Nervous Necrosis Virus Research Articles

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

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

Molecular characterization, immune expression, and functional delineation of peroxiredoxin 1 in Epinephelus akaara

Peroxiredoxin 1 is a member of the typical 2-Cys peroxiredoxin family, which serves diverse functions in gene expression, immune and inflammatory responses, and tumor progression. In this study, we aimed to analyze the structural, functional, and immunomodulatory properties of peroxiredoxin 1 from Epinephelus akaara (EaPrx1). The open reading frame of EaPrx1 is 597 base pairs in length, encoding 198 amino acids, with a molecular weight of approximately 22 kDa. The in silico analysis revealed that EaPrx1 shares a conserved thioredoxin fold and signature motifs that are critical for its catalytic activity and oligomerization. Further, EaPrx1 is closely related to Epinephelus lanceolatus Prx1 and clustered in the Fishes group of the vertebrate clade, revealing that EaPrx1 was conserved throughout evolution. In terms of tissue distribution, a high level of EaPrx1 expression was observed in the spleen, brain, and blood tissues. Likewise, in immune challenge experiments, significant transcriptional modulations of EaPrx1 upon lipopolysaccharide, polyinosinic:polycytidylic acid, and nervous necrosis virus injections were noted at different time points, indicating the immunological role of EaPrx1 against pathogenic infections. In the functional analysis, rEaPrx1 exhibited substantial DNA protection, insulin disulfide reduction, and tissue repair activities, which were concentration-dependent. EaPrx1/pcDNA™ 3.1 (+)-transfected fathead minnow cells revealed high cell viability upon arsenic toxicity, indicating the heavy metal detoxification activity of EaPrx1. Taken together, the transcriptional and functional studies imply critical roles of EaPrx1 in innate immunity, redox regulation, apoptosis, and tissue-repair processes in E. akaara.

Identification of B-Cell Epitopes on Capsid Protein Reveals Two Potential Neutralization Mechanisms in Red-Spotted Grouper Nervous Necrosis Virus.

Nervous necrosis virus (NNV), a formidable pathogen in marine and freshwater fish, has inflicted enormous financial tolls on the aquaculture industry worldwide. Although capsid protein (CP) is the sole structural protein with pathogenicity and antigenicity, public information on immunodominant regions remains extremely scarce. Here, we employed neutralizing monoclonal antibodies (MAbs) specific for red-spotted grouper NNV (RGNNV) CNPgg2018 in combination with partially overlapping truncated proteins and peptides to identify two minimal B-cell epitope clusters on CP, 122GYVAGFL128 and 227SLYNDSL233. Site-directed mutational analysis confirmed residues Y123, G126, and L128 and residues L228, Y229, N230, D231, and L233 as the critical residues responsible for the direct interaction with ligand, respectively. According to homologous modeling and bioinformatic evaluation, 122GYVAGFL128 is harbored at the groove of the CP junction with strict conservation among all NNV isolates, while 227SLYNDSL233 is localized in proximity to the tip of a viral protrusion having relatively high evolutionary dynamics in different genotypes. Additionally, 227SLYNDSL233 was shown to be a receptor-binding site, since the corresponding polypeptide could moderately suppress RGNNV multiplication by impeding virion entry. In contrast, 122GYVAGFL128 seemed dedicated only to stabilizing viral native conformation and not to assisting initial virus attachment. Altogether, these findings contribute to a novel understanding of the antigenic distribution pattern of NNV and the molecular basis for neutralization, thus advancing the development of biomedical products, especially epitope-based vaccines, against NNV. IMPORTANCE NNV is a common etiological agent associated with neurological virosis in multiple aquatic organisms, causing significant hazards to the host. However, licensed drugs or vaccines to combat NNV infection are very limited to date. Toward the advancement of broad-spectrum prophylaxis and therapeutics against NNV, elucidating the diversity of immunodominant regions within it is undoubtedly essential. Here, we identified two independent B-cell epitopes on NNV CP, followed by the confirmation of critical amino acid residues participating in direct interaction. These two sites were distributed on the shell and protrusion domains of the virion, respectively, and mediated the neutralization exerted by MAbs via drastically distinct mechanisms. Our work promotes new insights into NNV antigenicity as well as neutralization and, more importantly, offers promising targets for the development of antiviral countermeasures.

Novel bovine viral diarrhea virus (BVDV) virus-like particle vaccine candidates presenting the E2 protein using the SpyTag/SpyCatcher system induce a robust neutralizing antibody response in mice.

Bovine viral diarrhea virus (BVDV) is a pathogen of commercial consequence in cattle. Although many modified live and killed vaccines are commercially available, their drawbacks precipitate the need for new effective vaccines. Virus-like particles (VLPs) are a safe and powerful technology used in several human and veterinary vaccines; however, it is difficult to produce large amounts of BVDV VLPs. In this study, we generated red-spotted grouper nervous necrosis virus (RGNNV) VLPs presenting the BVDV E2 protein (domain I to IIIb) of the Nose (BVDV-1) or KZ-91-CP (BVDV-2) strain by exploiting SpyTag/SpyCatcher technology. Mice immunized twice with 30 μg of RGNNV VLPs conjugated with 10 μg of E2 proteins of the Nose or KZ-91-CP strain with a 14-day interval elicited high (1:512,000 to 1:1,024,000) and moderate (1:25,600 to 1:102,400) IgG titers against E2 proteins of homologous and heterologous strains, respectively. In addition, this prime-boost regimen induced strong (1:800 to 1:3,200) and weak (~1:10) neutralization titers against homologous and heterologous BVDV strains, respectively. Our results indicate that conjugation of the E2 protein to RGNNV VLPs strongly enhances the antigenicity of the E2 protein and that RGNNV VLPs presenting the E2 protein are promising BVDV vaccine candidates.

Preliminary Exploration of Transcriptome Comparative Analysis of Muscle Tissues from Asymptomatic and Diseased Epinephelus fuscoguttatus Infected with Nervous Necrosis Virus

Preliminary Exploration of Transcriptome Comparative Analysis of Muscle Tissues from Asymptomatic and Diseased Epinephelus fuscoguttatus Infected with Nervous Necrosis Virus

EVALUATION OF RESISTANCE AND GENE EXPRESSION OF BARRAMUNDI, Lates calcarifer POST-INFECTION OF NERVOUS NECROSIS VIRUS

The most common problem in barramundi Lates calcarifer seedling production is the high mortality (> 90%) caused by nervous necrosis virus (NNV) infection. This research aims to evaluate the resistance and gene expression of barramundi challenged by NNV. Two populations were used in this study, i.e., Australian, and Situbondo-originated barramundi populations. The immune-related gene expression levels in the liver, head of kidney, and spleen were observed at 48 and 96 hours after post-infection (hpi). Barramundi’s survival and blood parameters were evaluated post-NNV infection. The results showed that the highest survival was revealed in Situbondo’s barramundi (42.0±4.47%) compared to Australian barramundi (20.0±7.07%) and no mortality was observed in the control without NNV infection. The higher survival rate in barramundi from Situbondo was in line with the blood profile. The number of red blood cell from Situbondo barramundi post-NNV infection (ST) at 96 hpi was higher (P<0.05) than Australian barramundi post-NNV infection (AT). The number of white blood cell of ST at 48 hpi was higher (P<0.05) than AT, but started to decrease at 96 hpi in ST barramundi. The total white blood cell in AT barramundi increased from 48 to 192 hpi. TNFα and IL1-β gene expression levels were significantly higher in the liver, head kidney, and spleen of Situbondo compared to Australian barramundi at 48 hpi, while MHCIIα gene expression in Situbondo’s was significantly higher compared to Australian barramundi at 96 hpi. These results indicate the important roles of all the genes in the barramundi’s immune responses against viral infection. Based on the results of the research, Situbondo’s barramundi has the potential to be used as a candidate for generating broodstock of disease-resistant strain.

Immune response of European sea bass (Dicentrarchus labrax L.) against combination of antigens from three different pathogens

Three of the most important diseases of Mediterranean intensive European sea bass farming are, viral nervous necrosis (VNN) caused by the red grouper nervous necrosis virus (RGNNV) genotype of b-nodavirus, photobacteriosis caused by Photobacterium damselae subsp. piscicida (Phdp) and vibriosis caused mainly by the O1 serotype of Vibrio anguillarum (VaO1). Prevention against these diseases is performed through vaccination with a monovalent vaccine against the viral disease and, usually, with bivalent vaccines against the bacterial diseases. However, it is very difficult to program two vaccinations during the same season for the same fish stock and producers are forced to either vaccinate for the viral or the bacterial diseases or to perform double vaccination with both vaccines, without any prior knowledge on any interactions that may occur due to the plethora of antigens (Ag) injected. Ideally, therefore, a trivalent vaccine should be developed against all three diseases. The objective of this work was to analyse the immune response of sea bass against combinations of Ags from all three pathogens, namely viral particles, Phdp whole cells (WC), lipopolysaccharide (LPS), capsular polysaccharide (CPS) and extracellular products (ECPs) and VaO1 WC and ECPs in respect to the identification of any phenomena of immunodominance/immunosuppression between Ags with a view to select candidate Ags for inclusion in a trivalent vaccine formulation. Eight triplicate groups of fish were immunized with different combinations of the aforementioned Ags and another triplicate group served as negative control. Blood serum was isolated at various time-points post-immunization for the measurement of specific antibodies against each Ag and, in addition, leucocytes were isolated at day 29 post-immunization for analysis of various cellular activities. Results indicated that best levels of specific a-NNV virus antibodies (Abs) were produced when VaO1 ECPs were not included in the Ag combinations, in contrast to the leucocytes proliferation assay where best stimulation against NNV Ags was measured when VaO1 ECPs were present in Ag combinations. VaO1 ECPs apparently is a strong immunogen for both humoral and cellular responses but suppresses immunological reactions against the other Ags.VaO1 WC, Phdp LPS and ECPs raised good humoral immune responses in the groups with best responses against VNN Ags, but only VaO1 WC and Phdp ECPs provided good stimulation of leucocytes, with Phdp WC and CPS effecting either similar stimulation with untrained leucocytes (control groups) or down-stimulation. Results are discussed with a view to select Ags from all three pathogens for inclusion in trivalent vaccine against all three pathogens.

Identification of ISG15 in golden pompano, Trachinotus ovatus, and its role in virus and bacteria infections

Interferon (IFN)-stimulated gene product 15 (ISG15) is a ubiquitin-like protein critical for the control of microbial infections. Golden pompano, Trachinotus ovatus is one of the precious marine economic fish in the southern coast of China, always suffering from viruses, bacteria, and parasite infections. To date, the roles of golden pompano genes involved in viral and bacterial infections, especially IFN-related genes remained largely unknown. To identify the interferon system genes of golden pompano and explore their function, in this study, the ISG15 homolog (ToISG15) was cloned from golden pompano, and its role in response to grouper iridovirus (SGIV), nervous necrosis virus (NNV), and Aeromonas hydrophila infection was investigated. The whole ORF of ToISG15 was composed of 465 bp and encoded a polypeptide of 154 amino acids with different identity with the known ISG15 homologs from other fish species. Two conserved ubiquitin-like (UBL) domains and an Ub-conjugation domain (LRGG) were found in ToISG15 sequence. Expression analysis showed that ToISG15 was located mainly in the cytoplasm of golden pompano cells, and dramatically induced following SGIV, Aeromonas hydrophila, or poly I:C treatment, but little change was observed when NNV infection. Overexpression of ToISG15 in vitro significantly inhibited the replication of SGIV and NNV. Interestingly, ToISG15 possessed the ability to restrain the growth of Aeromonas hydrophila. Furthermore, To-ISG15 overexpression enhanced the expression of IFNc, IFNh, IRF3, IRF7, and viperin genes as well as, to a lesser extent, the IL-6 gene. Taken together, our results demonstrated the antiviral and antibacterial effect of To-ISG15, shedding light on the evolutionary conservation of ISG15 in the immune response to microbial infection.

Characterization of Nervous Necrosis Virus (NNV) Nonstructural Protein B2 and Its Enhancement on Virus Proliferation.

The nerve necrosis virus (NNV), a pathogen of viral nervous necrosis disease in several important mariculture economic fish species, causes economic loss. Its nonstructural protein B2 encoded by the sub-genomic RNA3 affects the amplification of the virus. In this study, the B2 protein was recombinantly expressed, the polyclonal antibodies were produced and the dynamics of the B2 protein and genomes were measured in vivo and in vitro after NNV infection. Then, the effects of the overexpressed B2 protein on virus proliferation were investigated. The results showed that the polyclonal antibodies can recognize the B2 protein in both SSN-1 cells and the brain/eye of the grouper. The RNA3 expression significantly increased at 12 h and kept rising till the end of the experiment; it was 106.9 copies/μL at 120 h. The B2 protein could be first detected at 3 h post-infection, which was earlier than the capsid protein was first detected (12 h post-infection). The B2 protein can be detected in the brain, eye and heart on day 3 and the copy number of genomes reached a maximum at 6 d post-infection. There was a low expression of NNV genomes in the liver, spleen and kidney, and no virus was detected in the gill, stomach and intestine. In the meantime, the B2 protein was successfully expressed in GF-1 cells and significantly enhanced virus proliferation, which produced an earlier cytopathic effect and higher cell death rates after 3 d post-infection than the control. In conclusion, the B2 protein acts as an early expressed protein during virus replication and proliferation and is involved in the early infection of NNV. The results may provide insight into the early stage of virus infection and prevention of the disease.

Pocket structures of surface protrusions shared among serologically distinct nervous necrosis viruses (NNVs) were predicted in silico to bind to sialylated N-glycans, a host cellular receptor

Nervous necrosis virus (NNV), one of the simplest spherical RNA-viruses, causes high mortality in aquaculture facilities worldwide. In this study, we investigated changes in the infectivity and reactivity of serologically distinct NNVs after treatment with 2.0 M urea, which was used as a means to predict a site or sites on surface protrusions responsible for NNV infection. Antigenicity of NNVs declined significantly after urea treatment but their infectivity was maintained, regardless of serological distinctions. The ability of urea-treated NNVs to be neutralized by antisera was also maintained, regardless of serological distinction. Moreover, weak but reliable cross-neutralizing reactions were observed between serologically distinct NNVs even after urea-treatment. These results suggested that common epitopes resistant to urea-treatment were shared among serologically distinct NNVs. Furthermore, two highly conserved amino-acid stretches among NNVs were revealed, and these stretches surround the pocket structures at the apex of surface protrusions. Molecular docking analysis showed that the pocket structure of surface protrusions was a site for maximally stable binding with the terminal moiety of sialylated N-glycans, a common cellular receptor for serologically distinct NNVs. Based on these results, the pocket structures on surface protrusions of NNVs, appear to be sites responsible for binding to sialylated N-glycans. Contrarily, a red-spotted grouper NNV (RGNNV)-specific MAb, neutralized naïve RGNNV but not the urea-treated RGNNV, suggesting that the epitope for this MAb was not present in the pocket structures. This suggests that disappearance of NNV infectivity due to binding with this MAb was the result of indirect inhibition of binding to the cellular receptor.

Expression profile and molecular function of beclin-1 in Epinephelus akaara in response to immune stimuli and oxidative stress

Beclin-1, the mammalian ortholog of the yeast autophagy-related gene 6 (Atg 6), is a key regulator of autophagy. A variety of health and disease conditions in mammals are intricately related to the broad spectrum of beclin-1 functions. Nevertheless, few studies have investigated the role of beclin-1 in fish. In this study, we identified and cloned the beclin-1 cDNA (EaBECN-1) of Epinephelus akaara (red-spotted grouper) and carried out in silico analysis, tissue-specific expression analysis, immune challenge experiment, and in vitro analysis of its roles against viral infection and oxidative stress. The open reading frame was 1344 bp long and encoded 447 amino acids with a molecular weight of 51.2 kDa. Beclin-1 consisted of a conserved N-terminal BH3 and APG6 domains, and shared more than 88% identity with other vertebrates, according to a pairwise sequence alignment. EaBECN-1 expression profile analysis in E. akaara revealed that it is mostly expressed in the blood. Moreover, transcriptional modulation of EaBECN-1 was observed following stimulation with lipopolysaccharide (LPS), polyinosinic-polycytidylic acid (poly (I:C)), and nervous necrosis virus. During the viral hemorrhagic septicemia virus challenge, increased viral gene expression was observed at 12 h post-infection in FHM cells ectopically expressing EaBECN-1, and decreased thereafter at 24 h post-infection compared to control cells. However, increased antiviral gene expression at 12 and 24 h confirmed the antiviral function of EaBECN-1. Furthermore, EaBECN-1 overexpression protected the cells against H2O2-mediated apoptosis, as evidenced by the MTT assay, analysis of mRNA expression levels of apoptotic genes, and AO-EtBr staining. Overall, our study demonstrated the protective role of EaBECN-1 against viral pathogenesis and oxidative stress through autophagy, increasing our understanding of the role of beclin-1 in fish.

First detection of Cyclopterus lumpus virus in England, following a mortality event in farmed cleaner fish

Lumpfish are used routinely as cleaner fish to delouse farmed Atlantic salmon (Salmo salar) and are often translocated globally. In September 2021, an aquaculture production business in Dorset, England experienced considerable mortalities in consignments of lumpfish (Cyclopterus lumpus) imported from Norway. Following suspicion of infection with Cyclopterus lumpus virus (CLuV) by a veterinarian, commercial testing provided presumptive confirmation of a CLuV infection using reverse-transcription real-time polymerase chain reaction (RT-rPCR). Affected fish were later resampled by the Centre of Environment, Fisheries, and Aquaculture Science, Fish Health Inspectorate for screening of CLuV by RT-rPCR, and confirmation by conventional RT-PCR and sequence analysis. Additional samples were tested using isolation of viruses on cell cultures, bacteriology, histopathology, and PCR/rPCR techniques to investigate whether other pathogens of concern to the salmon aquaculture industry were also present. All samples tested negative for the presence of viral haemorrhagic septicaemia virus, infectious haematopoietic necrosis virus , nervous necrosis virus, infectious pancreatic necrosis virus , infectious salmon anaemia virus , ranavirus, and salmonid alphavirus. Four pools of ten samples tested positive by RT-rPCR for CLuV (Ct values between 19 and 29) and was confirmed using conventional RT-PCR. Sequence analysis showed that amplicons shared a 99.63 % match with the published sequences for CLuV. The CLuV has only recently been reported in aquatic animals and its full impact is not yet known. To the best of our knowledge this is the first report of CLuV in England.

In silico characterisation, homology modelling and molecular docking of nervous necrosis virus capsid protein

In silico characterisation, homology modelling and molecular docking of nervous necrosis virus capsid protein

Thioredoxin domain-containing protein 12 (TXNDC12) in red spotted grouper (Epinephelus akaara): Molecular characteristics, disulfide reductase activities, and immune responses

Thioredoxins are small ubiquitous redox proteins that are involved in many biological processes. Proteins with thiol-disulfide bonds are essential regulators of cellular redox homeostasis and diagnostic markers for redox-dependent diseases. Here, we identified and characterized the thioredoxin domain-containing protein 12 (EaTXNDC12) gene in red spotted grouper (Epinephelus akaara), evaluated transcriptional responses, and investigated the activity of the recombinant protein using functional assays. EaTXNDC12 is a 19.22-kDa endoplasmic reticulum (ER)-resident protein with a 522-bp open reading frame and 173 amino acids, including a signal peptide. We identified a conserved active motif (66WCGAC70) and ER retention motif (170GDEL173) in the EaTXNDC12 amino acid sequence. Relative EaTXNDC12 mRNA expression was analyzed using 12 different tissues, with the highest expression seen in brain tissue, while skin tissue showed the lowest expression level. Furthermore, mRNA expression in response to immune challenges was analyzed in the head kidney, blood, and gill tissues. EaTXNDC12 was significantly modulated in response to bacterial endotoxin lipopolysaccharide (LPS), nervous necrosis virus (NNV), and polyinosinic:polycytidylic acid (poly(I:C)) challenges in all of the tested tissues. Recombinant EaTXNDC12 (rEaTXNDC12) displayed antioxidant ability in an insulin reductase assay, and a capacity for free radical inhibition in a 2,2-diphenyl-1-picryl-hydrazyl-hydrate assay. In addition, a DNA nicking assay revealed that purified rEaTXNDC12 exhibited concentration-dependent DNA protection activity, while results from 2-hydroxyethyl disulfide and L-dehydroascorbic assays indicated that rEaTXNDC12a possesses reducing ability. Furthermore, fathead minnow (FHM) cells transfected with EaTXNDC12-pcDNA demonstrated significantly upregulated cell survival against H2O2-induced apoptosis. Collectively, the results of this study strengthen our knowledge of EaTXNDC12 with respect to cellular redox hemostasis and immune regulation in Epinephelus akaara.

Differential gene expression of red-spotted grouper (Epinephelus akaara) in response to lipopolysaccharide, poly I:C, and nervous necrosis virus revealed by RNA-seq data

Red-spotted grouper (Epinephelus akaara) is a popular aquaculture species with high commercial value in the food industry. However, some infectious diseases may cause mass mortality in cultural practice. Therefore, it is important to understand the immune responses of red-spotted groupers upon pathogenic invasion to develop successful disease prevention mechanisms. Here, we analyzed the transcriptomic profiles of red-spotted grouper head kidney stimulated with lipopolysaccharide (LPS), polyinosinic:polycytidylic acid (poly I:C), and nervous necrosis virus (NNV) and identified differentially expressed genes (DEGs) using RNA-sequencing technology. Cluster analysis of the identified DEGs showed DEG distribution in nine separate clusters based on their expression patterns. However, significant upregulation of most DEGs was observed 6 h after poly I:C stimulation. The DEGs were functionally annotated using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, which revealed significant expression of many immune-related signaling pathways, including antiviral, protein translation, cellular protein catabolic process, inflammatory responses, DNA repair, and cell division. Furthermore, selected DEGs were validated by quantitative real-time PCR, confirming the reliability of our findings. Collectively, this study provides insight into the immune responses of red-spotted groupers, thereby expanding the understanding of fish immunity.

Temperature dependent cellular, and epigenetic regulatory mechanisms underlying the antiviral immunity in sevenband grouper to nervous necrosis virus infection

Changes in the thermal optima of fish impacts changes in the physiology and immune response associated with infections. The present study showed that at suboptimal temperatures (17 °C), the host tries to evade viral infection by downregulating the inflammatory response through enhanced neuronal protection. There was significantly less abundance of IgM + B cells in the 17 °C group compared to that in the 25 °C group. An increased macrophage population (Iba1+) during the survival phase in fish challenged at 25 °C demonstrated inflammation. Optimal temperature challenge activated virus-induced senescence in brain cells, demonstrated with a heterochromatin-associated H3K9me3 histone mark. There was an abundant expression of anti-inflammatory cytokines in the brain of fish at the suboptimal challenge. Besides the cytokines, the expression of BDNF was significantly higher in the suboptimally challenged group, suggesting that its neuronal protection activity following NNV infection is mediated through TGFβ. The suboptimal challenge resulted in H3k9ac displaying transcriptional competency, activation of trained immunity H3K4me3, and enrichment of H3 histone-lysine-4 monomethylation (H3K4me1), resulting in a robust re-stimulatory immune response. The observations from the H4 modifications showed that besides H4K12ac and H4K20m3, all the assayed modifications were significantly higher in suboptimal convalescent fishes. The suboptimally challenged fish acquired more methylation along cytosine residues than the optimally infected fish. Together, these observations suggest that optimal temperature results in an immune priming effect, whereas the protection enabled in suboptimal convalescent fishes is operated through epigenetically controlled trained immune functions.

The Analysis of Clinical sign, Mortality and Histopathology of Cantang Grouper Infected with VNN in Recirculating Aquaculture System

Hybrid grouper is a variety of grouper that has become increasingly popular in recent years. The development of grouper cultured in Indonesia utilizing the Recirculating Aquaculture System (RAS) is currently underway; nevertheless, fish cultured in the RAS system continue to struggle with infectious diseases, one of which is VNN (viral nervous necrosis) infection caused by piscine betanodavirus. The purpose of this study was to know the clinical sign, mortality, and histopathology of cantang grouper (Epinephelus fuscoguttatus x E. lanceolatus) infected with VNN in RAS. Fish samples were obtained from the hatchery using RAS. This research uses a mixed methods approach and the type of research used is exploratory research. The parameters observed were clinical signs, mortality, and histopathology. Water quality, including temperature, DO, salinity, and pH at the time of VNN infection, was used as secondary data. The results showed that clinical signs of sick fish were weakness, staying at the bottom of the rearing tank, decreased appetite, swimming with the stomach facing up and the body dark in color. Fish mortality occurred for ± 10 days with a peak of mortality reaching 26.86 ± 7.45% on day 9 from initial signs. Histopathology result, diseased fish showed vacuolization in the brain and retina of the eye.

Trace Determination of Grouper Nervous Necrosis Virus in Contaminated Larvae and Pond Water Samples Using Label-Free Fiber Optic Nanoplasmonic Biosensor.

We developed a fast (<20 min), label-free fiber optic particle plasmon resonance (FOPPR) immunosensing method to detect nervous necrosis virus (NNV), which often infects high-value economic aquatic species, such as grouper. Using spiked NNV particles in a phosphate buffer as samples, the standard calibration curve obtained was linear (R2 = 0.99) and the limit of detection (LOD) achieved was 2.75 × 104 TCID50/mL, which is superior to that obtained using enzyme-linked immunosorbent assay (ELISA). By using an enhancement method called fiber optic nanogold-linked immunosorbent assay (FONLISA), the LOD can be further improved to <1 TCID50/mL, which is comparable to that found by the conventional qPCR method. Employing the larvae homogenate samples of NNV-infected grouper, the results obtained by the FOPPR biosensor agree with those obtained by the quantitative polymerase chain reaction (qPCR) method. We also examined pond water samples from an infected container in an indoor aquaculture facility. The lowest detectable level of NNV coat protein was found to be 0.17 μg/mL, which is one order lower than the LOD reported by ELISA. Therefore, we demonstrated the potential of the FOPPR biosensor as an outbreak surveillance tool, which is able to give warning indication even when the trend of larvae death toll increment is still not clear.

A COMPARATIVE STUDY ON BRAIN-TROPISM OF BETANODAVIRUSES IN YOUNG MARINE FISH: A WAY TO PERFORM HOST SPECIFICITY

Investigation on the brain-tropism was carried out through direct injection of the virus into the vitreous body of the eye using five marine fish species: kelp grouper, striped jack, Japanese flounder, threeline grunt and yellow tail. When the brains of intravitreously-injected fish (106 TCID50 Fish-1) were examined at 72 h post infection, striped jack nervous necrosis virus (SJNNV; the type species of the genus Betanodavirus) was frequently detected from striped jack and flounder while kelp grouper nervous necrosis virus (KGNNV) were detected from kelp grouper, striped jack, and flounder. No constant growth of the virus was noticed in the brains of threeline grunt and yellow tail. Considering that natural outbreaks of VNN (viral nervous necrosis) have been frequently reported in the former three species but not in the later two. The present result supports that the brain cell-tropism might be one of the tools to perform the host-specificity of betanodavirus.

Silencing Asian Seabass gab3 Inhibits Nervous Necrosis Virus Replication.

The nervous necrosis virus (NNV) causes the viral nervous necrosis (VNN) disease in aquatic animals and has been a major threat in aquaculture. Thus, it is essential for the development of a prevention method to minimize economic losses caused by NNV such as the identification of NNV resistance genes and application of these genes in molecular breeding to increase disease resistance. gab3 is an important NNV resistance gene in Asian seabass. However, the mechanism of gab3 in NNV resistance has not been elucidated. In this study, knockdown of gab3 in NNV-infected Asian seabass cells resulted in a significant decrease in viral RNA and virus titers. Knockout of gab3 in zebrafish led to an increased survival rate and resistant time after NNV infection. Cellular localization of the GAB3 and NNV by immunofluorescence staining showed that the GAB3 was translocated from the nucleus to the cytoplasm, and finally reached the cell membrane of SB cells after 48h post NNV infection. Our study suggests that gab3 plays an important role in NNV replication and silencing gab3 can inhibit virus replication.