Red-spotted Grouper Nervous Necrosis Research Articles

Fish Snx27 promotes viral products by modulating the innate immune response and exosomal machinery.

Viral nervous necrosis caused by the nervous necrosis virus (NNV) poses a significant threat to the global aquaculture industry. Developing preventive methods to minimize economic losses due to NNV infections is crucial. This study explored the role of the sorting nexin 27 (Snx27) gene, encoded by the orange-spotted grouper (Epinephelus coioides) and referred to as EcSnx27, as an immune regulator affecting red-spotted grouper nervous necrosis virus (RGNNV) infection in vitro. Our findings revealed that EcSnx27 negatively regulates interferon (IFN)-related cytokines and the promoter activities of fish ISRE and NF-κB. Furthermore, we identified the SNX-FERM and SNX-FERM-like domains as responsible for the interaction between EcSnx27 and RGNNV coat protein. Through the detection of viable virions associated with EcSnx27-containing exosomes, we propose that EcSnx27 may contribute to the release process of RGNNV by influencing the apoptosis-linked gene 2-interacting protein X (ALIX)-associated exosomal pathway. Consequently, our study suggests that EcSnx27 promotes RGNNV replication by inhibiting the IFN immune response and facilitating virus production and release through ALIX-mediated exosomal machinery.IMPORTANCERed grouper nervous necrosis virus (RGNNV), a member of the Nodaviridae family, has emerged as a significant cause of fish diseases worldwide, leading to high morbidity and mortality rates. This study investigated the sorting nexin 27 (Snx27) gene encoded by the orange-spotted grouper (Epinephelus coioides) on RGNNV infection in grouper kidney cells. Our findings revealed that EcSnx27 negatively regulated the interferon pathway, resulting in the promotion of RGNNV replication. Additionally, we observed that EcSnx27 could interact with apoptosis-linked gene 2-interacting protein X (ALIX) and the RGNNV coat protein, suggesting its potential involvement in viral release processes through modulation of the exosomal pathway. Our study identified EcSnx27 as a key target that RGNNV exploits to enhance viral production. This finding offers valuable insights into the immune evasion and viral release mechanisms of non-enveloped RNA viruses.

Homemade isothermal amplification-initiated Cas14a assay for rapid quantitative detection of aquatic RNA virus gene with no PAM

The red-spotted grouper nervous necrosis virus (RGNNV) is the most widely distributed nervous necrosis virus (NNV) and causes a significant risk to aquatic food safety. Though a few CRISPR-based aquatic pathogen detection methods have been reported recently, the absence of the PAM site of RGNNV specific gene RNA2 impedes its CRISPR-based diagnostic. Herein, we developed a rapid and accurate homemade isothermal amplification-initiated Cas14a assay (HiaCas14a) for RGNNV detection. The introduction of the PAM site into RT-LAMP specific amplicons of RGNNV RNA2 unlocked its PAM restriction. Furthermore, fish genomic RNA purified by magnetic beads and polymerases used in RT-LAMP prepared by ourselves obviously gave the system an enhanced convenience and a reduced cost. HiaCas14a detected 63.4 aM RNA with excellent specificity and sensitivity quantitatively, and it demonstrated a high accuracy of field sample validation results compared with RT-qPCR. The method provided an accurate, low-cost, and sensitive tool for aquatic pathogen detection in resource-poor areas.

Nectin1 is a pivotal host factor involved in attachment and entry of red-spotted grouper nervous necrosis virus in the early stages of the viral life cycle.

Nervous necrosis virus (NNV) is a highly neurotropic virus that poses a persistent threat to the survival of multiple fish species. However, its inimitable neuropathogenesis remains largely elusive. To rummage potential partners germane to the nervous system, we investigated the interaction between red-spotted grouper NNV (RGNNV) and grouper brain by immunoprecipitation coupled with mass spectrometry and discerned Nectin1 as a novel host factor subtly involved in viral early invasion events. Nectin1 was abundant in neural tissues and implicated in the inception of tunnel nanotubes triggered by RGNNV. Its overexpression not only dramatically potentiated the replication dynamics of RGNNV in susceptible cells, but also empowered non-sensitive cells to expeditiously capture free virions within 2 min. This potency was impervious to low temperatures but was dose-dependently suppressed by soluble protein or specific antibody of Nectin1 ectodomain, indicating Nectin1 as an attachment receptor for RGNNV. Mechanistically, efficient hijacking of virions by Nectin1 strictly depended on intricate linkages to different modules of viral capsid protein, especially the direct binding between the IgC1 loop and P-domain. More strikingly, despite abortive proliferation in Nectin1-reconstructed CHSE-214 cells, a non-sensitive cell, RGNNV could gain access to the intracellular compartment by capitalizing on Nectin1, thereby inducing canonical cytoplasmic vacuolation. Altogether, our findings delineate a candidate entrance for RGNNV infiltration into the nervous system, which may shed unprecedented insights into the exploration and elucidation of RGNNV pathogenesis.IMPORTANCENervous necrosis virus (NNV) is one of the most virulent pathogens in the aquaculture industry, which inflicts catastrophic damage to ecology, environment, and economy annually around the world. Nevertheless, its idiosyncratic invasion and latency mechanisms pose enormous hardships to epidemic prevention and control. In this study, deploying grouper brain as a natural screening library, a single-transmembrane glycoprotein, Nectin1, was first identified as an emergent functional receptor for red-spotted grouper NNV (RGNNV) that widely allocated in nervous tissues and directly interacted with viral capsid protein through distinct Ig-like loops to bridge virus-host crosstalk, apprehend free virions, and concomitantly propel viral entry. Our findings illuminate the critical role of Nectin1 in RGNNV attachment and entry and provide a potential target for future clinical intervention strategies in the therapeutic race against RGNNV.

Molecular characterisation and expression analysis of an interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) homologue from Asian seabass (Lates calcarifer)

The interferon-induced protein with tetratricopeptide repeats 1(IFIT1) is an interferon-inducible protein that acts downstream of the interferon pathway in response to viral infection or an interferon-stimulation. Our study describes the isolation and characterization of an IFIT1 homologue from Lates calcarifer (named LcIFIT1), as well as its expression profile in response to viral mimetic poly(I:C) and Red-spotted grouper nervous necrosis virus (RGNNV). The complete ORF of LcIFIT1 encodes a 441 amino acid protein with nine tetratricopeptide repeat (TPR) domains. The protein-coding region of the LcIFIT1 gene was encoded by two exons separated by a small 157 bp intron. The phylogenetic analysis revealed that LcIFIT1 is evolutionarily conserved and forms a distinct clade with homologues from other teleosts. LcIFIT1 mRNA was constitutively expressed in all tissues examined, with predominant expression in blood. LcIFIT1 expression was upregulated following poly(I:C) and RGNNV challenges in different tissues examined. These results shed light on the antiviral role of LcIFIT1 in the host immune response against nodaviral infection.

Grouper OTUB1 and OTUB2 promote red-spotted grouper nervous necrosis virus (RGNNV) replication by inhibiting the host innate immune response

Red-spotted grouper nervous necrosis virus (RGNNV) is a major viral pathogen of grouper and is able to antagonize interferon responses through multiple strategies, particularly evading host immune responses by inhibiting interferon responses. Ovarian tumor (OTU) family proteins are an important class of DUBs and the underlying mechanisms used to inhibit interferon pathway activation are unknown. In the present study, primers were designed based on the transcriptome data, and the ovarian tumor (OTU) domain-containing ubiquitin aldehyde-binding protein 1 (OTUB1) and OTUB2 genes of Epinephelus coioides (EcOTUB1 and EcOTUB2) were cloned and characterized. The homology alignment showed that both EcOTUB1 and EcOTUB2 were most closely related to E. lanceolatus with 98 % identity. Both EcOTUB1 and EcOTUB2 were distributed to varying degrees in grouper tissues, and the transcript levels were significantly up-regulated following RGNNV stimulation. Both EcOTUB1 and EcOTUB2 promoted replication of RGNNV in vitro, and inhibited the promoter activities of interferon stimulated response element (ISRE), nuclear transcription factors kappaB (NF-κB) and IFN3, and the expression levels of interferon related genes and proinflammatory factors. Co-immunoprecipitation experiments showed that both EcOTUB1 and EcOTUB2 could interact with TRAF3 and TRAF6, indicating that EcOTUB1 and EcOTUB2 may play important roles in interferon signaling pathway. The results will provide a theoretical reference for the development of novel disease prevention and control techniques.

M1-type polarized macrophage contributes to brain damage through CXCR3.2/CXCL11 pathways after RGNNV infection in grouper

ABSTRACT The vertebrate central nervous system (CNS) is the most complex system of the body. The CNS, especially the brain, is generally regarded as immune-privileged. However, the specialized immune strategies in the brain and how immune cells, specifically macrophages in the brain, respond to virus invasion remain poorly understood. Therefore, this study aimed to examine the potential immune response of macrophages in the brain of orange-spotted groupers (Epinephelus coioides) following red-spotted grouper nervous necrosis virus (RGNNV) infection. We observed that RGNNV induced macrophages to produce an inflammatory response in the brain of orange-spotted grouper, and the macrophages exhibited M1-type polarization after RGNNV infection. In addition, we found RGNNV-induced macrophage M1 polarization via the CXCR3.2- CXCL11 pathway. Furthermore, we observed that RGNNV triggered M1 polarization in macrophages, resulting in substantial proinflammatory cytokine production and subsequent damage to brain tissue. These findings reveal a unique mechanism for brain macrophage polarization, emphasizing their role in contributing to nervous tissue damage following viral infection in the CNS.

Fish ELOVL7a is involved in virus replication via lipid metabolic reprogramming

The elongation of very long chain fatty acids (ELOVL) proteins are key rate-limiting enzymes that catalyze fatty acid synthesis to form long chain fatty acids. ELOVLs also play regulatory roles in the lipid metabolic reprogramming induced by mammalian viruses. However, little is known about the roles of fish ELOVLs during virus infection. Here, a homolog of ELOVL7 was cloned from Epinephelus coioides (EcELOVL7a), and its roles in red-spotted grouper nervous necrosis virus (RGNNV) and Singapore grouper iridovirus (SGIV) infection were investigated. The transcription level of EcELOVL7a was significantly increased upon RGNNV and SGIV infection or other pathogen-associated molecular patterns stimulation in grouper spleen (GS) cells. Subcellular localization analysis showed that EcELOVL7a encoded an endoplasmic reticulum (ER) related protein. Overexpression of EcELOVL7a promoted the viral production and virus release during SGIV and RGNNV infection. Furthermore, the lipidome profiling showed that EcELOVL7a overexpression reprogrammed cellular lipid components in vitro, evidenced by the increase of glycerophospholipids, sphingolipids and glycerides components. In addition, VLCFAs including FFA (20:2), FFA (20:4), FFA (22:4), FFA (22:5) and FFA (24:0), were enriched in EcELOVL7a overexpressed cells. Consistently, EcELOVL7a overexpression upregulated the transcription level of the key lipid metabolic enzymes, including fatty acid synthase (FASN), phospholipase A 2α (PLA 2α), and cyclooxygenases −2 (COX-2), LPIN1, and diacylglycerol acyltransferase 1α (DGAT1α). Together, our results firstly provided the evidence that fish ELOVL7a played an essential role in SGIV and RGNNV replication by reprogramming lipid metabolism.

Slc43a2+ T cell metastasis from spleen to brain in RGNNV infected teleost.

The origin of T cells in the teleost's brain is unclear. While viewing the central nervous system (CNS) as immune privileged has been widely accepted, previous studies suggest that T cells residing in the thymus but not in the spleen of the teleost play an essential role in communicating with the peripheral organs. Here, we identified nine T cell subpopulations in the thymus and spleen of orange-spotted grouper (Epinephelus coioices) through single-cell RNA-sequencing analysis. After viral CNS infection with red-spotted grouper nervous necrosis virus (RGNNV), the number of slc43a2+ T cells synchronously increased in the spleen and brain. During the infection tests in asplenic zebrafish (tlx1▲ zebrafish model), no increase in the number of slc43a2+ T cells was observed in the brain. Single-cell transcriptomic analysis indicated that slc43a2+ T cells mature and functionally differentiate within the spleen and then migrate into the brain to trigger an immune response. This study suggests a novel route for T cell migration from the spleen to the brain during viral infection in fish.

Characterization and functional analysis of SOCS9 from orange-spotted grouper (Epinephelus coioides) during virus infection

The suppressor of cytokine signaling (SOCS) proteins family have twelve members including eight known mammalian SOCS members (CISH, SOCS1-7) and four new discovery members (SOCS3b, SOCS5b, SOCS8 and SOCS9) that is regarded as a classic feedback inhibitor of cytokine signaling. Although the function of the mammalian SOCS proteins have been well studied, little is known about the roles of SOCS in fish during viral infection. In this study, the molecular characteristics of SOCS9 from orange-spotted grouper (Epinephelus coioides, EcSOCS9) is investigated. The EcSOCS9 protein encoded 543 amino acids with typical SH2 (389-475aa) and SOCS_box (491-527aa), sharing high identities with reported fish SOCS9. EcSOCS9 was expressed in all detected tissues and highly expressed in kidney. After red-spotted grouper nervous necrosis virus (RGNNV) infection, the expression of EcSOCS9 was significantly induced in vitro. Furthermore, EcSOCS9 overexpression enhanced RGNNV replication, promoted virus-induced mitophagy that evidenced by the increased level of LC3-Ⅱ, BCL2, PGAM5 and decreased level of BNIP3 and FUNDC1. Besides, EcSOCS9 overexpression suppressed the expression levels of ATP6, CYB, ND4, ATP level and induced ROS level. The expression levels of interferon (IFN) related factors (IRF1, IRF3, IRF7, P53), inflammatory factors (IL1-β, IL8, TLR2, TNF-α) and IFN-3, ISRE, NF-κB, AP1 activities were also reduced by overexpressing EcSOCS9. These date suggests that EcSOCS9 impacts RGNNV infection through modulating mitophagy, regulating the expression levels of IFN- related and inflammatory factors, which will expand our understanding of fish immune responses during viral infection.

Transcriptomic responses to RGNNV and Vibrio alginolyticus infection in the spleen of hybrid grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀)

The hybrid grouper (Epinephelus lanceolatus♂ × E. fuscoguttatus♀) is a novel and commercially important fish for mariculture in China. Recent years, the outbreak of viral and bacterial diseases have caused huge losses of hybrid grouper culture. However, the immune response of hybrid grouper against viral and bacterial infection remains unexplored. In this study, a total of 9 splenic transcriptomic libraries of hybrid grouper including untreated control, Red-spotted grouper nervous necrosis virus (RGNNV)-challenged, Vibrio alginolyticus-challenged were constructed. A sum of 6024 differentially expressed genes (DEGs) with significant differences were identified, of which the overlapped DEGs between RGNNV and V. alginolyticus challenged were significantly enriched in multiple immune-related pathway including interleukin-17 NOD-like receptor, C-type lectin receptor, chemokine signaling pathway and natural killer cell mediated cytotoxicity, indicating their important roles in immune defense of hybrid grouper against (both) viral and bacterial infection. To the best of our knowledge, this is the first report that investigated the immune response of hybrid grouper following viral and bacterial pathogen stimuli using transcriptomic analysis. These results provide comprehensive understandings of the innate immunity of hybrid grouper and valuable references to develop disease control strategy in grouper aquaculture.

Establishment and characterization of a muscle cell line from golden pompano (Trachinotus ovatus) and its application in viral susceptibility

A new cell line, golden pompano muscle (GPM), was developed and characterized from the muscle tissue of the golden pompano in this work. GPM cells were mostly fibroblast-like and proliferated well in Leibovitz's L-15 medium supplemented with fetal bovine serum (FBS) (concentration of 10% to 20%) at 28 °C. The cell line has been stably passed over 80 generations and has been successfully cryopreserved. Molecular characterization of cytochrome oxidase subunit I (COI) confirmed that the GPM cells used in this study were from the golden pompano. Chromosome analysis showed that the diploid chromosome number of GPM cells was 2 N = 48. The enhanced green fluorescent protein (EGFP) reporter gene was successfully expressed in the GPM cell line by liposome transfection technique, and the transfection efficiency of GPM cells was about 60%, which suggests that GPM cells are available for research on gene expression in vitro. Virus susceptibility assay showed that GPM cells were susceptible to red-spotted grouper nervous necrosis virus (RGNNV), evidenced by the occurrence of cytopathic effect (CPE) and the increased viral protein synthesis capacity. Furthermore, viral replication significantly increased the expression levels of interferon (IFN) related signaling molecules and pro-inflammatory cytokines in cells. Therefore, our work developed and characterized a new cell line from golden pompano muscle, which allowed for functional studies of genetic manipulation, viral pathogenesis, and the interactions of host and virus.

Immunoinformatics-guided selection of immunogenic peptides from betanodavirus induce antigen-specific antibody production in hybrid grouper, Epinephelus fuscoguttatus x Epinephelus lanceolatus

Betanodavirus is a significant pathogen that causes viral nervous necrosis (VNN) in a wide range of fish species. It could cause up to 100% mortality in the infected farmed fish, especially at the larval stage. Red-Spotted Grouper Nervous Necrosis Virus (RGNNV), one of the betanodavirus genotypes, has been proven to have a wide range of host-compatibility across warm-water fish species. To date, there is no viable strategy to combat this virus infection, but vaccination approach is known to be effective against infectious diseases. Thus, current study aimed to identify immunogenic peptides as potential subunit vaccine candidates against grouper betanodavirus. Nucleotide sequences of the grouper nervous necrosis virus were retrieved from the National Center for Biotechnology Information database. B- and T-cell epitopes were predicted using the Immune Epitope Database analysis resource and the NetCTL 1.2 server, respectively. The predicted B- and T-cell epitopes were analysed for its respective antigenicity, immunogenicity, physico-chemical properties, and surface accessibility. Selected epitopes were then synthesized for immunization assay to examine antigen-specific antibody production in the immunized grouper. Five epitopes (one B-cell and four T-cell epitopes) were selected based on the immunogenicity score. The B-cell epitope has the highest immunogenicity score of 0.9333. Physico-chemical properties analysis of the B-cell epitope shown highest GRAVY score of 0.516 and was predicted to possess the longest estimated half-life of 30 h. Structural analysis has shown that the B-cell epitope possesses the largest accessible surface on the virus particle. Immunization of grouper with the selected B-cell epitope induced the highest antigen-specific antibody production as compared to grouper that was immunized with the selected T-cell epitope. This study demonstrated the feasibility of reverse vaccinology approach in the selection and design of immunogenic peptides against NNV in grouper to serve as the potential vaccine candidate.

Grouper Rab1 inhibits nodovirus infection by affecting virus entry and host immune response

Rab1, a GTPase, is present in all eukaryotes, and is mainly involved in vesicle trafficking between the endoplasmic reticulum and Golgi, thereby regulating many cellular activities and pathogenic infections. However, little is known of how Rab1 functions in fish during virus infection. Groupers (Epinephelus spp.) are high in economic value and widely cultivated in China and Southeast Asia, although they often suffer from diseases. Red-spotted grouper nervous necrosis virus (RGNNV), a highly pathogenic RNA virus, is a major pathogen in cultured groupers, and causes huge economic losses. A series of host cellular proteins involved in RGNNV infection was identified. However, the impact of Rab1 on RGNNV infection has not yet been reported. In this study, a novel Rab1 homolog (EcRab1) from Epinephelus coioides was cloned, and its roles during virus infection and host immune responses were investigated. EcRab1 encoded a 202 amino acid polypeptide, showing 98% and 78% identity to Epinephelus lanceolatus and Homo sapiens, respectively. After challenge with RGNNV or poly(I:C), the transcription of EcRab1 was altered both in vitro and in vivo, implying that EcRab1 was involved in virus infection. Subcellular localization showed that EcRab1 was displayed as punctate structures in the cytoplasm, which was affected by EcRab1 mutants. The dominant negative (DN) EcRab1, enabling EcRab1 to remain in the GDP-binding state, caused EcRab1 to be diffusely distributed in the cytoplasm. Constitutively active (CA) EcRab1, enabling EcRab1 to remain in the GTP-binding state, induced larger cluster structures of EcRab1. During the late stage of RGNNV infection, some EcRab1 co-localized with RGNNV, and the size of EcRab1 clusters was enlarged. Importantly, overexpression of EcRab1 significantly inhibited RGNNV infection, and knockdown of EcRab1 promoted RGNNV infection. Furthermore, EcRab1 inhibited the entry of RGNNV to host cells. Compared with EcRab1, overexpression of DN EcRab1 or CA EcRab1 also promoted RGNNV infection, suggesting that EcRab1 regulated RGNNV infection, depending on the cycles of GTP- and GDP-binding states. In addition, EcRab1 positively regulated interferon (IFN) immune and inflammatory responses. Taken together, these results suggest that EcRab1 affects RGNNV infection, possibly by regulating host immunity. Our study furthers the understanding of Rab1 function during virus infection, thus helping to design new antiviral strategies.

Genome-wide association study identifies candidate SNPs and genes associated with red-spotted grouper nervous necrosis virus infection of the giant grouper (Epinephelus lanceolatus)

The giant grouper is one of the most important farmed marine animals worldwide; however, red-spotted grouper nervous necrosis virus (RGNNV) has seriously affected its survival. Here, we performed a genome-wide association study on the same pedigree population of 202 giant grouper individuals for full-genome screening to identify markers and candidate genes for RGNNV resistance. The results were used to screen for single-nucleotide polymorphism (SNP) loci and candidate genes that significantly correlated with RGNNV traits. We detected 2,360,340 SNP loci and used a general linear model to identify 54 SNP loci with significant anti-RGNNV associations. We screened for candidate genes in a 100-kb zone (50 kb upstream and 50 kb downstream) around each SNP locus and detected 25 candidate genes. Of these, G protein-coupled receptor 143 (gpr143) was a candidate gene for disease resistance in giant groupers. RT-qPCR was used to examine the expression of gpr143 in various tissues of healthy groupers and was found to be highly expressed in the eye and brain. Following RGNNV infection, gene expression analysis revealed that gpr143 mRNA expression was significantly upregulated in the brains of susceptible fish, suggesting that GPR143 plays a role in viral invasion. GPR143 overexpression in giant grouper cells significantly promoted RGNNV replication, accelerated the cytopathic effects of RGNNV infection, and increased viral gene transcription. Our results suggest that markers linked to RGNNV disease resistance, including SNP markers and genes, could be useful for marker-assisted introgression, allowing for the development of highly RGNNV-resistant giant groupers.

Alternative splicing variants of stimulator of interferon genes (STING) from Asian seabass (Lates calcarifer) and their immune response against red spotted grouper nervous necrosis virus (RGNNV)

The Stimulator of Interferon Genes (STING, also known as MITA/ERYS/MPYS) is an adaptor molecule that plays a crucial role in the RLR pathway and responds to DNA and RNA viruses. In the present study, we have identified two novel isoforms of STING (the canonical form named as LcSTINGa and its alternative splicing isoform named as LcSTINGb) from teleost Lates calcarifer. LcSTINGa has an ORF of 1230 bp, encoding a 409 amino acid protein, while its alternative splicing variant, LcSTINGb, features an ORF of 987 bp, encoding 328 amino acids. LcSTINGa is predicted to contain four transmembrane helices, whereas LcSTINGb has only two. The Lates STING protein showed about 86.85% identity with Perca flavescens, 86.45% with Seriola and 39.51% with Homo sapiens. The tissue distribution studies revealed that the STING variants were constitutively expressed in all the tissues examined, with the highest expression in blood. In-vivo upregulation of LcSTINGa and LcSTINGb mRNA following immune challenge with poly (I:C), Red-spotted grouper nervous necrosis virus (RGNNV) and zymosan A suggests its significance in the immune response.

Correlations of betanodavirus load in the brain of experimentally infected sea bass (Dicentrarchus labrax, L.) with varying levels of resistance to viral nervous necrosis

The red-spotted grouper nervous necrosis virus (RGNNV) genotype of Betanodavirus causes significant losses to the European sea bass aquaculture industry. The lack of widely available vaccines against this genotype has led researchers and the industry to investigate other avenues for reducing mortality caused by the disease, such as the selection of resistant stocks. The objectives of this study were: a) to assess the presence of natural resistance in sea bass families propagated in the genetic improvement program of Nireus S.A., Greece and, b) to test an ELISA method able to detect viral coat antigen and measure viral load in the brain of tested fish. The second objective was to investigate virus antigen fluctuations during the course of the infection and its correlation with disease development, mortality and resistance. Further aspects of disease pathogenesis were investigated. A population of sea bass consisting of 89 families was experimentally infected with an RGNNV genotype of Betanodavirus; mortalities were recorded and brain samples from dead and survived fish were collected. Experimental infection of fish resulted in a typical pattern of mortality development that reached 56%. Different levels of natural resistance between families were found with cumulative mortality ranging from 20% to 86.2%. There was no statistical difference between the weight of dead fish and the resistant and susceptible families, except when extreme phenotypes were tested, indicating that the weight of fish that died was not a significant factor of final mortality. Brain virus load in the population as a whole increased sharply until D6 post-infection and then gradually dropped until the end of the experiment. When the viral load in the population (dead and survived fish) was tested against time of death, no correlation was found. In fish that survived the infection, the virus load remained high. The viral load per mg of brain tissue in samples taken from dead fish was not a factor that influenced family cumulative % mortality. Brain samples from survived fish reproduced the disease after infection of healthy fish. This study revealed a natural resistance of sea bass to Betanodavirus infection. This important finding can be used as an additional tool in reducing the mortality of cultured stocks. Additionally, there was clear evidence that fish surviving infection become subclinical carriers of the pathogen. Apparently, survival may not be associated with mechanisms of viral clearance but to other mechanisms that may suspend viral replication or aid infected cell endurance. These findings may assist in developing future vaccines and stock selection. A combination of effective vaccination and resistant stocks could be the way ahead.

Methyltransferase-like 3 suppresses red spotted grouper nervous necrosis virus and viral hemorrhagic septicemia virus infection by enhancing type I interferon responses in sea perch.

Methylation at the N6 position of adenosine (m6A) is the most abundant internal mRNA modification in eukaryotes, tightly associating with regulation of viral life circles and immune responses. Here, a methyltransferase-like 3 homolog gene from sea perch (Lateolabrax japonicus), designated LjMETTL3, was cloned and characterized, and its negative role in fish virus pathogenesis was uncovered. The cDNA of LjMETTL3 encoded a 601-amino acid protein with a MT-A70 domain, which shared the closest genetic relationship with Echeneis naucrates METTL3. Spatial expression analysis revealed that LjMETTL3 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. LjMETTL3 expression was significantly upregulated at 12 and 24 h post RGNNV and VHSV infection in vitro. In addition, ectopic expression of LjMETTL3 inhibited RGNNV and VHSV infection in LJB cells at 12 and 24 h post infection, whereas knockdown of LjMETTL3 led to opposite effects. Furthermore, we found that LjMETTL3 may participate in boosting the type I interferon responses by interacting with TANK-binding kinase. Taken together, these results disclosed the antiviral role of fish METTL3 against RGNNV and VHSV and provided evidence for understanding the potential mechanisms of fish METTL3 in antiviral innate immunity.

Characterization of two tripartite motif-containing genes from Asian Seabass Lates calcarifer and their expression in response to virus infection and microbial molecular motifs.

We identified two tripartite motif (TRIM) genes, LcTRIM21 and LcTRIM39, from the Asian Seabass Lates calcarifer, and examined their responses to experimental betanodavirus infection and stimulation with microbial pathogen-associated molecular patterns. Genes encoding LcTRIM21 and LcTRIM39 were identified, cloned, and sequenced from the Asian Seabass. We analyzed the sequence using a variety of bioinformatics tools to determine protein structure, localization, and establish a phylogenetic tree. By using quantitative real-time PCR, we analyzed expression profiles of the LcTRIM21 and LcTRIM39 genes in response to betanodavirus challenge as well as molecular pathogen-associated molecular patterns like poly(I:C) and Zymosan A. The tissue distribution pattern of these genes was also examined in healthy animals. Asian Seabass homologues of the TRIM gene, LcTRIM21 and LcTRIM39, were cloned, both encoding proteins with 547 amino acids. LcTRIM21 is predicted to have an isoelectric point of 6.32 and a molecular mass of 62.11 kilodaltons, while LcTRIM39 has an isoelectric point of 5.57 and a molecular mass of 62.11 kilodaltons. LcTRIM21 and LcTRIM39 homologues were predicted to be localized in cytoplasm by in silico protein localization. Structurally, both proteins contain an N-terminal really interesting new gene (RING) zinc-finger domain, B-box domain, coiled-coil domain and C-terminal PRY/SPRY domain. Most tissues and organs examined showed constitutive expression of LcTRIM21 and LcTRIM39. Upon poly(I:C) challenge or red-spotted grouper nervous necrosis virus infection, LcTRIM21 and LcTRIM39 mRNA expression was significantly upregulated, suggesting that they may play a critical antiviral role against fish viruses. LcTRIM21 and LcTRIM39 expression were also upregulated by administration of the glucan Zymosan A. The TRIM-containing gene is an E3 ubiquitin ligase that exhibits antiviral activity by targeting viral proteins via proteasome-mediated ubiquitination. TRIM proteins can be explored for the discovery of antivirals and strategies to combat diseases like viral nervous necrosis, that threaten seabass aquaculture.

The genetic variability and evolution of red-spotted grouper nervous necrosis virus quasispecies can be associated with its virulence.

Nervous necrosis virus, NNV, is a neurotropic virus that causes viral nervous necrosis disease in a wide range of fish species, including European sea bass (Dicentrarchus labrax). NNV has a bisegmented (+) ssRNA genome consisting of RNA1, which encodes the RNA polymerase, and RNA2, encoding the capsid protein. The most prevalent NNV species in sea bass is red-spotted grouper nervous necrosis virus (RGNNV), causing high mortality in larvae and juveniles. Reverse genetics studies have associated amino acid 270 of the RGNNV capsid protein with RGNNV virulence in sea bass. NNV infection generates quasispecies and reassortants able to adapt to various selective pressures, such as host immune response or switching between host species. To better understand the variability of RGNNV populations and their association with RGNNV virulence, sea bass specimens were infected with two RGNNV recombinant viruses, a wild-type, rDl956, highly virulent to sea bass, and a single-mutant virus, Mut270Dl965, less virulent to this host. Both viral genome segments were quantified in brain by RT-qPCR, and genetic variability of whole-genome quasispecies was studied by Next Generation Sequencing (NGS). Copies of RNA1 and RNA2 in brains of fish infected with the low virulent virus were 1,000-fold lower than those in brains of fish infected with the virulent virus. In addition, differences between the two experimental groups in the Ts/Tv ratio, recombination frequency and genetic heterogeneity of the mutant spectra in the RNA2 segment were found. These results show that the entire quasispecies of a bisegmented RNA virus changes as a consequence of a single point mutation in the consensus sequence of one of its segments. Sea bream (Sparus aurata) is an asymptomatic carrier for RGNNV, thus rDl965 is considered a low-virulence isolate in this species. To assess whether the quasispecies characteristics of rDl965 were conserved in another host showing different susceptibility, juvenile sea bream were infected with rDl965 and analyzed as above described. Interestingly, both viral load and genetic variability of rDl965 in seabream were similar to those of Mut270Dl965 in sea bass. This result suggests that the genetic variability and evolution of RGNNV mutant spectra may be associated with its virulence.

Grouper annexin A2 affects RGNNV by regulating the host immune response.

Annexin A2 (AnxA2) is ubiquitous in vertebrates and has been identified as a multifunctional protein participating in a series of biological processes, such as endocytosis, exocytosis, signal transduction, transcription regulation, and immune responses. However, the function of AnxA2 in fish during virus infection still remains unknown. In this study, we identified and characterized AnxA2 (EcAnxA2) in Epinephelus coioides. EcAnxA2 encoded a 338 amino acid protein with four identical annexin superfamily conserved domains, which shared high identity with other AnxA2 of different species. EcAnxA2 was widely expressed in different tissues of healthy groupers, and its expression was significantly increased in grouper spleen cells infected with red-spotted grouper nervous necrosis virus (RGNNV). Subcellular locatio n analyses showed that EcAnxA2 diffusely distributed in the cytoplasm. After RGNNV infection, the spatial distribution of EcAnxA2 was unaltered, and a few EcAnxA2 co-localized with RGNNV during the late stage of infection. Furthermore, overexpression of EcAnxA2 significantly increased RGNNV infection, and knockdown of EcAnxA2 reduced RGNNV infection. In addition, overexpressed EcAnxA2 reduced the transcription of interferon (IFN)-related and inflammatory factors, including IFN regulatory factor 7 (IRF7), IFN stimulating gene 15 (ISG15), melanoma differentiation related gene 5 (MDA5), MAX interactor 1 (Mxi1) laboratory of genetics and physiology 2 (LGP2), IFN induced 35 kDa protein (IFP35), tumor necrosis factor receptor-associated factor 6 (TRAF6) and interleukin 6 (IL-6). The transcription of these genes was up-regulated when EcAnxA2 was inhibited by siRNA. Taken together, our results showed that EcAnxA2 affected RGNNV infection by down-regulating the host immune response in groupers, which provided new insights into the roles of AnxA2 in fish during virus infection.