Differential Immune Transcriptome and Modulated Signalling Pathways in Rainbow Trout Infected with Viral Haemorrhagic Septicaemia Virus (VHSV) and Its Derivative Non-Virion (NV) Gene Deleted.

Viral haemorrhagic septicaemia virus (VHSV) is one of the worst viral threats to fish farming. Non-virion (NV) gene-deleted VHSV (dNV-VHSV) has been postulated as an attenuated virus, because the absence of the NV gene leads to lower induced pathogenicity. However, little is known about the immune responses driven by dNV-VHSV and the wild-type (wt)-VHSV in the context of infection. Here, we obtained the immune transcriptome profiling in trout infected with dNV-VHSV and wt-VHSV and the pathways involved in immune responses. As general results, dNV-VHSV upregulated more trout immune genes than wt-VHSV (65.6% vs 45.7%, respectively), whereas wt-VHSV maintained more non-regulated genes than dNV-VHSV (45.7% vs 14.6%, respectively). The modulated pathways analysis (Gene-Set Enrichment Analysis, GSEA) showed that, when compared to wt-VHSV infected trout, the dNV-VHSV infected trout upregulated signalling pathways (n = 19) such as RIG-I (retinoic acid-inducible gene-I) like receptor signalling, Toll-like receptor signalling, type II interferon signalling, and nuclear factor kappa B (NF-kappa B) signalling, among others. The results from individual genes and GSEA demonstrated that wt-VHSV impaired the activation at short stages of infection of pro-inflammatory, antiviral, proliferation, and apoptosis pathways, delaying innate humoral response and cellular crosstalk, whereas dNV-VHSV promoted the opposite effects. Therefore, these results might support future studies on using dNV-VHSV as a potential live vaccine.

Herpesvirus Infection Induces both Specific and Heterologous Antiviral Antibodies in Carp.

IgM antibody diversity induced by viral infection in teleost fish sera remains largely unexplored despite several studies performed on their transcript counterparts in lymphoid organs. Here, IgM binding to microarrays containing ~20,000 human proteins was used to study sera from carp (Cyprinus carpio) populations having high titers of viral neutralization in vitro after surviving an experimental infection with cyprinid herpes virus 3 (CyHV-3). The range of diversity of the induced antibodies was unexpectedly high, showing CyHV-3 infection-dependent, non-specific IgM-binding activity of a ~20-fold wider variety than that found in sera from healthy carp (natural antibodies) with no anti-CyHV-3 neutralization titers. An inverse correlation between the IgM-binding levels in healthy versus infection-survivor/healthy ratios suggests that an infection-dependent feed back-like mechanism may control such clonal expansion. Surprisingly, among the infection-expanded levels, not only specific anti-frgIICyHV-3 and anti-CyHV-3 IgM-binding antibodies but also antibodies recognizing recombinant fragment epitopes from heterologous fish rhabdoviruses were detected in infection-survivor carp sera. Some alternative explanations for these findings in lower vertebrates are discussed.

Thyroid active agents T3 and PTU differentially affect immune gene transcripts in the head kidney of rainbow trout (Oncorynchus mykiss).

In mammals, numerous reports describe an immunomodulating effect of thyroid-active compounds. In contrast, only few reports have been published on this subject in fish. We previously demonstrated that immune cells of rainbow trout (Oncorhynchus mykiss) possess thyroid hormone receptors (THRs) and that exposure of trout to the thyroid hormone 3,3',5-triiodo-l-thyronine (T3) or the antithyroid drug propylthiouracil (PTU) alters immune cell transcript levels of THR and several immune genes. The present study aims to further characterize the immunomodulating action of thyroid-active compounds in trout immune cells. We report here the use of a custom-designed 60-mer oligo immune-targeted microarray for rainbow trout to analyze the gene expression profiles induced in the head kidney by T3 and PTU. Morphometric analyses of the thyroid showed that PTU exposure increased the size of the epithelial cells, whereas T3 induced no significant effects. Both T3 and PTU had diverse and partly contrasting effects on immune transcript profiles. The strongest differential effects of T3 and PTU on gene expressions were those targeting the Mitogen Associated Protein Kinase (MAPK), NFkB, Natural Killer (NK) and Toll-Like Receptor (TLR) pathways, a number of multipath genes (MPG) such as those encoding pleiotropic transcription factors (atf1, junb, myc), as well as important pro-inflammatory genes (tnfa, tnf6, il1b) and interferon-related genes (ifng, irf10). With these results we show for the first time in a fish species that the in vivo thyroidal status modulates a diversity of immune genes and pathways. This knowledge provides the basis to investigate both mechanisms and consequences of thyroid hormone- and thyroid disruptor-mediated immunomodulation for the immunocompetence of fish.

Transcriptome analysis of rainbow trout in response to non-virion (NV) protein of viral haemorrhagic septicaemia virus (VHSV).

The non-virion (NV) protein of viral haemorrhagic septicaemia virus (VHSV), an economically important fish novirhabdovirus, has been implicated in the interference of some host innate mechanisms (i.e. apoptosis) in vitro. This work aimed to characterise the immune-related transcriptome changes in rainbow trout induced by NV protein that have not yet been established in vivo. For that purpose, immune-targeted microarrays were used to analyse the transcriptomes from head kidney and spleen of rainbow trout (Oncorhynchus mykiss) after injection of recombinant NV (rNV). Results showed the extensive downregulation (and in some cases upregulation) of many innate and adaptive immune response genes not related previously to VHSV infection. The newly identified genes belonged to VHSV-induced genes (vigs), tumour necrosis factors, Toll-like receptors, antigen processing and presentation, immune co-stimulatory molecules, interleukins, macrophage chemotaxis, transcription factors, etc. Classification of differentially downregulated genes into rainbow trout immune pathways identified stat1 and jun/atf1 transcription factor genes as the most representative of the multipath gene targets of rNV. Altogether, these results contribute to define the role and effects of NV in trout by orchestrating an immunosuppression of the innate immune responses for favouring viral replication upon VHSV infection. Finally, these transcriptome results open up the possibility to find out new strategies against VHSV and better understand the interrelationships between some immune pathways in trout.

Autophagy-inducing peptides from mammalian VSV and fish VHSV rhabdoviral G glycoproteins (G) as models for the development of new therapeutic molecules.

It has not been elucidated whether or not autophagy is induced by rhabdoviral G glycoproteins (G) in vertebrate organisms for which rhabdovirus infection is lethal. Our work provides the first evidence that both mammalian (vesicular stomatitis virus, VSV) and fish (viral hemorrhagic septicemia virus, VHSV, and spring viremia carp virus, SVCV) rhabdoviral Gs induce an autophagic antiviral program in vertebrate cell lines. The transcriptomic profiles obtained from zebrafish genetically immunized with either Gsvcv or Gvhsv suggest that autophagy is induced shortly after immunization and therefore, it may be an important component of the strong antiviral immune responses elicited by these viral proteins. Pepscan mapping of autophagy-inducing linear determinants of Gvhsv and Gvsv showed that peptides located in their fusion domains induce autophagy. Altogether these results suggest that strategies aimed at modulating autophagy could be used for the prevention and treatment of rhabdoviral infections such as rabies, which causes thousands of human deaths every year.

Antibodies against Marinobacter algicola and Salmonella typhimurium flagellins do not cross-neutralize TLR5 activation.

Flagellins evoke strong innate and adaptive immune responses. These proteins may play a key role as radioprotectors, exert antitumoral activity in certain types of tumor and reduce graft-versus-host disease in allogeneic hematopoietic stem cell transplant recipients. Notwithstanding, flagellins are highly immunogenic, and repeated use leads to their neutralization by systemic antibodies. This neutralization is not prevented by using functional deleted flagellins. These observations led us to explore the possibility of preventing initial neutralization by means of another functional flagellin that does not belong to common pathogenic bacteria but that has the capacity to activate TLR5. Here we characterized the functional capacity of the two-phase Marinobacter algicola (MA)-derived flagellins (F and FR) as systemic and mucosal adjuvants and compared their performance with that of Salmonella typhimurium (STF) flagellins (FljB and FliC). We also report for the first time on the in vitro and in vivo capacity of various flagellins to trigger TLR5 activation in the presence of species-specific anti-flagellin antibodies, the cross-neutralization mediated by these antibodies, and the sequential use of these flagellins for TLR5 activation. Our results showed that MA flagellins behave in a similar way to STF ones, inducing pro-inflammatory cytokines (IL8, CCL20, CCL2) and evoking a strong in vivo antibody response against a model epitope. More importantly, MA flagellins were fully functional, in vitro or in vivo, in the presence of a high concentration of neutralizing anti-flagellin STF antibodies, and STF flagellin was not inhibited by neutralizing anti-flagellin MA antibodies. The use of active flagellins from distinct bacteria could be a useful approach to prevent systemic neutralization of this group of adjuvants and to facilitate the rational design of flagellin-based vaccines and/or other therapeutic treatments (against ischemia, acute renal failure, tumors, ionizing radiations and also to improve the outcome of bone marrow transplants).

Trout oral VP2 DNA vaccination mimics transcriptional responses occurring after infection with infectious pancreatic necrosis virus (IPNV).

Time-course and organ transcriptional response profiles in rainbow trout Oncorhynchus mykiss were studied after oral DNA-vaccination with the VP2 gene of the infectious pancreatic necrosis virus (IPNV) encapsulated in alginates. The profiles were also compared with those obtained after infection with IPNV. A group of immune-related genes (stat1, ifn1, ifng, mx1, mx3, il8, il10, il11, il12b, tnf2, mhc1uda, igm and igt) previously selected from microarray analysis of successful oral vaccination of rainbow trout, were used for the RTqPCR analysis. The results showed that oral VP2-vaccination qualitatively mimicked both the time-course and organ (head kidney, spleen, intestine, pyloric ceca, and thymus) transcriptional profiles obtained after IPNV-infection. Highest transcriptional differential expression levels after oral vaccination were obtained in thymus, suggesting those might be important for subsequent protection against IPNV challenges. However, transcriptional differential expression levels of most of the genes mentioned above were lower in VP2-vaccinated than in IPNV-infected trout, except for ifn1 which were similar. Together all the results suggest that the oral-alginate VP2-vaccination procedure immunizes trout against IPNV in a similar way as IPNV-infection does while there is still room for additional improvements in the oral vaccination procedure. Some of the genes described here could be used as markers to further optimize the oral immunization method.

Oral immunization of rainbow trout to infectious pancreatic necrosis virus (Ipnv) induces different immune gene expression profiles in head kidney and pyloric ceca.

Induction of neutralizing antibodies and protection by oral vaccination with DNA-alginates of rainbow trout Oncorhynchus mykiss against infectious pancreatic necrosis virus (IPNV) was recently reported. Because orally induced immune response transcript gene profiles had not been described yet neither in fish, nor after IPNV vaccination, we studied them in head kidney (an immune response internal organ) and a vaccine entry tissue (pyloric ceca). By using an oligo microarray enriched in immune-related genes validated by RTqPCR, the number of increased transcripts in head kidney was higher than in pyloric ceca while the number of decreased transcripts was higher in pyloric ceca than in head kidney. Confirming previous reports on intramuscular DNA vaccination or viral infection, mx genes increased their transcription in head kidney. Other transcript responses such as those corresponding to interferons, their receptors and induced proteins (n=91 genes), VHSV-induced genes (n=25), macrophage-related genes (n=125), complement component genes (n=176), toll-like receptors (n=31), tumor necrosis factors (n=32), chemokines and their receptors (n=121), interleukines and their receptors (n=119), antimicrobial peptides (n=59), and cluster differentiation antigens (n=58) showed a contrasting and often complementary behavior when head kidney and pyloric ceca were compared. For instance, classical complement component transcripts increased in head kidney while only alternative pathway transcripts increased in pyloric ceca, different ß-defensins increased in head kidney but remained constant in pyloric ceca. The identification of new gene markers on head kidney/pyloric ceca could be used to follow up and/or to improve immunity during fish oral vaccination.