Red-spotted Grouper Nervous Necrosis Virus Infection 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.

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.

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.

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.

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.

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.

Comparative transcriptomic analysis reveals different host cell responses to Singapore grouper iridovirus and red-spotted grouper nervous necrosis virus

Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) are important pathogens that cause high mortality and heavy economic losses in grouper aquaculture. Interestingly, SGIV infection in grouper cells induces paraptosis-like cell death, while RGNNV infection induces autophagy and necrosis characterized morphologically by vacuolation of lysosome. Here, a comparative transcriptomic analysis was carried out to identify the different molecular events during SGIV and RGNNV infection in grouper spleen (EAGS) cells. The functional enrichment analysis of DEGs suggested that several signaling pathways were involved in CPE progression and host immune response against SGIV or RGNNV. Most of DEGs featured in the KEGG “lysosome pathway” were up-regulated in RGNNV-infected cells, indicating that RGNNV induced lysosomal vacuolization and autophagy might be due to the disturbance of lysosomal function. More than 100 DEGs in cytoskeleton pathway and mitogen-activated protein kinase (MAPK) signal pathway were identified during SGIV infection, providing additional evidence for the roles of cytoskeleton remodeling in cell rounding during CPE progression and MAPK signaling in SGIV induced cell death. Of note, consistent with changes at the transcriptional levels, the post-translational modifications of MAPK signaling-related proteins were also detected during RGNNV infection, and the inhibitors of extracellular signal-regulated kinase (ERK) and p38 MAPK significantly suppressed viral replication and virus induced vacuoles formation. Moreover, the majority of DEGs in interferon and inflammation signaling were obviously up-regulated during RGNNV infection, but down-regulated during SGIV infection, suggesting that SGIV and RGNNV differently manipulated host immune response in vitro. In addition, purine and pyrimidine metabolism pathways were also differently regulated in SGIV and RGNNV-infection cells. Taken together, our data will provide new insights into understanding the potential mechanisms underlying different host cell responses against fish DNA and RNA virus.

Critical Roles of G3BP1 in Red-Spotted Grouper Nervous Necrosis Virus-Induced Stress Granule Formation and Viral Replication in Orange-Spotted Grouper (Epinephelus coioides).

Viral infection causes changes in the internal environment of host cells, and a series of stress responses are generated to respond to these changes and help the cell survive. Stress granule (SG) formation is a type of cellular stress response that inhibits viral replication. However, the relationship between red-spotted grouper nervous necrosis virus (RGNNV) infection and SGs, and the roles of the SG marker protein RAS GTPase-activating protein (SH3 domain)-binding protein 1 (G3BP1) in viral infection remain unclear. In this study, RGNNV infection induced grouper spleen (GS) cells to produce SGs. The SGs particles co-located with the classic SG marker protein eIF3η, and some SGs depolymerized under treatment with the translation inhibitor, cycloheximide (CHX). In addition, when the four kinases of the eukaryotic translation initiation factor 2α (eIF2α)-dependent pathway were inhibited, knockdown of HRI and GCN2 with small interfering RNAs and inhibition of PKR with 2-aminopurine had little effect on the formation of SGs, but the PERK inhibitor significantly inhibited the formation of SGs and decreased the phosphorylation of eIF2α. G3BP1 of Epinephelus coioides (named as EcG3BP1) encodes 495 amino acids with a predicted molecular weight of 54.12 kDa and 65.9% homology with humans. Overexpression of EcG3BP1 inhibited the replication of RGNNV in vitro by up-regulating the interferon and inflammatory response, whereas knockdown of EcG3BP1 promoted the replication of RGNNV. These results provide a better understanding of the relationship between SGs and viral infection in fish.

Sea perch (Lateolabrax japonicus) autophagy related gene 5 promotes RGNNV infection via inhibiting RLRs-interferon signaling pathway

Autophagy-related gene 5 (Atg5), an essential component of autophagy machinery, is associated with innate immune responses. Here, the Atg5 of sea perch (Lateolabrax japonicus) (LjAtg5) was cloned and its role in regulating autophagy and interferon (IFN) response during red-spotted grouper nervous necrosis virus (RGNNV) infection was investigated. The LjAtg5 cDNA encoded a polypeptide of 275 amino acids with an APG5 domain, and had the closet genetic relationship with Micropterus salmoides Atg5. Autophagic detection showed LjAtg5 was conserved in inducing cell autophagy. Spatial expression analysis revealed LjAtg5 had a higher expression level in liver, brain, and kidney tissues of RGNNV-infected sea perch compared with the control group. In RGNNV-infected LJB cells, overexpression of LjAtg5 significantly increased the mRNA and protein levels of capsid protein, whereas knockdown of LjAtg5 led to the opposite effect, indicating LjAtg5 played a pro-viral role during RGNNV infection. Furthermore, dual luciferase reporter assay revealed LjAtg5 significantly suppressed the activation of sea perch type I IFN promoter in vitro, and overexpression of LjAtg5 strongly weaken the expression of genes related to the RIG-I-like receptors (RLRs) signaling pathway and IFN stimulated genes. These results suggested LjAtg5 promoted RGNNV infection by negatively regulating RLRs-IFN signaling pathway.

High-density lipoproteins negatively regulate innate immunity and facilitate red-spotted grouper nervous necrosis virus entry via scavenger receptor B type 1

Lipid metabolism plays an important role in viral infections, and it can directly or indirectly affect various stages of viral infection in cells. As an important component of lipid metabolism, high-density lipoprotein (HDL) plays crucial roles in inflammation, immunity, and viral infections. Scavenger receptor B type 1 (SR-B1), a receptor of HDL, cannot be ignored in the regulation of lipid metabolism. Here, we investigate, for the first time, the role of Epinephelus coioides SR-B1 (Ec-SR-B1) in red-spotted grouper nervous necrosis virus (RGNNV) infection. Our results indicate that Ec-SR-B1 could promote RGNNV infection. We also demonstrate that Ec-SR-B1 could facilitate viral entry and interact with capsid protein (CP) of RGNNV. As the natural ligand of SR-B1, HDL significantly increased RGNNV entry in a dose-dependent manner. However, we observed no effect of HDL on Ec-SR-B1 expression. The results of the micro-scale thermophoresis assay did not reveal an association between HDL and CP, suggesting that RGNNV does not enter target cells by using HDL as a ligand to bind to its receptor. In addition, block lipid transport-1, a compound that inhibits HDL-mediated cholesterol transfer, reduced the HDL-induced enhancement of RGNNV infection, indicating a role for lipid transfer in facilitating RGNNV entry. Furthermore, HDL inhibited the expression of pro-inflammatory factors and antiviral genes in a dose-dependent manner. These findings suggest that the HDL-induced enhancement of RGNNV entry involves the complex interplay between Ec-SR-B1, HDL, and RGNNV, as well as the regulation of innate antiviral responses by HDL. In summary, we highlight the crucial role of HDL in RGNNV entry, identify a possible molecular connection between RGNNV and lipoprotein metabolism, and indicate the role of Ec-SR-B1 in RGNNV infection.

Establishment and characterization of a liver cell line from black porgy, Acanthopagrus schlegelii

In this study, a liver cell line (ASL) derived from black porgy (Acanthopagrus schlegelii) was established and characterised. After the initial primary culture, the cell line has been cultured for over 40 passages at 28 °C in Dulbecco's modified Eagle's medium (DMEM) with 15 % foetal bovine serum (FBS). Immunostaining results indicated that ASL cells contained at least two cytotypes in the initial culture stage; however, epithelioid cells dominated and replaced the other type during passage 6–20. ASL cells grow in temperature range 20–28 °C in DMEM containing 15–20 % FBS. Cytogenetic analysis showed that the modal chromosome number in ASL cells was 48. Partial amplification of the cytochrome c oxidase subunit I gene indicates that ASL originates from black porgy. ASL cells showed a transfection efficiency of approximately 15 % after transfection with the pEGFP-N3 plasmid. Cytopathic effects were observed in ASL cells after red-spotted grouper nervous necrosis virus (RGNNV) infection, and viral replication in RGNNV-infected ASL cells was confirmed using quantitative reverse transcription polymerase chain reaction, virus titre assays, western blot, and transmission electron microscopy assays. Furthermore, several antiviral genes were induced to varying degrees in RGNNV-infected ASL cells, indicating that the immune response was activated in ASL cells post RGNNV infection. In conclusion, the newly established ASL cell line is an ideal in vitro tool for studying NNV-black porgy interactions.

Oral immunizations with Bacillus subtilis spores expressing MCP protein provide protection against red-spotted grouper nervous necrosis virus (RGNNV) infection in juvenile grouper, Epinephelus coioides

Nervous necrosis virus (NNV) causes severe viral disease in grouper, resulting in major economic losses in the global grouper aquaculture industry as a result of the high mortality rate. In the present study, we aimed to develop a safe and efficient vaccine to protect grouper against NNV infection. To achieve effective oral and bath immune responses, spores of Bacillus subtilis (B. subtilis) WB600 were utilized as the vehicle to deliver MCP (major capsid protein of RGNNV) partnered with CotC, one of the coat proteins of B. subtilis, to the gastrointestinal tract. After routine culturing for 8–12 h in LB medium, B. subtilis containing CotC-MCP (B.s-CotC-MCP) was transferred into the sporulation culture medium. Western blot analysis and the growth curve indicated that the best sporulation time for recombinant WB600 was 24–30 h at 37 °C with continuous shaking (250 rpm). The grouper then received surface displaying B·S-CotC-MCP vaccine against RGNNV via either oral or bath immunization and their immune responses were monitored. The results revealed that some non-specific immune parameters (acid phosphatase (ACP), alkaline phosphatase (AKP) and total antioxidant capacity (T-AOC)) were strongly enhanced in juvenile grouper following oral or bath immunizations (1 × 106, 1 × 107, and 1 × 108 CFU g−1). The levels of specific antibodies against RGNNV and the transcription of immune-related genes (TNF-α, IL-1β, MHCI and IgM) were also significantly enhanced in juvenile grouper flowing oral or bath immunizations (1 × 106, 1 × 107, and 1 × 108 CFU g−1). However, only the highest dose of bath vaccination induced the production of specific antibodies significantly and up-regulated transcriptions of several immune-related genes (IgM and MHCI). The lower cumulative mortality in vaccinated groups after RGNNV challenge clearly demonstrated that surface-displayed MCP vaccine could protect fish against RGNNV infection. The relative percentage survival (RPS) value in the orally vaccinated group (88.89%) was much higher than the bath group (18.89%), which was consistent with the production of specific serum antibodies, non-specific immune response and immune related gene expression. Therefore, this study demonstrated that the CotC-MCP spores can trigger high levels of mucosal and humoral immunity, and represent a promising candidate vaccine against NNV infection.

Inhibition of RGNNV by dsRNA in the orange-spotted grouper

Nervous necrosis virus (NNV) is highly contagious and virulent and causes neural disease in many marine and brackish water fish. To date, there is no effective treatment for NNV. In this study, we evaluated the antiviral potential of long double-strand RNA (dsRNA) against red-spotted grouper nervous necrosis virus (RGNNV). Plasmids containing the fish U6 promoter were engineered to express dsRNA targeting the RGNNV capsid RNA. In vitro and in vivo data show that our engineered dsRNA system successfully inhibited RGNNV infection in grouper, which indicates that this method may be a potential novel approach for RNA-guided immunity against other RNA viruses in fish.

Establishment and characterization of a fin cell line from yellowfin sea bream (Acanthopagrus latus) and its application to fish virology and toxicology

Fish cell lines are ideal tools in vitro for virology and toxicology. Yellowfin sea bream ( Acanthopagrus latus ) is an economically important marine fish suffering from virus infections and is also a reliable indicator in toxicological studies. However, no continuous cell line from A. latus has been developed. Here, a new cell line (ALF) derived from the fin tissue of A. latus was established and identified. Up to now, the cell line has been subcultured for more than 50 times in Dulbecco's modified Eagle's medium. ALF cells could grow well at a range of temperature from 20 to 28 °C and had a maximum growth rate at 28 °C, and grow fastest in DMEM with 20% FBS concentration (in the range of 10% to 20%). Partial sequence amplification and sequencing of cytochrome B gene indicated that the cell line was originated from A. latus . The chromosome number of ALF cell was 48. ALF was susceptible to red-spotted grouper nervous necrosis virus (RGNNV), as suggested by the typical cytopathic effects and the amplification of RGNNV RNA-dependent RNA polymerase gene. Virus replication in ALF cells was confirmed by qRT-PCR, virus titer assay, western blot and transmission electron microscopy assays. Furthermore, the expression of four immune-related genes, TRAF3 , NF-κB1 , STAT1 and STAT2 , was varied changed in ALF cells upon RGNNV infection, indicating the induction of cellular antiviral response. In addition, p,p′-DDT was cytotoxic to ALF cells and induced cell apoptosis as evidenced by the results of annexin V-FITC/PI double staining assay, DNA fragmentation assay and the differential expression of apoptosis-related genes. Moreover, p,p'-DDT exposure increased the sensitivity of ALF cells to RGNNV via inhibiting the antiviral response in ALF cells. In conclusion, the ALF cell line will be a good in vitro platform for fish virology and toxicology studies. • A cell line (ALF) derived from the fin tissue of yellowfin sea bream ( Acanthopagrus latus ) was established and identified. • ALF is susceptible to red-spotted grouper nervous necrosis virus (RGNNV). • p,p′-DDT was cytotoxic to ALF cells and induced apoptosis in ALF cells. • p,p′-DDT increased the susceptibility of ALF cells to RGNNV via inhibiting the antiviral response in ALF cells.

Grouper USP12 exerts antiviral activity against nodavirus infection

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

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

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

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

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

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

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