Nervous Necrosis Virus Infection Research Articles

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.

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.

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.

Functional characterization of Malabar grouper (Epinephelus malabaricus) interferon regulatory factor 9 involved in antiviral response

IRF9 is a crucial component in the JAK-STAT pathway. IRF9 interacts with STAT1 and STAT2 to form IFN-I-stimulated gene factor 3 (ISGF3) in response to type I IFN stimulation, which promotes ISG transcription. However, the mechanism by which IFN signaling regulates Malabar grouper (Epinephelus malabaricus) IRF9 is still elusive. Here, we explored the nd tissue-specific mRNA distribution of the MgIRF9 gene, as well as its antiviral function in E. malabaricus. MgIRF9 encodes a protein of 438 amino acids with an open reading frame of 1317 base pairs. MgIRF9 mRNA was detected in all tissues of a healthy M. grouper, with the highest concentrations in the muscle, gills, and brain. It was significantly up-regulated by nervous necrosis virus infection and poly (I:C) stimulation. The gel mobility shift test demonstrated a high-affinity association between MgIRF9 and the promoter of zfIFN in vitro. In GK cells, grouper recombinant IFN-treated samples showed a significant response in ISGs and exhibited antiviral function. Subsequently, overexpression of MgIRF9 resulted in a considerable increase in IFN and ISGs mRNA expression (ADAR1, ADAR1-Like, and ADAR2). Co-immunoprecipitation studies demonstrated that MgIRF9 and STAT2 can interact in vivo. According to the findings, M. grouper IRF9 may play a role in how IFN signaling induces ISG gene expression in grouper species.

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.

Identification of candidate SNPs and genes associated with resistance to nervous necrosis virus in leopard coral grouper (Plectropomus leopardus) using GWAS

The leopard coral grouper (Plectropomus leopardus), which has become increasingly popular in consumption due to its bright body color and great nutritional, holds a high economic and breeding potential. However, in recent years, the P.leopardus aquaculture industry has been impeded by the nervous necrosis virus (NNV) outbreak, leading to widespread mortality among fry and juvenile grouper. However, the genetic basis of resistance to NNV in P. leopardus remains to be investigated. In the present study, we conducted a genome-wide association analysis (GWAS) on 100 resistant and 100 susceptible samples to discover variants and potential genes linked with NNV resistance. For this study, 157,926 high-quality single nucleotide polymorphisms (SNPs) based on whole genome resequencing were discovered, and eighteen SNPs loci linked to disease resistance were discovered. We annotated six relevant candidate genes, including sik2, herc2, pip5k1c, npr1, mybpc3, and arhgap9, which showed important roles in lipid metabolism, oxidative stress, and neuronal survival. In the brain tissues of resistant and susceptible groups, candidate genes against NNV infection showed significant differential expression. The results indicate that regulating neuronal survival or pathways involved in lipid metabolism may result in increased resistance to NNV. Understanding the molecular mechanisms that lead to NNV resistance will be beneficial for the growth of the P. leopardus breeding sector. Additionally, the identified SNPs could be employed as biomarkers of disease resistance in P. leopardus, which will facilitate the selective breeding of grouper.

Molecular and functional characterization of golden pompano (Trachinotus blochii) TBK1 on IFN regulation

The golden pompano (Trachinotus blochii), a pivotal commercial marine species in China, has gained significant popularity worldwide. However, accompanied with rapid growth and high density aquaculture, golden pompano has been seriously threatened by Nervous necrosis virus (NNV), while its molecular biology research regarding the innate immune system remains unexplored, which is crucial for understanding the activation of interferon (IFN) production and antiviral responses. In this study, we aimed to identify the characterization and function of golden pompano TANK-binding kinase 1 (gpTBK1), thereby providing evidence of the conservation of this classical factor in the RLR pathway among marine fish. Initially, we found the expression of gpTBK1 upregulation in diseased golden pompano with NNV infection and we successfully cloned the full-length open reading frame (ORF) of gpTBK1, consisting of 2172 nucleotides encoding 723 amino acids, from the head kidney. Subsequent analysis of the amino acid sequence revealed homology between gpTBK1 and other fish TBK1 proteins, with conserved N-terminal Serine/Threonine protein kinases catalytic domain (S_TKc) and C-terminal coiled coil domain (CCD). Moreover, the expression pattern showed that gpTBK1 exhibited ubiquitous expression across all evaluated tissues. Furthermore, functional identification experiments indicated that gpTBK1 activated interferon promoters’ activity in golden pompano and induced the expression of downstream IFN-stimulated genes (ISGs). Notably, gpTBK1 was found to co-localize and interact with gpIRF3 in the cytoplasm. Collectively, these data provide a comprehensive analysis of the characterization and functional role of gpTBK1 in promoting interferon production. This research may facilitate the further study of the innate antiviral response, particularly the anti-NNV mechanisms, in golden pompano.

Nervous necrosis virus (NNV) vaccination of carrier Senegalese sole (Solea senegalensis)

Viral encephalopathy and retinopathy (VER), caused by nervous necrosis virus (NNV), is one of the most threatening diseases affecting marine farmed fish, especially in early developmental stages. In addition, subclinical infections are commonly detected in both farmed and wild fish. In recent years numerous efforts have been made to achieve effective vaccines for the protection of different fish species against NNV infection. Vaccination experiments are always conducted on pathogen-free animals, but subclinically infected individuals may respond differently to immunization. In this study, we have observed a different response between experimentally-induced-NNV carrier Senegalese sole and NNV-free animals when immunized with a BEI-inactivated vaccine, whereas mock-vaccinated carrier fish behaved like the NNV-free group. Analysis of the immune response after vaccination showed specific antibody production as well as up-regulation of the genes coding for the proinflammatory cytokines IL-6 and IL-8, the antiviral protein HERC4 and the cytotoxic T lymphocyte (CTL) and T helper markers CD8 and CD4 in vaccinated NNV-free and mock-vaccinated carrier individuals. However, no significant antibody production was detected and only cd4 up-regulation was recorded in the vaccinated carrier fish. After challenge, viral replication was similar in the vaccinated groups up to 15 days post-challenge (dpc), although thereafter the viral load in the vaccinated carrier fish reached values similar to the control fish, whereas in the vaccinated NNV-free sole remained 1.5–2 log lower. At 2 dpc 10% mortality was recorded in the vaccinated carrier group that did not correlate with a higher viral titre, but with an overexpression of inflammation-related genes. These results suggest that an acute inflammatory reaction could have been the cause of the fish death. Excepting the values recorded in this episode, poor modulation of the immune response was observed at the early days post-challenge. However, at 30 dpc the IgM levels in the vaccinated carrier fish were significantly higher than those of the NNV-free sole. Longer post-vaccination studies would be necessary to clarify if this antibody increase may be relevant for fish protection.

Molecular and functional characterization of viperin in golden pompano, Trachinotus ovatus

The radical S-adenosyl methionine domain-containing protein 2 (RSAD2), also known as viperin, plays a momentous and multifaceted role in antiviral immunity. However, the function of viperin is uninvestigated in golden pompano, Trachinotus ovatus. In the present study, a viperin homolog, named To-viperin, was cloned and characterized from golden pompano, and its role in response to grouper iridovirus (SGIV) and nervous necrosis virus (NNV) infection was investigated. The whole open reading frame (ORF) of To-viperin was composed of 1050 bp and encoded a polypeptide of 349 amino acids with 70.66%–83.51% identity with the known viperin homologs from other fish species. A variable N-terminal domain, a highly conserved C-terminal domain, and a conserved middle radical SAM domain (aa 61–271) with the three-cysteine motif CxxCxxC was found in To-viperin sequence. Expression analysis showed that To-viperin was constitutively expressed in all tested organs and was located mainly in the ER of golden pompano cells. Treatments with SGIV, poly I: C, or NNV could induce the up-regulation of viperin to varying degrees. The ectopic expression of To-viperin in vitro significantly reduced the viral titer of SGIV and NNV. Furthermore, To-viperin overexpression enhanced the expression of IFNc, IRF3, and ISG15 genes as well as, to a lesser extent, the IL-6 gene. In summary, our results suggested that the function of viperin is likely to be conserved in fish specise, as observed in other vertebrates, shedding light on the evolutionary conservation of viperin.

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.

Regulatory effects of potassium channel blockers on potassium channel genes upon nervous necrosis virus infection in sevenband grouper Hyporthodus septumfasciatus

Ion channels in fishes regulate the flow of important ions that play an active role in the excitation and transmission of impulses through neuronal cells. Specific housekeeping genes translates into proteins and selectively permeabilize and facilitate ion crossover transmissions. Potassium (K+) channels play a crucial role in a wide range of functions such as cell volume regulation, hormone secretion, synaptic transmission and muscle contraction. The dysfunction of ion channels result in channelopathies, which hinder critical cellular activities. Recent studies have indicated that viral pathogens tend to regulate cellular ion channels for entry into host cells. Hence, the present study aimed to elucidate the role of K+ channels during nervous necrosis virus (NNV) infections in the sevenband grouper (Hyporthodus septumfasciatus). Real-time PCR with the standardized potassium genes revealed the downregulation of potassium two pore domain channel subfamily member - KCNK10, KCNK9, KCNK2, and KCNK1 genes post infection at both 17 °C and 25 °C whereas an upregulation was noted in the case of gill tissues. SMART analysis revealed a transmembrane region in all genes. Multiple sequence alignment using MultAlin and phylogenetic analysis revealed true homology of potassium genes with other higher vertebrates. In vitro and in vivo challenge study of NNV using Tetra ethyl ammonium (TEA) as potential drug showed inverse relation to that of viral replication and a corresponding downregulation of K+ channel gene expression was observed which was further confirmed by an immunofluorescence assay. These findings indicate that K+ channels play a crucial role during viral infection. Moreover, the observed downregulation can be related to rapid endocytosis resulting from recycling endosomes during a viral infection. Hence, further studies are warranted to better understand the role of K+ channel genes during NNV infection.

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.

Ring Finger Protein 34 Facilitates Nervous Necrosis Virus Evasion of Antiviral Innate Immunity by Targeting TBK1 and IRF3 for Ubiquitination and Degradation in Teleost Fish.

Ubiquitination, as one of the most prevalent posttranslational modifications of proteins, enables a tight control of host immune responses. Many viruses hijack the host ubiquitin system to regulate host antiviral responses for their survival. Here, we found that the fish pathogen nervous necrosis virus (NNV) recruited Lateolabrax japonicus E3 ubiquitin ligase ring finger protein 34 (LjRNF34) to inhibit the RIG-I-like receptor (RLR)-mediated interferon (IFN) response via ubiquitinating Lateolabrax japonicus TANK-binding kinase 1 (LjTBK1) and interferon regulatory factor 3 (LjIRF3). Ectopic expression of LjRNF34 greatly enhanced NNV replication and prevented IFN production, while deficiency of LjRNF34 led to the opposite effect. Furthermore, LjRNF34 targeted LjTBK1 and LjIRF3 via its RING domain. Of note, the interactions between LjRNF34 and LjTBK1 or LjIRF3 were conserved in different cellular models derived from fish. Mechanically, LjRNF34 promoted K27- and K48-linked ubiquitination and degradation of LjTBK1 and LjIRF3, which in turn diminished LjTBK1-induced translocation of LjIRF3 from the cytoplasm to the nucleus. Ultimately, NNV capsid protein (CP) was found to bind with LjRNF34, CP induced LjTBK1 and LjIRF3 degradation, and IFN suppression depended on LjRNF34. Our finding demonstrates a novel mechanism by which NNV CP evaded host innate immunity via LjRNF34 and provides a potential drug target for the control of NNV infection. IMPORTANCE Ubiquitination plays an essential role in the regulation of innate immune responses to pathogens. NNV, a type of RNA virus, is the causal agent of a highly destructive disease in a variety of marine and freshwater fish. A previous study reported NNV could hijack the ubiquitin system to manipulate the host's immune responses; however, how NNV utilizes ubiquitination to facilitate its own replication is not well understood. Here, we identified a novel distinct role of E3 ubiquitin ligase LjRNF34 as an IFN antagonist to promote NNV infection. NNV capsid protein utilized LjRNF34 to target LjTBK1 and LjIRF3 for K27- and K48-linked ubiquitination and degradation. Importantly, the interactions between LjRNF34 and CP, LjTBK1, or LjIRF3 are conserved in different cellular models derived from fish, suggesting it is a general immune evasion strategy exploited by NNV to target the IFN response via RNF34.

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.

Recombinant viral hemorrhagic septicemia virus with rearranged genomes as vaccine vectors to protect against lethal betanodavirus infection.

The outbreaks of viral hemorrhagic septicemia (VHS) and viral encephalopathy and retinopathy (VER) caused by the enveloped novirhabdovirus VHSV, and the non-enveloped betanodavirus nervous necrosis virus (NNV), respectively, represent two of the main viral infectious threats for aquaculture worldwide. Non-segmented negative-strand RNA viruses such as VHSV are subject to a transcription gradient dictated by the order of the genes in their genomes. With the goal of developing a bivalent vaccine against VHSV and NNV infection, the genome of VHSV has been engineered to modify the gene order and to introduce an expression cassette encoding the major protective antigen domain of NNV capsid protein. The NNV Linker-P specific domain was duplicated and fused to the signal peptide (SP) and the transmembrane domain (TM) derived from novirhabdovirus glycoprotein to obtain expression of antigen at the surface of infected cells and its incorporation into viral particles. By reverse genetics, eight recombinant VHSVs (rVHSV), termed NxGyCz according to the respective positions of the genes encoding the nucleoprotein (N) and glycoprotein (G) as well as the expression cassette (C) along the genome, have been successfully recovered. All rVHSVs have been fully characterized in vitro for NNV epitope expression in fish cells and incorporation into VHSV virions. Safety, immunogenicity and protective efficacy of rVHSVs has been tested in vivo in trout (Oncorhynchus mykiss) and sole (Solea senegalensis). Following bath immersion administration of the various rVHSVs to juvenile trout, some of the rVHSVs were attenuated and protective against a lethal VHSV challenge. Results indicate that rVHSV N2G1C4 is safe and protective against VHSV challenge in trout. In parallel, juvenile sole were injected with rVHSVs and challenged with NNV. The rVHSV N2G1C4 is also safe, immunogenic and efficiently protects sole against a lethal NNV challenge, thus presenting a promising starting point for the development of a bivalent live attenuated vaccine candidate for the protection of these two commercially valuable fish species against two major diseases in aquaculture.

Identification and characterization of immunoglobulin tau (IgT) in Asian Seabass (Lates calcarifer) and mucosal immune response to nervous necrosis virus.

Mucosal immunity plays a critical role in the protection of teleost fish against infection, but mucosal immunoglobulin of important aquaculture species unique to Southeast Asia remained greatly understudied. In this study, the sequence of immunoglobulin T (IgT) from Asian sea bass (ASB) is described for the first time. IgT of ASB possesses the characteristic structure of immunoglobulin with a variable heavy chain and four CH4 domains. The CH2-CH4 domains and full-length IgT were expressed and CH2-CH4 specific antibody was validated against full-length IgT expressed in Sf9 III cells. Subsequent use of the anti-CH2-CH4 antibody in immunofluorescence staining confirmed the presence of IgT-positive cells in the ASB gill and intestine. The constitutive expression of ASB IgT was characterized in different tissues and in response to red-spotted grouper nervous necrosis virus (RGNNV) infection. The highest basal expression of secretory IgT (sIgT) was observed in the mucosal and lymphoid tissues such as the gills, intestine and head kidney. Following NNV infection, IgT expression was upregulated in the head kidney and mucosal tissues. Moreover, a significant increase in localized IgT was found in gills and intestines of infected fish on day 14 post-infection. Interestingly, a significant increase in NNV-specific IgT secretion was only observed in the gills of the infected group. Our results suggest that ASB IgT may play an important role in the adaptive mucosal immune responses against viral infection and could potentially be adapted as a tool for the evaluation of prospective mucosal vaccines and adjuvants for the species.

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.

EVALUATION OF RESISTANCE AND GENE EXPRESSION OF BARRAMUNDI, Lates calcarifer POST-INFECTION OF NERVOUS NECROSIS VIRUS

The most common problem in barramundi Lates calcarifer seedling production is the high mortality (> 90%) caused by nervous necrosis virus (NNV) infection. This research aims to evaluate the resistance and gene expression of barramundi challenged by NNV. Two populations were used in this study, i.e., Australian, and Situbondo-originated barramundi populations. The immune-related gene expression levels in the liver, head of kidney, and spleen were observed at 48 and 96 hours after post-infection (hpi). Barramundi’s survival and blood parameters were evaluated post-NNV infection. The results showed that the highest survival was revealed in Situbondo’s barramundi (42.0±4.47%) compared to Australian barramundi (20.0±7.07%) and no mortality was observed in the control without NNV infection. The higher survival rate in barramundi from Situbondo was in line with the blood profile. The number of red blood cell from Situbondo barramundi post-NNV infection (ST) at 96 hpi was higher (P<0.05) than Australian barramundi post-NNV infection (AT). The number of white blood cell of ST at 48 hpi was higher (P<0.05) than AT, but started to decrease at 96 hpi in ST barramundi. The total white blood cell in AT barramundi increased from 48 to 192 hpi. TNFα and IL1-β gene expression levels were significantly higher in the liver, head kidney, and spleen of Situbondo compared to Australian barramundi at 48 hpi, while MHCIIα gene expression in Situbondo’s was significantly higher compared to Australian barramundi at 96 hpi. These results indicate the important roles of all the genes in the barramundi’s immune responses against viral infection. Based on the results of the research, Situbondo’s barramundi has the potential to be used as a candidate for generating broodstock of disease-resistant strain.