Red-spotted Grouper Nervous Necrosis Virus Research Articles

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.

Nervous necrosis virus induced vacuolization is a Rab5- and actin-dependent process

ABSTRACT Cytoplasmic vacuolization is commonly induced by bacteria and viruses, reflecting the complex interactions between pathogens and the host. However, their characteristics and formation remain unclear. Nervous necrosis virus (NNV) infects more than 100 global fish species, causing enormous economic losses. Vacuolization is a hallmark of NNV infection in host cells, but remains a mystery. In this study, we developed a simple aptamer labelling technique to identify red-spotted grouper NNV (RGNNV) particles in fixed and live cells to explore RGNNV-induced vacuolization. We observed that RGNNV-induced vacuolization was positively associated with the infection time and virus uptake. During infection, most RGNNV particles, as well as viral genes, colocalized with vacuoles, but not giant vacuoles > 3 μm in diameter. Although the capsid protein (CP) is the only structural protein of RGNNV, its overexpression did not induce vacuolization, suggesting that vacuole formation probably requires virus entry and replication. Given that small Rab proteins and the cytoskeleton are key factors in regulating cellular vesicles, we further investigated their roles in RGNNV-induced vacuolization. Using live cell imaging, Rab5, a marker of early endosomes, was continuously located in vacuoles bearing RGNNV during giant vacuole formation. Rab5 is required for vacuole formation and interacts with CP according to siRNA interference and Co-IP analysis. Furthermore, actin formed distinct rings around small vacuoles, while vacuoles were located near microtubules. Actin, but not microtubules, plays an important role in vacuole formation using chemical inhibitors. These results provide valuable insights into the pathogenesis and control of RGNNV infections.

Generation and Characterization of ORF55/ORF57-Deleted Recombinant Cyprinid herpesvirus 2 Mutants with Chimeric Capsid Protein Gene of Grouper Nervous Necrosis Virus.

Cyprinid herpesvirus 2 (CyHV-2) is a pathogen that causes significant losses to the global aquaculture industry due to mass mortality in crucian carp and goldfish. This study demonstrates that the ORF55/ORF57 deletion mutants CyHV-2-Δ55-CP and CyHV-2-Δ57-CP obtained through homologous recombination replicate effectively within the caudal fin of Carassius auratus gibelio (GiCF) cells and exhibit morphologies similar to the CyHV-2 wild-type strain. Both mutants demonstrated a decrease in virulence, with CyHV-2-Δ57-CP exhibiting a more significant reduction. This serves as a reference for the subsequent development of recombinant attenuated vaccines against CyHV-2. Additionally, both mutants expressed the inserted RGNNV-CP (capsid protein of Redspotted grouper nervous necrosis virus) fusion protein gene, and inoculation with CyHV-2-Δ57-CP-infected GiCF cell lysates elicited an antibody response in the grouper. These results indicate that, while ORF55 and ORF57 genes of CyHV-2 are not required for viral replication in vitro, they do play a role in virulence in vivo. Additionally, expression of foreign protein in CyHV-2 suggests that the fully attenuated mutant of CyHV-2 could potentially function as a viral vector for developing subunit vaccines or multivalent recombinant attenuated vaccines.

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.

Whole‐genome sequencing identifies interferon-induced protein IFI6/IFI27-like as a strong candidate gene for VNN resistance in European sea bass

BackgroundViral nervous necrosis (VNN) is a major disease that affects European sea bass, and understanding the biological mechanisms that underlie VNN resistance is important for the welfare of farmed fish and sustainability of production systems. The aim of this study was to identify genomic regions and genes that are associated with VNN resistance in sea bass.ResultsWe generated a dataset of 838,451 single nucleotide polymorphisms (SNPs) identified from whole-genome sequencing (WGS) in the parental generation of two commercial populations (A: 2371 individuals and B: 3428 individuals) of European sea bass with phenotypic records for binary survival in a VNN challenge. For each population, three cohorts were submitted to a red-spotted grouper nervous necrosis virus (RGNNV) challenge by immersion and genotyped on a 57K SNP chip. After imputation of WGS SNPs from their parents, quantitative trait loci (QTL) were mapped using a Bayesian sparse linear mixed model (BSLMM). We found several QTL regions that were specific to one of the populations on different linkage groups (LG), and one 127-kb QTL region on LG12 that was shared by both populations and included the genes ZDHHC14, which encodes a palmitoyltransferase, and IFI6/IFI27-like, which encodes an interferon-alpha induced protein. The most significant SNP in this QTL region was only 1.9 kb downstream of the coding sequence of the IFI6/IFI27-like gene. An unrelated population of four large families was used to validate the effect of the QTL. Survival rates of susceptible genotypes were 40.6% and 45.4% in populations A and B, respectively, while that of the resistant genotype was 66.2% in population B and 78% in population A.ConclusionsWe have identified a genomic region that carries a major QTL for resistance to VNN and includes the ZDHHC14 and IFI6/IFI27-like genes. The potential involvement of the interferon pathway, a well-known anti-viral defense mechanism in several organisms (chicken, human, or fish), in survival to VNN infection is of particular interest. Our results can lead to major improvements for sea bass breeding programs through marker-assisted genomic selection to obtain more resistant fish.

Characterization of SNX5 in Orange-Spotted Grouper (Epinephelus coioides) during In Vitro Viral Infection

SNX5 is a protein that is involved in endosomal sorting, signal transduction and endocytosis pathways. However, the roles of fish SNX5 were largely unknown. In this study, we identified an SNX5 homolog (EcSNX5) from an orange-spotted grouper (E. coioides) and investigated its role during viral infection. EcSNX5 encoded 412 amino acids with a PX domain and a BAR domain. In addition, it shared high identities with other known fish SNX5. Through quantitative real-time polymerase chain reaction (qRT-PCR), the high expression of EcSNX5 was observed in the head, kidney and heart. After stimulation with the red-spotted grouper nervous necrosis virus (RGNNV) in vitro, EcSNX5 expression was significantly induced. After RGNNV infection in vitro, EcSNX5 overexpression enhanced the expression of RGNNV genes, including coat protein (CP) and RNA-dependent RNA polymerase (RdRp). EcSNX5 knockdown downregulates expression of CP and RdRp. The TCID50 assay showed a higher viral titer when EcSNX5 is over expressed. Moreover, EcSNX5 overexpression could reduce the expression of interferon genes (IRF1, IRF3, IRF7, MX1, ISG15, ISG56, MDA5 and TRIF) and inflammatory genes (IL6, IL8, IL-1β and TNF-α). EcSNX5 knockdown could promote the expression of interferon factors and inflammatory factors. Moreover, EcSNX5 overexpression suppresses the expression of autophagy genes (LC3-II, BECN1, ATG5 and ATG16L1) and upregulates the expression of apoptosis genes (Bax, BNIP3), but EcSNX5 knockdown had the opposite effect. According to the subcellular localization, EcSNX5 is localized in the cytoplasm and co-localizaed with RGNNV CP protein. The results showed EcSNX5 can influence viral infections by regulating the expression of interferon factors and inflammatory factors as well as adjusting virus-induced autophagy. These data will contribute to a better understanding of the immune response of fish during virus infection.

An improved oral vaccine with molecular adjuvant β-defensin protects grouper against nervous necrosis virus infection

Nervous necrosis virus (NNV) is one of the most important fish viral pathogens infecting more than 120 fish species worldwide. Due to the mass mortality rates often seen among larvae and juveniles, few effective vaccines against NNV were developed up to now. Here, the protective effect of recombinant coat protein (CP) from red-spotted grouper nervous necrosis virus (RGNNV) fused with grouper β-defensin (DEFB) as an oral vaccine was evaluated using Artemia as a biocarrier delivery system in pearl gentian grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀). Feeding with Artemia encapsulated with E. coli expressing control vector (control group), CP, or CP-DEFB showed no obvious side effects on the growth of groupers. ELISA and antibody neutralization assay showed that CP-DEFB oral vaccination group induced higher anti-RGNNV CP specific antibodies and exhibited higher neutralization potency than the CP and control group. Meanwhile, the expression levels of several immune and inflammatory factors in the spleen and kidney after feeding with CP-DEFB were also significantly increased compared with the CP group. Consistently, after challenge with RGNNV, groupers fed CP-DEFB and CP exhibited 100% and 88.23% relative percentage survival (RPS), respectively. Moreover, the lower transcription levels of viral genes and milder pathological changes in CP-DEFB group were detected compared with the CP and control group. Thus, we proposed that grouper β-defensin functioned as an efficient molecular adjuvant for an improved oral vaccine against nervous necrosis virus infection.