Grouper TIM-1 promotes nodavirus infection by inhibiting immune and inflammation response.

T-cell/transmembrane immunoglobulin and mucin domain-containing (TIM) protein family has attracted particular attention because of their broad immune functions and the response to viral infections. TIM-1, a member of the TIM family, has been demonstrated to play an important role in viral infections. However, its roles during fish nodavirus infection still remained largely unknown. In this study, a homolog of TIM-1 from orange-spotted grouper (Epinephelus coioides) (EcTIM-1) was identified, and characterized. EcTIM-1 encoded a 217-amino acids protein, containing one Immunoglobulin domain. Homology analysis showed that EcTIM-1 shared 98.62 % and 42.99 % identity to giant grouper (E. lanceolatus) and human (Homo sapiens). Quantitative Real-time PCR analyses indicated that EcTIM-1 was expressed in all examined tissues, with higher expression in liver, spleen, skin, and heart, and was significantly up-regulated in response to red-spotted grouper nervous necrosis virus (RGNNV) infection. EcTIM-1 was distributed in the cytoplasm, and partly co-localized with Golgi apparatus and lysosomes in vitro. The ectopic expression of EcTIM-1 promoted RGNNV replication by increasing the level of viral genes transcription and protein synthesis. Besides, overexpression of EcTIM-1 decreased the luciferase activity of type I interferon (IFN1), interferon stimulated response elements (ISRE) and nuclear factor kappa-B (NF-κB) promoters, as well as the transcription of pro-inflammatory factors and interferon related genes. EcTIM-1 significantly suppressed the luciferase activity of IFN1, ISRE and NF-κB promoters evoked by Epinephelus coioides melanoma differentiation-associated gene 5 (EcMDA5), mitochondrial antiviral signaling protein (EcMAVS), stimulator of IFN genes (EcSTING) or TANK-binding kinase 1 (EcTBK1). Collectively, EcTIM-1 negatively regulated interferon and inflammatory response to promote RGNNV infection. These results provide a basis for a better understanding of the innate immune response of TIM-1 in fish.

Nervous necrosis virus capsid protein and Protein A dynamically modulate the fish cGAS-mediated IFN signal pathway to facilitate viral evasion.

Nervous necrosis virus (NNV), an aquatic RNA virus belonging to Betanodavirus, infects a variety of marine and freshwater fishes, leading to massive mortality of cultured larvae and juveniles and substantial economic losses. The enzyme cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) is widely recognized as a central component in the innate immune response to cytosolic DNA derived from different pathogens. However, little is known about the response of cGAS to aquatic RNA viruses. This study found that Epinephelus coioides cGAS (EccGAS) overexpression inhibited NNV replication, whereas EccGAS silencing promoted NNV replication. The anti-NNV activity of EccGAS was involved in interferon (IFN) signaling activation including tumor necrosis factor receptor-associated factor family member-associated NF-kappa-B activator-binding kinase 1 (TBK1) phosphorylation, interferon regulatory factor 3 (IRF3) nuclear translocation, and the subsequent induction of IFNc and ISGs. Interestingly, NNV employed its capsid protein (CP) or Protein A (ProA) to negatively or positively modulate EccGAS-mediated IFN signaling by simultaneously targeting EccGAS. CP interacted with EccGAS via the arm-P, S-P, and SD structural domains and promoted its polyubiquitination with K48 and K63 linkages in an EcUBE3C (the ubiquitin ligase)-dependent manner, ultimately leading to EccGAS degradation. Conversely, ProA bound to EccGAS and inhibited its ubiquitination and degradation. In regulating EccGAS protein content, CP's inhibitory action was more pronounced than ProA's protective effect, allowing successful NNV replication. These novel findings suggest that NNV CP and ProA dynamically modulate the EccGAS-mediated IFN signaling pathway to facilitate the immune escape of NNV. Our findings shed light on a novel mechanism of virus-host interaction and provide a theoretical basis for the prevention and control of NNV.IMPORTANCEAs a well-known DNA sensor, cGAS is a pivotal component in innate anti-viral immunity to anti-DNA viruses. Although there is growing evidence regarding the function of cGAS in the resistance to RNA viruses, the mechanisms by which cGAS participates in RNA virus-induced immune responses in fish and how aquatic viruses evade cGAS-mediated immune surveillance remain elusive. Here, we investigated the detailed mechanism by which EccGAS positively regulates the anti-NNV response. Furthermore, NNV CP and ProA interacted with EccGAS, regulating its protein levels through ubiquitin-proteasome pathways, to dynamically modulate the EccGAS-mediated IFN signaling pathway and facilitate viral evasion. Notably, NNV CP was identified to promote the ubiquitination of EccGAS via ubiquitin ligase EcUBE3C. These findings unveil a novel strategy for aquatic RNA viruses to evade cGAS-mediated innate immunity, enhancing our understanding of virus-host interactions.

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.

Plant Expression of Trans-Encapsidated Chimeric Viral Vaccines with Animal RNA Replicons: An Update.

In this protocol, we outline how to produce a chimeric viral vaccine in a biosafety level 1 (BSL1) environment. An animal viral vector RNA encapsidated with tobacco mosaic virus (TMV) coat protein can be fully assembled in planta. Agrobacterium cultures containing each component are inoculated together into tobacco leaves and the self-assembled hybrid chimeric viral vaccine is harvested 4 days later and purified with a simple PEG precipitation. The viral RNA delivery vector is derived from the BSL1 insect virus, Flock House virus (FHV), and replicates in human and animal cells but does not spread systemically. A polyethylene glycol purification protocol is also provided to collect and purify these vaccines for immunological tests. In this update, we also provide a protocol for in trans co-inoculation of a modified FHV protein A, which significantly increased the yield of in planta chimeric viral vaccine.

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

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.

Water-in-oil adjuvant challenges in fish vaccination: An experimental inactivated adjuvanted vaccine against betanodavirus infection in Senegalese sole.

The extensive growth of intensive fish farming has led to a massive spread of infectious diseases. Nervous necrosis virus (NNV) is the causative agent of the viral encephalo- and retinopathy disease which has become a major threat for fish farming all over the globe. The devastating mortality rates recorded in disease outbreaks, especially when infected specimens are at early stages of development, have a high economic impact on the sector. Currently, vaccines are the most cost-effective preventing tool in the fight against viruses. Inactivated vaccines have the advantage of simplicity in their development at the same time as present the antigen in a similar manner than the natural infection in the host. Nevertheless, they usually trigger weaker immune responses needing adjuvants to boost their effectiveness. In this work, we have intraperitoneally vaccinated Senegalese sole juveniles (Solea senegalensis) with a previously designed inactivated vaccine against NNV based on binary ethylenimine (BEI), mixed or not with an oil-adjuvant. Our results demonstrated the potential activation of different immune pathways when the vaccine was administered alone compared to the oil-adjuvanted vaccine, both resulting in an equivalent partial improvement in survival following a NNV challenge. However, whilst the vaccine alone led to a significant increase in specific antibodies, in the adjuvanted version those antibodies were kept basal although with a slight improvement in their neutralization capacity. At transcriptional level, neither vaccine (adjuvanted or not) triggered the immune system activation during the vaccination period. However, after NNV infection, the BEI-inactivated vaccines alone and oil-adjuvanted both elicited the stimulation of antiviral responsive genes (rtp3, herc4), antigen presentation molecules (mhcii) and T-cell markers (cd8a) in the head-kidney. Additionally, the oil-adjuvanted vaccine appears to stimulate mediator cytokines (il6) and B-cell markers (ight and ighm). Surprisingly, when the adjuvant was administered alone, fish showed the highest survival rates concomitantly with a lack of NNV-IgM production, pointing to the possible induction of different immune pathways than the B-cell responses via antibodies by the adjuvant. Since this combined vaccine did not succeed in the full extension of protection against the pathogen, further studies should be performed focusing on unravelling the molecular mechanisms through which adjuvants trigger the immune response, both independently and when added to a vaccine antigen.

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.

Sea perch (Lateolabrax japonicus) UBC9 augments RGNNV infection by hindering RLRs-interferon response.

Small ubiquitin-like modifier (SUMO) is a reversible post-translational modification that regulates various biological processes in eukaryotes. Ubiquitin-conjugating enzyme 9 (UBC9) is the sole E2-conjugating enzyme responsible for SUMOylation and plays an important role in essential cellular functions. Here, we cloned the UBC9 gene from sea perch (Lateolabrax japonicus) (LjUBC9) and investigated its role in regulating the IFN response during red-spotted grouper nervous necrosis virus (RGNNV) infection. The LjUBC9 gene consisted of 477 base pairs and encoded a polypeptide of 158 amino acids with an active site cysteine residue and a UBCc domain. Phylogenetic analysis showed that LjUBC9 shared the closest evolutionary relationship with UBC9 from Paralichthys olivaceus. Tissue expression profile analysis demonstrated that LjUBC9 was significantly increased in multiple tissues of sea perch following RGNNV infection. Further experiments showed that overexpression of LjUBC9 significantly increased the mRNA and protein levels of RGNNV capsid protein in LJB cells infected with RGNNV, nevertheless knockdown of LjUBC9 had the opposite effect, suggesting that LjUBC9 exerted a pro-viral effect during RGNNV infection. More importantly, we found that the 93rd cysteine is crucial for its pro-viral effect. Additionally, dual luciferase assays revealed that LjUBC9 prominently attenuated the promoter activities of sea perch type Ⅰ interferon (IFN) in RGNNV-infected cells, and overexpression of LjUBC9 markedly suppressed the transcription of key genes associated with RLRs-IFN pathway. In summary, these findings elucidate that LjUBC9 impairs the RLRs-IFN response, resulting in enhanced RGNNV infection.

Orange-spotted grouper IFNh response to NNV or MSRV and its potential antiviral activities.

Type I interferon (IFN) plays a crucial role in the antiviral immune response. Nervous necrosis virus (NNV) and Micropterus salmoides rhabdovirus (MSRV) are the most important viruses in cultured larvae and juveniles, causing great economic losses to fish farming. To better understand the antiviral activities and immunoregulatory role of IFN from orange-spotted grouper (Epinephelus coioides), EcIFNh was cloned from NNV infected sample. EcIFNh has an open reading frame (ORF) of 552 bp and encodes a polypeptide of 183 amino acids. Phylogenetic tree analysis showed that EcIFNh was clustered into the IFNh branch. The tissue distribution analysis revealed that EcIFNh was highly expressed in the liver and brain of healthy orange-spotted grouper. The mRNA levels of EcIFNh were significantly upregulated after poly (I:C) stimulation and NNV or MSRV infection. Furthermore, the promoter of EcIFNh was characterized and significantly activated by EcMDA5, EcMAVS, EcSTING, EcIRF3, and EcIRF7 in the luciferase activity assays. We found that EcIFNh overexpression resisted the replication of NNV and MSRV, while EcIFNh silencing facilitated NNV replication in GB cells. In addition, EcIFNh recombinant protein (rEcIFNh) enhanced the immune response by inducing the expression of ISGs in vivo and in vitro, suggesting the potential application of rEcIFNh for anti-NNV and anti-MSRV. Taken together, our research may offer the foundation for virus-IFN system interaction in orange-spotted grouper.

Cytotoxic activity and gene expression during in vitro adaptive cell-mediated cytotoxicity of head-kidney cells from betanodavirus-infected European sea bass.

Cell-mediated cytotoxicity (CMC) is essential in eradicating virus-infected cells, involving CD8+ T lymphocytes (CTLs) and natural killer (NK) cells, through the activation of different pathways. This immune response is well-studied in mammals but scarcely in teleost fish. Our aim was to investigate the adaptive CMC using head-kidney (HK) cells from European sea bass infected at different times with nodavirus (NNV), as effector cells, and the European sea bass brain cell line (DLB-1) infected with different NNV genotypes, as target cells. Results showed low and unaltered innate cytotoxic activity through the infection time. However, adaptive CMC against RGNNV and SJNNV/RGNNV-infected target cells increased from 7 to 30 days post-infection, peaking at 15 days, demonstrating the specificity of the cytotoxic activity and suggesting the involvement of CTLs. At transcriptomic level, we observed up-regulation of genes related to T cell activation, perforin/granzyme and Fas/FasL effector pathways as well as apoptotic cell death. Further studies are necessary to understand the adaptive role of European sea bass CTLs in the elimination of NNV-infected cells.

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.

The Viromes of Mosquitoes from the Natural Landscapes of Western Siberia.

The metagenomic analysis of mosquitoes allows for the genetic characterization of mosquito-associated viruses in different regions of the world. This study applied a metagenomic approach to identify novel viral sequences in seven species of mosquitoes collected from the Novosibirsk region of western Siberia. Using NGS sequencing, we identified 15 coding-complete viral polyproteins (genomes) and 15 viral-like partial sequences in mosquitoes. The complete sequences for novel viruses or the partial sequences of capsid proteins, hypothetical viral proteins, and RdRps were used to identify their taxonomy. The novel viral sequences were classified within the orders Tymovirales and Picornavirales and the families Partitiviridae, Totiviridae, Tombusviridae, Iflaviridae, Nodaviridae, Permutotetraviridae, and Solemoviridae, with several attributed to four unclassified RNA viruses. Interestingly, the novel putative viruses and viral sequences were mainly associated with the mosquito Coquillettidia richardii. This study aimed to increase our understanding of the viral diversity in mosquitoes found in the natural habitats of Siberia, which is characterized by very long, snowy, and cold winters.

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 acids 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.

Identification of B-Cell Epitopes on Capsid Protein Reveals Two Potential Neutralization Mechanisms in Red-Spotted Grouper Nervous Necrosis Virus.

Nervous necrosis virus (NNV), a formidable pathogen in marine and freshwater fish, has inflicted enormous financial tolls on the aquaculture industry worldwide. Although capsid protein (CP) is the sole structural protein with pathogenicity and antigenicity, public information on immunodominant regions remains extremely scarce. Here, we employed neutralizing monoclonal antibodies (MAbs) specific for red-spotted grouper NNV (RGNNV) CNPgg2018 in combination with partially overlapping truncated proteins and peptides to identify two minimal B-cell epitope clusters on CP, 122GYVAGFL128 and 227SLYNDSL233. Site-directed mutational analysis confirmed residues Y123, G126, and L128 and residues L228, Y229, N230, D231, and L233 as the critical residues responsible for the direct interaction with ligand, respectively. According to homologous modeling and bioinformatic evaluation, 122GYVAGFL128 is harbored at the groove of the CP junction with strict conservation among all NNV isolates, while 227SLYNDSL233 is localized in proximity to the tip of a viral protrusion having relatively high evolutionary dynamics in different genotypes. Additionally, 227SLYNDSL233 was shown to be a receptor-binding site, since the corresponding polypeptide could moderately suppress RGNNV multiplication by impeding virion entry. In contrast, 122GYVAGFL128 seemed dedicated only to stabilizing viral native conformation and not to assisting initial virus attachment. Altogether, these findings contribute to a novel understanding of the antigenic distribution pattern of NNV and the molecular basis for neutralization, thus advancing the development of biomedical products, especially epitope-based vaccines, against NNV. IMPORTANCE NNV is a common etiological agent associated with neurological virosis in multiple aquatic organisms, causing significant hazards to the host. However, licensed drugs or vaccines to combat NNV infection are very limited to date. Toward the advancement of broad-spectrum prophylaxis and therapeutics against NNV, elucidating the diversity of immunodominant regions within it is undoubtedly essential. Here, we identified two independent B-cell epitopes on NNV CP, followed by the confirmation of critical amino acid residues participating in direct interaction. These two sites were distributed on the shell and protrusion domains of the virion, respectively, and mediated the neutralization exerted by MAbs via drastically distinct mechanisms. Our work promotes new insights into NNV antigenicity as well as neutralization and, more importantly, offers promising targets for the development of antiviral countermeasures.

Grouper TRIM23 exerts antiviral activity against iridovirus and nodavirus.

TRIM (tripartite motif) proteins have been demonstrated to exert critical roles in host defense against different microbial pathogens. Among them, TRIM23 acts as an important regulatory factor in antiviral immune and inflammatory responses, but the roles of fish TRIM23 against virus infection still remain largely unknown. Here, we investigated the characteristics of TRIM23 homolog from orange spotted grouper (Epinephelus coioides) (EcTRIM23). EcTRIM23 encoded a 580 amino acid peptide, which shared 93.1%, 89.73% and 86.36% identity with golden perch (Perca flavescens), zebrafish (Danio rerio) and human (Homo sapiens), respectively. The transcription levels of EcTRIM23 were significantly up-regulated in response to Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) infection. EcTRIM23 overexpression in vitro significantly inhibited RGNNV and SGIV replication, evidenced by the delayed cytopathic effect (CPE) progression and the decreased expression of viral core genes. EcTRIM23 significantly increased the expression levels of interferon (IFN) related signaling molecules and pro-inflammatory cytokines, as well as the promoter activities of IFN and NF-κB, suggesting that EcTRIM23 exerted antiviral function by positively regulating host IFN response. Exogenous EcTRIM23 exhibited either diffuse or aggregated localization in grouper cells. After co-transfection, TANK binding kinase 1 (TBK1), TNF receptor associated factor (TRAF) 3 and TRAF4, TRAF5 and TRAF6 were found to interact with EcTRIM23 in grouper cells. Moreover, these proteins could be recruited and co-localized with EcTRIM23 in vitro. Together, our results demonstrated that fish TRIM23 exerted antiviral activity against fish viruses by interacting with multiple host proteins to regulate immune responses.

Grouper ATF1 plays an antiviral role in response to iridovirus and nodavirus infection.

Transcription factor ATF1 is a member of the ATF/CREB family of the CREB subfamily and is involved in physiological processes such as tumorigenesis, organ development, reproduction, cell survival, and apoptosis in mammals. However, studies on ATF1 in fish have been relatively poorly reported, especially on its role in antiviral immunity in fish. In this study, ATF1 from orange-spotted grouper (named EcATF1) were cloned and characterized. Molecular characterization analysis showed that EcATF1 encodes a 307-amino-acid protein, containing PKID and bZIP_CREB1 domains. Homology analysis showed that had the highest homology with E. lanceolatus(88.93%). Tissue expression pattern showed that EcATF1 was extensively distributed in twelve selected tissues, with higher expression in the skin, gill, liver and spleen. Subcellular localization analysis showed that EcATF1 was distributed in the nucleus of GS cells. EcATF1 overexpression inhibits Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) replication, as evidenced by a diminished degree of CPE induced by SGIV and RGNNV and a reduction in the level of viral gene transcription and viral capsid protein expression. Furthermore, EcATF1 overexpression upregulated interferon pathway-related genes and proinflammatory factors, and increased the promoter activities of IFN, IFN stimulated response element (ISRE), and nuclear factor κB(NFκB). Meanwhile, EcATF1 overexpression positive regulate the MHC-I signaling pathway, and upregulated the promoter activity of MHC-I. Collectively, these data demonstrate that EcATF1 plays an important role during the host antiviral immune response. This study provides insights into the function of ATF1 in the immune system of lower vertebrates.

Grouper interferon-induced protein 35, a CP-interacting protein, inhibits fish nodavirus replication via positively regulating host interferon and inflammatory immune response.

Interferon (IFN)-induced protein 35 (IFI35, also known as IFP35), a member of IFN induced genes (ISGs), participates in virus infection, cancer progression and the chronic inflammatory diseases. However, its roles during fish nodavirus infection still remained largely unknown. In the present study, a homolog of IFI35 from orange spotted grouper (Epinephelus coioides) (EcIFI35) was cloned and characterized. The open reading frame of EcIFI35 was composed of 1,128 bp, and encoded a 375 amino acid polypeptide, which contained two conserved N-myc-interactor (Nmi)/IFP35 domains (NIDs). Homology analysis indicated that EcIFI35 shared 95.73% and 31.96% identity with homologs of giant grouper (E. lanceolatus) and human (Homo sapiens), respectively. The transcription of EcIFI35 was significantly up-regulated in grouper spleen (GS) cells after challenged with red-spotted grouper nervous necrosis virus (RGNNV), polyinosinic:polycytidylic acid [poly(I:C)] or lipopolysaccharide (LPS). The subcellular localization analysis showed that EcIFI35 encoded a cytoplasmic protein. The ectopic expression of EcIFI35 inhibited RGNNV replication by reducing viral genes transcription and protein synthesis. Co-immunoprecipitation (Co-IP) assay demonstrated that EcIFI35 interacted with RGNNV coat protein (CP), and partly co-localized with CP. EcIFI35 overexpression promoted the expression of IFN-related molecules and pro-inflammatory factors, including IFN regulatory factor 7 (IRF7), mitochondrial antiviral signaling protein (MAVS) and myxovirus resistance gene I (MxI), nuclear factor κB (NF-κB), interleukin 6 (IL-6) and IL-8. Together, our results revealed that EcIFI35 interacted with CP and inhibited fish nodavirus replication through positively regulated host innate immune response.

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.

Grouper TRAF3 inhibits nodavirus infection by regulating the STING-mediated antiviral signaling pathway.

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are major signal transducers for the TNF and interleukin-1/Toll-like receptor superfamilies that transduce signals from various immune receptors. To investigate the interaction of TRAF3 and other proteins in signaling pathways and to identify its antiviral function in teleosts, we cloned and characterized a TRAF3 homolog from orange-spotted grouper (Epinephelus coioides) (EcTRAF3). The open reading frame of EcTRAF3 consists of 1767 base pairs encoding a 588 amino acid protein, and the predicted molecular mass is 66.71 kDa EcTRAF3 shares 99.83% identity with TRAF3 of Epinephelus lanceolatus. Expression analysis revealed that EcTRAF3 was broadly distributed in examined tissues and was up-regulated under polyinosinic-polycytidylic acid and red-spotted grouper nervous necrosis virus (RGNNV) stimulation in vivo. EcTRAF3 was identified as a cytosolic protein based on fluorescence microscopy analysis. Overexpression of EcTRAF3 inhibited RGNNV replication in grouper spleen cells, and it interacted with the coat protein of RGNNV. Overexpression of EcTRAF3 also induced the activation of interferon ß (IFN-ß), IFN-stimulated response element (ISRE), and nuclear factor-κB (NF-κB). EcTRAF3 co-transfected with Stimulator of Interferon Genes (STING) of grouper (EcSTING) induced a significantly higher level of IFN-ß promoter activity. Moreover, EcTRAF3 interacted with EcSTING, implying that EcTRAF3 may function as an enhancer in EcSTING-mediated signaling. Taken together, our results suggest that EcTRAF3 negatively regulates the RGNNV-induced cellular antiviral response and plays an important role in the immune response system of fish.