Red-spotted Grouper Nervous Necrosis Virus Research Articles

Grouper USP12 exerts antiviral activity against nodavirus infection

The ubiquitin-specific proteases (USPs) have attracted particular attention due to their multiple functions in different biological processes. USP12, a member of the USP family, has been demonstrated to exert critical roles in diverse cellular processes, including cell death, cancer and antiviral immunity. Here, we cloned a USP12 homolog from orange spotted grouper (Epinephelus coioides, E. coioides), and its roles in fish RNA virus replication were investigated. EcUSP12 contained a 1119-bp open reading frame (ORF) encoding a 372-amino acid polypeptide, which shared 100.00% and 91.32% identity with USP12 homolog of Etheostoma cragini and Homo sapiens, respectively. Sequence analysis indicated that EcUSP12 contained a conserved peptidase-C19G domain (aa 40–369). qPCR analysis showed that EcUSP12 transcript was most abundant in head kidney and spleen of grouper E. coioides. The expression of EcUSP12 was significantly upregulated in grouper spleen (GS) cells in response to red-spotted grouper nervous necrosis virus (RGNNV) infection. Subcellular localization analysis showed that EcUSP12 was evenly distributed throughout the cytoplasm, and mainly co-localized with endoplasmic reticulum (ER). Interestingly, during RGNNV infection, the endogenous distribution of EcUSP12 was obviously altered, and mostly overlapped with viral coat protein (CP). Co-Immunoprecipitation (Co-IP) assay indicated that EcUSP12 interacted with viral CP. In addition, overexpression of EcUSP12 significantly inhibited the replication of RGNNV in vitro, as evidenced by the decrease in viral gene transcription and protein synthesis during infection. Consistently, knockdown of EcUSP12 by small interfering RNA (siRNA) promoted the replication of RGNNV. Furthermore, EcUSP12 overexpression also increased the transcription level of inflammatory factors and interferon-related genes, including tumor necrosis factor α (TNF-α), interleukin (IL)-1β, IL-6, IL-8, interferon regulatory factor 3 (IRF3), and IRF7. Taken together, our results demonstrated that EcUSP12, as a positive regulator of IFN signaling, interacted with viral CP to inhibit virus infection.

Capsid protein from red-spotted grouper nervous necrosis virus induces incomplete autophagy by inactivating the HSP90ab1-AKT-MTOR pathway.

As a highly important fish virus, nervous necrosis virus (NNV) has caused severe economic losses to the aquaculture industry worldwide. Autophagy, an evolutionarily conserved intracellular degradation process, is involved in the pathogenesis of several viruses. Although NNV can induce autophagy to facilitate infection in grouper fish spleen cells, how it initiates and mediates autophagy pathways during the initial stage of infection is still unclear. Here, we found that red-spotted grouper NNV (RGNNV) induced autophagosome formation in two fish cell lines at 1.5 and 3 h post infection, indicating that autophagy is activated upon entry of RGNNV. Moreover, autophagic detection showed that RGNNV entry induced incomplete autophagy by impairing the fusion of autophagosomes with lysosomes. Further investigation revealed that binding of the RGNNV capsid protein (CP) to the Lateolabrax japonicus heat shock protein HSP90ab1 (LjHSP90ab1), a cell surface receptor of RGNNV, contributed to RGNNV invasion-induced autophagy. Finally, we found that CP blocked the interaction of L. japonicus protein kinase B (AKT) with LjHSP90ab1 by competitively binding the NM domain of LjHSP90ab1 to inhibit the AKT-mechanistic target of the rapamycin (MTOR) pathway. This study provides novel insight into the relationship between NNV receptors and autophagy, which may help clarify the pathogenesis of NNV.

Functional characterization of myosin III light chain b of sea perch (Lateolabrax japonicus) in response to fish virus infection

Myosins are key motor proteins of vertebrates that are important in cell-cell adhesion and migration; they are associated with viral infections. Herein, a myosin III light chain b gene from sea perch (Lateolabrax japonicus), designated Ljmyo3b, was characterized and its positive role in fish virus pathogenesis was revealed. The cDNA of Ljmyo3b encoded a 150-amino acid protein with two classical EF-hand domains; it shared the closest genetic relationship with Larimichthys crocea myo3b. Cellular distribution analysis showed that Ljmyo3b was localized in the cytoplasm and nucleus of LJB cells, and also on the cell surface, which was further confirmed by the protease protection assay. Spatial expression analysis revealed that Ljmyo3b was more abundant in the immune tissues of sea perch post red spotted grouper nervous necrosis virus (RGNNV) or viral hemorrhagic septicemia virus (VHSV) infection. In RGNNV- and VHSV-treated LJB cells, Ljmyo3b expression was significantly upregulated at 2, 4, 24, and 48 hpi. Furthermore, the ectopic expression of Ljmyo3b facilitated RGNNV and VHSV infection in LJB cells at 2, 4, and 24 hpi. Taken together, these results suggest that Ljmyo3b is involved in mediating RGNNV and VHSV infection in fish.

Update on the Inactivation Procedures for the Vaccine Development Prospects of a New Highly Virulent RGNNV Isolate.

Viral nervous necrosis (VNN) caused by the nervous necrosis virus (NNV) affects a broad range of primarily marine fish species, with mass mortality rates often seen among larvae and juveniles. Its genetic diversification may hinder the effective implementation of preventive measures such as vaccines. The present study describes different inactivation procedures for developing an inactivated vaccine against a new NNV isolate confirmed to possess deadly effects upon the European seabass (Dicentrarchus labrax), an important Mediterranean farmed fish species that is highly susceptible to this disease. First, an NNV isolate from seabass adults diagnosed with VNN was rescued and the sequences of its two genome segments (RNA1 and RNA2) were classified into the red-spotted grouper NNV (RGNNV) genotype, closely clustering to the highly pathogenic 283.2009 isolate. The testing of different inactivation procedures revealed that the virus particles of this isolate showed a marked resistance to heat (for at least 60 °C for 120 min with and without 1% BSA) but that they were fully inactivated by 3 mJ/cm2 UV-C irradiation and 24 h 0.2% formalin treatment, which stood out as promising NNV-inactivation procedures for potential vaccine candidates. Therefore, these procedures are feasible, effective, and rapid response strategies for VNN control in aquaculture.

Chitosan Treatment of E-11 Cells Modulates Transcription of Nonspecific Immune Genes and Reduces Nodavirus Capsid Protein Gene Expression.

Simple SummaryViral Encephalopathy and Retinopathy (VER) is caused by an RNA virus named Betanodavirus representing a major problem in Mediterranean marine aquaculture resulting in great economic losses. Control of VER currently relies on effective biosecurity measures and vaccines. Natural non-toxic biopolymers such as chitosan are produced commercially from crab and shrimp shell waste offering useful biomolecules for a wide range of applications for their antimicrobial and antioxidant activity, immunostimulant effect and bioactive coating. This study evaluates the in-vitro antiviral activity of chitosan extracted from the exo-skeleton of Parapenaeus longirostris, focusing on its ability to modulate the innate immune response in E-11 cells challenged with nodavirus as well as its impact on nodavirus RNA2 gene expression. Using qPCR, results reviewed here demonstrated a modulation of the innate immune gene expression and a reduction in virus load suggesting that chitosan was found to be a suppressor of nodaviral infection in fish cell systems.This study explores whether crustacean products inhibit viral infections in aquaculture. Chitosan (CHT) was extracted from waste products of Parapenaeus longirostris. Biochemical composition, viscosity measurement, molecular weight, structure and cytotoxicity tests were used to characterize the extracted chitosan. Cultures of E-11 cells derived from snakehead Ophicephalus striatus were inoculated with 106.74 TCID50 of an isolate of betanodavirus genotype RGNNV (redspotted grouper nervous necrosis virus) after being treated with solutions of 0.3% CHT for 1 h at room temperature. The antiviral effect of CHT was assessed by comparing the ability of RGNVV to replicate and produce cytopathic effects on CHT-treated cell cultures. The change in RNA expression levels of the nodavirus capsid protein gene and three mediator genes in infected cells with or without CHT treatment was evaluated by qPCR. Changes in gene expression compared to control groups were monitored at 6, 24, 48 and 71 h post treatment in all target gene transcripts. The CCR3 expression in CHT treated cells showed a significant increase (p < 0.05) until day 3. On the other hand, the expression of TNF-α decreased significantly (p < 0.05) in CHT treated cells throughout the experimental period. Likewise, the expression of the IL-10 gene showed a significant downregulation in CHT treated cells at all time points (p ≤ 0.05). As further evidence of an antiviral effect, CHT treatment of cells produced a reduction in virus load as measured by a reduced expression of the viral capsid gene and the increase in RQ values from 406 ± 1.9 at hour 1 to 695 ± 3.27 at 72 h post inoculation. Statistical analysis showed that the expression of the viral capsid gene was significantly lower in cells treated with chitosan (p ≤ 0.05). These results improve our knowledge about the antiviral activity of this bioactive molecule and highlight its potential use in fish feed industry.

Immune Response of Senegalese Sole against Betanodavirus Mutants with Modified Virulence.

Nervous necrosis virus (NNV), genus Betanodavirus, the etiological agent of the viral encephalopathy and retinopathy (VER), presents a genome with two positive-sense single-stranded RNA segments. Striped jack nervous necrosis virus (SJNNV) and red-spotted grouper nervous necrosis virus (RGNNV), together with reassortants RGNNV/SJNNV, are the betanodaviruses predominantly isolated in Southern Europe. An RGNNV/SJNNV reassortant isolated from Senegalese sole (wt160) causes high mortalities in this fish species. This virus presents differences in the sequence of the 3’ non-coding region (NCR) of both segments compared to RGNNV and SJNNV reference strains. Previously, it has been reported that the reversion of two of these differences (nucleotides 1408 and 1412) in the RNA2 3’NCR to the SJNNV-type (recombinant r1408-1412) resulted in a decrease in sole mortality. In the present study, we have applied an OpenArray® to analyse the involvement of sole immune response in the virulence of several recombinants: the r1408-1412 and two recombinants, developed in the present study, harbouring mutations at positions 3073 and 3093 of RNA1 3’NCR to revert them to RGNNV-type. According to the correlation values and to the number of expressed genes, the infection with the RNA2-mutant provoked the most different immune response compared to the immune response triggered after the infection with the rest of the viruses, and the exclusive and high upregulation of genes related to the complement system. The infection with the RNA1-mutants also provoked a decrease in mortality and their replication was delayed at least 24 h compared to the wt160 replication, which could provoke the lag observed in the immune response. Furthermore, the infection with the RNA1-mutants provoked the exclusive expression of pkr and the downregulation of il17rc.

Establishment and characterization of a liver cell line, ALL, derived from yellowfin sea bream, Acanthopagrus latus, and its application to fish virology.

Yellowfin sea bream (Acanthopagrus latus) is an important economic fish, which is seriously threatened by various fish viruses. In this study, a cell line designated as ALL derived from the liver of yellowfin sea bream was developed and characterized. The cell line grew well in Dulbecco's modified Eagle's medium containing 10%-20% foetal bovine serum at 28°C. Amplification of the cytochrome B gene indicated that ALL cells originated from yellowfin sea bream. The modal chromosome number of ALL cells was 48. ALL cells were efficiently transfected with pEGFP-N3 plasmids, indicating the potential application of ALL cells in exogenous gene manipulation studies. ALL cells were susceptive to three main fish viruses, including viral haemorrhagic septicaemia virus (VHSV), red-spotted grouper nervous necrosis virus (RGNNV) and largemouth bass virus (LMBV). The replication of VHSV, RGNNV and LMBV in ALL cells was confirmed by quantitative real-time polymerase chain reaction, virus titre and transmission electron microscopy assays. Moreover, ALL cells could respond to VHSV, RGNNV and LMBV infections, as indicated by the differential expression of antiviral genes involving in the innate immune response. In conclusion, the newly established ALL cell line will be an excellent in vitro platform for the study of the virus-yellowfin sea bream interaction.

Establishment of a cell line from the head kidney of giant grouper (Epinephelus lanceolatus) and its susceptibility to fish viruses

Grouper is an important marine fish mainly cultured in China and Southeast Asia. Here, a continuous cell line, named as ELHK, was established from head kidney of giant grouper (Epinephelus lanceolatus). The origin of cell line was confirmed by sequencing of 18s rRNA genes. ELHK cells consisted predominantly of fibroblast-like cells, and multiplied well in Leibovitz’s L-15 medium supplemented with 10% fetal bovine serum at 28 °C. Karyotyping analysis indicated that the modal chromosome number of ELHK cell was 60 at 50 passages. The cells were successfully transfected with GFP reporter gene suggesting that ELHK cell could be used for exogenous gene expression in vitro. Moreover, ELHK cells was susceptible to Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV), demonstrated by the occurrence of cytopathic effects (CPEs) and increased transcription of the viral genes during virus infection. Furthermore, numerous viral particles were observed in SGIV- or RGNNV-infected ELHK cells using electron microscopy, which further confirmed that ELHK cells could be used for fish virus propagation in vitro. In addition, quantitative PCR analysis indicated that SGIV and RGNNV infection in ELHK cells significantly increased the transcripts of several interferon- or pro-inflammation-related cytokines, suggested that both of them up-regulated host interferon and inflammatory immune response in vitro. Taken together, our established ELHK cell line could be used as an in vitro tool for gene expression, as well as virus propagation and virus-host interaction studies.

Investigation of betanodavirus in sea bass (Dicentrarchus labrax) at all production stages in all hatcheries and on selected farms in Turkey.

Viral nervous necrosis (VNN) is one of the most important problems in sea bass culture. Although there have been many studies on detection and molecular characterization of betanodavirus, the causative agent of VNN, there has been little focus on understanding its prevalence to create epidemiological maps. The purpose of this study was to investigate the prevalence of betanodavirus in active sea bass hatcheries and on selected farms in Turkey by RT-qPCR. A total of 2460 samples, including fertilized eggs, prelarvae, postlarvae, fry, and fingerlings, were collected from 16 hatcheries to cover all production stages. A total of 600 sea bass were also collected from 20 farms. Betanodavirus was detected in one hatchery (1/16) in fingerling-sized sea bass, and the prevalence of betanodavirus at the hatchery level was calculated to be 6.25%. Betanodavirus was also detected on one farm (1/20) in fingerling-sized sea bass, and the prevalence of betanodavirus at the farm level was calculated to be 5%. Virus isolation initially could not be achieved in E-11 cells, but later, SSN-1 cells were used successfully. Partial genome sequence analysis of the RNA1 and RNA2 segments of the viruses revealed that they were of the red-spotted grouper nervous necrosis virus genotype, which is endemic in the Mediterranean basin. The absence of mortality related to VNN in the hatcheries and on the farms, the healthy appearance of the sea bass, the low viral load detected, and the results of retrospective epidemiological studies indicated that the infection was subclinical. Not detecting betanodavirus in other age groups where biosecurity was implemented indicates that there was no active infection. In light of these findings, it can be concluded that there was no betanodavirus circulating in hatcheries, and the virus might have been of seawater origin.

Emergence of Reassortment between a New and Reported Types of Betanodavirus in Shellfish.

Recently, three types of betanodavirus including red spotted grouper nervous necrosis virus (RGNNV), barfin flounder nervous necrosis virus (BFNNV), and Korean shellfish nervous necrosis virus (KSNNV) (proposed as a new fifth type) have been detected in shellfish in the marine environment around Korea. To investigate the presence of reassortment between betanodavirus types, the type based on the RNA2 segment of betanodaviruses carried in 420 domestic shellfish (n = 306) and finfish (n = 35), as well as imported shellfish (n = 79), was compared with the type identified by reverse-transcriptase polymerase chain reaction (RT-PCR) for RNA1 segment. Only five samples carrying reassortant betanodaviruses were found, appearing as RG/KSNNV (n = 2), KS/RGNNV (n = 1), and SJ/RGNNV (n = 2) types. From these samples, we successfully isolated two reassortant strains from Korean and Chinese shellfish in E-11 cells and called them KG1-reKS/RG and CM1-reRG/KS, respectively. In the full genome sequences, each RNA segment of the reassortant strains exhibited the same gene length and high sequence homology (≥98%) with the reference strains corresponding to the type of each segment. Both these reassortant strains induced high mortality to sevenband grouper (Epinephelus septemfasciatus) larvae with high viral concentrations in the body (109 viral particles/mg) and severe vacuolation in the retina and brain. These are the first results showing the involvement of the KSNNV type in the reassortment of RNA segments in the reported types of betanodavirus, which could represent a new potential risk in fish.

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

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

Mannose receptor of Epinephelus coioides exerts antiviral activity against red-spotted grouper nervous necrosis virus and regulates apoptosis and inflammation

Mannose receptor (MR), a pattern recognition receptor, plays a critical role in innate immune responses by binding to pathogen-associated molecular patterns. The antiviral role of MR has been described extensively in mammals but not well characterized in teleosts. In this study, we investigated the role of Epinephelus coioides MR in red spotted grouper nervous necrosis virus (RGNNV) infection by cloning and characterizing EcMR. The sequence analysis showed that EcMR contained a signal peptide, ricin-like β-type clover domain (RICIN), fibronectin type II domain (FN II), eight tandem C-type lectin-like domains (CLECTs), and a transmembrane domain. The function of EcMR was analyzed using constructs expressing three segments of the full-length protein including EcMR1 (RICIN-FN II), EcMR2 (CLECT 1–3), and EcMR3 (CLECT 4–8). Overexpression of EcMR1, EcMR2, and EcMR3 significantly reduced RGNNV coat protein expression at both mRNA and protein levels. Conversely, RGNNV replication was increased by EcMR knockdown. EcMR was found to inhibit apoptosis signaling and inflammation in GF-1 grouper fin cells, as determined by immunofluorescence analysis and flow cytometry. The increases in apoptosis-related gene expression (FADD, Fas, Bax, and p53) and apoptosis-related enzyme activities (caspase-3, −8, and − 9) in cells overexpressing EcMR1, EcMR2, and EcMR3 and the contrary effect observed upon EcMR knockdown indicated that EcMR blocks apoptosis induced by RGNNV. EcMR also exhibited an anti-inflammatory function through suppression of proinflammatory cytokine production. These results demonstrate that EcMR contributes to the response of E. coioides to virus infection by modulating virus-induced apoptosis and inflammation, which may have important implications for commercial aquaculture practices.

Protective effects of egg yolk immunoglobulins (IgY) on juvenile groupers (Epinephelus fuscoguttatus × Epinephelus lanceolatus) with red-spotted grouper nervous necrosis virus infection

Red-spotted grouper nervous necrosis virus (RGNNV) is a highly infectious, explosive, and lethal virus that mainly infects marine fish larvae. Currently, there is no effective method to mitigate the impact of RGNNV on aquaculture production. Previous studies have shown that yolk immunoglobulin (IgY) can neutralize RGNNV in grouper fin-1 (GF-1) cells. In this study, RGNNV propagated in GF-1 cells was harvested and purified successfully. The purified RGNNV was used as an immunogen to immunize laying hens. Water-soluble fractions were separated from egg yolk, and IgY was harvested using a two-step precipitation method with saturated ammonium sulfate salt. In vitro analysis showed that the purified IgY could neutralize RGNNV. An in vivo protection assay with groupers revealed that fish fed with 10% lyophilized egg yolk powder from immunized hens were significantly protected during RGNNV challenge. The relative protection rates of groupers under treatment after RGNNV infection and continuous feeding during RGNNV infection were 68% and 70%, respectively, when fed with 10% specific egg yolk powder. This is the first study to validate that anti-RGNNV IgY can effectively protect groupers from RGNNV infection, laying a foundation for the development of anti-RGNNV products.

Single-cell RNA-seq landscape midbrain cell responses to red spotted grouper nervous necrosis virus infection.

Viral nervous necrosis (VNN) is an acute and serious fish disease caused by nervous necrosis virus (NNV) which has been reported massive mortality in more than fifty teleost species worldwide. VNN causes damage of necrosis and vacuolation to central nervous system (CNS) cells in fish. It is difficult to identify the specific type of cell targeted by NNV, and to decipher the host immune response because of the functional diversity and highly complex anatomical and cellular composition of the CNS. In this study, we found that the red spotted grouper NNV (RGNNV) mainly attacked the midbrain of orange-spotted grouper (Epinephelus coioides). We conducted single-cell RNA-seq analysis of the midbrain of healthy and RGNNV-infected fish and identified 35 transcriptionally distinct cell subtypes, including 28 neuronal and 7 non-neuronal cell types. An evaluation of the subpopulations of immune cells revealed that macrophages were enriched in RGNNV-infected fish, and the transcriptional profiles of macrophages indicated an acute cytokine and inflammatory response. Unsupervised pseudotime analysis of immune cells showed that microglia transformed into M1-type activated macrophages to produce cytokines to reduce the damage to nerve tissue caused by the virus. We also found that RGNNV targeted neuronal cell types was GLU1 and GLU3, and we found that the key genes and pathways by which causes cell cytoplasmic vacuoles and autophagy significant enrichment, this may be the major route viruses cause cell death. These data provided a comprehensive transcriptional perspective of the grouper midbrain and the basis for further research on how viruses infect the teleost CNS.

Grouper TRAF4, a Novel, CP-Interacting Protein That Promotes Red-Spotted Grouper Nervous Necrosis Virus Replication.

Tumor necrosis factor receptor-associated factors (TRAFs) play important roles in the biological processes of immune regulation, the inflammatory response, and apoptosis. TRAF4 belongs to the TRAF family and plays a major role in many biological processes. Compared with other TRAF proteins, the functions of TRAF4 in teleosts have been largely unknown. In the present study, the TRAF4 homologue (EcTRAF4) of the orange-spotted grouper was characterized. EcTRAF4 consisted of 1413 bp encoding a 471-amino-acid protein, and the predicted molecular mass was 54.27 kDa. EcTRAF4 shares 99.79% of its identity with TRAF4 of the giant grouper (E. lanceolatus). EcTRAF4 transcripts were ubiquitously and differentially expressed in all the examined tissues. EcTRAF4 expression in GS cells was significantly upregulated after stimulation with red-spotted grouper nervous necrosis virus (RGNNV). EcTRAF4 protein was distributed in the cytoplasm of GS cells. Overexpressed EcTRAF4 promoted RGNNV replication during viral infection in vitro. Yeast two-hybrid and coimmunoprecipitation assays showed that EcTRAF4 interacted with the coat protein (CP) of RGNNV. EcTRAF4 inhibited the activation of IFN3, IFN-stimulated response element (ISRE), and nuclear factor-κB (NF-κB). Overexpressed EcTRAF4 also reduced the expression of interferon (IFN)-related molecules and pro-inflammatory factors. Together, these results demonstrate that EcTRAF4 plays crucial roles in RGNNV infection.

MicroRNA expression profiling of sea perch brain cells reveals the roles of microRNAs in autophagy induced by RGNNV infection.

Nervous necrosis virus (NNV) is one of the most destructive fish viruses and affects more than 120 marine and freshwater teleost species. However, the pathogenesis of NNV has not been made clear. MicroRNAs (miRNAs) play important roles in the regulation of viral infection. To understand the roles and regulation patterns of miRNAs in NNV infection, high-throughput sequencing was carried out in Lateolabrax japonicus brain (LJB) cells with or without red-spotted grouper NNV (RGNNV) infection at 12 and 24hr. Here, we identified 59 known and 61 novel differentially expressed miRNAs (DE miRNAs) between mock and RGNNV-infected LJB cells. KEGG pathway analysis showed that the target genes of DE miRNAs were significantly enriched in immune-related signalling pathways, such as autophagy, mitophagy and TGF-beta signalling pathways. The expression patterns of four DE miRNAs (lja-miR-145, lja-miR-182, lja-miR-183 and lja-miR-187) were verified by qRT-PCR both in vivo and in vitro. We found that lja-miR-145 promoted RGNNV proliferation, while lja-miR-183 suppressed RGNNV proliferation. Furthermore, lja-miR-145 facilitated RGNNV-induced autophagy activation, whereas lja-miR-183 repressed autophagy in LJB cells as measured by LC3B-II/I and p62 protein levels. All these results indicate the involvement of lja-miR-145 and lja-miR-183 in RGNNV-induced autophagy. In conclusion, this study provides evidence for the important roles of miRNAs in NNV infection and a basis for uncovering the molecular regulation mechanism of NNV-induced autophagy.

Development of cross-priming amplification (CPA) combined with colorimetric and lateral flow dipstick visualization for scale drop disease virus (SDDV) detection.

Scale drop disease virus (SDDV) is one of the most important pathogens that causes scale drop disease (SDD) in Asian sea bass (Lates calcarifer). The outbreaks of this disease are one of the factors causing substantial losses in Asian sea bass aquaculture. In this study, the uracil-DNA glycosylase (UDG)-supplemented cross-priming amplification (UCPA) combined with a colorimetric detection method using the hydroxynaphthol blue (HNB) and lateral flow dipstick (LFD) for detection of SDDV was developed. The UDG was utilized to prevent carryover contamination, and the CPA reactions can be readily observed by HNB and LFD. The CPA primers and probe were designed to target the major capsid protein (MCP) gene of the SDDV. The optimized UCPA conditions were performed at the temperature of 61°C for 60min. The UCPA assays demonstrated specificity to SDDV without cross-reaction to other tested viruses including red-spotted grouper nervous necrosis virus (RGNNV), infectious spleen and kidney necrosis virus (ISKNV) and Lates calcarifer herpes virus (LCHV), and other bacterial species commonly found in aquatic animals. The sensitivity of the UCPA-HNB and UCPA-LFD was 100 viral copies/µl and 10pg of extracted total DNA, which was 10-fold more sensitive than that of conventional PCR. The UCPA-HNB and UCPA-LFD assays could be used to detect the SDDV infection in all 25 confirmed SDDV-infected fish samples. Therefore, the UCPA coupled with HNB and LFD was rapid, simple and effective and might be applied for diagnosis of SDDV infection.

Non-lethal screening of Asian seabass (Lates calcarifer) by monoclonal antibody based indirect enzyme linked immunosorbent assay for viral nervous necrosis

Viral nervous necrosis (VNN) is a serious viral disease of several species of farmed and wild fishes. Adult fish are asymptomatic and become carriers of the virus when infected with nervous necrosis virus (NNV) and they transmit the virus to the offspring through eggs. ELISA is ideal for non-lethal screening of adult fish for VNN. Asian seabass (Lates calcarifer) IgM was purified using Protein A affinity column and hybridoma clones secreting monoclonal antibodies (MAb) specific to the heavy chain of IgM was developed. An Indirect ELISA using anti-seabass IgM MAb was developed by optimizing all the reagents. The assay was used to screen adult Asian seabass from grow-out farms in comparison to RT-PCR. The assay was also used to assess the immune response in Asian seabass immunized with inactivated Red-spotted grouper NNV (RGNNV). Seabass IgM on SDS-PAGE analysis revealed three heavy chain bands of size 96, 82 and 76 kDa and a single light chain of size 25 kDa. Out of 18 positive hybridoma clones, two selected clones reacted specifically with the 76 kDa heavy chain band. Out of 28 serum samples of Asian seabass from grow-out farms 26 were positive for NNV antibodies while 22 were positive by RT-PCR. Fish immunized with inactivated RGNNV showed immune response by one week post-immunization, and the peak immune response was observed four weeks post-immunization. The assay developed can be used for non-lethal screening of adult Asian seabass for VNN and to assess the immune response after vaccination.

Long-Term Protection and Serologic Response of European Sea Bass Vaccinated with a Betanodavirus Virus-Like Particle Produced in Pichia pastoris.

Viral Nervous Necrosis (VNN) causes high mortality and reduced growth in farmed European sea bass (Dicentrarchus labrax) in the Mediterranean. In the current studies, we tested a novel Pichia-produced virus-like particle (VLP) vaccine against VNN in European sea bass, caused by the betanodavirus “Red-Spotted Grouper Nervous Necrosis Virus” (RGNNV). European sea bass were immunized with a VLP-based vaccine formulated with different concentrations of antigen and with or without adjuvant. Antibody response was evaluated by ELISA and serum neutralization. The efficacy of these VLP-vaccine formulations was evaluated by an intramuscular challenge with RGNNV at different time points (1, 2 and 10 months post-vaccination) and both dead and surviving fish were sampled to evaluate the level of viable virus in the brain. The VLP-based vaccines induced an effective protective immunity against experimental infection at 2 months post-vaccination, and even to some degree at 10 months post-vaccination. Furthermore, the vaccine formulations triggered a dose-dependent response in neutralizing antibodies. Serologic response and clinical efficacy, measured as relative percent survival (RPS), seem to be correlated with the administered dose, although for the individual fish, a high titer of neutralizing antibodies prior to challenge was not always enough to protect against disease. The efficacy of the VLP vaccine could not be improved by formulation with a water-in-oil (W/O) adjuvant. The developed RGNNV-VLPs show a promising effect as a vaccine candidate, even without adjuvant, to protect sea bass against disease caused by RGNNV. However, detection of virus in vaccinated survivors means that it cannot be ruled out that survivors can transmit the virus.

Characterization of red-spotted grouper nervous necrosis virus isolated from ovarian fluids of asymptomatic wild Asian seabass, Lates calcarifer

Viral nervous necrosis (VNN) caused by nervous necrosis virus is a serious disease affecting a wide range of marine, brackishwater and freshwater fishes. The disease causes acute mortalities in larval and early juvenile stages while adult fishes are asymptomatic and remain as carriers of the virus. In the present study, nervous necrosis virus was isolated from ovarian fluids of asymptomatic wild-collected Asian seabass, Lates calcarifer in SSN-1 cell line. The complete genome of the virus was amplified by PCR from plaque purified virus and sequenced. Bioinformatic analysis of the sequence revealed that the isolate belonged to red-spotted grouper nervous necrosis virus. The virus purified from infected SSN-1 cell culture supernatants by cesium chloride gradient ultracentrifugation resulted in two bands in the gradient. Both the bands on SDS-PAGE analysis and western blot revealed a single 40 kDa capsid protein. The virus replication was optimum at 26–28 °C in vitro and the virus can be completely inactivated using 3 mM binary ethylenimine in 32 h at 25 °C. The isolated virus was infectious and caused 100% mortality in Asian seabass larvae. The inactivated virus when injected to Asian seabass fingerlings produced neutralizing antibody titre ranging from 1:320 to 1: 1280. The isolate is a potential candidate for production of an inactivated vaccine against VNN.