Project description:In recent years, the innate immune response has gained importance since evidence indicates that after an adequate priming protocol, it is possible to obtain some prolonged and enhanced immune responses. Nevertheless, several factors, such as the timing and method of administration of the immunostimulants, must be carefully considered. An inappropriate protocol can transform the treatments into a double-edged sword for the teleost immune system, resulting in a stressful and immunosuppressive state. In this work, we analyzed the long-term effects of different stimuli (?-glucans, lipopolysaccharide, and polyinosinic:polycytidylic acid) on the transcriptome modulation induced by Spring Viremia Carp Virus (SVCV) in adult zebrafish (Danio rerio) and on the mortality caused by this infection. At 35?days post-immunostimulation, the transcriptome was found to be highly altered compared to that of the control fish, and these stimuli also conditioned the response to SVCV challenge, especially in the case of ?-glucans. No protection against SVCV was found with any of the stimuli, and non-significant higher mortalities were even observed, especially with ?-glucans. However, in the short term (pre-stimulation with ?-glucan and infection after 7?days), slight protection was observed after infection. The transcriptome response in the zebrafish kidney at 35?days posttreatment with ?-glucans revealed a significant response associated with stress and immunosuppression. The identification of genes that were differentially expressed before and after the infection seemed to indicate a high energy cost of the immunostimulation that was prolonged over time and could explain the lack of protection against SVCV. Differential responses to stress and alterations in lipid metabolism, the tryptophan-kynurenine pathway, and interferon-gamma signaling seem to be some of the mechanisms involved in this response, which represents the end of trained immunity and the beginning of a stressful state characterized by immunosuppression.
Project description:Tripartite motif (TRIM) proteins were shown to play an important role in innate antiviral immunity. FinTRIM (ftr) is a new subset of TRIM genes that do not possess obvious orthologs in higher vertebrates. However, little is known about its function. In this study, we used bioinformatic analysis to examine the phylogenetic relationships and conserved domains of zebrafish (Danio rerio) ftr01, ftr42, and ftr58, as well as qualitative real-time PCR to examine their expression patterns in zebrafish embryonic fibroblast (ZF4) cells and zebrafish tissues. Sequence analysis showed that the three finTRIMs are highly conserved, and all contain a RING domain, B-box domain, and SPRY-PRY domain. In addition, ftr42 and ftr58 had one coiled-coil domain (CCD), whereas ftr01 had two CCDs. Tissue expression analysis revealed that the mRNA level of ftr01 was the highest in the liver, whereas those of ftr42 and ftr58 were the highest in the gill; the expression of these finTRIMs was clearly upregulated not in the eyes, but in the liver, spleen, kidney, gill, and brain of zebrafish following spring viremia of carp virus (SVCV) infection. Similarly, the expression of these three finTRIM genes also increased in ZF4 cells after SVCV infection. Our study revealed that ftr01, ftr42, and ftr58 may play an important role in antiviral immune responses, and these findings validate the need for more in-depth research on the finTRIM family in the future.
Project description:Spring viraemia of carp (SVC) caused by spring viraemia of carp virus (SVCV) is an acute and highly lethal viral disease of cyprinid fish. However, effective therapy for SVC is still scarce until now. Here we evaluated the inhibition of anisomycin (Ani), a metabolite produced by Streptomyces griseolus, on the replication of SVCV in vitro and in vivo. Our results demonstrated that Ani could suppress SVCV replication with the maximum inhibitory rate > 95% in epithelioma papulosum cyprini (EPC) cells. And the half maximal inhibitory concentrations (IC50) of Ani on SVCV glycoprotein (G), nucleoprotein (N) and phosphoprotein mRNA expressions were 21.79, 13.13 and 12.24?nM, respectively. Besides, Ani decreased SVCV-induced cytopathic effects and nucleus damages. As expected, Ani also showed a strong anti-SVCV activity in vivo, as indicated by inhibiting viral gene expression and increasing the survival rate of zebrafish. Intraperitoneal injection of Ani increased the survival rate of zebrafish by 30% and markedly inhibited the expressions of G and N mRNA by > 60% in kidney and spleen at day 1 and day 4 post-infection. Results so far suggest that Ani as a powerful agent against SVCV can be applied to the control of SVC in aquaculture.
Project description:In the last years, the innate immune response has gained importance since evidences indicate that, after an adequate priming protocol, it is possible to obtain some prolonged and enhanced immune response. Nevertheless, several factors, such as timing and method of administration of the immunostimulants need to be carefully considered. An inappropriate protocol can transform the treatments into a double-edged sword for the teleost immune system, resulting in a stressful and immunosuppressive status. In this work, we analyzed the long-term effect of different stimuli (β-glucans, lipopolysaccharide and Polyinosinic:polycytidylic acid) on the transcriptome modulation induced by Spring Viraemia Carp Virus (SVCV) in adult zebrafish (Danio rerio) and on the mortalities caused by this infection. At 35 days post-immunostimulation the transcriptome was found to be highly altered compared to the control fish, and these stimuli also conditioned the response to SVCV challenge, especially in the case of β-glucans. No protection against SVCV was found with any of the stimuli and even non-significant higher mortalities were observed, especially with β-glucans. However, at short-term (a pre-stimulation with β-glucan and infection after 7 days) a slight protection was observed after infection. The transcriptome response in zebrafish kidney at 35 days post-treatment with β-glucans revealed a significant response associated to stress and immunosuppression. The identification of genes differentially expressed before and after the infection seem to indicate a high energy cost of the immunostimulation prolonged in time that could explain the lack of protection against the SVCV. The differential response to stress, alterations in the lipid metabolism, the tryptophan-kynurenine pathway and the interferon-gamma signaling seem to be some of the mechanisms involved in this particular response, which means the end of the trained immunity and the beginning of a stressful status characterized by immunosuppression. Overall design: Four groups composed of 92 adult zebrafish/each were intraperitoneally (i.p.) injected with 20 µl of one of the following treatments: β-glucans (1 mg/ml), LPS (0.75 mg/ml), Poly(I:C) (1 mg/ml) and the control group with phosphate-buffered saline (PBS). After 35 days, half of the individuals were i.p. infected with 20 µl of a SVCV suspension (3 × 102 TDCI50/ml) and the remaining fish, which served as uninfected control, were inoculated with viral medium (MEM + 2% FBS + Primocin). For microarray hybridization, a total of 16 fish from each treatment (β-glucans-control, LPS-control, Poly(I:C)-control, β-glucans-SVCV, LPS-SVCV and Poly(I:C)-SVCV) were sacrificed at 24h post-infection and the kidney was removed, obtaining 4 pooled biological replicates (four fish/replicate).
Project description: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.
Project description:Although the modulation of immune-related genes after viral infection has been widely described in vertebrates, the potential implications of non-coding RNAs (ncRNAs), especially long non-coding RNAs (lncRNAs), in immunity are still a nascent research field. The model species zebrafish could serve as a useful organism for studying the functionality of lncRNAs due to the numerous advantages of this teleost, including the existence of numerous mutant lines. In this work, we conducted a whole-transcriptome analysis of wild-type (WT) and heterozygous rag1 mutant (rag1+/-) zebrafish after infection with the pathogen spring viraemia of carp virus (SVCV). WT and rag1+/- zebrafish were infected with SVCV for 24?h. Kidney samples were sampled from infected and uninfected fish for transcriptome sequencing. From a total of 198,540 contigs, 12,165 putative lncRNAs were identified in zebrafish. Most of the putative lncRNAs were shared by the two zebrafish lines. However, by comparing the lncRNA profiles induced after SVCV infection in WT and rag1+/- fish, most of the lncRNAs that were significantly induced after viral challenge were exclusive to each line, reflecting a highly differential response to the virus. Analysis of the neighboring genes of lncRNAs that were exclusively modulated in WT revealed high representation of metabolism-related terms, whereas those from rag1+/- fish showed enrichment in terms related to the adaptive immune response, among others. On the other hand, genes involved in numerous antiviral processes surrounded commonly modulated lncRNAs, as expected. These results clearly indicate that after SVCV infection in zebrafish, the expression of an array of lncRNAs with functions in different aspects of immunity is induced.
Project description:Because fin base is supposed to be the entry zone of some fish virus, we wanted to know which transcripts are induced after infection of zebrafish with Spring Viremia Carp Virus (SVCV). Two days after infection, differentially expressed transcript levels from selected immune-related zebrafish genes were studied in zebrafish fins. Also transcripts from resistant fishes to viral infection one month after inoculation were studied. Overall design: Three different experiments were performed to get three biological replicates. Fishes were divided in two groups in each experiment. First group was infected by immersion with SVCV 107 pfu/ml, second group was used as a control of non-infected fishes. 6 fishes per group were sacrified two days post infection, whereas the rest of infected fishes from the three experiments were maintained for 30 days in the aquariums and then survivors (six for experiment) were sacrified.
Project description:BACKGROUND: Spring viraemia of carp virus (SVCV) has been identified as the causative agent of spring viraemia of carp (SVC) and it has caused significant losses in the cultured common carp (Cyprinus carpio) industry. The molecular mechanisms that underlie the pathogenesis of the disease remain poorly understood. In this study, deep RNA sequencing was used to analyse the transcriptome and gene expression profile of EPC cells at progressive times after SVCV infection. This study addressed the complexity of virus-cell interactions and added knowledge that may help to understand SVCV. RESULTS: A total of 33,849,764 clean data from 36,000,000 sequence reads, with a mean read length 100 bp, were obtained. These raw data were assembled into 88,772 contigs. Of these contigs, 19,642 and 25,966 had significant hits to the NR and Uniprot databases where they matched 17,642 and 13,351 unique protein accessions, respectively. At 24 h post SVCV infection (1.0 MOI), a total of 623 genes were differentially expressed in EPC cells compared to non-infected cells, including 288 up-regulated genes and 335 down-regulated genes. These regulated genes were primarily involved in pathways of apoptosis, oxidative stress and the interferon system, all of which may be involved in viral pathogenesis. In addition, 8 differentially expressed genes (DEGs) were validated by quantitative PCR. CONCLUSIONS: Our findings demonstrate previously unrecognised changes in gene transcription that are associated with SVCV infection in vitro, and many potential cascades identified in the study clearly warrant further experimental investigation. Our data provide new clues to the mechanism of viral susceptibility in EPC cells.
Project description:BACKGROUND:Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) had been demonstrated to prime interferon (IFN) response against viral infection via the conserved RLR signaling in fish, and a novel fish-specific gene, the grass carp reovirus (GCRV)-induced gene 2 (Gig2), had been suggested to play important role in host antiviral response. METHODOLOGY/PRINCIPAL FINDINGS:In this study, we cloned and characterized zebrafish Gig2 homolog (named Danio rerio Gig2-I, DreI), and revealed its antiviral role and expressional regulation signaling pathway. RT-PCR, Western blot and promoter activity assay indicate that DreI can be induced by poly I:C, spring viremia of carp virus (SVCV) and recombinant IFN (rIFN), showing that DreI is a typical ISG. Using the pivotal signaling molecules of RLR pathway, including RIG-I, MDA5 and IRF3 from crucian carp, it is found that DreI expression is regulated by RLR cascade and IRF3 plays an important role in this regulation. Furthermore, promoter mutation assay confirms that the IFN-stimulated regulatory elements (ISRE) in the 5' flanking region of DreI is essential for its induction. Finally, overexpression of DreI leads to establish a strong antiviral state against SVCV and Rana grylio virus (RGV) infection in EPC (Epithelioma papulosum cyprinid) cells. CONCLUSIONS/SIGNIFICANCE:These data indicate that DreI is an antiviral protein, which is regulated by RLR signaling pathway.