Gene expression profile after injection of recombinant deleted non-virion protein gene (NV) VHSV
ABSTRACT: The function of the non-virion protein (NV) of the Viral hemorrhagic septicemia virus (VHSV) has been long questioned but it still remains unknown. We report here the differences in gene expression profiles of trouts infected with deleted NV VHSV or wild type VHSV so that it could shed some light on the issue. Eight fingerlings of rainbow trout were used for the experiment. Four of them were injected with deleted NV VHSV and the rest were injected with wild type VHSV as a control. Head kidney and spleen of each trout were collected 2 days post-injection and total RNA was extracted.
Project description:Due to the common presence of a non-virion NV gene absent from other fish rhabdoviruses, NV-coding rhabdoviruses, such as the viral haemorrhagic septicemia (VHSV), were placed into a new Novirhabdovirus genus. Because conflicting results do exist on the importance of NV for VHSV trout pathogenicity and the NV function is still unclear, the effects of intraperitoneal injection of recombinant NV protein were studied by using home-designed immune-related microarrays from rainbow trout Oncorhynchus mykiss. Trouts were separated in two groups. One group was injected with PBS and it was used as control group. Second group was injected with soluble VHSV non-virion (NV) recombinant protein
Project description:Due to the common presence of a non-virion NV gene absent from other fish rhabdoviruses, NV-coding rhabdoviruses, such as the viral haemorrhagic septicemia (VHSV), were placed into a new Novirhabdovirus genus. Because conflicting results do exist on the importance of NV for VHSV trout pathogenicity and the NV function is still unclear, the effects of intraperitoneal injection of recombinant NV protein were studied by using home-designed immune-related microarrays from rainbow trout Oncorhynchus mykiss. The results were compared with parallel experiments using bacterial flagellins, a well know mammalian agonist of toll receptors (tlr5) with adjuvant activity recently described also in fish Four experimental groups were formed with four biological replicates each, 16 samples in total. First one was injected with NV recombinant protein in PBS, second one was a control group injected with PBS, the third group was injected with bacterial flagellins in PBS and the fourth group was injected with polihistidine peptide as a reference group because the recombinant proteins have a polihistidine tail. There is a technical replicate within each array.
Project description:We describe here transcripts induced after intraperitoneal injection of rainbow trout with 2 different viruses, both belonging to strain 23.75 of viral hemorrhagic septicemia virus (VHSV): a deleted Nv gene (dNV) virus and a wild type (wt) virus. Two days after infection, differentially expressed transcript levels from selected immune-related trout genes were studied in internal organs (spleen and head kidney). Fishes were divided in two groups (3 fishes per group). The first group was intraperitoneally injected with 100000 pfu per trout of dNV VHSV, while the second group was injected with 100000 pfu/trout of wt VHSV. All fishes were sacrificed two days post infection.
Project description:Oral immunization of trout against infectious pancreatic necrosis virus (IPNV) have been recently reported by using a DNA vector coding the viral capsid VP2 gene encapsulated in alginate microspheres. We report here an study of the transcripts in head kidney and pyloric ceca 7 days after oral vaccination of rainbow trout by using a newly designed rainbow trout 60-mer oligo microarray focused in their immune-related genes. Trout were obtained from 4 different farms, one group of trout was defined as belonging to one of the farms. Each of the trout 4 groups were divided into two subgroups of 6 trout for each subgroup. Subgroup I was orally vaccinated with 10 µl of suspension of the vaccine microspheres each containing 10 µg of pcDNA-VP2 diluted in 10µl of PBS, while subgroup II received similar microspheres suspension but pcDNA. Head kidney and pyloric ceca were collected from each subgroup and RNA was extracted.
Project description:In mammals, increasing data suggests that there exists a complex and bi-directional relationship between thyroid and immune systems. However the existence of such interactions in fish is unknown. Here, we administered the biologically active hormone 3.3′.5-triiodo-L-thyronine (T3) and the anti-thyroid drug, propilthiouracil (PTU) to juvenile rainbow trout and examined the head kidney expression profile with a custom-made microarray enriched in immune-related genes. A seven day-experiment was performed. Fish were divided in 3 groups. First group received 20 µg/g fish feed of T3; second group received 5000 µg/g fish feed of PTU; third group served as control. After 7 days orally administering T3 or PTU, differentially expressed transcript levels from selected immune-related rainbow trout genes were studied in head kidney. Three groups were studied (T3-treated, PTU treated and control), each group having 4 biological replicates (each replicate consisting of 2 pooled fish).
Project description:NV is a viral protein only present in the genus Novirhabdovirus (fam Rhabdoviridae), its function is not well characterized yet. In this study we try to shed some light in the biological function of this viral protein in the zebrafish. We describe here transcripts induced after injection of adult zebrafish with NV recombinant protein of VHSV. Two days after infection, differentially expressed transcript levels from selected immune-related zebrafish genes were studied in zebrafish internal organs (pooled spleen and head kidney). 6 zebrafish were divided in two groups, 3 per group . First group was injected with recombinant NV protein (tag:polyhistidine tail) and second group was used as a control and injected with polyhistidine protein. Two days after injection, zebrafish were sacrified and head kidney and spleen of each individual were extracted and pooled between this group. This experiment was repeated three times to get four biological replicates. RNA was extracted and submitted to quality controls. Lately, RNA was labelled with Cy3 and used in the Agilent Danio rerio custom array zfin 2.2 ID: 47562.
Project description:We describe here the transcripts induced after infection of zebrafish with viral haemorrhagic septicemia rhabdovirus (VHSV). Two days after infection, differentially expressed transcript levels from selected immune-related zebrafish genes were studied in both fins and internal organs (pooled spleen, head kidney and liver). VHSV-induced gene expression in zebrafish fins and organs was measured at 2 days after infection. Four independent experiments were performed for each treatment and their corresponding controls
Project description:In order to explore the relative importance of adult zebrafish innate and adaptive immune responses to different kinds of infections, we compared the gene expression profile of VHSV-challenge-survivors, bacterial infection survivors and control non infected zebrafish. VHSV -infection-survivors zebrafish were divided in two groups of 12 individuals, first group was infected with VHSV (samples named A) and second group was mock-infected (samples named B). Two days after challenge, zebrafish of each group were divided in four subgroups (3 fishes per group) and head kidney and spleen of each individual were sampled. For each group, internal organs of three fishes were pooled, so four biological replicates were generated . Zebrafish surviving a natural infection caused by Aeromonas hydrophila and Vibrio fluvialis (samples named C) were divided in four groups (3 fishes per group) and head kidney and spleen were extracted. Finally, 12 healthy zebrafish were used as control group (samples named D) and processed in the same way as group C.
Project description:The relevance of immune-endocrine interactions to the regulation of ovarian function in teleosts is virtually unexplored. As part of the innate immune response during infection, a number of cytokines such as tumor necrosis factor α (TNFα) and other immune factors, are produced and act on the reproductive system. However, TNFα is also an important physiological player in the ovulatory process in mammals. In the present study, we have examined for the first time the effects of TNFα in vitro in preovulatory ovarian follicles of a teleost fish, the brown trout (Salmo trutta). In control and recombinant trout TNFα (rtTNFα)-treated granulosa cells, we examined the percentage of apoptosis by flow cytometry analysis and cell viability by propidium iodide (PI) staining. Furthermore, we determined the in vitro effects of rtTNFα on follicle contraction and testosterone production in preovulatory trout ovarian follicles. In addition, we analyzed the gene expression profiles of control and rtTNFα-treated ovarian tissue by microarray and real-time PCR (qPCR) analyses.Treatment with rtTNFα induces ovarian cell apoptosis, decreases granulosa cell viability and stimulates the expression of genes known to be involved in the normal ovulatory process in trout. In addition, rtTNFα causes a significant increase in follicle contraction and testosterone production. Also, using a salmonid-specific microarray platform (SFA2.0 immunochip) we observed that rtTNFα induces the expression of genes known to be involved in inflammation, proteolysis and tissue remodeling. In view of these results, we propose that TNFα could have an important role in the biomechanics of follicle weakening, ovarian rupture and oocyte expulsion during ovulation in trout, primarily through its stimulation of follicular cell apoptosis and the expression of genes involved in follicle wall proteolysis and contraction. Reproductively mature female brown trout (Salmo trutta) from a cultured stock at the Piscifactoria de Bagà (Generalitat de Catalunya, Bagà, Spain) were kept under natural conditions of temperature and photoperiod. Fish at the preovulatory stage (according to the position of the germinal vesicle (GV) in oocytes that were cleared using a solution previously described), were anesthesized in 3-aminobenzoic acid ethyl ester (0.1 g/l; Sigma, Alcobendas, Spain) dissolved in fresh water, and the fish were sacrificed by concussion prior to the collection of the ovaries. The dissected ovaries were immediately used for the various in vitro assays. After dissection, brown trout preovulatory ovaries were placed in Hank´s balanced salt solution (HBSS) and individual ovarian follicles were manually separated with forceps from each ovary on ice, as previously described. To collect ovarian tissue for RNA extraction, preovulatory follicles from each of a total of three females were incubated (400 follicles/50 ml) in HBSS-BSA in the absence or presence of rtTNFα (100 ng/ ml, dissolved directly in HBSS-BSA), at 15ºC for 24 h with gentle shaking (100 rpm). At the end of the incubation follicles (previously de-yolked by gentle pressure) were removed, flash frozen in liquid nitrogen and stored at -80ºC until assayed.
Project description:The focus of this study was to investigate the response of cultured rainbow trout erythrocytes to lipopolysaccharide (LPS) and to the analog viral dsRNA Poly(I:C) using a salmonid-specific microarray platform enriched with immune-related genes. Although the transcriptomic response measured as the total number of differentially expressed genes was higher in LPS, the intensity response, in terms of genes with a FC>2, was greater in Poly(I:C) treated cells. These two treatments shared the regulation of some genes, but not alls followed the same pattern. Over representation of Gene Ontology functional categories showed us that Poly(I:C) treatment produced a immune response whereas LPS stimulation affect biological processes specially. Trout erythrocytes were isolated from blood by twice Ficoll 1.007 density gradient centrifugations. For primary cell culture, erythrocytes were resuspended in DMEM (PAA Laboratories) supplemented with 10% heat-inactivated fetal bovine serum (FBS, PAA Laboratories) and the antibiotic Primocin (100 μg/ml, Invivogen) at a density of 7.5x106 cells/ml in six well cell culture plates (NUNC) and cultured at 18 ºC and 5% CO2. Erythrocytes were stimulated with LPS from E. coli (serotype 026:B6, Sigma) and Poly(I:C) (Invivogen) at 50 μg/ml, each treatment had their own control plate. All culture plates were incubated for 24h. Total RNA was extracted from the cultures using 1 ml of Tri Reagent (Sigma) following the manufacturer’s instructions with minor modifications. Quantity and integrity was analyzed by Experion RNA StdSens Analysis Kit (Bio-Rad). The design of the microarray posses 1818 genes printed in six replicates each, including random clones from common and subtracted cDNA libraries which were compared with known vertebrate proteins using blastx. The platform was enriched in immune-related genes.