BackgroundDespite rhabdoviral infections being one of the best known fish diseases, the gene expression changes induced at the surface tissues after the natural route of infection (infection-by-immersion) have not been described yet. This work describes the differential infected versus non-infected expression of proteins and immune-related transcripts in fins and organs of zebrafish Danio rerio shortly after infection-by-immersion with viral haemorrhagic septicemia virus (VHSV).ResultsTwo-dimensional differential gel electrophoresis detected variations on the protein levels of the enzymes of the glycolytic pathway and cytoskeleton components but it detected very few immune-related proteins. Differential expression of immune-related gene transcripts estimated by quantitative polymerase chain reaction arrays and hybridization to oligo microarrays showed that while more transcripts increased in fins than in organs (spleen, head kidney and liver), more transcripts decreased in organs than in fins. Increased differential transcript levels in fins detected by both arrays corresponded to previously described infection-related genes such as complement components (c3b, c8 and c9) or class I histocompatibility antigens (mhc1) and to newly described genes such as secreted immunoglobulin domain (sid4), macrophage stimulating factor (mst1) and a cluster differentiation antigen (cd36).ConclusionsThe genes described would contribute to the knowledge of the earliest molecular events occurring in the fish surfaces at the beginning of natural rhabdoviral infections and/or might be new candidates to be tested as adjuvants for fish vaccines.

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It has been described that fish nucleated red blood cells (RBCs) Background: generate a wide variety of immune-related gene transcripts when viruses highly replicate inside them and are their main target cell. The immune response and mechanisms of fish RBCs against viruses targeting other cells or tissues has not yet been explored and is the objective of our study.Trout RBCs were obtained from peripheral blood, ficoll purified and Methods: exposed to (VHSV). Immune response Viral Haemorrhagic Septicaemia virus was evaluated by means of RT-qPCR, flow cytometry, immunofluorescence and isobaric tag for relative and absolute quantification (iTRAQ) protein profiling VHSV N gene transcripts incremented early postexposure and were Results: drastically decreased after 6 hours postexposure (hpe). The expression of the type I interferon ( ) gene was significantly downregulated at early ifn1 postexposure (3 hpe), together with a gradual downregulation of interferon-inducible and genes until 72 hpe. Type I IFN protein was mx pkr downregulated and interferon-inducible Mx protein was maintained at basal levels. Co-culture assays of RBCs with TSS (stromal cell line from spleen) revealed the IFN crosstalk between both cell types. On the other hand, anti-microbial peptide β-defensin 1 and neutrophil chemotactic factor interleukin 8 were slightly upregulated in VHSV-exposed RBCs Isobaric tag for relative and absolute quantification (iTRAQ) revealed that VHSV exposure can induce a global protein downregulation in trout RBCs, mainly related to RNA stability and proteasome pathways. The antioxidant/antiviral response is also suggested to be involved in the response of trout RBCs to VHSV.

Rhabdoviruses show an RNA-containing helically-wound nucleocapsid either enclosed by or enclosing a membrane M protein, surrounded by a lipid bilayer through which dynamic protein trimers made up of non-covalently associated monomers of glycoprotein G (G) project outside. Mature monomeric rhabdoviral G has more than 500 amino acids, 2-6 potential glycosylation sites, 12-16 highly conserved cysteine residues, 2-3 stretches of a-d hydrophobic heptad-repeats, a removed amino terminal hydrophobic signal peptide, a close to the carboxy terminal hydrophobic transmembrane sequence and a carboxy terminal short hydrophylic cytoplasmic domain. Association-dissociation between monomers-trimers and displacement of the trimers along the plane of the lipid membrane, are induced by changes in the external conditions (pH, temperature, detergents, etc.). Throughout conformational changes the G trimers are responsible for the virus attachment to cell receptors, for low-pH membrane fusion and for reacting with host neutralizing monoclonal antibodies (MAbs). Antigenic differences could exist between monomers and trimers, which may have implications for future vaccine developments. The family Rhabdoviridae is made up of the Lyssavirus (rabies), the Vesiculovirus (vesicular stomatitis virus, VSV) and many rhabdoviruses infecting fish, plants, and arthropod insects. All these reasons make the G of rhabdoviruses an ideal subject to study comparative virology and to investigate new vaccine technologies.

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Agitation with glass beads, electroporation and microparticle bombardment are all used to transfer exogenous genes into unicellular eukaryotic algae (microalgae). For nuclear transformation most researchers use glass beads techniques or, to a lesser extent, electroporation, while for chloroplast transformation bombardment is often used. Glass bead agitation and electroporation require the removal of the cell wall while bombardment can be performed with intact microalgae. Chlamydomonas reinhardtii has been the microalga most commonly transformed, but success has also been reported with Volvox carteri, Chlorella spp., Dunaliella salina, Haematococcus pluvialis, Euglena gracilis, diatoms (Phaeodactylum tricornutum, Navicula saprophila, Cyclotella cryptica and Thalassiosira weissflogii), dinoflagellates (Amphidinium klebsii and Symbiodinium microadriaticum) and red algae (Porphyridium spp and Cyanidioschyzon merolae). The first C. reinhardtii transformants produced by making use of Agrobacterium tumefaciens have also been reported. A comparison of the above methods might help provide the information necessary to successfully transform further microalgal species.

Spring viremia carp virus (SVCV) is a rhabdovirus seasonally affecting warm-water cyprinid fish farming causing high impacts in worldwide economy. Because of the lack of effective preventive treatments, the identification of multipath genes involved in SVCV infection might be an alternative to explore the possibilities of using drugs for seasonal prevention of this fish disease. Because the zebrafish (Danio rerio) is a cyprinid susceptible to SVCV and their genetics and genome sequence are well advanced, it has been chosen as a model for SVCV infections. We have used newly designed pathway-targeted microarrays 3-4-fold enriched for immune/infection functional-relevant probes by using zebrafish orthologous to human genes from selected pathways of the Kyoto Encyclopedia of Genes and Genomes (KEGG). The comparative analysis of differential expression of genes through 20 pathways in 2-day exposed or 30-day survivors of SVCV infection allowed the identification of 16 multipath genes common to more than 6 pathways. In addition, receptors (Toll-like, B-cell, T-cell, RIG1-like) as well as viral RNA infection pathways were identified as the most important human-like pathways targeted by SVCV infection. Furthermore, by using bioinformatic tools to compare the promoter sequences corresponding to up and downregulated multipath gene groups, we identified putative common transcription factors which might be controlling such responses in a coordinated manner. Possible drug candidates to be tested in fish, can be identified now through search of data bases among those associated with the human orthologous to the zebrafish multipath genes. With the use of pathway-targeted microarrays, we identified some of the most important genes and transcription factors which might be implicated in viral shutoff and/or host survival responses after SVCV infection. These results could contribute to develop novel drug-based prevention methods and consolidate the zebrafish/SVCV as a model for vertebrate viral diseases.