).\u003c/p\u003e \u003cp\u003eFurthermore, no vaccine is available against the reassortant RGNNV/SJNNV nodavirus strain in the Mediterranean Sea. This virus in the recent years is causing severe mortalities in some of the most important species for the aquaculture: gilthead sea bream, European sea bass, and Senegale sole (Toffan et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Agrebi et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo address these challenges, our study aimed to investigate gamma irradiation as a novel and promising method for inactivating \u003cem\u003eBetanodavirus\u003c/em\u003e while preserving its structural and antigenic integrity, two essential factors for generating an effective immune response. The selected \u003cem\u003eBetanodavirus\u003c/em\u003e strain was recently isolated of RGNNV/SJNNV, a strain which lacks a commercially available vaccine. \u003cem\u003eBetanodavirus\u003c/em\u003e are renowned for their extreme resistance to inactivation (Arimoto et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Frerichs et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Nu\u0026ntilde;ez-Ortiz et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Falco et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Traditional virus inactivation methods, such as heat or formalin treatment, are commonly used in VNN vaccine production but are fraught with limitations, including the potential for residual toxicity and the degradation of viral epitopes, which can undermine vaccine efficacy(Falco et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In contrast, gamma irradiation provides an effective alternative by inactivating the virus while preserving its antigenic properties, often through the use of radioprotectants such as trehalose, which are considered essential for inducing long-term protective immunity (Seo \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Turan et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGamma irradiation, particularly with cobalt-60 as the radiation source, has been shown to be effective in ionizing radiation inactivation of a wide range of pathogens. One of its key advantages is the ability to minimally disrupt protein structures and antigenic epitopes, making it an ideal candidate for virus sterilization without compromising vaccine quality. The high penetration of gamma radiation, coupled with its efficacy in frozen conditions, reduces the damage caused by free radical generation during water radiolysis (Stauffer et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; David et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Indeed, our results show that gamma irradiation effectively inactivated \u003cem\u003eBetanodavirus\u003c/em\u003e too without altering its antigenicity, as confirmed by ELISA, TEM and WB, proving that capsid proteins of the irradiated virus retained their immunoreactivity. These results are consistent with previous studies on other viruses, such as foot-and-mouth disease virus (FMDV), herpes simplex virus (HSV) and white spot syndrome virus (WSSV), where gamma irradiation preserved viral antigenicity, further supporting its potential for vaccine development (Motamedi-Sedeh et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt has to be recall that the dose of radiation used to inactivate the virus is a critical factor. Determining the optimal dose of gamma irradiation that effectively inactivates the virus without compromising its antigenicity is a primary objective in many irradiated vaccine studies (Sedeh et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Syaifudin et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The optimal dose depends on several factors, including irradiation temperature, size and structural configuration of the viral genome, presence of oxygen during irradiation, water content and post-irradiation conditions (Whitby JL and Gelda AK 1979; Silva Aquino \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The sterilizing dose for γ-irradiated materials is determined using the SAL, which quantifies the probability of a single viable pathogen surviving the sterilization process. For products intended to contact compromised tissues, such as vaccines, the International Atomic Energy Agency (IAEA) recommends a SAL of 10⁻⁶, corresponding to a one-in-a-million chance of pathogen survival. The irradiation dose required to meet this standard, DS\u003csub\u003eSAL\u003c/sub\u003e, can be calculated using established mathematical models (Singleton et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, previous research suggested that practical validation may take precedence over theoretical dose estimations to confirm the reliability and safety of the inactivation process (Leung et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Singleton et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). To support this point, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e illustrates the distribution of estimated minimum and maximum D₁₀ values across selected viral families within a genome-based phylogenetic framework. Herein, the SAL-related gamma irradiation dose was calculated by adding twice the D₁₀ value (7.55 kGy) to the inactivation dose of 55.35 kGy, resulting in a final SAL-related dose of 70.45 kGy. Based on this, a SAL dose of 70.45 kGy was sufficient to ensure complete virus inactivation, which was further confirmed by the absence of cytopathic effects (CPE) in SSN-1 cells after three passages. These results are consistent with established standards for microbial inactivation and confirm gamma irradiation as a reliable and safe method for viral inactivation (Wang et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Hume et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTrehalose is a disaccharide sugar that acts as a cryoprotectant and free radical quencher, stabilizing proteins and preserving vital biological processes (Richards et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Martinon et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Motamedi-sedeh et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This sugar however, presents some limitations: it increases the viscosity of the solution in which it is added and it has a high commercial price. For this reason, our study also evaluated the advantage of adding different concentration of trehalose to preserve \u003cem\u003eBetanodavirus\u003c/em\u003e integrity during freezing and irradiation. Trehalose did not significantly affect the shape and the WB results while a slight reduction in antibody binding efficiency was observed as proved by ELISA and by IgM production in fish. This effect may be explained by the fact that trehalose forms a rigid hydrogen-bonded matrix around proteins, inducing steric hindrance that masks hydrophobic regions and consequently reduces the accessibility of immunogenic epitopes as already reported (Jonsson et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eStructural preservation is critical for the immune system to recognize the virus and mount an appropriate immune response. Transmission electron microscopy (TEM) supported our findings, showing that the irradiated \u003cem\u003eBetanodavirus\u003c/em\u003e retained its characteristic morphology, with no significant changes in size, shape, or surface features. The combination of intact viral structure and preserved antigenicity support gamma irradiation as an ideal method for the inactivation of \u003cem\u003eBetanodavirus\u003c/em\u003e without compromising vaccine quality.\u003c/p\u003e \u003cp\u003eThe preservation of antigenicity in irradiated \u003cem\u003eBetanodavirus\u003c/em\u003e resulted in a high immune response in the European sea bass group immunised with irradiated \u003cem\u003eBetanodavirus\u003c/em\u003e without trehalose, with 92% of the fish-testing positive for IgM at 42 days post-vaccination. In contrast, the addition of trehalose to the vaccine formulation did not significantly enhance the immune response compared to irradiated \u003cem\u003eBetanodavirus\u003c/em\u003e alone. Although a gradual increase in the number of IgM-positive fish was observed over time, the addition of either 6 or 12% trehalose did not result in a significant improvement in antibody production. These results suggest that trehalose may not significantly affect the immunogenicity of the irradiated \u003cem\u003eBetanodavirus\u003c/em\u003e vaccine in European seabass. This is in contrast to other studies where trehalose has been reported to improve immunogenicity, suggesting that its effects may depend on specific vaccine formulations or experimental conditions (Motamedi-Sedeh et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGenome-based phylogenetic trees\u0026mdash;including accession numbers, viral family names, and D₁₀ values (kGy)\u0026mdash;were downloaded from the Virus Variation Resource \u003cem\u003e(\u003c/em\u003eHatcher et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The trees were visualized and annotated with corresponding datasets (minimum and maximum D₁₀ values) using EvolView \u003cem\u003e(\u003c/em\u003eZhang et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). D₁₀ values were retrieved from published data sources \u003cem\u003e(\u003c/em\u003eMietanko et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Al-Hadyan et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e)).\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003e \u003cem\u003eBetanodavirus\u003c/em\u003e remains a critical threat to Mediterranean aquaculture, particularly affecting the early developmental stages of European seabass (\u003cem\u003eD. labrax)\u003c/em\u003e and causing substantial economic losses to fish farms. Emergence of reassortant strains for which no commercial vaccines are available is of particular concern. Given the limitations and safety concerns associated with conventional methods, there is an urgent need for alternative virus inactivation strategies that ensure both safety and efficacy. Our study demonstrates that gamma irradiation, specifically using cobalt-60, effectively inactivates \u003cem\u003eBetanodavirus\u003c/em\u003e while preserving its structural integrity and antigenicity. The absence of cytopathic effects in SSN-1 cells following irradiation at a SAL-related dose of 70.45 kGy confirms the reliability and safety of this virus inactivation method. Transmission electron microscopy further corroborated the preservation of viral morphology post-irradiation. The use of trehalose as a radioprotectant, although potentially beneficial in stabilizing viral proteins during freezing and irradiation, in our study resulted in a slight but non-significant reduction in antibody binding efficiency and did not enhance the immunogenicity of the irradiated virus in European seabass. Therefore, we conclude that trehalose is not an essential ingredient of a future irradiate vaccine against VNN.\u003c/p\u003e \u003cp\u003eOverall, gamma irradiation emerges as a promising, non-chemical approach for producing \u003cem\u003eBetanodavirus\u003c/em\u003e vaccines that maintain immunogenic properties and induce robust humoral immune responses without the drawbacks of traditional inactivation techniques. This technology holds substantial potential for advancing vaccine development against \u003cem\u003eBetanodavirus\u003c/em\u003e and other aquaculture pathogens, thereby supporting sustainable and safe fish farming practices globally.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicts of Interest:\u003c/h2\u003e \u003cp\u003eThe authors declare the absence of any conflicts of interest, whether financial or non-financial, that could influence the research.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eEthic statement:\u003c/h2\u003e \u003cp\u003e All animal experimental trials were carried out in compliance with European Directive 2010/63/EU, 2010. The experimental protocol was authorized by the Italian Ministry of Health (Law decree 529/2020-PR on 26/5/2022).\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization, experimental design, C.N, S.H. and T.A .; *in vivo* and *in vitro* experiments, A.H, B.A. and B.P .; Irradiation experiments ,T.K.R. and W.V .; Data analysis and interpretation , A.H, B.A. and N.S.; Writing original draft preparation, A.H.; Writing, review and editing of the manuscript, C.N, S.H. T.A, B.Z.B., coordination of financial and technical support, H.S., R.T.K., and V.W. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe sincerely acknowledge the financial support provided by the Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture through the International Atomic Energy Agency (IAEA) Technical Cooperation (TC) Project TUN5032 (AquaVac-ir, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.aquavac-ir.tn/\u003c/span\u003e\u003cspan address=\"http://www.aquavac-ir.tn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and the IAEA CRP 2296 (Project Code: D3.20.37). This research was conducted under Contract Number 26187 (Immune2AquaVac-ir).\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003eAgrebi, Houda, Miriam Abbadi, Alessandra Buratin, et al. 2025. \u0026laquo; Near-Complete Genome Sequence of a Reassortant Fish Nervous Necrosis Virus Isolated from a European Sea Bass ( \u003cem\u003eDicentrarchus Labrax\u003c/em\u003e ) in Tunisia \u0026raquo;. \u003cem\u003eMicrobiology Resource Announcements\u003c/em\u003e 14 (10): e00058-25. https://doi.org/10.1128/mra.00058-25.\u003c/p\u003e\n\u003cp\u003eAl-Hadyan, Khaled, Ghazi Alsbeih, Najla Al-Harbi, et al. 2021. \u0026laquo; Effect of Gamma Irradiation on Filtering Facepiece Respirators and SARS-CoV-2 Detection \u0026raquo;. \u003cem\u003eScientific Reports\u003c/em\u003e 11 (1): 19888. https://doi.org/10.1038/s41598-021-99414-6.\u003c/p\u003e\n\u003cp\u003eArimoto, Misao, Jun Sato, Keigo Maruyama, Gen Mimura, et Iwao Furusawa. 1996. \u0026laquo; Effect of Chemical and Physical Treatments on the Inactivation of Striped Jack Nervous Necrosis Virus (SJNNV) \u0026raquo;. \u003cem\u003eAquaculture\u003c/em\u003e 143 (1): 15‑22. https://doi.org/10.1016/0044-8486(96)01261-6.\u003c/p\u003e\n\u003cp\u003eBand\u0026iacute;n, Isabel, et Sandra Souto. 2020. \u0026laquo; Betanodavirus and VER Disease: A 30-Year Research Review \u0026raquo;. \u003cem\u003ePathogens\u003c/em\u003e 9 (2): 106. https://doi.org/10.3390/pathogens9020106.\u003c/p\u003e\n\u003cp\u003eBars\u0026oslash;e, Sofie. 2021. \u003cem\u003eVaccination of European Sea Bass (Dicentrarchus labrax) against Viral Nervous Necrosis and Characterization of Protective Immunity\u003c/em\u003e. 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Caffara, et al. 2020. \u0026laquo; Viral Nervous Necrosis Outbreaks Caused by the RGNNV/SJNNV Reassortant Betanodavirus in Gilthead Sea Bream (Sparus Aurata) and European Sea Bass (Dicentrarchus Labrax) \u0026raquo;. \u003cem\u003eAquaculture\u003c/em\u003e 523 (juin): 735155. https://doi.org/10.1016/j.aquaculture.2020.735155.\u003c/p\u003e\n\u003cp\u003eWang, Wei, Zengliang Yu, et Wenhui Su. 2010. \u0026laquo; Ion irradiation and biomolecular radiation damage II. Indirect effect \u0026raquo;. Version 1. Pr\u0026eacute;publication, arXiv. https://doi.org/10.48550/ARXIV.1004.4394.\u003c/p\u003e\n\u003cp\u003eWhitby JL, et Gelda AK. 1979. \u0026laquo; Use of incremental doses of cobalt 60 radiation as a means to determine radiation sterilization dose. \u0026raquo; \u003cem\u003eJournal of the Parenteral Drug Association\u003c/em\u003e 33 (3): 144‑55.\u003c/p\u003e\n\u003cp\u003eZhang, Huangkai, Shenghan Gao, Martin J. Lercher, Songnian Hu, et Wei-Hua Chen. 2012. \u0026laquo; EvolView, an Online Tool for Visualizing, Annotating and Managing Phylogenetic Trees \u0026raquo;. \u003cem\u003eNucleic Acids Research\u003c/em\u003e 40 (W1): W569‑72. https://doi.org/10.1093/nar/gks576.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Antigenicity, Aquaculture, Betanodavirus, Dicentrarchus labrax, Gamma irradiation, Inactivated vaccine, RGNNV/SJNNV, Sterility Assurance Level, Trehalose, Viral nervous necrosis","lastPublishedDoi":"10.21203/rs.3.rs-8549652/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8549652/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eViral nervous necrosis (VNN), caused by \u003cem\u003eBetanodavirus\u003c/em\u003e, poses a significant challenge to Mediterranean aquaculture, especially for European sea bass (\u003cem\u003eDicentrarchus labrax\u003c/em\u003e). Conventional chemical inactivation methods often compromise viral immunogenicity or introduce cytotoxic effects, highlighting the need for safer and more effective alternatives for virus inactivation. This study explores gamma irradiation as an alternative approach to inactivate reassortant \u003cem\u003eBetanodavirus\u003c/em\u003e (RGNNV/SJNNV) while preserving its antigenic properties for vaccine development. Virus samples were exposed to gamma radiation doses ranging from 5 to 60 kGy. The D₁₀ value (7.55 kGy), the inactivation dose (55.35 kGy), and the gamma radiation dose (70.45 kGy) ensuring a Sterility Assurance Level (SAL) of the Tunisian \u003cem\u003eBetanodavirus\u003c/em\u003e were established based on viral infectivity reduction. \u003cem\u003eIn vitro\u003c/em\u003e immunological assays, including enzyme-linked immunosorbent assay (ELISA) demonstrated that the structural proteins of the virus retained antigenicity post-irradiation, as evidenced by consistent IgM antibody recognition titers. Transmission electron microscopy confirmed that irradiation did not alter the viral shape or structural integrity. The addition of trehalose as a radioprotectant prior to irradiation did not enhance the immunogenic response, as measured by ELISA. Notably, the highest immune response characterized by 92% IgM-positive individuals, was observed in fish vaccinated with irradiated virus in absence of trehalose. These results highlight gamma irradiation as a promising, non-chemical method for the development of safe and immunogenic inactivated vaccines against \u003cem\u003eBetanodavirus\u003c/em\u003e in aquaculture.\u003c/p\u003e","manuscriptTitle":"Gamma-Ray Inactivation of Betanodavirus: Antigen Preservation and Vaccine Potential","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-22 06:00:17","doi":"10.21203/rs.3.rs-8549652/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"69e9e57a-d507-48a7-8cc4-7ec9786c6938","owner":[],"postedDate":"January 22nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-29T18:25:33+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-22 06:00:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8549652","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8549652","identity":"rs-8549652","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}