Red-spotted Grouper Nervous Necrosis Virus Research Articles

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.

Detection of RGNNV genotype betanodavirus in the Black Sea and monitoring studies.

Viral nervous necrosis (VNN), caused by betanodavirus, is a significant viral infection that threatens marine aquaculture. Freshwater and marine fish farms in Turkey are subjected to annual pathogen screenings. In 2016, during the Nervous Necrosis Virus screening program conducted in the Black Sea, betanodavirus was unexpectedly detected using real-time reverse transcription-polymerase chain reaction in apparently healthy sea bass. Phylogenetic analysis of both the RNA1 and RNA2 segments of the virus determined that the betanodavirus detected was red-spotted grouper nervous necrosis virus genotype (RGNNV). Following the initial discovery of betanodavirus in the Black Sea, monitoring studies performed over a 3 yr period have not indicated any additional presence of the virus. The absence of clinical symptoms related to VNN disease in the area's marine fish farms and the surrounding detection zone, and the fact that the virus has not been detected anew in monitoring programmes conducted following the initial detection, indicate that there is no virus circulation in the detection zone.

Polystyrene nanoplastics alter virus replication in orange-spotted grouper (Epinephelus coioides) spleen and brain tissues and spleen cells

Polystyrene nanoplastics (PS-NPs) are known to impair the function of the digestive system, intestinal flora, immune system, and nervous system of marine organisms. We tested whether PS-NPs influence viral infection of orange-spotted grouper (Epinephelus coioides). We found that grouper spleen (GS) cells took up PS-NPs at exposure concentrations of 5, 50, and 500 μg/mL and experienced cytotoxicity at 50 and 500 μg/mL concentrations. At 12 h after exposure to 50 μg/mL of PS-NPs, the replication of Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) increased in GS cells after their invasion. Juvenile fish exposed to 300 and 3000 μg/L of PS-NPs for 7 d showed PS-NPs uptake to the spleen and vacuole formation in brain tissue. Moreover, PS-NPs exposure accelerated SGIV replication in the spleen and RGNNV replication in the brain. PS-NP exposure also decreased the expression of toll-like receptor genes and interferon-related genes before and after virus invasion in vitro and in vivo, thus reducing the resistance of cells and tissues to viral replication. This is the first report that PS-NPs have toxic effects on GS cells and spleen and brain tissues, and it provides new insights into assessing the impact of PS-NPs on marine fish.

Grouper Interferon-Induced Transmembrane Protein 1 Inhibits Iridovirus and Nodavirus Replication by Regulating Virus Entry and Host Lipid Metabolism.

Interferon-induced transmembrane proteins (IFITMs) are novel viral restriction factors which inhibit numerous virus infections by impeding viral entry into target cells. To investigate the roles of IFITMs during fish virus infection, we cloned and characterized an IFITM1 homolog from orange spotted grouper (Epinephelus coioides) (EcIFITM1) in this study. EcIFITM1 encodes a 131-amino-acid polypeptide, which shares 64 and 43% identity with Seriola dumerili and Homo sapiens, respectively. The multiple sequence alignment showed that EcIFITM1 contained five domains, including NTD (aa 1–45), IMD (aa 46–67), CIL (aa 68–93), TMD (aa 94–119), and CTD (aa 120–131). In vitro, the level of EcIFITM1 mRNA expression was significantly up-regulated in response to Singapore grouper iridovirus (SGIV), or red-spotted grouper nervous necrosis virus (RGNNV) infection. EcIFITM1 encoded a cytoplasmic protein, which was partly colocalized with early endosomes, late endosomes, and lysosomes. The ectopic expression of EcIFITM1 significantly inhibited the replication of SGIV or RGNNV, which was demonstrated by the reduced virus production, as well as the levels of viral gene transcription and protein expression. In contrast, knockdown of EcIFITM1 using small interfering RNAs (siRNAs) promoted the replication of both viruses. Notably, EcIFITM1 exerted its antiviral activity in the step of viral entry into the host cells. Furthermore, the results of non-targeted lipometabolomics showed that EcIFITM1 overexpression induced lipid metabolism remodeling in vitro. All of the detected ceramides were significantly increased following EcIFITM1 overexpression, suggesting that EcIFITM1 may suppress SGIV entry by regulating the level of ceramide in the lysosomal system. In addition, EcIFITM1 overexpression positively regulated both interferon-related molecules and ceramide synthesis-related genes. Taken together, our results demonstrated that EcIFITM1 exerted a bi-functional role, including immune regulation and lipid metabolism in response to fish virus infections.

Identification of candidate SNPs and genes associated with anti-RGNNV using GWAS in the red-spotted grouper, Epinephelus akaara

The red-spotted grouper, Epinephelus akaara, has been cultured widely in China, and in several countries of Southeast Asia, due to its important economic value. However, in recent years the outbreak of disease caused by red-spotted grouper nervous necrosis virus (RGNNV) has caused mass mortality in the stage of the grouper lifecycle from fry to juvenile, resulting in considerable economic loss in commercial aquaculture. However, the molecular mechanism underlying anti-RGNNV infection in red-spotted grouper has never been fully understood. To identify the anti-RGNNV related markers and candidate genes, we performed a genome-wide association study (GWAS) on a natural population of 100 individuals for a full-genome screen of the red-spotted grouper. In this research, 36,311 single, high quality nucleotide polymorphisms (SNPs) were developed. Two significantly associated SNPs and three suggestively associated SNPs were identified at the genome level. From these identified SNPs, five candidate genes were annotated: EPHA7, Osbpl2, GPC5, CDH4 and Pou3f1. These genes are involved in nervous system development, retinal formation, and lipid metabolism regulation. In combination with studies on the characteristics of NNV infection, it was speculated that in the fry stage of the grouper lifecycle, the immune system is not fully developed. Therefore, improved resistance to RGNNV may come through regulating nervous system development or lipid metabolism related pathways. In addition, the genotypes of SNPs associated with disease-resistant traits were analyzed. The markers and genes obtained in this study may facilitate a marker-assisted selection for red-spotted grouper aiming at disease resistance to RGNNV.

Different survival of three populations of European sea bass (Dicentrarchus labrax) following challenge with two variants of nervous necrosis virus (NNV)

Viral Nervous Necrosis (VNN, also called viral encephalo- and retinopathy (VER)), is a widespread disease of marine aquaculture caused by betanodavirus (or nervous necrosis virus - NNV), a segmented positive sense RNA virus, member of the nodaviridae family. VNN affects predominantly marine fish and cause significant losses to the Mediterranean fish farming industry, including the production of European sea bass (Dicentrarchus labrax). Of the four circulating genotypes of betanodavirus, red-spotted grouper NNV (RGNNV) and the reassortant genotype red-spotted grouper/striped jack NNV (RG/SJNNV) are most prevalent in the Mediterranean. Inheritable resistance against VNN has been detected in sea bass, and selective breeding could be a mean to limit this untreatable disease. In the current study, we compare resistance to disease among three populations from the Atlantic Ocean (AT), Eastern Mediterranean (EM) and Western Mediterranean (WM), by challenge trials using both a highly pathogenic isolate of RGNNV and a lower pathogenic reassortant isolate of RG/SJNNV. The survival of the three populations were modelled with a logistic regression, and the odds ratio (OR) of surviving was calculated. The challenge with RG/SJNNV reduced the odds of surviving three-fold (OR = 0.29 [0.07-0.87]), whereas the challenge with RGNNV reduced the odds of surviving 100-fold (OR = 0.01 [0.00-0.03]). Overall, the EM population had 3.32 (1.92–5.86) times higher odds of surviving the challenge than the AT and WM stocks. All survivors were harboring viral RNA in the brain, as demonstrated by RT-qPCR. However, viral RNA levels were in average lower in survivors from the EM population in both challenges, though only significantly lower in the challenge with RG/SJNNV (p < 0.01). The survival results combined with the RT-qPCR results indicate that the EM sea bass population has a natural resistance to disease caused by RGNNV, possibly associated with limited viral entry into and/or replication in the brain.

E3 Ubiquitin Ligase RNF114 Inhibits Innate Immune Response to Red-Spotted Grouper Nervous Necrosis Virus Infection in Sea Perch by Targeting MAVS and TRAF3 to Mediate Their Degradation.

RIG-I-like receptor (RLR)-mediated antiviral signaling is critical to trigger the immune response to virus infection; however, the antiviral responses are also tightly regulated to avoid uncontrolled production of type I IFN by various mechanisms, including ubiquitination. In this study, an E3 ubiquitin ligase ring finger protein 114 (RNF114) from sea perch (Lateolabrax japonicus) (LjRNF114) was identified as a suppressor of RLR signaling pathways during red-spotted grouper nervous necrosis virus (RGNNV) infection. RGNNV infection promoted the expression of LjRNF114. Overexpression of LjRNF114 enhanced RGNNV replication, whereas knockdown of LjRNF114 led to opposite effects. Type I IFN production induced by RGNNV was suppressed by LjRNF114, which is dependent on its ubiquitin ligase activity. Moreover, LjRNF114 inhibited IFN promoter activation induced by key signaling molecules in RLR signaling pathways. We observed the interactions between LjRNF114 and both sea perch mitochondrial antiviral signaling protein (MAVS) and TNFR-associated factor 3 (TRAF3). Domain mapping experiments indicated that the RING and ubiquitin interacting motif domains of LjRNF114 were required for its interaction with TRAF3 and MAVS. We found that LjRNF114 targeted MAVS and TRAF3 for K27- and K48-linked ubiquitination and degradation, resulting in the inhibition of IFN production. Taken together, our study reveals, to our knowledge, a novel mechanism that LjRNF114 targets and promotes K27- and K48-linked ubiquitination of MAVS and TRAF3 to negatively regulate the RLR signaling pathways, promoting viral infection.

Antiviral activities of sea perch type I and type II IFNs against RGNNV and their different roles in antigen presentation

Viral nervous necrosis disease, caused by nervous necrosis virus (NNV), is one of the most serious diseases in many marine and freshwater fish species worldwide. To better understand the antiviral ability and immunoregulatory function of sea perch (Lateolabrax japonicus) interferon (IFN) to NNV infection, three type I IFN genes (IFNc, IFNd and IFNh) and two type II IFN genes (IFNγ and IFNγ rel) were cloned and characterized in this study. Tissue distribution analysis showed that all five IFNs were high expressed in immune organs of sea perch. The transcription levels of IFNc, IFNd and IFNh were significantly up-regulated in both LJB cells in response to Poly I:C treatment or red-spotted grouper nervous necrosis virus (RGNNV) infection and head kidney leukocytes (HKLs) treated with Poly I:C, while the expression of IFNγ and IFNγ rel were only detected in HKLs. Furthermore, promoter activity analysis showed that overexpression of IRF7 significantly enhanced the promoter activities of IFNc, IFNd and IFNh, whereas IRF1 and IRF3 overexpression only induced IFNh promoter activity. In vitro study, IFNc, IFNd, IFNh and IFNγ possessed significant antiviral activities against RGNNV. In addition, overexpressed IFNc and IFNγ up-regulated the expression of antigen presentation cell markers MHCII-α and MHCII-β in LJF cells, indicating IFNc and IFNγ might be involved in antigen presentation. Taken together, our study may provide the basis for exploring the interaction between viruses and the IFN system in sea perch.

Betanodavirus infection in Kuhlia rupestris and Ambassis marianus and isolation of betanodavirus from infected pond water.

This is the first report of betanodavirus infection in 2 species of finfish, Kuhlia rupestris (jungle perch) and Ambassis marianus (estuary perchlet). This report also describes isolation of betanodavirus from infected pond water using the SSN-1 cell line. Histopathology of K. rupestris larvae revealed vacuolation in the eye and brain, which was confirmed using betanodavirus-specific immunohistochemistry. The eye and brain from A. marianus and betanodavirus isolated from pond water were confirmed using real-time PCR and Sanger sequencing. High throughput sequencing was used to obtain betanodavirus sequences from paraffin blocks containing infected K. rupestris. The phylogenetic analysis of betanodavirus RNA1 and RNA2 sequences from all 3 sources were associated with the red-spotted grouper nervous necrosis virus (RGNNV) genotype. The RNA1 nucleotide sequence from jungle perch showed 100% identity with the betanodavirus water isolate and 99.37% identity with A. marianus. Furthermore, we have demonstrated the usefulness of combining recovery of viable virus from environmental samples through fish cell line culture with PCR testing as a means of validating the efficacy of chlorination to eradicate betanodavirus from the pond environment.

Development of a sea perch (Lateolabrax japonicus) fry cell line and its application to the study of fish virus-host interactions

We developed and characterized a new cell line from sea perch (Lateolabrax japonicus) fry (SPF). SPF cells were subcultured over 100 passages, and sequencing of the mitochondrial 18 s rRNA gene confirmed that SPF cells derived from Lateolabrax japonicus. SPF cells consisted predominantly of epithelial-like cells, and proliferated well in Leibovitz's L-15 medium supplemented with 15% fetal bovine serum at 28 °C. Karyotyping analysis indicated that the modal chromosome number was 48 at passage 55. Green fluorescence could be observed in pEGFP-N3 transfected SPF cells at passages 55. SPF cells showed susceptibility to sea perch iridovirus (SPIV), Singapore grouper iridovirus (SGIV), and red-spotted grouper nervous necrosis virus (RGNNV), which was demonstrated by the occurrence of severe cytopathic effects, increased viral protein synthesis, and abundant virus particles in the cytoplasm. Moreover, SPIV and SGIV infection in SPF cells caused profound alterations in the actin cytoskeleton, as evidenced by a significant decrease in the number of actin stress fibers. Of note, no apoptotic bodies were observed in either SGIV- or SPIV-infected cells, suggesting that these two viruses induced non-apoptotic cell death in vitro. Interferon-related genes were up-regulated in SPIV- or RGNNV-infected SPF cells, suggesting that the host interferon immune response was involved in SPIV or RGNNV infection. These results showed that our newly established SPF cell line was a useful tool for genetic manipulation, virus isolation, and functional studies of host-virus interactions.

Identification of BAG5 from orange-spotted grouper (Epinephelus coioides) involved in viral infection

Bcl-2-associated athanogene 5 (BAG5) is a kind of molecular chaperone that can bind to the Bcl-2 and modulate cell survival. However, little is known about the functions of fish BAG5. In this study, we characterized a BAG5 homolog from orange-spotted grouper (Epinephelus coioides) gene (Ec-BAG5) and investigated its roles during viral infection. The Ec-BAG5 protein encoded 468 amino acids with four BAG domains, which shared high identities with reported BAG5. The highest transcriptional level of Ec-BAG5 was found in the peripheral blood lymphocyte (PBL). And the Ec-BAG5 expression were significantly up-regulated after red-spotted grouper nervous necrosis virus (RGNNV) or Lipopolysaccharide (LPS) stimulation in vitro. Furthermore, Ec-BAG5 overexpression could inhibited viral replication and the expression of viral genes (coat protein (CP) and RNA-dependent RNA polymerase (RdRp)). Also, overexpression of Ec-BAG5 significantly increased the expression of interferon pathway-related factors including interferon regulatory factor 3 (IRF3), interferon-stimulated gene 15 (ISG15), interferon-induced protein 35 (IFP35), myxovirus resistance gene 1 (Mx1) and inflammatory-related factors including tumor necrosis factor receptor-associated factor 6 (TRAF6), tumor necrosis factor-α (TNF-α), interleukin-1 beta (IL-1β), as well as the activities of NF-κB, ISRE and IFN-1. These data indicate that Ec-BAG5 can affect viral infection through regulating the expression of IFN- and inflammation-related factors, which provide useful information to better understand the immune response against viral infection.

Transcriptomic analysis of red-spotted grouper nervous necrosis virus infected Asian seabass Lates calcarifer (Bloch, 1790)

Red-spotted grouper nervous necrosis virus (RGNNV) has threatened aquaculture of the Asian seabass (Lates calcarifer) in many countries of the Asia-Pacific for decades. To understand the pathogenesis of RGNNV, a transcriptome analysis of the head kidney, a major fish lymphoid tissue which performs important immune functions and is involved in the hematopoiesis was undertaken. RNA from the head kidney of RGNNV infected fish was sequenced and compared RNA from uninfected control fish at two and 15-days post infection. Functional analysis revealed a number of differentially expressed genes implicated in autophagy (beclin-1 and inositol 1,4,5-trisphosphate receptor), superoxide production (cytochrome b-245 light chain, C3a anaphylatoxin chemotactic receptor and neutrophil cytosol factor 2-like), inflammation (leukocyte surface antigen CD53), antigen presentation (MHC class I alpha chain), antiviral activity (cholesterol 25-hydroxylase) and other immune functions (oxysterol-binding protein 1, dipeptidyl peptidase I, interleukin-5 receptor subunit alpha-like and tyrosine-protein kinase BTK). This is the first description of the change in L. calcarifer head kidney mRNA expression as a consequence of RGNNV infection and the information will serve as the rational basis for future investigations into the pathophysiology of NNV infection of fish.

Expression profile of selected genes of the E-11 cell line in response to red-spotted grouper nervous necrosis virus infection

Most fish species are susceptible to red-spotted grouper nervous necrosis virus (RGNNV) which causes viral encephalopathy and retinopathy leading to huge financial losses in the aquaculture farming industry for decades. A knowledge of RGNNV replication and the host response in depth is warranted to understand the host-virus interaction and to develop an effective approach to inhibit this virus. This study therefore aimed to investigate the expression of selected immune and stress-related genes of the E-11 clonal cell line upon RGNNV infection. The results showed significant upregulation of viperin, hsp70 and tnfaip3 transcripts at 48 h post infection, while the transcript abundance of irf3, cc chemokine, hsp30 showed increases which did not reach significance and tnf-α showed no change in expression in response to infection. This study highlights the involvement of viperin, hsp70 and tnfaip3 during RGNNV infection of E-11 cells which provides a clearer understanding of the pathogenic mechanism of the disease.

Genome-wide association studies for resistance to viral nervous necrosis in three populations of European sea bass (Dicentrarchus labrax) using a novel 57k SNP array DlabChip

Viral Nervous Necrosis (VNN) is a major threat for the European sea bass (Dicentrarchus labrax) aquaculture industry. The improvement of disease resistance through selective breeding is a promising option to reduce outbreaks. With the development of high-throughput genotyping technologies, identification of genomic regions involved in the resistance could improve the efficiency of selective breeding. The aim of this study was to identify quantitative trait loci (QTL) involved in VNN resistance and to quantify their effect.Four experimental backcross families comprising 378, 454, 291 and 211 individuals and two commercial populations A and B comprising 1027 and 1042 individuals obtained from partial factorial crosses (59♂ x 20♀ for pop A; 39♂ x 14♀ for pop B) were submitted to a redspotted grouper nervous necrosis virus (RGNNV) challenge by bath. A high-density single nucleotide polymorphism (SNP) chip panel was designed to develop the ThermoFisher Axiom™ 57k SNP DlabChip, which was used for genotyping all individuals and building a high quality linkage map. In the backcross families, composite interval mapping was performed on 30,917, 23,592, 30,656 and 31,490 markers, respectively. In the commercial populations, 40,263 markers in pop A and 41,166 markers in pop B were used to perform genome-wide association studies (GWAS) using a GBLUP and a BayesCπ approach.One QTL was identified on chromosome LG12 in three of the four experimental backcross families, and one additional QTL on LG8 was detected in only one family. In commercial populations, QTL mapping revealed a total of seven QTLs, among which the previously mentioned QTL on LG12 was detected in both. This QTL, which was mapped to an interval of 3.45 cM, explained 9.21% of the total genetic variance in pop A, while other identified QTLs individually explained less than 1% of the total genetic variance.The identification of QTL regions involved in VNN resistance in European sea bass, with one having a strong effect, should have a great impact on the aquaculture industry. Future work could focus on the fine mapping of the causal mutation present on LG12 using whole genome sequencing.

Investigation of routes of entry and dispersal pattern of RGNNV in tissues of European sea bass, Dicentrarchus labrax.

Viral encephalopathy and retinopathy (VER) is a serious neuropathological fish disease affecting in the Mediterranean aquaculture mainly European sea bass, Dicentrarchus labrax. It is well known that betanodaviruses are neurotropic viruses that replicate in nerve tissues, preferentially brain and retina. However, routes of entry and progression of the virus in the central nervous system (CNS) remain unclear. The role of four tissues-eye, oesophagus, gills and skin-as possible gateways of a betanodavirus, the redspotted grouper nervous necrosis virus (RGNNV), was investigated after experimental challenges performed on European seabass juveniles. The dispersal pattern of Betanodavirus at primarily stages of the disease was also assessed, using a real-time qPCR assay. The development of typical clinical signs of VER, the presence of characteristic histopathological lesions in the brain and retina and the detection of viral RNA in the tissues of all experimental groups ascertained that successful invasion of RGNNV under all experimental routes was achieved. Transneuronal spread along pathways known to be connected to the initial site of entry seems to be the predominant scenario of viral progression in the CNS. Furthermore, viraemia appeared only after the installation of the infection in the brain.

Fish RIP1 Mediates Innate Antiviral Immune Responses Induced by SGIV and RGNNV Infection.

Receptor interacting protein 1 (RIP1) is an essential sensor of cellular stress, which may respond to apoptosis or cell survival and participate in antiviral pathways. To investigate the roles of fish RIP1 in Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) infection, a RIP1 homolog from orange-spotted grouper (Epinephelus coioides) (EcRIP1) was cloned and characterized. EcRIP1 encoded a 679 amino acid protein that shares 83.28% identity with that of Perca flavescens and contained a homologous N-terminal kinase (S-TKc) domain, a RIP isotype interaction motif (RHIM), and a C-terminal domain (DD). EcRIP1 was predominantly detected in immune tissues, and its expression was induced by RGNNV or SGIV infection in vitro. Subcellular localization showed that EcRIP1 was distributed in the cytoplasm with point-like uniform and dot-like aggregation forms. Overexpression of EcRIP1 inhibited SGIV and RGNNV replication and positively regulated the expression levels of interferon (IFN) and IFN-stimulated genes and pro-inflammatory factors. EcRIP1 may interact with grouper tumor necrosis factor receptor type 1-associated DEATH domain protein (EcTRADD) to promote SGIV-induced apoptosis, and interact with grouper Toll/interleukin-1 receptor (TIR) domain containing adapter inducing interferon-β (EcTRIF) and participate in Myeloid Differentiation Factor 88 (MyD88)-independent toll-like receptor (TLR) signaling. EcRIP1 may also interact with grouper tumor necrosis factor receptor-associated factors (TRAFs) as intracellular linker proteins and mediate the signaling of various downstream signaling pathways, including NF-κB and IFN. These results suggest that EcRIP1 may inhibit SGIV and RGNNV infection by regulating apoptosis and various signaling molecules. Our study offers new insights into the regulatory mechanism of RIP1-related signaling, and provides a novel perspective on fish diseases mediated by RIP1.

Marine medaka heat shock protein 90ab1 is a receptor for red-spotted grouper nervous necrosis virus and promotes virus internalization through clathrin-mediated endocytosis.

Nervous necrosis virus (NNV) can infect many species of fish and causes serious acute or persistent infection. However, its pathogenic mechanism is still far from clear. Specific cellular surface receptors are crucial determinants of the species tropism of a virus and its pathogenesis. Here, the heat shock protein 90ab1 of marine model fish species marine medaka (MmHSP90ab1) was identified as a novel receptor of red-spotted grouper NNV (RGNNV). MmHSP90ab1 interacted directly with RGNNV capsid protein (CP). Specifically, MmHSP90ab1 bound to the linker region (LR) of CP through its NM domain. Inhibition of MmHSP90ab1 by HSP90-specific inhibitors or MmHSP90ab1 siRNA caused significant inhibition of viral binding and entry, whereas its overexpression led to the opposite effect. The binding of RGNNV to cultured marine medaka hMMES1 cells was inhibited by blocking cell surface-localized MmHSP90ab1 with anti-HSP90β antibodies or pretreating virus with recombinant MmHSP90ab1 or MmHSP90ab1-NM protein, indicating MmHSP90ab1 was an attachment receptor for RGNNV. Furthermore, we found that MmHSP90ab1 formed a complex with CP and marine medaka heat shock cognate 70, a known NNV receptor. Exogenous expression of MmHSP90ab1 independently facilitated the internalization of RGNNV into RGNNV impenetrable cells (HEK293T), which was blocked by chlorpromazine, an inhibitor of clathrin-dependent endocytosis. Further study revealed that MmHSP90ab1 interacted with the marine medaka clathrin heavy chain. Collectively, these data suggest that MmHSP90ab1 is a functional part of the RGNNV receptor complex and involved in the internalization of RGNNV via the clathrin endocytosis pathway.

First isolation and identification of red-grouper nervous necrosis virus (RGNNV) from adult hybrid Hulong grouper (Epinephelus fuscoguttatus × Epinephelus lanceolatus) in China

With the sharp development of present aquaculture, outbreaks of viral diseases have become a major obstacle for the sustainable development of the aquaculture industry with massive economic losses. Viral encephalopathy and retinopathy (VER) represents one of the main viral threats to world aquaculture, which is caused by nervous necrosis virus (NNV). In the present study, a new RGNNV strain named RGNNV-HL was isolated from cultured adult hybrid Hulong grouper (Epinephelus fuscoguttatus(♀) × Epinephelus lanceolatus(♂)) in Guangdong province, China. Cell culture-based virus isolation, RT-PCR detection, tissue section, electron microscopy, and complete genome sequence were used for virus identification. Furthermore, an artificial infection experiment was conducted for virulence testing. Gross examination of affected fish showed no noticeable lesions, no parasites in the gills or on the body surfaces, and no pathogenic bacteria. Histopathological examination revealed severe vacuolations in the brain of diseased groupers. Inoculation of the tissue homogenates of diseased fish induced obvious cytopathic effects in SSN-1, GF-1, and GS cell cultures. Electron microscopic observation revealed the presence of small-sized icosahedral-shaped viral particles (~30 nm) with no viral envelope, similar to betanodaviruses. RT-PCR and sequencing analysis confirmed the presence of red-spotted grouper nervous necrosis virus (RGNNV) capsid protein sequences. The virus clustered with RGNNV genotype, genus Betanodavirus within the Nodaviridae family on an evolutionary tree constructed based on the complete genome sequence. Thus, this newly isolated virus from adult Hulong grouper is tentatively designated RGNNV-HL. The artificial infection experiment showed that groupers began to die at 4 and 5 days after intraperitoneal injection and bath immersion, respectively. The cumulative mortality rates by injection and bath were 90% and 83.33%, respectively. To the best of our acknowledge, this is the first report of the isolation and characterization of RGNNV from Hulong grouper. Findings from this study will provide a candidate material and useful epidemiology information for further study on the infection mechanism and prevention and control strategies for RGNNV.

The roles of grouper TANK in innate immune defense against iridovirus and nodavirus infections

The TRAF family member-associated nuclear factor (NF)-κB activator (TANK) was first identified as a TRAF-binding protein with both stimulatory and inhibitory properties in host innate immune activation. To elucidate the roles of TANK in teleosts, we cloned and characterized the TANK homologue of orange-spotted grouper (Epinephelus coioides). The open reading frame (ORF) of EcTANK consists of 1026 nucleotides encoding a 342 amino acid protein with a predicted molecular mass of 38.24 kDa. EcTANK shares 89.47% and 88.89% identity with Larimichthys crocea TANK and Lates calcarifer TANK, respectively. EcTANK was distributed in all 11 examined tissues. The expression of EcTANK in the spleen increased after infection with Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV). EcTANK was mainly located in the cytoplasm of grouper spleen cells. EcTANK enhanced SGIV and RGNNV replication during viral infection in vitro. Overexpression EcTANK decreased the expression levels of interferon-associated cytokines and pro-inflammatory factors, and enhanced activation of NF-κB. Taken together, these results suggest that EcTANK may play an important role in antiviral innate immune activation in grouper.