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.

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.

Fish RIP1 Mediates Innate Antiviral Immune Responses Induced by SGIV and RGNNV Infection.

Receptor interacting protein 1 (RIP1) is an essential sensor of cellular stress, which may respond to apoptosis or cell survival and participate in antiviral pathways. To investigate the roles of fish RIP1 in Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) infection, a RIP1 homolog from orange-spotted grouper (Epinephelus coioides) (EcRIP1) was cloned and characterized. EcRIP1 encoded a 679 amino acid protein that shares 83.28% identity with that of Perca flavescens and contained a homologous N-terminal kinase (S-TKc) domain, a RIP isotype interaction motif (RHIM), and a C-terminal domain (DD). EcRIP1 was predominantly detected in immune tissues, and its expression was induced by RGNNV or SGIV infection in vitro. Subcellular localization showed that EcRIP1 was distributed in the cytoplasm with point-like uniform and dot-like aggregation forms. Overexpression of EcRIP1 inhibited SGIV and RGNNV replication and positively regulated the expression levels of interferon (IFN) and IFN-stimulated genes and pro-inflammatory factors. EcRIP1 may interact with grouper tumor necrosis factor receptor type 1-associated DEATH domain protein (EcTRADD) to promote SGIV-induced apoptosis, and interact with grouper Toll/interleukin-1 receptor (TIR) domain containing adapter inducing interferon-ß (EcTRIF) and participate in Myeloid Differentiation Factor 88 (MyD88)-independent toll-like receptor (TLR) signaling. EcRIP1 may also interact with grouper tumor necrosis factor receptor-associated factors (TRAFs) as intracellular linker proteins and mediate the signaling of various downstream signaling pathways, including NF-κB and IFN. These results suggest that EcRIP1 may inhibit SGIV and RGNNV infection by regulating apoptosis and various signaling molecules. Our study offers new insights into the regulatory mechanism of RIP1-related signaling, and provides a novel perspective on fish diseases mediated by RIP1.

Fish Granzyme A Shows a Greater Role Than Granzyme B in Fish Innate Cell-Mediated Cytotoxicity.

Granzymes (Gzm) are serine proteases, contained into the secretory granules of cytotoxic cells, responsible for the cell-mediated cytotoxicity (CMC) against tumor cells and intracellular pathogens such as virus and bacteria. In fish, they have received little attention to their existence, classification or functional characterization. Therefore, we aimed to identify and evaluate their functional and transcriptomic relevance in the innate CMC activity of two relevant teleost fish species, gilthead seabream and European sea bass. Afterwards, we wanted to focus on their regulation upon nodavirus (NNV) infection, a virus that causes great mortalities to sea bass specimens while seabream is resistant. In this study, we have identified genes encoding GzmA and GzmB in both seabream and sea bass, as well as GzmM in seabream, which showed good phylogenetic relation to their mammalian orthologs. In addition, we found enzymatic activity related to tryptase (GzmA and/or GzmK), aspartase (GzmB), metase (GzmM), or chymase (GzmH) in resting head-kidney leucocytes (HKLs), with the following order of activity: GzmA/K ~ GzmM >> GzmH >>> GzmB. In addition, during innate CMC assays consisting on HKLs exposed to either mock- or NNV-infected target cells, though all the granzyme transcripts were increased only the tryptase activity did. Thus, our data suggest a high functional activity of GzmA/K in the innate CMC and a marginal one for GzmB. Moreover, GzmB activity was detected into target cells during the CMC assays. However, the percentage of target cells with GzmB activity after the CMC assays was about 10-fold lower than the death target cells, demonstrating that GzmB is not the main inductor of cell death. Moreover, in in vivo infection with NNV, gzm transcription is differently regulated depending on the fish species, genes and tissues. However, the immunohistochemistry study revealed an increased number of GzmB stained cells and areas in the brain of seabream after NNV infection, which was mainly associated with the lesions detected. Further studies are needed to ascertain the molecular nature, biological function and implication of fish granzymes in the CMC activity, and in the antiviral defense in particular.

Oral immunization with recombinant protein antigen expressed in tobacco against fish nervous necrosis virus.

Nervous necrosis virus (NNV), also known as betanodavirus, has been recently implicated in mass mortalities of cultured marine fish. An effective vaccine is urgently needed to protect fish against this virus. However, parenteral immunization methods are very stressful. Individual immunization for thousands of fish is very labor intensive and expensive. Therefore, we expressed NNV coat protein in tobacco chloroplasts and used it as an oral vaccine to induce immunities in fish followed by challenges with NNV. Our results revealed that mice (IgG and IgA) and fish (IgM) immunized with the oral vaccine developed significantly higher antibody titers against the NNV coat protein. Fish were partially protected against viral challenge. Taken together, our results demonstrated that a plant-based vaccine could effectively induce immune response and protect groupers against NNV. The present method could be used to develop oral fish vaccine in the future.

Immunoglobulin T from sea bass (Dicentrarchus labrax L.): molecular characterization, tissue localization and expression after nodavirus infection.

BACKGROUND: Immunoglobulins (Igs) are fundamental components of the adaptive immune system of vertebrates, with the IgT/IgZ isotype specific of Teleosts. In this paper we describe the identification of an IgT heavy chain from the European sea bass (Dicentrarchus labrax L.), its molecular characterization and tissue mRNA localization by in situ hybridization. RESULTS: Sea bass IgT consists of 552 aa (Accession Number KM410929) and it contains a putative 19 amino acids long signal peptide and one potential N-glycosylation site. The C-region consists of four CH domains; each contains the cysteine and tryptophan residues required for their correct folding. Based on the recent sequencing of sea bass genome, we have identified five different genomic contigs bearing exons unequivocally pertaining to IgT (CH2, CH3 and CH4), but none corresponded to a complete IgH locus as IgT sequences were found in the highly fragmented assembled genomic regions which could not be assigned to any major scaffold. The 3D structure of sea bass IgT has been modelled using the crystal structure of a mouse Ig gamma as a template, thus showing that the amino acid sequence is suitable for the expected topology referred to an immunoglobulin-like architecture. The basal expression of sea bass IgT and IgM in different organs has been analysed: gut and gills, important mucosal organs, showed high IgT transcripts levels and this was the first indication of the possible involvement of sea bass IgT in mucosal immune responses. Moreover, sea bass IgT expression increased in gills and spleen after infection with nodavirus, highlighting the importance of IgT in sea bass immune responses. In situ hybridization confirmed the presence of IgT transcripts in the gut and it revealed a differential expression along the intestinal tract, with a major expression in the posterior intestine, suggesting the hindgut as a site for the recruitment of IgT+ cells in this species. IgT transcripts were also found in gill filaments and parallel lamellae and, for the first time, we identified scattered IgT positive cells in the liver, with a strong signal in the hepatic parenchyma. CONCLUSIONS: In conclusion, we performed a full molecular characterization of IgT in sea bass that points out its possible involvement in mucosal immune responses of this species.

Interleukin-1ß secreted from betanodavirus-infected microglia caused the death of neurons in giant grouper brains.

High interleukin (IL)-1ß gene expression was observed in dead giant grouper brains after nervous necrosis virus (NNV) infection. To investigate the neuronal death caused by NNV infection, primary tissue culture of giant grouper brains (pGB) was performed. In NNV-infected pGB cells, the viral capsid protein was detected in both neurons and microglia; furthermore, microglial proliferation and neuronal death were observed. The culture supernatant (CS) of NNV-infected pGB cells contained IL-1ß and tumor necrosis factor-α, which were mainly released from the microglia. A new batch of pGB cells was treated with CS, resulting in neuronal death, which could be prevented by blocking the IL-1ß in the CS by using anti-IL-1ß polyclonal antibodies. Moreover, pGB cells treated with recombinant IL-1ß showed microglial proliferation and neuronal death. Thus, NNV infection may activate microglial proliferation and stimulate microglial secretion of IL-1ß, which is a critical cytokine responsible for neuronal death in NNV-infected grouper brains.

Identification of sea perch (Lateolabrax japonicus) ribonucleoprotein PTB-Binding 1 involved in antiviral immune response against RGNNV.

RIG-I-like receptors (RLRs) can recognize viral RNA and initiate innate antiviral response. In earlier studies, we demonstrated that RLRs were implicated in the antiviral immunity against RGNNV in the seawater fish sea perch (Lateolabrax japonicus). However, potential regulators of RLRs-mediated signaling pathways involved in RGNNV infection remain unclear. In this study, a novel ribonucleoprotein PTB-binding 1 (Raver1) of sea perch (LjRAVER1) was identified for the first time. The cDNA of LjRAVER1 was 4066 bp in length and encoded a deduced polypeptide of 733 amino acids. Phylogenetic analysis revealed a closer affinity of LjRAVER1 with Larimichthys Crocea Raver1. LjRAVER1 mRNA was constitutively expressed in all 10 sampled tissues, and rapidly and significantly increased in vivo upon RGNNV infection. Time course analysis showed that LjRAVER1 transcripts were significantly increased both in vivo and in vitro after RGNNV infection. Viral infection and poly I:C treatment caused translocation of LjRAVER1 from the nucleus to the cytoplasm. Ectopic expression of LjRAVER1 increased the transcription level of several RLR signaling pathway related genes inducible by poly I:C treatment in vitro. Moreover, the viral gene transcription and virus production of RGNNV were significantly decreased in LjRAVER1 overexpressing cells. Luciferase reporter assays demonstrated that overexpression of LjRAVER1 significantly increased the promoter activity of zebrafish IFN1. Taken together, these findings indicated that LjRAVER1 might be an important component of RLR signaling pathway and involved in RLR pathway-mediated IFN response in sea perch.

Molecular Basis for Antigenic Diversity of Genus Betanodavirus.

Betanodaviruses are the causative agents of viral nervous necrosis (VNN), a devastating disease for the Mediterranean mariculture. Four different betanodavirus species are recognized, Striped jack-, Redspotted grouper-, Tiger puffer-, and Barfin flounder nervous necrosis virus (SJNNV, RGNNV, TPNNV and BFNNV), but there is little knowledge on their antigenic properties. In order to describe the serological relationships among different betanodavirus genotypes, serum neutralization assays were performed using rabbit polyclonal antisera against eight fish nodaviruses that cover a wide species-, temporal-, spatial- and genetic range. The results indicate that the SJNNV and RGNNV are antigenically distinct, constituting serotypes A and C, respectively. The TPNNV and BFNNV, the latter representing cold-water betanodaviruses, are antigenically related and cluster within serotype B. The reassortant viruses RGNNV/SJNNV and SJNNV/RGNNV group within serotypes A and C, respectively, indicating that the coat protein encoded by RNA2 acts as major immunoreactivity determinant. Immunostaining of in vitro expressed wild type and chimeric capsid proteins between the RGNNV and the SJNNV species indicated that the C-terminal part of the capsid protein retains the immunoreactive portion. The amino acid (aa) residues determining RGNNV and SJNNV antigenic diversity were mapped to aa residues 217-256 and aa 257-341, respectively. Neutralization of reverse genetics derived chimeric viruses indicated that these areas determine the neutralizing epitopes. The data obtained are crucial for the development of targeted serological tests for the diagnosis of VNN, and informative for development of cross-protective vaccines against various betanodavirus genotypes.

An oral chitosan DNA vaccine against nodavirus improves transcription of cell-mediated cytotoxicity and interferon genes in the European sea bass juveniles gut and survival upon infection.

Vaccines for fish need to be improved for the aquaculture sector, with DNA vaccines and the oral administration route providing the most promising improvements. In this study, we have created an oral chitosan-encapsulated DNA vaccine (CP-pNNV) for the nodavirus (NNV) in order to protect the very susceptible European sea bass (Dicentrarchus labrax). Our data show that the oral CP-pNNV vaccine failed to induce serum circulating or neutralizing specific antibodies (immunoglobulin M) or to up-regulate their gene expression in the posterior gut. However, the vaccine up-regulated the expression of genes related to the cell-mediated cytotoxicity (CMC; tcrb and cd8a) and the interferon pathway (IFN; ifn, mx and ifng). In addition, 3 months after vaccination, challenged fish showed a retarded onset of fish death and lower cumulative mortality with a relative survival of 45%. Thus, we created a chitosan-encapsulated DNA vaccine against NNV that is partly protective to European sea bass juveniles and up-regulates the transcription of genes related to CMC and IFN. However, further studies are needed to improve the anti-NNV vaccine and to understand its mechanisms.

Identification and characterization of the melanoma differentiation - associated gene 5 in sea perch, Lateolabrax japonicus.

The RIG-I-like receptors family is a group of cytosolic RNA helicase proteins that can recognize viral RNA via binding to pathogen associated molecular pattern motifs within RNA ligands. A novel vertebrate RLR counterpart named LjMDA5 was firstly identified from the marine fish sea perch Lateolabrax japonicus in this study. The full-length cDNA of LjMDA5 is 3750 bp and encodes a polypeptide of 988 amino acids, containing two N-terminal tandem caspase activation and recruitment domains, a DExH (Asp-Glu-X-His) box domain, an HELICc domain, and a C-terminal domain RIG-I. Phylogenetic analysis showed that LjMDA5 shared the closest genetic relationship with the MDA5 of Larimichthys crocea. Quantitative RT-PCR analysis showed that LjMDA5 was ubiquitously expressed and up-regulated significantly in all selected tissues in vivo post NNV infection. Time course analysis showed that LjMDA5 transcripts significantly increased in spleen and kidney. We found LjMDA5 could be regulated in the sea perch LJB and LJF cell lines after lipopolysaccharide, polyinosinic-polycytidylic acid treatment and NNV challenge. RNA interference experiment indicated that silencing of LjMDA5 significantly increased RGNNV replication and virus production in NNV infected LJF cells. Our results revealed that MDA5 was essential for host defense against NNV, which provided new insights into the function of RLR signaling pathway during NNV infection in fish.

Molecular characterization and expression analysis of mitochondrial antiviral signaling protein gene in sea perch, Lateolabrax japonicus.

The mitochondrial antiviral signaling protein (MAVS) is vital for host defenses against viral infection by inducing expression of type I interferon. Here, the MAVS of sea perch (Lateolabrax japonicus) (LjMAVS) was cloned and analyzed. The complete cDNA sequence of LjMAVS was 3207 bp and encoded a polypeptide of 601 amino acids. LjMAVS contains an N-terminal CARD-like domain, a central proline-rich domain and a C-terminal transmembrane domain. Phylogenetic analysis indicated that LjMAVS exhibited the closest relationship to O. fasciatus MAVS. LjMAVS was ubiquitously expressed in all tested tissues of healthy fish. The expression of LjMAVS was significantly increased post nervous necrosis virus (NNV) infection in vivo in all the selected tissues. Furthermore, time course analysis showed that LjMAVS transcripts significantly increased in the brain, spleen and kidney tissues after NNV infection. LjMAVS mRNA expression was significantly up-regulated in vitro after poly I:C stimulation. The viral gene transcription of RGNNV was significantly decreased in LjMAVS over-expressing LJB cells. These findings provide useful information for further elucidating the function ofLjMAVS in antiviral innate immune against NNV in sea perch.

Recognition of Linear B-Cell Epitope of Betanodavirus Coat Protein by RG-M18 Neutralizing mAB Inhibits Giant Grouper Nervous Necrosis Virus (GGNNV) Infection.

Betanodavirus is a causative agent of viral nervous necrosis syndrome in many important aquaculture marine fish larvae, resulting in high global mortality. The coat protein of Betanodavirus is the sole structural protein, and it can assemble the virion particle by itself. In this study, we used a high-titer neutralizing mAB, RG-M18, to identify the linear B-cell epitope on the viral coat protein. By mapping a series of recombinant proteins generated using the E. coli PET expression system, we demonstrated that the linear epitope recognized by RG-M18 is located at the C-terminus of the coat protein, between amino acid residues 195 and 338. To define the minimal epitope region, a set of overlapping peptides were synthesized and evaluated for RG-M18 binding. Such analysis identified the 195VNVSVLCR202 motif as the minimal epitope. Comparative analysis of Alanine scanning mutagenesis with dot-blotting and ELISA revealed that Valine197, Valine199, and Cysteine201 are critical for antibody binding. Substitution of Leucine200 in the RGNNV, BFNNV, and TPNNV genotypes with Methionine200 (thereby simulating the SJNNV genotype) did not affect binding affinity, implying that RG-M18 can recognize all genotypes of Betanodaviruses. In competition experiments, synthetic multiple antigen peptides of this epitope dramatically suppressed giant grouper nervous necrosis virus (GGNNV) propagation in grouper brain cells. The data provide new insights into the protective mechanism of this neutralizing mAB, with broader implications for Betanodavirus vaccinology and antiviral peptide drug development.

Characterization of the IFN pathway in the teleost fish gonad against vertically transmitted viral nervous necrosis virus.

One of the most powerful innate immune responses against viruses is mediated by type I IFN. In teleost fish, it is known that virus infection triggers the expression of ifn and many IFN-stimulated genes, but the viral RNA sensors and mediators leading to IFN production are scarcely known. Thus, we have searched for the presence of these genes in gilt-head sea bream (Sparus aurata) and European sea bass (Dicentrarchus labrax), and evaluated their expression after infection with viral nervous necrosis virus (VNNV) in the brain, the main viral target tissue, and the gonad, used to transmit the virus vertically. In sea bream, a fish species resistant to the VNNV strain used, we found an upregulation of the genes encoding MDA5 (melanoma differentiation-associated gene 5), TBK1 (TANK-binding kinase 1), IRF3 (IFN regulatory factor 3), IFN, Mx [myxovirus (influenza) resistance protein] and PKR (dsRNA-dependent protein kinase receptor) proteins in the brain, which were unaltered in the gonad and could favour the dissemination by gonad fluids or gametes. Strikingly, in European sea bass, a very susceptible species, we also identified, transcripts coding for LGP2 (Laboratory of Genetics and Physiology 2), MAVS (mitochondrial antiviral signalling), TRAF3 (TNF receptor-associated factor 3), TANK (TRAF family member-associated NFκB activator) and IRF7 (IFN regulatory factor 7), and found that all the genes analysed were upregulated in the gonad, but only mda5, lgp2, irf3, mx and pkr were upregulated in the brain. These findings supported the notion that the European sea bass brain innate immune response is unable to clear the virus and pointed to the importance of gonad immunity to control the dissemination of VNNV to the progeny--an aspect that is worth investigating in aquatic animals.

Functional analysis of an orange-spotted grouper (Epinephelus coioides) interferon gene and characterisation of its expression in response to nodavirus infection.

We cloned and sequenced 2C I-IFN, a two-cysteine containing type I interferon (I-IFN) gene, in orange-spotted grouper (Epinephelus coioides). The cDNA has 769 base pairs, the protein has 172 amino acids, and the predicted signal peptide has 18 amino acids with two cysteines. This gene is similar to I-FNs from sea bass and other teleosts. 2C I-IFN has 5 exons and 4 introns, also similar to other teleost I-IFNs. Immunohistochemical (IHC) analysis indicated that expression is predominantly membrane-localized in healthy grouper, but has a zonal distribution in nodavirus-infected grouper. Grouper infected with nodavirus had elevated levels of 2C I-IFN at 72 h and Mx at days 6-7. Recombinant 2C I-IFN activated grouper Mx, leading to upregulated antiviral activity. The grouper Mx promoter was highly induced after treatment with recombinant 2C I-IFN. The present results suggest that expression of grouper 2C I-IFN may participate in the immunologic barrier function against nodavirus.

Molecular characterization and functional analysis of Toll-like receptor 3 gene in orange-spotted grouper (Epinephelus coioides).

Toll-like receptor 3 (TLR3) plays an important role in activating innate immune responses during viral infection. In this report, TLR3 (EcTLR3) was characterized and analyzed for the first time in Epinephelus coioides. The full-length EcTLR3 cDNA is predicted to encode a 909 amino acid polypeptide that contains a signal peptide sequence, 18 leucine-rich repeat (LRR) motifs, a transmembrane region and a Toll/interleukin-1 receptor (TIR) domain. Quantitative real-time PCR revealed that the EcTLR3 mRNA was much more abundant in the liver than in other immune organs, and that the expression levels were very low in hemocyte and muscle. During development of the grouper, the levels of EcTLR3 transcripts increased with age, with very low expression levels at the early stages of development. EcTLR3 mRNA levels were examined in the liver at different times after treatment with polyriboinosinic polyribocytidylic acid (Poly I:C), and in nervous necrosis virus (NNV)-infected larval groupers. The results suggested that EcTLR3 plays an important role in a fish's defense against viral infection.

Atlantic halibut (Hippoglossus hippoglossus L.) T-cell and cytokine response after vaccination and challenge with nodavirus.

Viral encephalopathy and retinopathy (VER), caused by nodavirus, is one of the major infectious diseases affecting the marine fish farming industry, yet no effective vaccine is available. In this study, we examined the halibut immune response following administration of an experimental vaccine comprising a recombinant nodavirus capsid protein in combination with an oil adjuvant (OA). Four groups of halibut were injected with either: PBS alone, PBS plus OA, 10µg recCP plus OA, or 50µg recCP plus OA. 15 weeks later, half the fish in each group were challenged with nodavirus and the immune response investigated by analysis of: serum levels of recCP-specific halibut immunoglobulins (Igs), and mRNA transcript levels of several T-cell markers (CD3ɛ, Lck, CD4, CD4-2, CD8α and CD8ß) and cytokines (IL-1ß, IL-6, IL-12ßc and IFNγ). Additionally, the presence of nodaviral RNA2 transcripts in the brains of infected halibut was analysed. After vaccination, the level of IL-6 was consistently elevated in the spleens of fish given injections containing the OA. The combination of recCP and OA increased the expression of IL-1ß and IFNγ, as well as the level of recCP-specific Igs in blood plasma. Following challenge with nodavirus, IL-1ß and IFNγ were elevated in halibut spleens after 24h in all groups that had received OA with or without recCP antigen. In brain, a general increase in the expression levels of all T-cell markers and IFNγ was observed following challenge with nodavirus. The viral load at 8 weeks post-challenge was lower in the fish that received 50µg recCP, with 5 out of 8 individuals being negative for nodavirus. Additionally, a better correlation between these markers (apart from the CD8 markers), and the viral RNA2 was also observed in this group, suggesting that the activation of CD4+T-cells might be important in reducing the viral load. In conclusion, this study identifies recCP as a promising candidate antigen for the future development of a vaccine against nodavirus.

Molecular cloning, expression and functional analysis of ISG15 in orange-spotted grouper, Epinephelus coioides.

Interferon-stimulated gene 15 (ISG15) is an ubiquitin homolog that is significantly induced by type I interferons or viral infections. Groupers, Epinephelus spp. being maricultured in China and Southeast Asian countries, always suffer from virus infection, including iridovirus and nodavirus. To date, the roles of grouper genes, especially interferon related genes in virus infection remained largely unknown. Here, the ISG15 homolog (EcISG15) was cloned from grouper Epinephelus coioides and its immune response to Singapore grouper iridovirus (SGIV) and grouper nervous necrosis virus (GNNV) was investigated. The full-length EcISG15 cDNA was composed of 948 bp and encoded a polypeptide of 155 amino acids with 37-68% identity with the known ISG15 homologs from other fish species. Amino acid alignment analysis indicated that EcISG15 contained two ubiquitin-like (UBL) domains and an Ub-conjugation domain (LRGG). Expressional analysis showed that EcISG15 was dramatically induced by GNNV infection, poly I:C or poly dA-dT treatment, but no obvious changes were observed during SGIV infection. Immunofluorescence assay showed that EcISG15 localized mainly in the cytoplasm of grouper cells in response to poly I:C stimulation or GNNV infection, but not in mock or SGIV infected cells. Western blot analysis indicated that the ISGylation was absent in SGIV-infected cells, but significantly enhanced in GNNV-infected or poly I:C transfected cells, suggesting that EcISG15 might play different roles in SGIV and GNNV infection. Furthermore, overexpression of EcISG15 in vitro inhibited the transcription of GNNV genes significantly. Taken together, the results indicated that fish ISG15 might exert important roles against RNA virus infection.

Genomic analysis of the host response to nervous necrosis virus in Atlantic cod (Gadus morhua) brain.

Genome sequencing combined with transcriptome profiling promotes exploration of defence against pathogens and discovery of immune genes. Based on sequences from the recently released genome of Atlantic cod, a genome-wide oligonucleotide microarray (ACIQ-1) was designed and used for analyses of gene expression in the brain during infection with nervous necrosis virus (NNV). A challenge experiment with NNV was performed with Atlantic cod juveniles and brain samples from virus infected and uninfected fish were used for microarray analysis. Expression of virus induced genes increased at 5 days post challenge and persisted at stable level to the last sampling at 25 days post challenge. A large fraction of the up-regulated genes (546 features) were known or expected to have immune functions and most of these have not previously been characterized in Atlantic cod. Transcriptomic changes induced by the virus involved strong activation of genes associated with interferon and tumour necrosis factor related responses and acute inflammation. Up-regulation of genes involved in adaptive immunity suggested a rapid recruitment of B and T lymphocytes to the NNV infected brain. QPCR analyses of 15 candidate genes of innate immunity showed rapid induction by poly(I:C) in Atlantic cod larvae cells suggesting an antiviral role. Earliest and greatest expression changes after poly I:C stimulation was observed for interferon regulatory factors IRF4 and IRF7. Comparative studies between teleost species provided new knowledge about the evolution of innate antiviral immunity in fish. A number of genes is present or responds to viruses only in fish. Innate immunity of Atlantic cod is characterized by selective expansion of several medium-sized multigene families with ribose binding domains. An interesting finding was the high representation of three large gene families among the early antiviral genes, including tripartite motif proteins (TRIM) and proteins with PRY-SPRY and NACHT domains. The latter two with respectively 52 and 114 members in Atlantic cod have gone through expansions in different groups of fish. These proteins most likely have ligand binding properties and their propagation could be linked to the loss of MHC class II in the Atlantic cod genome.

A CD83-like molecule in sea bass (Dicentrarchus labrax): molecular characterization and modulation by viral and bacterial infection.

The CD83 cell surface marker is an important and intriguing component of immune system. It is considered the best marker for mature human dendritic cells, but it is also important for thymic development of T cells, and it also plays a role as a regulator of peripheral B-cell function and homeostasis. A CD83-like molecule was identified in sea bass (Dicentrarchus labrax) by EST sequencing of a thymus cDNA library; the CD83 cDNA is composed of 816 bp and the mature CD83 peptide consists of 195 amino acids, with a putative signal peptide of 18 amino acids and two possible N-glycosylation sites. The comparison of sea bass CD83 sequence with its homologues in other fish species and mammals shows some differences, with two cysteine residues conserved from fish to mammals and a high variability both in the total number of cysteines and in mature CD83 sequence polypeptide length. Basal transcripts levels of CD83 mRNA are highest in liver, followed by thymus. The in vitro treatment of head kidney leukocytes with LPS resulted in a down-regulation on CD83 mRNA leves both after 4 and 24 h, whereas with poly I:C an up-regulation after 4h followed by a down-regulation at 24 h was observed. An in vivo infection of sea bass juveniles with nodavirus induced an increase of CD83 expression on head kidney leukocytes both after 6 and 24 h and a decrease after 72 h. On the other hand, an in vivo infection with Photobacterium damselae bacteria induced a decrease of CD83 transcript levels after 6 and 24 h and an increase after 72 h. These findings suggest in sea bass CD83 expression could be modulated by viral and bacterial immune response.