M. Carmen Alonso

Bacteriophages presence in human faeces of healthy subjects and patients with gastrointestinal disturbances

The variation of the content of enteric bacteria and their bacteriophages in faeces from the different types of diarrhoeal processes has been studied. A total of 122 samples of human faeces from both healthy individuals and patients with diarrhoeal diseases of functional or infectious origin were tested. Detection rates for all microbial parameters tested decreased in the faeces of individuals with functional gastrointestinal disturbances. On the contrary, no significant differences of the microbial detection frequency was observed in faeces containing pathogenic microorganisms compared to faeces of healthy subjects. Human faeces were a poor source of F-specific, Salmonella, and Bacteroides bacteriophages, whereas specific Escherichia coli phages were isolated in most samples tested. Coliphage concentrations in faeces of healthy individuals were not directly correlated with levels of faecal coliforms. On the basis of their high correlation, faecal streptococci and coliphages were the most adequate indicators of the intestinal ecosystem variations in subjects with diarrhoeal processes.

Antiviral Specificity of the Solea senegalensis Mx Protein Constitutively Expressed in CHSE-214 Cells

Interferons play a key role in fish resistance to viral infections by inducing the expression of antiviral proteins, such as Mx. The aim of the present study was to test the antiviral activity of the Senegalese sole Mx protein (SsMx) against RNA and DNA viruses pathogenic to fish, i.e. the infectious pancreatic necrosis virus (IPNV, dsRNA), the viral haemorrhagic septicaemia virus (VHSV, ssRNA), and the European sheatfish virus (ESV, dsDNA), using a CHSE-214 cell clone expressing this antiviral protein. A strong inhibition of IPNV and VHSV replication was recorded in SsMx-expressing cells, as has been shown by the virus yield reduction and the decrease in the synthesis of the viral RNA encoding the polyprotein (for IPNV) and the nucleoprotein (for VHSV). The titres of these viruses replicating on SsMx-expressing cells were 100 times lower than those recorded on non-transfected cells. In contrast, SsMx did not inhibit ESV replication since no significant differences were observed regarding the virus yield or the major capsid protein gene transcription in transfected and non-transfected cells.

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.

Are the defined substrate-based methods adequate to determine the microbiological quality of natural recreational waters?

Monitoring the microbiological quality of water used for recreational activities is very important to human public health. Although the sanitary quality of recreational marine waters could be evaluated by standard methods, they are time-consuming and need confirmation. For these reasons, faster and more sensitive methods, such as the defined substrate-based technology, have been developed. In the present work, we have compared the standard method of membrane filtration using Tergitol-TTC agar for total coliforms and Escherichia coli, and Slanetz and Bartley agar for enterococci, and the IDEXX defined substrate technology for these faecal pollution indicators to determine the microbiological quality of natural recreational waters. ISO 17994:2004 standard was used to compare these methods. The IDEXX for total coliforms and E. coli, Colilert, showed higher values than those obtained by the standard method. Enterolert test, for the enumeration of enterococci, showed lower values when compared with the standard method. It may be concluded that more studies to evaluate the precision and accuracy of the rapid tests are required in order to apply them for routine monitoring of marine and freshwater recreational bathing areas. The main advantages of these methods are that they are more specific, feasible and simpler than the standard methodology.

Structural and functional characterization of the Senegalese sole (Solea senegalensis) Mx promoter.

Mx proteins are one of the most studied interferon-stimulated genes (ISGs). The antiviral activity against different fish viruses has been demonstrated for diverse fish Mx proteins, including the Senegalese sole (Solea senegalensis) Mx protein (SsMx). The aim of the current study is to characterize the structure and functional activity of the SsMx promoter. Several polyclonal cell populations expressing the luciferase reporter gene under the control of the SsMx promoter have been used to determine the ability of this promoter to drive the expression of the luciferase gene after poly I:C stimulation. In addition, the implication of each interferon-stimulated response element (ISRE) in the activation of the promoter has also been analysed. The genomic structure of the Senegalese sole and Japanese flounder Mx promoters (containing three ISREs) differs from the rest of the fish Mx promoters described to date. The ISRE1, the one closest to the start codon, is the main ISRE involved in the SsMx promoter activity, whereas ISRE2 and ISRE3 show a minor additive effect on this activity. Another feature differing SsMx promoter from the rest of the fish Mx promoters is the presence of a 24-bp GC island close to the ATG codon, including one Sp1 binding site, which may constitute the transcriptional start site. Furthermore, the SsMx promoter contains a gamma interferon activation site (GAS) element.

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

Abstract 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. HighlightsSenegalese sole isg15 gene (ssisg15) contains a 402‐bp intron in the 5′‐UTR.Maximum levels of ssisg15 transcripts were reached in brain, head kidney and gills after poly I:C induction.RGNNV/SJNNV induced the highest levels of ssisg15 transcripts with a delayed onset.The RGNNV/SJNNV isolate reduces poly I:C induced ssisg15 transcript levels.

Differential response of the Senegalese sole (Solea senegalensis) Mx promoter to viral infections in two salmonid cell lines

Mx proteins are main effectors of the antiviral innate immune defence mediated by type I interferon (IFN I). The IFN I response is under a complex regulation; hence, one of the key issues in understanding virus-host interaction is the knowledge of the regulatory mechanisms governing this response. With this purpose, in this study Chinook salmon embryo cells (CHSE-214) and rainbow trout gonad cells (RTG-2) were transiently transfected with a vector containing the luciferase reporter gene under the control of the Senegalese sole Mx promoter. These transfected cells were infected with infectious pancreatic necrosis virus (IPNV), viral haemorrhagic septicaemia virus (VHSV) and epizootic haematopoietic necrosis virus (EHNV) at different doses in order to study the luciferase fold induction in response to viral infections. Transfected CHSE-214 cells infected with EHNV showed significant induction of the luciferase reporter gene, compared to control non-infected cells, at different times post infection (p.i.). The maximum expression was recorded at 24h p.i. in cells inoculated with 5 × 10(2)TCID50/mL (2.17 folds compared to control cells). In these cells, the infection with IPNV and VHSV did not result in the luciferase expression at any time and doses tested. In transfected RTG-2 cells, VHSV stimulated luciferase expression, obtaining a maximum activity at 48 h p.i. in cells infected with 5 × 10(2)TCID50/mL (2.9 folds compared to control cells), whereas RTG-2 cells infected with IPNV and EHNV did not show significant luciferase activity at any time point. The different induction of the Senegalese sole Mx promoter in CHSE-214 and RTG-2 cells after infection with the same viruses indicates that cell-specific factors are significantly involved in the IFN-signalling response, and, probably, on the success of the strategies of these viruses to escape the IFN mechanisms. The use of these two different cellular systems might be an interesting approach to identify such cellular factors.

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

ABSTRACT 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‐&agr; 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‐&ggr; 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. HIGHLIGHTSIFN I system response against RGNNV is more intense, especially in kidney.Transcription of inflammatory genes is higher in brain, with RGNNV being a stronger inductor.Antibody response against SJNNV is more intense.The early TR‐&ggr; response in brain suggests a key role of acquired immunity in controlling viral replication.

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.

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

Abstract 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. HighlightsIntracellular sea bass ISG15 exerts an antiviral role against SJNNV infection.The intracellular anti‐RGNNV activity of the sea bass ISG15 has not been recorded.Intracellular sea bass ISG15 modulates gene transcription in RGNNV‐inoculated cells.Medium containing sea bass ISG15 confers protection from RGNNV and SJNNV.