Genomic characterization and transcription analysis of European sea bass (Dicentrarchus labrax) rtp3 genes.

Fish RTP3, belonging to the receptor-transporting protein family, display several functions, including a putative antiviral role as virus-responsive gene. In this work, we have identified and characterized two different European sea bass rtp3 genes. In addition, an in vivo transcription analysis in response to LPS, poly I:C and betanodavirus infection (RGNNV genotype) has been performed. The sequence analysis showed that European sea bass displays two rtp3 genes, X1 and X2, composed of two exons and a single intron (1007-bp and 888-bp long, respectively), located within the ORF sequence. The full-length cDNA is 1969 bp for rtp3 X1, and 1491 bp for rtp3 X2. Several ATTTA motifs have been found in the intron sequence of both genes, whereas rtp3 X1 also contains this motif in both untranslated regions. The transcription analyses revealed significant level of rtp3 X2 mRNA in brain and head kidney after LPS and poly I:C inoculation; however, the induction elicited by RGNNV infection was much higher, suggesting an essential role for this protein in controlling NNV infections.

The genetic variability and evolution of red-spotted grouper nervous necrosis virus quasispecies can be associated with its virulence.

Nervous necrosis virus, NNV, is a neurotropic virus that causes viral nervous necrosis disease in a wide range of fish species, including European sea bass (Dicentrarchus labrax). NNV has a bisegmented (+) ssRNA genome consisting of RNA1, which encodes the RNA polymerase, and RNA2, encoding the capsid protein. The most prevalent NNV species in sea bass is red-spotted grouper nervous necrosis virus (RGNNV), causing high mortality in larvae and juveniles. Reverse genetics studies have associated amino acid 270 of the RGNNV capsid protein with RGNNV virulence in sea bass. NNV infection generates quasispecies and reassortants able to adapt to various selective pressures, such as host immune response or switching between host species. To better understand the variability of RGNNV populations and their association with RGNNV virulence, sea bass specimens were infected with two RGNNV recombinant viruses, a wild-type, rDl956, highly virulent to sea bass, and a single-mutant virus, Mut270Dl965, less virulent to this host. Both viral genome segments were quantified in brain by RT-qPCR, and genetic variability of whole-genome quasispecies was studied by Next Generation Sequencing (NGS). Copies of RNA1 and RNA2 in brains of fish infected with the low virulent virus were 1,000-fold lower than those in brains of fish infected with the virulent virus. In addition, differences between the two experimental groups in the Ts/Tv ratio, recombination frequency and genetic heterogeneity of the mutant spectra in the RNA2 segment were found. These results show that the entire quasispecies of a bisegmented RNA virus changes as a consequence of a single point mutation in the consensus sequence of one of its segments. Sea bream (Sparus aurata) is an asymptomatic carrier for RGNNV, thus rDl965 is considered a low-virulence isolate in this species. To assess whether the quasispecies characteristics of rDl965 were conserved in another host showing different susceptibility, juvenile sea bream were infected with rDl965 and analyzed as above described. Interestingly, both viral load and genetic variability of rDl965 in seabream were similar to those of Mut270Dl965 in sea bass. This result suggests that the genetic variability and evolution of RGNNV mutant spectra may be associated with its virulence.

Cytokine-like activity of European sea bass ISG15 protein on RGNNV-infected E-11 cells.

IFN-I generates an antiviral state by inducing the expression of numerous genes, called IFN-stimulated genes, ISGs, including ISG15, which is the only ISG with cytokine-like activity. In a previous study, we developed the Dl_ISG15_E11 cell line, which consisted of E11 cells able to express and secrete sea bass ISG15. The current study is a step forward, analysing the effect of secreted sea bass ISG15 on RGNNV replication in E11 cells, and looking into its immunomodulatory activity in order to corroborate its cytokine-like activity. The medium from ISG15-produccing cells compromised RGNNV replication, as it has been demonstrated both, by reduction in the viral genome synthesis and, specially, in the yield of infective viral particles. The implication of sea bass ISG15 in this protection has been demonstrated by ISG15 removal, which decreased the percentage of surviving cells upon viral infection, and by incubation of RGNNV-infected cells with a recombinant sea bass ISG15 protein, which resulted in almost full protection. Furthermore, the immunomodulatory activity of extracellular sea bass ISG15 has been demonstrated, which reaffirms a cytokine-like role for this protein.

Relationship of healthy vascular aging with lifestyle and metabolic syndrome in the general Spanish population. The EVA study.

INTRODUCTION AND OBJECTIVES: Our objective was to study the relationship of healthy vascular aging (HVA) with lifestyle and the components of metabolic syndrome. We also analyzed the differences between chronological age and heart age (HA) and vascular age (VA) in the Spanish adult population without cardiovascular disease. METHODS: This descriptive cross-sectional study selected 501 individuals without cardiovascular disease (mean age, 55.9 years; 50.3% women) via random sampling stratified by age and sex. HA was estimated with the Framingham equation, whereas VA was estimated with the VaSera VS-1500 device. HVA was defined as a <5-year difference between the chronological age and the HA or VA and the absence of a vascular lesion, hypertension, and diabetes mellitus. RESULTS: Compared with the chronological age, the mean HA and VA were 2.98±10.13 and 3.08±10.15 years lower, respectively. Smoking (OR, 0.23), blood pressure ≥ 130/85mmHg (OR, 0.11), altered baseline blood glucose (OR, 0.45), abdominal obesity (OR, 0.58), triglycerides ≥ 150mg/dL (OR, 0.17), and metabolic syndrome (OR, 0.13) decreased the probability of HVA estimated by HA; an active lifestyle (OR, 1.84) and elevated high-density lipoprotein-cholesterol (OR, 3.26) increased the probability of HVA estimated by HA. Smoking (OR, 0.45), blood pressure ≥ 130/85mmHg (OR, 0.26), altered baseline blood glucose (OR, 0.42), and metabolic syndrome (OR, 0.40) decreased the probability of HVA estimated by VA; abdominal obesity (OR, 1.81) had the opposite effect. CONCLUSIONS: HA and VA were 3 years lower than the chronological age. HA was associated with tobacco consumption, physical activity, and the components of metabolic syndrome. Meanwhile, VA was associated with tobacco consumption, blood pressure, waist circumference, and altered baseline glycemia. CLINICAL TRIAL REGISTRATION: http://www.clinicaltrials.gov. Identifier: NCT02623894.

Differential immunogene expression profile of European sea bass (Dicentrarchus labrax, L.) in response to highly and low virulent NNV.

European sea bass is highly susceptible to the nervous necrosis virus, RGNNV genotype, whereas natural outbreaks caused by the SJNNV genotype have not been recorded. The onset and severity of an infectious disease depend on pathogen virulence factors and the host immune response. The importance of RGNNV capsid protein amino acids 247 and 270 as virulence factors has been previously demonstrated in European sea bass; however, sea bass immune response against nodaviruses with different levels of virulence has been poorly characterized. Knowing the differences between the immune response against both kinds of isolates may be key to get more insight into the host mechanisms responsible for NNV virulence. For this reason, this study analyses the transcription of immunogenes differentially expressed in European sea bass inoculated with nodaviruses with different virulence: a RGNNV virus obtained by reverse genetics (rDl956), highly virulent to sea bass, and a mutated virus (Mut247+270Dl956, RGNNV virus displaying SJNNV-type amino acids at positions 247 and 270 of the capsid protein), presenting lower virulence. This study has been performed in brain and head kidney, and the main differences between the immunogene responses triggered by both viruses have been observed in brain. The immunogene response in this organ is stronger after inoculation with the most virulent virus, and the main differences involved genes related with IFN I system, inflammatory response, cell-mediated response, and apoptosis. The lower virulence of Mut247+270Dl956 to European sea bass can be associated with a delayed IFN I response, as well as an early and transitory inflammation and cell-mediated responses, suggesting that those can be pivotal elements in controlling the viral infection, and therefore, their functional activity could be analysed in future studies. In addition, this study supports the role of capsid amino acids at positions 247 and 270 as important determinants of RGNNV virulence to European sea bass.

Comparative analysis of marine and freshwater viral haemorrhagic septicaemia virus (VHSV) isolates antagonistic activity.

Viral Haemorrhagic Septicaemia Virus (VHSV) isolates virulent to marine fish species can replicate in freshwater species, although producing little or no mortality. Conversely, isolates from freshwater fish do not cause disease in marine species. An inverse relationship between VHSV virulence and host mx gene up-regulation has been described for several fish species, suggesting that differences between the antagonistic activity exerted by these isolates might be involved in the outcome of infections. In this study, the antagonistic activity against the type I interferon system of two representative marine and freshwater VHSV isolates has been characterised using RTG-2 cells stably transfected with the luciferase gene under the control of the Senegalese sole mx (ssmx) promoter, RTG pssmx-luc cells. Both isolates exerted a dose-dependent negative effect on the activation of ssmx promoter, showing a notably different minimal viral dose to exert the antagonism. In particular, an inverse relationship between the minimal MOI required and the viral virulence to sole has been recorded, which suggests this parameter as a possible in vivo VHSV virulence marker. Furthermore, the quantification of the endogenous inf I, mx1 and mx3 mRNA has demonstrated differences between both isolates in their antagonistic activity. Besides, a different nv RNA kinetics, which seems to depend on specific cellular factors, has been recorded for both isolates. This knowledge could contribute to the development of efficient tools to fight against viral infections in fish farming. For that purpose, the RTG pssmx-luc cells may be a suitable in vitro tool to identify the molecular mechanisms underlying VHSV-host interactions.

Reference values of arterial stiffness parameters and their association with cardiovascular risk factors in the Spanish population. The EVA Study.

INTRODUCTION AND OBJECTIVES: To describe, for the first time, reference values for the cardio-ankle vascular index (CAVI), brachial-ankle pulse wave velocity (BA-PWV), carotid-femoral pulse wave velocity (CF-PWV), and the central augmentation index and to establish their association with cardiovascular risk factors in the Spanish adult population aged 35 to 75 years without cardiovascular disease. METHODS: We conducted a cross-sectional study. Through random sampling stratified by age and sex, we included 501 participants without cardiovascular disease. The mean age was 55.9 years and 50.3% were women. The measurements were taken using the SphigmoCor and Vasera VS-1500 devices. RESULTS: Values for all measures, except those for the central augmentation index, were higher in men and increased with age and blood pressure. The mean values were as follows: CAVI, 8.01±1.44; BA-PWV, 12.93±2.68m/s; CF-PWV, 6.53±2.03 m/s, and central augmentation index, 26.84±12.79. On multiple regression analysis, mean blood pressure was associated with the 4 measures, glycated hemoglobin was associated with all measures except the central augmentation index, and body mass index showed an inverse association with CAVI. The explanatory capacity of age, sex, and mean blood pressure was 62% for BA-PWV, 49% for CF-PWV 49%, 54% for the CAVI, and 38% for the central augmentation index. On logistic regression, hypertension was associated with the CAVI (OR=3.45), VOP-BT (OR=3.44), VOP-CF (OR=3.38) and with the central augmentation index (OR=3.73). CONCLUSIONS: All arterial stiffness measures increased with age. The CAVI and CF-PWV were higher in men and the central augmentation index was higher in women, with no differences in BA-PWV. This study is registered at ClinicalTrials.gov. Identifier NCT02623894.

Off-label use of dupilumab for pediatric patients with atopic dermatitis: A multicenter retrospective review.

BACKGROUND: Atopic dermatitis (AD) is a common, chronic type 2 inflammatory skin disease, typically starting in infancy, with increased risk for subsequent extracutaneous atopic morbidities. Dupilumab is the first biologic agent targeting type 2 inflammation approved by the U.S. Food and Drug Administration (USFDA); it was licensed in 2017 for adults with moderate to severe AD and 2 years later for adolescents. Systemic treatment for pediatric AD remains a significant unmet medical need. OBJECTIVE: To analyze off-label use of dupilumab in children with AD. METHODS: Multicenter retrospective review that evaluated children who were prescribed dupilumab for moderate to severe AD. RESULTS: One hundred eleven of 124 patients (89.5%) gained access to dupilumab after a mean of 9 weeks. The dosing range was 4 to 15.5 mg/kg for the loading dose and 2.0 to 15.3 mg/kg every other week for maintenance. The range was widest for 6- to 11-year-olds and was related to use of either full or half of adult dosing. Associated morbidities, treatment response, and adverse events were comparable to those in previous adolescent and adult trials. LIMITATIONS: The retrospective design of the study limited uniform data collection. CONCLUSION: Access to dupilumab was achievable for the majority of children after a mean 9-week delay because of insurance payment denial. This review supports dupilumab response and tolerability in children. Optimal dosing for patients younger than 12 years has not been defined. Availability of the drug in 2 different concentrations is an important safety issue.

Capsid amino acids at positions 247 and 270 are involved in the virulence of betanodaviruses to European sea bass.

European sea bass (Dicentrarchus labrax) is severely affected by nervous necrosis disease, caused by nervous necrosis virus (NNV). Two out of the four genotypes of this virus (red-spotted grouper nervous necrosis virus, RGNNV; and striped jack nervous necrosis virus, SJNNV) have been detected in sea bass, although showing different levels of virulence to this fish species. Thus, sea bass is highly susceptible to RGNNV, whereas outbreaks caused by SJNNV have not been reported in this fish species. The role of the capsid protein (Cp) amino acids 247 and 270 in the virulence of a RGNNV isolate to sea bass has been evaluated by the generation of recombinant RGNNV viruses harbouring SJNNV-type amino acids in the above mentioned positions (Mut247Dl965, Mut270Dl965 and Mut247 + 270Dl965). Viral in vitro and in vivo replication, virus virulence and fish immune response triggered by these viruses have been analysed. Mutated viruses replicated on E-11 cells, although showing some differences compared to the wild type virus, suggesting that the mutations can affect the viral cell recognition and entry. In vivo, fish mortality caused by mutated viruses was 75% lower, and viral replication in sea bass brain was altered compared to non-mutated virus. Regarding sea bass immune response, mutated viruses triggered a lower induction of IFN I system and inflammatory response-related genes. Furthermore, mutations caused changes in viral serological properties (especially the mutation in amino acid 270), inducing higher seroconversion and changing antigen recognition.

Differential antiviral activity of European sea bass interferon-stimulated 15 protein (ISG15) against RGNNV and SJNNV betanodaviruses.

ISG15 is an antiviral protein acting intracellularly, by conjugation to viral or cellular proteins, or extracellularly, as cytokine. In this work, an in vitro system, consisting of E-11 cells over-expressing European sea bass ISG15 (Dl_ISG15_E11 cells), has been developed to evaluate the European sea bass ISG15 protein activity against RGNNV and SJNNV isolates. Regarding RGNNV, RNA2 copy number and viral titres were similar in E-11 and Dl_ISG15_E11 cells, and the cellular survival analyses demonstrated that Dl_ISG15_E11 cells were not protected from this virus. In contrast, ISG15 compromises SJNNV replication, since a reduction of the SJNNV genome synthesis has been recorded. The ISG15 anti-SJNNV activity was confirmed by viral titration and survival assays. In addition, a role of the intracellular ISG15 in modulating the transcription of endogenous genes has being recorded, with tlr3 gene being knocked out and e3 gene being up-regulated in RGNNV-inoculated Dl_ISG15_E11 cells. Sea bass ISG15 has also been detected extracellularly, and its activity has been evaluated by co-culture. The survival rate of RGNNV-inoculated E-11 cells increased from 25% to 46% when they were co-cultured with ISG15-producing cells. Similarly, the survival rate of SJNNV-inoculated E-11 cells increased from 27% to 51% in co-culture with ISG15-producing cells. To our knowledge, this is the first description of a differential antiviral activity of an ISG15 protein against two betanodavirus species, and the first evaluation of the cytokine-like activity of a fish ISG15 protein on non-immune cells.

Transcriptomic Profiles of Senegalese Sole Infected With Nervous Necrosis Virus Reassortants Presenting Different Degree of Virulence.

Betanodaviruses [nervous necrosis virus (NNV)] are the causative agent of the viral encephalopathy and retinopathy, a disease that affects cultured Senegalese sole (Solea senegalensis). NNV reassortants, combining genomic segments from redspotted grouper nervous necrosis virus (RGNNV) and striped jack nervous necrosis virus (SJNNV) genotypes, have been previously isolated from several fish species. The wild-type reassortant wSs160.03, isolated from Senegalese sole, has been proven to be more virulent to sole than the parental genotypes (RGNNV and SJNNV), causing 100% mortality. Mutations at amino acids 247 (serine to alanine) and 270 (serine to asparagine) in the wSs160.03 capsid protein have allowed us to obtain a mutant reassortant (rSs160.03247+270), which provokes a 40% mortality decrease. In this study, the RNA-Seq technology has been used to comparatively analyze Senegalese sole transcriptomes in two organs (head kidney and eye/brain) after infection with wild-type and mutant strains. A total of 633 genes were differentially expressed (DEGs) in animals infected with the wild-type isolate (with higher virulence), whereas 393 genes were differentially expressed in animals infected with the mutant strain (37.9% decrease in the number of DEGs). To study the biological functions of detected DEGs involved in NNV infection, a gene ontology (GO) enrichment analysis was performed. Different GO profiles were obtained in the following subclasses: (i) biological process; (ii) cellular component; and (iii) molecular function, for each viral strain tested. Immune response and proteolysis have been the predominant biological process after the infection with the wild-type isolate, whereas the infection with the mutant strain induces proteolysis in head kidney and inhibition of vasculogenesis in nervous tissue. Regarding the immune response, genes coding for proteins acting as mediators of type I IFN expression (DHX58, IRF3, IRF7) and IFN-stimulated genes (ISG15, Mx, PKR, Gig1, ISG12, IFI44, IFIT-1, to name a few) were upregulated in animals infected with the wild-type isolate, whereas no-differential expression of these genes was observed in samples inoculated with the mutant strain. The different transcriptomic profiles obtained could help to better understand the NNV pathogenesis in Senegalese sole, setting up the importance as virulence determinants of amino acids at positions 247 and 270 within the RNA2 segment.

Betanodavirus infection in bath-challenged Solea senegalensis juveniles: A comparative analysis of RGNNV, SJNNV and reassortant strains.

Senegalese sole has been shown to be highly susceptible to betanodavirus infection, although virulence differences were observed between strains. To study the mechanisms involved in these differences, we have analysed the replication in brain tissue of three strains with different genotypes during 15 days after bath infection. In addition, possible portals of entry for betanodavirus into sole were investigated. The reassortant RGNNV/SJNNV and the SJNNV strain reached the brain after 1 and 2 days postinfection, respectively. Although no RGNNV replication was detected until day 3-4 postinfection, at the end of the experiment this strain yielded the highest viral load; this is in accordance with previous studies in which sole infected with the reassortant showed more acute signs and earlier mortality than the RGNNV and SJNNV strains. Differences between strains were also observed in the possible portals of entry. Thus, whereas the reassortant strain could infect sole mainly through the skin or the oral route, and, to a minor extent, through the gills, the SJNNV strain seems to enter fish only through the gills and the RGNNV strain could use all tissues indistinctly. Taken together, all these results support the hypothesis that reassortment has improved betanodavirus infectivity for sole.

Immuno-related gene transcription and antibody response in nodavirus (RGNNV and SJNNV)-infected European sea bass (Dicentrarchus labrax L.).

The immune response of European sea bass to RGNNV and SJNNV infections has been evaluated by quantifying the transcription of some genes involved in the IFN I system, as well as in the inflammatory and adaptive immune mechanisms. The transcription of IFN-I, ISG-12, ISG-15 and MxA genes has been analyzed in brain and head kidney, showing that RGNNV genotype induces a more intense response of the IFN I system than SJNNV in both organs. In addition, the results obtained indicate the importance of the inflammatory response in nodavirus pathogenesis, with the transcription of IL-8 and TNF-α significantly higher in brain than in head kidney, being RGNNV the strongest inductor. An important difference between the immune response induced by both genotypes refers to the IgM titre in sera, which was higher in SJNNV-inoculated fish. The acquired response is also important locally, since TR-γ transcription is higher in brain than in head kidney (especially in the RGNNV-inoculated group). To our knowledge, this is the first study addressing the sea bass anti-SJNNV immune response.

Molecular characterization and expression analyses of the Solea senegalensis interferon-stimulated gene 15 (isg15) following NNV infections.

Interferons are essential in fish resistance to viral infections. They induce interferon-stimulated genes, such as isg15. In this study, the Senegalese sole isg15 gene (ssisg15) has been characterized. As other isg15, ssisg15 contains a 402-bp intron sited in the 5'-UTR, and the full length cDNA is 1492-bp, including a 480-bp ORF. The expression analyses revealed basal levels of isg15 transcripts, and a clear induction after poly I:C injection, that reached maximum values in brain, head kidney and gills. The ssisg15 induction patterns were similar in RGNNV- and SJNNV-inoculated fish, whereas the reassortant (RG/SJ) isolate, which has higher replication fitness, triggered delayed but higher transcript levels. Furthermore, RG/SJ infection after poly I:C treatment reduced the induction of ssisg15 transcripts, suggesting an antagonistic mechanism against interferon type I system, that might allow an efficient viral replication at the initial steps of the infective process.

Association of metabolic syndrome and its components with arterial stiffness in Caucasian subjects of the MARK study: a cross-sectional trial.

BACKGROUND: The cardio-ankle vascular index (CAVI) and brachial-ankle pulse wave velocity (baPWV) can reflect both central and peripheral arterial stiffness. Metabolic syndrome (MetS) and its components may increase arterial stiffness and the risk of cardiovascular diseases. However, the correlation of MetS and its components with arterial stiffness is still not clear. The primary aim of this study is thus the relationship using baPWV and CAVI in Caucasian adults with intermediate cardiovascular risk. The secondary aim is to analyze sex differences. METHODS: This study analyzed 2351 subjects aged 35-74 years (mean, 61.4 ± 7.7 years) comprising 61.7 % males and enrolled in the improving interMediAte Risk management (MARK) study. CAVI was measured using a VaSera VS-1500 ® device, and baPWV was calculated using a validated equation. MetS was defined based on the Joint Scientific Statement National Cholesterol Education Program III. Waist circumference, blood pressure, fasting plasma glucose, and lipid profile were measured. RESULTS: MetS was found in 51.9 % of the subjects. All MetS components except reduced HDL-cholesterol (p = 0.578) were associated with CAVI. High density lipoprotein cholesterol (p = 0.075) and waist circumference (p = 0.315) were associated with baPWV. The different MetS components that assess dyslipidemia using the stiffness measures show different associations according to patient sex. The high blood pressure component had a greater odds ratio (OR) for both baPWV ≥ 17.5 m/sec (OR = 6.90, 95 % CI 3.52-13.519) and CAVI ≥ 9 (OR = 2.20, 95 % CI 1.63-1.90). CONCLUSIONS: MetS and all its components (except HDL-cholesterol with baPWV and CAVI and WC with baPWV) were associated with baPWV and CAVI. However, there were sex differences in the association of MetS and its components with baPWV and CAVI. Data from this study suggest a greater association of CAVI and baPWV values with MetS components in males than in females and indicate greater arterial stiffness in the event of simultaneously elevated blood pressure, fasting plasma glucose, and waist circumference. Trial Registration Clinical Trials.gov Identifier: https://clinicaltrials.gov/ct2/show/ NCT01428934. Registered 2 September 2011. Last updated September 8, 2016.

Genetic characterization and transcription analyses of the European sea bass (Dicentrarchus labrax) isg15 gene.

Fish interferons are cytokines involved in its resistance to viral infections by inducing the transcription of several interferon-induced genes, such as isg15. The aim of the present study was the genetic characterization of the European sea bass isg15 gene, describing the regulatory motifs found in its sequence. In addition, an in vivo analysis of transcription in response to betanodavirus (RGNNV genotype) and poly I:C has been performed. The analysis of the resulting sequences showed that sea bass isg15 gene is composed of two exons and a single 276-bp intron located at the 5'-UTR region. The full length cDNA is 1143-bp, including a 102-bp 5'-UTR region, a 474-bp ORF, and a 291-bp 3'-UTR region. Several mRNA-regulatory elements, including three unusual ATTTA instability motifs in the intron, and four ATTTA motifs along with a cytoplasmic polyadenylation element in the 3'-UTR region, have been found in this sequence. The in vivo analyses revealed a similar kinetics and level of transcription in fish brain and head kidney after poly I:C inoculation; however, the induction caused by RGNNV started earlier in brain, where the upregulation of isg15 gene transcription was high. The present study contributes to further characterize the European sea bass IFN I response against RGNNV infections.

SJNNV down-regulates RGNNV replication in European sea bass by the induction of the type I interferon system.

European sea bass is highly susceptible to the betanodavirus RGNNV genotype, although the SJNNV genotype has also been detected in this fish species. The coexistence of both genotypes may affect the replication of both viruses by viral interaction or by stimulation of the host antiviral defense system in which the IFN I system plays a key role. IFN I triggers the transcription of interferon-stimulated genes, including Mx genes, whose expression has been used as a reporter of IFN I activity. The present study evaluated the effect of a primary exposure to an SJNNV isolate on a subsequent RGNNV infection and analyzed the role of the IFN I system in controlling VNNV infections in sea bass using different in vivo approaches. VNNV infection and Mx transcription were comparatively evaluated after single infections, superinfection (SJ+RG) and co-infection (poly I:C+RG). The single RGNNV infection resulted in a 24% survival rate, whereas the previous SJNNV or poly I:C inoculation increased the survival rate up to 96 and 100%, respectively. RGNNV replication in superinfection was reduced compared with RGNNV replication after a single inoculation. Mx transcription analysis shows differential induction of the IFN I system by both isolates. SJNNV was a potent Mx inducer, whereas RGNNV induced lower Mx transcription and did not interfere with the IFN I system triggered by SJNNV or poly I:C. This study demonstrates that an antiviral state exists after SJNNV and poly I:C injection, suggesting that the IFN I system plays an important role against VNNV infections in sea bass.

Role of the IFN I system against the VHSV infection in juvenile Senegalese sole (Solea senegalensis).

Senegalese sole is susceptible to marine VHSV isolates but is not affected by freshwater isolates, which may indicate differences regarding virus-host immune system interaction. IFN I induces an antiviral state in fish, stimulating the expression of genes encoding antiviral proteins (ISG). In this study, the stimulation of the Senegalese sole IFN I by VHSV infections has been evaluated by the relative quantification of the transcription of several ISG (Mx, Isg15 and Pkr) after inoculation with marine (pathogenic) and freshwater (non-pathogenic) VHSV isolates. Compared to marine VHSV, lower levels of RNA of the freshwater VHSV induced transcription of ISG to similar levels, with the Isg15 showing the highest fold induction. The protective role of the IFN I system was evaluated in poly I:C-inoculated animals subsequently challenged with VHSV isolates. The cumulative mortality caused by the marine isolate in the control group was 68%, whereas in the poly I:C-stimulated group was 5%. The freshwater VHSV isolate did not cause any mortality. Furthermore, viral RNA fold change and viral titers were lower in animals from the poly I:C + VHSV groups than in the controls. The implication of the IFN I system in the protection observed was confirmed by the transcription of the ISG in animals from the poly I:C + VHSV groups. However, the marine VHSV isolate exerts a negative effect on the ISG transcription at 3 and 6 h post-inoculation (hpi), which is not observed for the freshwater isolate. This difference might be partly responsible for the virulence shown by the marine isolate.

Monitoring Glycan-Protein Interactions by NMR Spectroscopic Analysis: A Simple Chemical Tag That Mimics Natural CH-π Interactions.

Detection of molecular recognition processes requires robust, specific, and easily implementable sensing methods, especially for screening applications. Here, we propose the difluoroacetamide moiety (an acetamide bioisoster) as a novel tag for detecting by NMR analysis those glycan-protein interactions that involve N-acetylated sugars. Although difluoroacetamide has been used previously as a substituent in medicinal chemistry, here we employ it as a specific sensor to monitor interactions between GlcNAc-containing glycans and a model lectin (wheat germ agglutinin). In contrast to the widely employed trifluoroacetamide group, the difluoroacetamide tag contains geminal (1) H and (19) F atoms that allow both (1) H and (19) F NMR methods for easy and robust detection of molecular recognition processes involving GlcNAc- (or GalNAc-) moieties over a range of binding affinities. The CHF2 CONH- moiety behaves in a manner that is very similar to that of the natural acetamide fragment in the involved aromatic-sugar interactions, providing analogous binding energy and conformations, whereas the perfluorinated CF3 CONH- analogue differs more significantly.

Cooperative hydrogen bonding in glyco-oligoamides: DNA minor groove binders in aqueous media.

A strategy to create cooperative hydrogen-bonding centers by using strong and directional intramolecular hydrogen-bonding motifs that can survive in aqueous media is presented. In particular, glyco-oligoamides, a family of DNA minor groove binders, with cooperative and non-cooperative hydrogen-bonding donor centers in the carbohydrate residues have been designed, synthesized, and studied by means of NMR spectroscopy and molecular modeling methods. Indeed, two different sugar moieties, namely, ß-D-Man-Py-γ-Py-Ind (1; Ind=indole, Man=mannose, Py=pyrrole) and ß-D-Tal-Py-γ-Py-Ind (2; Tal=talose), were chosen according to our design. These sugar molecules should present one- or two-directional intramolecular hydrogen bonds. The challenge has been to study the conformation of the glyco-oligoamides at low temperature in physiological media by detecting the exchangeable protons (amide NH and OH resonances) by means of NMR spectroscopic analysis. In addition, two more glyco-oligoamides with non-cooperative hydrogen-bonding centers, that is, ß-D-Glc-Py-γ-Py-Ind (3; Glc=glucose), ß-D-Gal-Py-γ-Py-Ind (4; Gal=galactose), and the model compounds ß-D-Man-Py-NHAc (5) and ß-D-Tal-Py-NHAc (6) were synthesized and studied for comparison. We have demonstrated the existence of directional intramolecular hydrogen bonds in 1 and 2 in aqueous media. The unexpected differences in terms of stabilization of the intramolecular hydrogen bonds in 1 and 2 relative to 5 and 6 promoted us to evaluate the influence of CH-π interactions on the establishment of intramolecular hydrogen bonds by using computational methods. Initial binding studies of 1 and 2 with calf-thymus DNA and poly(dA-dT)2 by NMR spectroscopic analysis and molecular dynamics simulations were also carried out. Both new sugar-oligoamides are bound in the minor groove of DNA, thus keeping a stable hairpin structure, as in the free state, in which both intramolecular hydrogen-bonding and CH-π interactions are present.