Dataset Information

Antiviral Activity of a Turbot (Scophthalmus maximus) NK-Lysin Peptide by Inhibition of Low-pH Virus-Induced Membrane Fusion.

ABSTRACT: Global health is under attack by increasingly-frequent pandemics of viral origin. Antimicrobial peptides are a valuable tool to combat pathogenic microorganisms. Previous studies from our group have shown that the membrane-lytic region of turbot (Scophthalmus maximus) NK-lysine short peptide (Nkl71?100) exerts an anti-protozoal activity, probably due to membrane rupture. In addition, NK-lysine protein is highly expressed in zebrafish in response to viral infections. In this work several biophysical methods, such as vesicle aggregation, leakage and fluorescence anisotropy, are employed to investigate the interaction of Nkl71?100 with different glycerophospholipid vesicles. At acidic pH, Nkl71?100 preferably interacts with phosphatidylserine (PS), disrupts PS membranes, and allows the content leakage from vesicles. Furthermore, Nkl71?100 exerts strong antiviral activity against spring viremia of carp virus (SVCV) by inhibiting not only the binding of viral particles to host cells, but also the fusion of virus and cell membranes, which requires a low pH context. Such antiviral activity seems to be related to the important role that PS plays in these steps of the replication cycle of SVCV, a feature that is shared by other families of virus-comprising members with health and veterinary relevance. Consequently, Nkl71?100 is shown as a promising broad-spectrum antiviral candidate.

SUBMITTER: Falco A

PROVIDER: S-EPMC6410327 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Antiviral Activity of a Turbot (Scophthalmus maximus) NK-Lysin Peptide by Inhibition of Low-pH Virus-Induced Membrane Fusion.

Falco Alberto A Medina-Gali Regla María RM Poveda José Antonio JA Bello-Perez Melissa M Novoa Beatriz B Encinar José Antonio JA

Marine drugs 20190201 2

Global health is under attack by increasingly-frequent pandemics of viral origin. Antimicrobial peptides are a valuable tool to combat pathogenic microorganisms. Previous studies from our group have shown that the membrane-lytic region of turbot (Scophthalmus maximus) NK-lysine short peptide (Nkl71⁻100) exerts an anti-protozoal activity, probably due to membrane rupture. In addition, NK-lysine protein is highly expressed in zebrafish in response to viral infections. In this wor ...[more]

PMID: 30717094

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Project description:BackgroundTurbot (Scophthalmus maximus L.) is an important aquacultural resource both in Europe and Asia. However, there is little information on gene sequences available in public databases. Currently, one of the main problems affecting the culture of this flatfish is mortality due to several pathogens, especially viral diseases which are not treatable. In order to identify new genes involved in immune defense, we conducted 454-pyrosequencing of the turbot transcriptome after different immune stimulations.Methodology/principal findingsTurbot were injected with viral stimuli to increase the expression level of immune-related genes. High-throughput deep sequencing using 454-pyrosequencing technology yielded 915,256 high-quality reads. These sequences were assembled into 55,404 contigs that were subjected to annotation steps. Intriguingly, 55.16% of the deduced protein was not significantly similar to any sequences in the databases used for the annotation and only 0.85% of the BLASTx top-hits matched S. maximus protein sequences. This relatively low level of annotation is possibly due to the limited information for this specie and other flatfish in the database. These results suggest the identification of a large number of new genes in turbot and in fish in general. A more detailed analysis showed the presence of putative members of several innate and specific immune pathways.Conclusions/significanceTo our knowledge, this study is the first transcriptome analysis using 454-pyrosequencing for turbot. Previously, there were only 12,471 EST and less of 1,500 nucleotide sequences for S. maximus in NCBI database. Our results provide a rich source of data (55,404 contigs and 181,845 singletons) for discovering and identifying new genes, which will serve as a basis for microarray construction, gene expression characterization and for identification of genetic markers to be used in several applications. Immune stimulation in turbot was very effective, obtaining an enormous variety of sequences belonging to genes involved in the defense mechanisms.

Project description:DNA vaccines encoding the viral G glycoprotein show the most successful protection capability against fish rhabdoviruses. Nowadays, the molecular mechanisms underlying the protective response remain still poorly understood. With the aim of shedding light on the protection conferred by the DNA vaccines based in the G glycoprotein of viral haemorrhagic septicaemia virus (VHSV) in turbot (Scophthalmus maximus) we have used a specific microarray highly enriched in antiviral sequences to carry out the transcriptomic study associated to VHSV DNA vaccination/infection. The differential gene expression pattern in response to empty plasmid (pMCV1.4) and DNA vaccine (pMCV1.4-G860) intramuscular administration with regard to non-stimulated turbot was analyzed in head kidney at 8, 24 and 72 hours post-vaccination. Moreover, the effect of VHSV infection one month after immunization was also analyzed in vaccinated and non-vaccinated fish at the same time points. Genes implicated in the Toll-like receptor signalling pathway, IFN inducible/regulatory proteins, numerous sequences implicated in apoptosis and cytotoxic pathways, MHC class I antigens, as well as complement and coagulation cascades among others were analyzed in the different experimental groups. Fish receiving the pMCV1.4-G860 vaccine showed transcriptomic patterns very different to the ones observed in pMCV1.4-injected turbot after 72 h. On the other hand, VHSV challenge in vaccinated and non-vaccinated turbot induced a highly different response at the transcriptome level, indicating a very relevant role of the acquired immunity in vaccinated fish able to alter the typical innate immune response profile observed in non-vaccinated individuals. This exhaustive transcriptome study will serve as a complete overview for a better understanding of the crosstalk between the innate and adaptive immune response in fish after viral infection/vaccination. Moreover, it provides interesting clues about molecules with a potential use as vaccine adjuvants, antiviral treatments or markers for vaccine efficiency monitoring.

Project description:In mammals, forkhead box O3 (foxo3) plays important roles in liver immune system. The foxo3 can regulate cell cycle, DNA repair, hypoxia, apoptosis and so on. However, as such an important transcription factor, few studies on foxo3 in fish have been reported. The present study characterized the foxo3 in turbot (Scophthalmus maximus L.). Lipopolysaccharide (LPS) incubated in vitro (hepatocytes) and injected in vivo (turbot liver) were used to construct inflammatory models. The foxo3 was interfered and overexpressed to investigate its functions in liver inflammation. The open reading frame (ORF) of foxo3 was 1998 bp (base pair), encoding 665 amino acids. Sequence analysis showed that foxo3 of turbot was highly homologous to other fishes. Tissue distribution analysis revealed that the highest expression of foxo3 was in muscle. Immunofluorescence result showed that foxo3 was expressed in cytoplasm and nucleus. Knockdown of foxo3 significantly increased mRNA levels of tumor necrosis factor-α (tnf-α), interleukin-1β (il-1β), interleukin-6 (il-6), myeloid-differentiation factor 88 (myd88), cd83, toll-like receptor 2 (tlr-2) and protein level of c-Jun N-terminal kinase (JNK) in sifoxo3 + LPS (siRNA of foxo3+ LPS) group compared with NC + LPS (negative control + LPS) group in turbot hepatocytes. Overexpressed foxo3 significantly decreased mRNA levels of tnf-α, il-6, nuclear transcription factor-kappa B (nf-κb), cd83, tlr-2 and the protein level of JNK in vitro. In vivo analysis, foxo3 knockdown significantly increased levels of GOT in serum after LPS injection compared with NC+LPS group. Overexpressed foxo3 significantly decreased levels of GPT and GOT in pcDNA3.1-foxo3+LPS group compared with pcDNA3.1+LPS group in vivo. Foxo3 knockdown significantly increased mRNA levels of tnf-α, il-1β, il-6, nf-κb, myd88 and protein level of JNK in vivo in sifoxo3+LPS group compared with NC+LPS group in turbot liver. Overexpressed foxo3 significantly decreased mRNA levels of il-1β, il-6, myd88, cd83, jnk and protein level of JNK in pcDNA3.1-foxo3+LPS group compared with pcDNA3.1+LPS group in turbot liver. The results indicated that foxo3 might modulate LPS-activated hepatic inflammation in turbot by decreasing the proinflammatory cytokines, the levels of GOT and GPT as well as activating JNK/caspase-3 and tlr-2/myd88/nf-κb pathways. Taken together, these findings indicated that FoxO3 may play important roles in liver immune responses to LPS in turbot and the research of FoxO3 in liver immunity enriches the studies on immune regulation, and provides theoretical basis and molecular targets for solving liver inflammation and liver injury in fish.

Project description:BACKGROUND: Enteromyxosis caused by the intestinal myxozoan parasite Enteromyxum scophthalmi is a serious threat for turbot (Scophthalmus maximus, L.) aquaculture, causing severe catarrhal enteritis leading to a cachectic syndrome, with no therapeutic options available. There are still many aspects of host-parasite interaction and disease pathogenesis that are yet to be elucidated, and to date, no analysis of the transcriptomic changes induced by E. scophthalmi in turbot organs has been conducted. In this study, RNA-seq technology was applied to head kidney, spleen and pyloric caeca of severely infected turbot with the aim of furthering our understanding of the pathogenetic mechanisms and turbot immune response against enteromyxosis. RESULTS: A huge amount of information was generated with more than 23,000 identified genes in the three organs, amongst which 4,762 were differently expressed (DE) between infected and control fish. Associate gene functions were studied based on gene ontology terms and available literature, and the most interesting DE genes were classified into five categories: 1) immune and defence response; 2) apoptosis and cell proliferation; 3) iron metabolism and erythropoiesis; 4) cytoskeleton and extracellular matrix and 5) metabolism and digestive function. The analysis of down-regulated genes of the first category revealed evidences of a connexion failure between innate and adaptive immune response, especially represented by a high number of DE interferon-related genes in the three organs. Furthermore, we found an intense activation of local immune response at intestinal level that appeared exacerbated, whereas in kidney and spleen genes involved in adaptive immune response were mainly down-regulated. The apoptotic machinery was only clearly activated in pyloric caeca, while kidney and spleen showed a marked depression of genes related to erythropoiesis, probably related to disorders in iron homeostasis. The genetic signature of the causes and consequences of cachexia was also demonstrated by the down-regulation of the genes encoding structural proteins and those involved in the digestive metabolism. CONCLUSIONS: This transcriptomic study has enabled us to gain a better understanding of the pathogenesis of enteromyxosis and identify a large number of DE target genes that bring us closer to the development of strategies designed to effectively combat this pathogen.

Dataset Information

Antiviral Activity of a Turbot (Scophthalmus maximus) NK-Lysin Peptide by Inhibition of Low-pH Virus-Induced Membrane Fusion.

Publications

Antiviral Activity of a Turbot (<i>Scophthalmus maximus</i>) NK-Lysin Peptide by Inhibition of Low-pH Virus-Induced Membrane Fusion.

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