Innate immune response of rainbow trout erythrocytes to spinycterins expressing a downsized viral fragment of viral haemorrhagic septicaemia virus.

Recent studies have reported on the importance of RBCs in fish responses to viral infections and DNA vaccines. Surface-displaying recombinant bacterins (spinycterins) are a safe and adaptable prototype for viral vaccination of fish and represent an alternative method of aquaculture prophylaxis, since have been reported to enhance fish immune response. We evaluated the innate immune response of rainbow trout (Oncorhynchus mykiss) red blood cells (RBCs), head kidney, and spleen to spinycterins expressing a fragment of the glycoprotein G of viral haemorrhagic septicemia virus (VHSV), one of the most devastating world-wide diseases in farmed salmonids. We first selected an immunorelevant downsized viral fragment of VHSV glycoprotein G (frg16252-450). Then, spinycterins expressing frg16252-450 fused to Nmistic anchor-motif (Nmistic+frg16252-450) were compared to spinycterins expressing frg16252-450 internally without the anchor motif. Nmistic+frg16252-450 spinycterins showed increased attachment to RBCs in vitro and modulated the expression of interferon- and antigen presentation-related genes in RBCs in vitro and in vivo, after intravenous injection. In contrast, the head kidney and spleen of fish injected with frg16252-450, but not Nmistic+frg16252-450, spinycterins demonstrated upregulation of interferon and antigen-presenting genes. Intravenous injection of Nmistic+frg16252-450 spinycterins resulted in a higher innate immune response in RBCs while frg16252-450 spinycterins increased the immune response in head kidney and spleen. Although more studies are required to evaluate the practicality of using spinycterins as fish viral vaccines, these results highlight the important contribution of RBCs to the fish innate immune response to antiviral prophylactics.

Residuos de celulares y tabletas: incidencia del nivel de escolaridad en las actitudes y percepciones frente a su manejo / Residues of cell phones and tablets: incidence of the school level in the attitudes and perceptions in front of its handling

RESUMEN El avance tecnológico ha traído consigo la generación de residuos de celulares y tabletas. Esta tendencia seguirá en aumento, dado el fácil acceso a estos dispositivos. En el siguiente estudio, se relaciona la incidencia del nivel de escolaridad, sobre las actitudes y las percepciones, que se tienen sobre el manejo de los residuos provenientes de celulares y de tabletas, con el fin de ahondar en esta problemática y aportar insumos para la creación de estrategias y de políticas encaminadas al desarrollo sustentable. Para esto, se aplicó una encuesta a 450 personas que habitan en la ciudad de Florencia - Caquetá, Colombia. Se realizó un test de independencia, que permitió decidir si el nivel de escolaridad incide sobre las actitudes y las percepciones. Los resultados obtenidos indicaron que no existe tal dependencia con las actitudes, pero sí con las percepciones, lo que demuestra la inconsistencia del ser humano entre lo que piensa y cómo actúa. Un hallazgo relevante en el presente estudio es que, en su gran mayoría, estos dispositivos o sus partes, después de un tiempo, terminan en la basura, junto con los residuos convencionales.

SUMMARY The technological advance has brought with it the generation of cell and tablet waste. This trend will continue to increase, given the easy access to these devices. In the following study, the incidence of schooling level is related to attitudes and perceptions about the handling of cell and tablet waste, in order to deepen this problem and provide inputs for the creation of strategies and policies aimed at sustainable development. For this, a survey was applied to 450 people living in the city of Florencia Caquetá Colombia. An independence test was carried out, which allowed to decide if the level of schooling affects attitudes and perceptions. The results obtained indicated that there is no such dependence on attitudes, but with perceptions, this shows the human being's lack of balance between what he thinks and how he acts. A relevant finding in the present study, is that the vast majority of these devices or their parts, after a while end up in the trash, along with conventional waste.

Development of new therapeutical/adjuvant molecules by pepscan mapping of autophagy and IFN inducing determinants of rhabdoviral G proteins.

Surface glycoproteins of enveloped virus are potent elicitors of both innate and adaptive host immune responses. Therefore, the identification of viral glycoprotein determinants directly implicated in the induction of these responses might be of special interest for designing new therapeutical/adjuvant molecules. In this work we review the contribution of the "pepscan" approach to the screening of viral functions in the sequence of glycoprotein G (gpG) of the fish rhabdovirus of viral hemorrhagic septicemia (VHSV). Among others, by scanning gpG peptides, it has been possible to identify and validate minimal determinants for gpG directly implicated in initiating the fish type I Interferon-associated immune responses as well as in the antiviral autophagy program. Further fine-tunning of the identified peptides in the gpG of VHSV has allowed designing novel adjuvants that decrease DNA vaccine requirements and identify possible innovative antiviral molecules. In addition, these results have also contributed to improve our knowledge on how to stimulate the fish immune system.

VHSV G glycoprotein major determinants implicated in triggering the host type I IFN antiviral response as DNA vaccine molecular adjuvants.

We have recently identified the two major determinants of the glycoprotein G of the viral hemorrhagic septicaemia rhabdovirus (gpGVHSV), peptides p31 and p33 implicated in triggering the host type I IFN antiviral response associated to these rhabdoviral antigens. With the aim to investigate the properties of these viral glycoprotein regions as DNA molecular adjuvants, their corresponding cDNA sequences were cloned into a plasmid (pMCV1.4) flanked by the signal peptide and transmembrane sequences of gpGVHSV. In addition, a plasmid construct encoding both sequences p31 and p33 (pMCV1.4-p31+p33) was also designed. In vitro transitory cell transfection assays showed that these VHSV gpG regions were able to induce the expression of type I IFN stimulated genes as well as to confer resistance to the infection with a different fish rhabdovirus, the spring viremia of carp virus (SVCV). In vivo, zebrafish intramuscular injection of only 1µg of the construct pMCV1.4-p31+p33 conferred fish protection against SVCV lethal challenge up to 45 days post-immunization. Moreover, pMCV1.4-p31+p33 construct was assayed for molecular adjuvantcity's for a DNA vaccine against SVCV based in the surface antigen of this virus (pAE6-GSVCV). The results showed that the co-injection of the SVCV DNA vaccine and the molecular adjuvant allowed (i) a ten-fold reduction in the dose of pAE6-Gsvcv without compromising its efficacy (ii) an increase in the duration of protection, and (iii) an increase in the survival rate. To our knowledge, this is the first report in which specific IFN-inducing regions from a viral gpG are used to design more-efficient and cost-effective viral vaccines, as well as to improve our knowledge on how to stimulate the innate immune system.

Ex vivo transfection of trout pronephros leukocytes, a model for cell culture screening of fish DNA vaccine candidates.

DNA vaccination opened a new era in controlling and preventing viral diseases since DNA vaccines have shown to be very efficacious where some conventional vaccines have failed, as it occurs in the case of the vaccines against fish novirhabdoviruses. However, there is a big lack of in vitro model assays with immune-related cells for preliminary screening of in vivo DNA vaccine candidates. In an attempt to solve this problem, rainbow trout pronephros cells in early primary culture were transfected with two plasmid DNA constructions, one encoding the green fluorescent protein (GFP) and another encoding the viral haemorrhagic septicaemia virus (VHSV) glycoprotein G (G(VHSV)) - the only viral antigen which has conferred in vivo protection. After assessing the presence of GFP- and G(VHSV)-expressing cells, at transcription and protein levels, the immune response in transfected pronephros cells was evaluated. At 24h post-transfection, G(VHSV) up-regulated migm and tcr transcripts expression, suggesting activation of B and T cells, as well, a high up-regulation of tnfα gene was observed. Seventy-two hours post-transfection, we detected the up-regulation of mx and tnfα genes transcripts and Mx protein which correlated with the induction of an anti-VHSV state. All together we have gathered evidence for successful transfection of pronephros cells with pAE6G, which correlates with in vivo protection results, and is less time-consuming and more rapid than in vivo assays. Therefore, this outcome opens the possibility to use pronephros cells in early primary culture for preliminary screening fish DNA vaccines as well as to further investigate the function that these cells perform in fish immune response orchestration after DNA immunisation.

In vitro analysis of the factors contributing to the antiviral state induced by a plasmid encoding the viral haemorrhagic septicaemia virus glycoprotein G in transfected trout cells.

We have found out that transfection of the RTG-2 cell line with the viral haemorrhagic septicaemia virus (VHSV) glycoprotein G (G(VHSV))-coding plasmid induces an anti-VHSV state, similar to that induced by poly I:C. Taking the advantage of the constitutive expression of toll-like receptor 9 gene (tlr9) in RTG-2 cells, we have investigated whether this antiviral state was induced by the cytosine-phosphodiester-guanine (CpG) motifs present in the plasmid DNA, by the endogenous expression of G(VHSV) protein or by both elements. For that, we have analysed the expression profile of the rainbow trout tlr9 and several genes related to TLR9-mediated immune response in the absence or presence of a lysosomotropic drug that specifically blocks TLR9-CpG DNA interaction. The results suggested that the high levels of cell protection conferred by a plasmid encoding G(VHSV) gene are due to G(VHSV) rather than to the CpG motifs within plasmid DNA. Therefore, plasmid DNA might not play a key role in the immune response elicited by DNA vaccines or perhaps other receptors instead TLR9 could be implicated in CpG motifs recognition and signalling. In addition, since RTG-2 cells express tlr9 gene, this cell line could be a good tool for screening TLR9 agonists, such as the immunomodulatory oligonucleotides (IMOs), as fish DNA vaccine adjuvants.

Pepscan mapping of viral hemorrhagic septicemia virus glycoprotein G major lineal determinants implicated in triggering host cell antiviral responses mediated by type I interferon.

Surface glycoproteins of enveloped virus are potent elicitors of type I interferon (IFN)-mediated antiviral responses in a way that may be independent of the well-studied genome-mediated route. However, the viral glycoprotein determinants responsible for initiating the IFN response remain unidentified. In this study, we have used a collection of 60 synthetic 20-mer overlapping peptides (pepscan) spanning the full length of glycoprotein G (gpG) of viral hemorrhagic septicemia virus (VHSV) to investigate what regions of this protein are implicated in triggering the type I IFN-associated immune responses. Briefly, two regions with ability to increase severalfold the basal expression level of the IFN-stimulated mx gene and to restrict the spread of virus among responder cells were mapped to amino acid residues 280 to 310 and 340 to 370 of the gpG protein of VHSV. In addition, the results obtained suggest that an interaction between VHSV gpG and integrins might trigger the host IFN-mediated antiviral response after VHSV infection. Since it is known that type I IFN plays an important role in determining/modulating the protective-antigen-specific immune responses, the identification of viral glycoprotein determinants directly implicated in the type I IFN induction might be of special interest for designing new adjuvants and/or more-efficient and cost-effective viral vaccines as well as for improving our knowledge on how to stimulate the innate immune system.

Antimicrobial peptides as model molecules for the development of novel antiviral agents in aquaculture.

Antimicrobial peptides (AMPs) are one of the components of the non-specific immune system that operate first lines of protection in many animal species including fish. They exert broad-spectrum antimicrobial activity, apart from many other potential roles in innate immunity, and represent a promising class of antiviral agents. Recent advances in understanding the mechanisms of their antiviral action(s) indicate that they have a dual role in antiviral defence, acting not only directly on the virion but also on the host cell. Despite the acute problems of viral diseases and restrictions in using chemicals in aquaculture, few but successful attempts to assess the antiviral activities of fish AMPs have been reported. This review focuses on the antiviral activities and mechanisms of action of some AMPs, and their potential relevance in the aquaculture industry, one of the most important sources of fishery products in the near future. It is a matter of notable concern to understand whether the AMPs can be used as model molecules for designing antiviral drugs that might help to solve the problems with viruses in the fish farming industry worldwide. In addition, because fish rely more heavily on their innate immune defences than mammals, they might constitute a potential rich source of antiviral compounds for fighting against mammalian viral infections.

The immunogenicity of viral haemorragic septicaemia rhabdovirus (VHSV) DNA vaccines can depend on plasmid regulatory sequences.

A plasmid DNA encoding the viral hemorrhagic septicaemia virus (VHSV)-G glycoprotein under the control of 5' sequences (enhancer/promoter sequence plus both non-coding 1st exon and 1st intron sequences) from carp beta-actin gene (pAE6-G(VHSV)) was compared to the vaccine plasmid usually described the gene expression is regulated by the human cytomegalovirus (CMV) immediate-early promoter (pMCV1.4-G(VHSV)). We observed that these two plasmids produced a markedly different profile in the level and time of expression of the encoded-antigen, and this may have a direct effect upon the intensity and suitability of the in vivo immune response. Thus, fish genetic immunisation assays were carried out to study the immune response of both plasmids. A significantly enhanced specific-antibody response against the viral glycoprotein was found in the fish immunised with pAE6-G(VHSV). However, the protective efficacy against VHSV challenge conferred by both plasmids was similar. Later analysis of the transcription profile of a set of representative immune-related genes in the DNA immunized fish suggested that depending on the plasmid-related regulatory sequences controlling its expression, the plasmid might activate distinct patterns of the immune system. All together, the results from this study mainly point out that the selection of a determinate encoded-antigen/vector combination for genetic immunisation is of extraordinary importance in designing optimised DNA vaccines that, when required for inducing protective immune response, could elicit responses biased to antigen-specific antibodies or cytotoxic T cells generation.

The rainbow trout TLR9 gene and its role in the immune responses elicited by a plasmid encoding the glycoprotein G of the viral haemorrhagic septicaemia rhabdovirus (VHSV).

The aim of this work was to improve the knowledge about the factors contributing to the immunogenicity of the DNA vaccines based on the viral haemorrhagic septicaemia virus glycoprotein G gene, through identifying the rainbow trout Toll-like receptor 9 (Omtlr9) gene that curiously contains an insertion of an incomplete transposon at the 5'-end of the third intron. Concerning the role played by this receptor in the fish innate defence, in response to the injection of a plasmid (pAE6) encoding or not the viral haemorrhagic septicaemia rhabdovirus (VHSV) glycoprotein G gene (pAE6-G), the presence of Omtlr9 transcripts remained unchanged in the fish secondary lymphoid organs while was highly increased at the injection site (muscle). The level of Omtlr9 transcripts correlated with those of cluster of differentiation 83 (cd83) and CXC chemokine receptor 4 (cxcr4), suggesting the recruitment of dendritic-like cells into the muscle as the source of Omtlr9 expressing cells. Transcription of tumour necrosis factor-alpha (tnf alpha) and interleukin-6 (il6) genes, two cytokines directly related to TLR9 induction with unmethylated CpG oligodeoxynucleotides (CpG ODNs), was solely observed in head kidney and spleen of the fish immunised with pAE6-G. Thus, the glycoprotein G of VHSV could be more implicated in triggering the pathways for TNF-alpha and IL6 production than the recognition of the unmethylated CpG motifs of the plasmid backbone by OmTLR9. Therefore, our results seem to indicate that OmTLR9-mediated recognition of plasmid DNA is not the key of the innate immune recognition of the adjuvant elements of fish DNA vaccines.

Expression and antiviral activity of a beta-defensin-like peptide identified in the rainbow trout (Oncorhynchus mykiss) EST sequences.

The in silico identification of a beta-defensin-like peptide sequence (omBD-1) in the rainbow trout (Oncorhynchuss mykiss) database of salmonid EST is reported here. We have studied the transcript expression of this beta-defensin-like sequence in different organs and expressed the recombinant peptide in a fish cell line. Finally, we have demonstrated the in vitro antiviral activity of the recombinant trout beta-defensin-like peptide against viral haemorrhagic septicaemia rhabdovirus (VHSV), one of the most devastating viruses for worldwide aquaculture. Thus, the resistance to VHSV infection of EPC cells transfected with pMCV 1.4-omBD-1 has been shown. Since EPC cells transfected with omBD-1 produced acid and heat stable antiviral activity and up regulation of Mx, a type I IFN-mediated mechanism of antiviral action is suggested. To our knowledge, this is the first report showing biological activity of a beta-defensin-like peptide from any fish.

In vitro and in vivo differential expression of rainbow trout (Oncorhynchus mykiss) Mx isoforms in response to viral haemorrhagic septicaemia virus (VHSV) G gene, poly I:C and VHSV.

Three different Mx isoforms are known to be present in rainbow trout, however, to date, neither their mechanism of action nor their regulation have been established. Because most previous studies have focused only on one Mx isoform of the three present in rainbow trout, the expression of all isoforms was simultaneously studied in this work in response to the viral haemorrhagic septicaemia virus (VHSV) G gene, poly I:C or VHSV. Thus, RT-PCR assays were specifically designed to amplify each of the Mx1, Mx2 and Mx3 transcripts induced both in vitro (RTG-2 cell line and head kidney leucocytes) and in vivo (muscle, head kidney, spleen and liver). Regardless of the inducer used, in vitro results showed that while in RTG-2 cells Mx3 was predominantly induced, all three isoforms were similarly induced in head kidney leukocytes. In vivo, regardless of the inducer used a predominant expression of Mx3 transcripts was also observed in muscle but expression of all three Mx isoforms or predominantly Mx1 and Mx2 was found in head kidney and spleen. Mx expression in the liver was however more dependent on the inducer used. In conclusion, the results obtained demonstrated, for the first time, that both in vitro and in vivo the expression of the different Mx genes is differentially regulated. Moreover, this is also the first report showing Mx induction after cell transfection with a plasmid coding for the VHSV-G protein.