RESUMEN

Antimicrobial resistance is a major global health problem, and, among Gram-positive bacteria, methicillin-resistant Staphylococcus aureus (MRSA) represents a serious threat. MRSA causes a wide range of infections, including bacteremia, which, due to the limited use of ß-lactams, is difficult to treat. This study aimed to analyze 51 MRSA isolates collected in 2018 from samples of patients with bacteremia from two hospitals of the Metropolitan Health Service of Santiago, Chile, both in their resistance profile and in the identification of virulence factors. In addition, genomic characterization was carried out by the WGS of an isolate that was shown to be the one of greatest concern (N°. 42) due to its intermediate resistance to vancomycin, multiple virulence factors and being classified as ST8 PVL-positive. In our study, most of the isolates turned out to be multidrug-resistant, but there are still therapeutic options, such as tetracycline, rifampicin, chloramphenicol and vancomycin, which are currently used for MRSA infections; however, 18% were PVL positive, which suggests greater virulence of these isolates. It was determined that isolate N°42 is grouped within the USA300-LV strains (ST8, PVL+, COMER+); however, it has been suggested that, in Chile, a complete displacement of the PVL-negative ST5 clone has not occurred.

RESUMEN

The COVID-19 pandemic has resulted in millions of deaths globally, and while several diagnostic systems were proposed, real-time reverse transcription polymerase chain reaction (RT-PCR) remains the gold standard. However, diagnostic reagents, including enzymes used in RT-PCR, are subject to centralized production models and intellectual property restrictions, which present a challenge for less developed countries. With the aim of generating a standardized One-Step open RT-qPCR protocol to detect SARS-CoV-2 RNA in clinical samples, we purified and tested recombinant enzymes and a non-proprietary buffer. The protocol utilized M-MLV RT and Taq DNA pol enzymes to perform a Taqman probe-based assay. Synthetic RNA samples were used to validate the One-Step RT-qPCR components, demonstrating sensitivity comparable to a commercial kit routinely employed in clinical settings for patient diagnosis. Further evaluation on 40 clinical samples (20 positive and 20 negative) confirmed its comparable diagnostic accuracy. This study represents a proof of concept for an open approach to developing diagnostic kits for viral infections and diseases, which could provide a cost-effective and accessible solution for less developed countries.

Asunto(s)

COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/diagnóstico , Prueba de COVID-19 , ARN Viral/genética , ARN Viral/análisis , Pandemias , Técnicas de Laboratorio Clínico/métodos , Sensibilidad y Especificidad

RESUMEN

The piscine orthomyxovirus called infectious salmon anemia virus (ISAV) is one of the most important emerging pathogens affecting the salmon industry worldwide. The first reverse genetics system for ISAV, which allows the generation of recombinant ISA virus (rISAV), is an important tool for the characterization and study of this virus. The plasmid-based reverse genetics system for ISAV includes the use of a novel fish promoter, the Atlantic salmon internal transcribed spacer region 1 (ITS-1). The salmon, viral, and mammalian genetic elements included in the pSS-URG vectors allow the expression of the eight viral RNA segments. In addition to four cytomegalovirus (CMV)-based vectors that express the four proteins of the ISAV ribonucleoprotein complex, the eight pSS-URG vectors allowed the generation of infectious rISAV in salmon cells.

Asunto(s)

Enfermedades de los Peces , Isavirus , Infecciones por Orthomyxoviridae , Orthomyxoviridae , Animales , Isavirus/genética , ADN Complementario/genética , Línea Celular , Orthomyxoviridae/genética , ARN Viral/genética , Infecciones por Orthomyxoviridae/veterinaria , Salmón/genética , Mamíferos/genética

RESUMEN

Infectious salmon anemia virus (ISAV) is the etiological agent of infectious salmon anemia. It belongs to the genus isavirus, one of the genera of the Orthomyxoviridae family, as does Influenzavirus A. The ISAV genome comprises eight negative-sense single-stranded RNA segments that code for at least 10 proteins. Although some ISAV strains can reach 100% mortality rates, the factors that determine isavirus infectivity remain unknown. However, some studies suggest that segments 5 and 6 are responsible for the different degrees of virulence and infectivity among ISAV subtypes, unlike the influenza A virus, where most segments are involved in the virus infectivity. In this work, synthetic reassortant viruses for the eight segments of ISAV were generated by reverse genetics, combining a highly virulent virus, ISAV 752_09 (HPR7b), and an avirulent strain, SK779/06 (HPR0). We characterized the rescued viruses and their capacity to replicate and infect different cell lines, produce plaques in ASK cells, and their ability to induce and modulate the cellular immune response in vitro. Our results show that the majority of ISAV segments are involved in at least one of the analyzed characteristics, segment 5 being one of the most important, allowing HPR0 viruses, among other things, to produce plaques and replicate in CHSE-214 cells. We determined that segments 5 and 6 participate in different stages of the viral cycle, and their compatibility is critical for viral infection. Additionally, we demonstrated that segment 2 can modulate the cellular immune response. Our results indicate a high degree of genetic compatibility between the genomic segments of HPR7b and HPR0, representing a latent risk of reassortant that would give rise to a new virus with an unknown phenotype.

Asunto(s)

Enfermedades de los Peces , Isavirus , Infecciones por Orthomyxoviridae , Salmo salar , Animales , Isavirus/genética , Infecciones por Orthomyxoviridae/veterinaria , Filogenia , Salmo salar/genética , Análisis de Secuencia de ADN

RESUMEN

The ISAV has a genome composed of eight segments of (-)ssRNA, segment 6 codes for the hemagglutinin-esterase protein, and has the most variable region of the genome, the highly polymorphic region (HPR), which is unique among orthomyxoviruses. The HPR has been associated with virulence, infectivity and pathogenicity. The full length of the HPR is called HPR0 and the strain with this HPR is avirulent, in contrast to strains with deleted HPR that are virulent to varying degrees. The molecular mechanism that gives rise to the different HPRs remains unclear. Here, we studied in vitro the evolution of reassortant recombinant ISAV (rISAV) in Atlantic salmon head kidney (ASK) cells. To this end, we rescued and cultivated a set of rISAV with different segment 6-HPR genotypes using a reverse genetics system and then sequencing HPR regions of the viruses. Our results show rapid multiple recombination events in ISAV, with sequence insertions and deletions in the HPR, indicating a dynamic process. Inserted sequences can be found in four segments of the ISAV genome (segments 1, 5, 6, and 8). The results suggest intra-segmental heterologous recombination, probably by class I and class II template switching, similar to the proposed segment 5 recombination mechanism.

Asunto(s)

Isavirus/genética , Isavirus/patogenicidad , Recombinación Genética , Animales , Línea Celular , Enfermedades de los Peces/virología , Genotipo , Hemaglutininas Virales/genética , Infecciones por Orthomyxoviridae/virología , Salmo salar , Análisis de Secuencia de ADN , Proteínas Virales de Fusión/genética , Virulencia/genética

RESUMEN

Flavanones (-)-(2S)-5,4'-dihydroxy-7-methoxyflavanone (1) and (-)-(2S)-5,3',4'-trihydroxy-7-methoxyflavanone (2) were isolated from the extracts of Calceolaria thyrsiflora Graham, an endemic perennial small shrub growing in the central zone of Chile. The absolute configuration of these compounds was resolved by optical rotation experiments and in silico calculations. Three analogs (3, 4, and 5) were synthesized to do structure-activity relationships with the biological assays studied. Biological tests revealed that only flavanone 2 exhibited a moderate inhibitory activity against the methicillin-resistant strain S. aureus MRSA 97-77 (MIC value of 50 µg/ml). In addition, flavanone 2 showed a potent, selective, and competitive inhibition of 5-hLOX, which supports the traditional use of this plant as an anti-inflammatory in diseases of the respiratory tract. Also, 2 exhibited cytotoxic and selective effects against B16-F10 (8.07 ± 1.61 µM) but 4.6- and 17-fold lesser activity than etoposide and taxol.

RESUMEN

Reovirus is known to have an anticancer effect in both the preclinical and clinical assays. Current evidence suggests that the reovirus-mediated impact on tumor growth depends on the activation of specific antitumor immune responses. A feasible explanation for the oncolytic effects and immune system activation is through the expression of the fusogenic reovirus protein. In this work, we evaluated the in vivo antitumor effects of the expression of fusogenic protein p10 of avian reovirus (ARV-p10). We used chitosan nanoparticles (CH-NPs) as a vehicle for the ARV-p10 DNA in murine B16 melanoma models both in vitro and in vivo. We confirmed that ARV-p10 delivery through a chitosan-based formulation (ARV-p10 CH-NPs) was capable of inducing cell fusion in cultured melanoma cells, showing a mild cytotoxic effect. Interestingly, intratumor injection of ARV-p10 CH-NPs delayed tumor growth, without changing lymphoid populations in the tumor tissue and spleen. The injection of chitosan nanoparticles (CH-NPs) also delayed tumor growth, suggesting the nanoparticle itself would attack tumor cells. In conclusion, we proved that in vitro ARV-p10 protein expression using CH-NPs in murine melanoma cells induces a cytotoxic effect associated with its cell fusion. Further studies are necessary for establishing a protocol for efficient in vivo DNA delivery of fusion proteins to produce an antitumoral effect.

Asunto(s)

Vacunas contra el Cáncer , Melanoma Experimental , Orthoreovirus Aviar , Proteínas Recombinantes de Fusión , Proteínas Virales , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Vacunas contra el Cáncer/química , Vacunas contra el Cáncer/genética , Vacunas contra el Cáncer/farmacología , Supervivencia Celular/efectos de los fármacos , Quitosano/química , Sistemas de Liberación de Medicamentos/métodos , Ratones , Ratones Endogámicos C57BL , Nanopartículas/química , Orthoreovirus Aviar/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Transfección , Proteínas Virales/química , Proteínas Virales/genética

RESUMEN

Recombinant vaccines have low-cost manufacturing, regulatory requirements, and reduced side effects compared to attenuated or inactivated vaccines. In the porcine industry, post-weaning multisystemic disease syndrome generates economic losses, characterized by progressive weight loss and weakness in piglets, and it is caused by porcine circovirus type 2 (PCV2). We designed a chimeric antigen (Qm1) to assemble the main exposed epitopes of the Cap-PCV2 protein on the capsid protein of the tobacco necrosis virus (TNV). This design was based on the Cap-N-terminal of an isolated PCV2 virus obtained in Chile. The virus was characterized, and the sequence was clustered within the PCV2 genotype b clade. This chimeric protein was expressed as inclusion bodies in both monomeric and multimeric forms, suggesting a high-molecular-weight aggregate formation. Pigs immunized with Qm1 elicited a strong and specific antibody response, which reduced the viral loads after the PCV2 challenge. In conclusion, the implemented design allowed for the generation of an effective vaccine candidate. Our proposal could be used to express the domains or fragments of antigenic proteins, whose structural complexity does not allow for low-cost production in Escherichia coli. Hence, other antigen domains could be integrated into the TNV backbone for suitable antigenicity and immunogenicity. This work represents new biotechnological strategies, with a reduction in the costs associated with vaccine development.

Asunto(s)

Antígenos Virales/genética , Proteínas de la Cápside/genética , Circovirus/inmunología , Vacunas Virales/genética , Animales , Anticuerpos Antivirales/sangre , Antígenos Virales/inmunología , Proteínas de la Cápside/inmunología , Chile/epidemiología , Infecciones por Circoviridae/epidemiología , Infecciones por Circoviridae/prevención & control , Infecciones por Circoviridae/veterinaria , Circovirus/clasificación , Circovirus/genética , Epítopos , Fermentación , Filogenia , Síndrome Multisistémico de Emaciación Posdestete Porcino/epidemiología , Síndrome Multisistémico de Emaciación Posdestete Porcino/prevención & control , Porcinos , Tombusviridae/genética , Vacunación/veterinaria , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/metabolismo , Vacunas Virales/administración & dosificación , Vacunas Virales/inmunología , Vacunas Virales/metabolismo

RESUMEN

One slightly beige-white pigmented, Gram-stain-negative, rod-shaped bacterium, strain I-STPP5bT, was isolated from the trachea of a Gentoo penguin chick individual (Pygoscelin papua) investigated in Fildes Bay, Chilean Antarctic (62° 12' S, 58° 57' W). I-STPP5bT consists of a 3.4 Mb chromosome with a DNA G+C content of 44.4 mol%. Of the 3056 predicted genes, 1206 were annotated as hypothetical proteins and 51 were tRNAs. Phylogenetic analysis based on nearly full-length 16S rRNA gene sequences showed that the isolate shared a 16S rRNA gene sequence identity to the type strains of Psychrobacter phenylpyruvicus (98.8 %), Psychrobacter arenosus and Psychrobacter pasteurii (both 98.3 %), Psychrobacter piechaudii (98.2 %) and Psychrobacter sanguinis (98.1 %), but 16S rRNA gene sequence similarities to all other Psychrobacter species were ≤98.0 %. Partial gyrB nucleotide and amino acid sequence similarities among strain STPP5bT and the next related type strains were all below 81.8 and 92.9%, respectively. DNA-DNA hybridisation (DDH) with P. phenylpyruvicus LMG 5372T, P. arenosus DSM 15389T and P. sanguinis DSM 23635T also showed low values (all below 30 %). The main cellular fatty acids of the strain were C18 : 1ω9c and C16 : 1ω7c and/or C16 : 1ω6c. Based on phylogenetic, chemotaxonomic, genomic and phenotypic analyses we propose a new species of the genus Psychrobacter, with the name Psychrobacter pygoscelis sp. nov. and strain I-STPP5bT (=CIP 111410T= CCM 8799T=LMG 30301T) as type strain.

Asunto(s)

Filogenia , Psychrobacter/clasificación , Spheniscidae/microbiología , Tráquea/microbiología , Animales , Regiones Antárticas , Técnicas de Tipificación Bacteriana , Composición de Base , Chile , ADN Bacteriano/genética , Ácidos Grasos/química , Hibridación de Ácido Nucleico , Psychrobacter/aislamiento & purificación , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN

RESUMEN

The Isavirus is an orthomyxovirus with a genome composed of eight segments of negative single-strand RNA (-ssRNA). It has been proposed that the eight genomic segments of the Isavirus are organized as a ribonucleoprotein (RNP) complex called a minigenome, which contains all the viral RNA segments, a viral heterotrimeric polymerase and multiple copies of the viral nucleoprotein (NP). Here, we develop an Isavirus minigenome system and show the importance of the formation of active RNPs and the role of viral NP R189, R194, R302 and K325 residues in the NP RNA-binding domain in the context of RNPs. The results indicate it is possible to generate a minigenome in salmon cells, a composite ISAV RNPs with EGFP-based chimeric vRNA with heterotrimeric polymerase (PB1, PB2, PA) and NP protein using CMV-based auxiliary plasmids. It was also shown that NP R189, R194, R302 and K325 residues are important to generate viral mRNA from the constituted RNPs and a detectable reporter protein. This work is the first salmon cell-based minigenome assay for the Isavirus, which was evaluated by a bioinformatic and functional study of the NP protein in viral RNPs, which showed that correct NP-vRNA interaction is key to the functioning of RNPs.

Asunto(s)

Genoma Viral , Isavirus/genética , Motivos de Unión al ARN/genética , Ribonucleoproteínas/genética , Salmo salar/virología , Proteínas Virales/genética , Animales , Genómica

RESUMEN

We investigated twelve benzyl phenyl ketone derivatives which are synthetic precursors of isoflavonoids that are shown be good 5-hLOX inhibitors, especially those that have the catechol group, but these precursors never have been assayed as 5-hLOX inhibitors being a novelty as inhibitors of the enzyme, due to sharing important structural characteristics. Screening assays, half maximal inhibitory concentration (IC50) and kinetic assays of all the studied molecules (5 µg/ml in media assay) showed that 1-(2,4-dihydroxy-3-methylphenyl)-2-(3-chlorophenyl)-ethanone (K205; IC50 = 3.5 µM; Ki = 4.8 µM) and 1-(2,4-dihydroxy-3-methylphenyl)-2-(2-nitrophenyl)-ethanone (K206; IC50 = 2.3 µM; Ki = 0.7 µM) were potent, selective, competitive and nonredox inhibitors of 5-hLOX. Antioxidant behavior was also assayed by DPPH, FRAP, and assessing ROS production, and those with antibacterial and antiproliferative properties relating to 1-(2,4-dihydroxy-3-methylphenyl)-2-(2-chlorophenyl)-ethanone (K208) established it as the most interesting and relevant compound studied, as it showed nearly 100% inhibition of bacterial growth of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Finally, docking studies were done that helped to characterize how the inhibitor structures correlated to decreased 5-hLOX activity.

Asunto(s)

Antibacterianos/farmacología , Benzoína/análogos & derivados , Benzoína/farmacología , Inhibidores de la Lipooxigenasa/farmacología , Animales , Antibacterianos/síntesis química , Antibacterianos/química , Araquidonato 5-Lipooxigenasa/química , Araquidonato 5-Lipooxigenasa/metabolismo , Benzoína/síntesis química , Dominio Catalítico , Línea Celular Tumoral , Sinergismo Farmacológico , Escherichia coli/efectos de los fármacos , Humanos , Inhibidores de la Lipooxigenasa/síntesis química , Inhibidores de la Lipooxigenasa/química , Meticilina/farmacología , Ratones , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Especies Reactivas de Oxígeno/metabolismo , Staphylococcus aureus/efectos de los fármacos

RESUMEN

Immunosenescence has been described as age-associated changes in the immune function which are thought to be responsible for the increased morbidity with age. Human Natural Killer (NK) cells are a specialized heterogeneous subpopulation of lymphocytes involved in immune defense against tumor and microbial diseases. Interestingly, aging-related NK cell dysfunction is associated with features of aging such as tumor incidence, reduced vaccination efficacy, and short survival due to infection. It is known that NK cell effector functions are critically dependent on cytokines and metabolic activity. Our aim was to determine whether there is a difference in purified human NK cell function in response to high concentration of IL-2 between young and elder donors. Here, we report that the stimulation of human NK cells with IL-2 (2000 U/mL) enhance NK cell cytotoxic activity from both young and elderly donors. However, while NK cells from young people responded to IL-2 signaling by increasing mitochondrial mass and mitochondrial membrane potential, no increase in these mitochondrial functional parameters was seen in purified NK cells from elderly subjects. Moreover, as purified NK cells from the young exhibited an almost three-fold increase in PGC-1α expression after IL-2 (2000 U/mL) stimulation, PGC-1α expression was inhibited in purified NK cells from elders. Furthermore, this response upon PGC-1α expression after IL-2 stimulation promoted an increase in ROS production in NK cells from elderly humans, while no increase in ROS production was observed in NK cells of young donors. Our data show that IL-2 stimulates NK cell effector function through a signaling pathway which involves a PGC-1α-dependent mitochondrial function in young NK cells, however it seems that NK cells from older donors exhibit an altered IL-2 signaling which affects mitochondrial function associated with an increased production of ROS which could represent a feature of NK cell senescence.

Asunto(s)

Células Asesinas Naturales/metabolismo , Mitocondrias/metabolismo , Biogénesis de Organelos , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Anciano , Anciano de 80 o más Años , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Interleucina-2/farmacología , Células K562 , Células Asesinas Naturales/efectos de los fármacos , Masculino , Potencial de la Membrana Mitocondrial , Persona de Mediana Edad , Mitocondrias/efectos de los fármacos , ATPasas de Translocación de Protón Mitocondriales/antagonistas & inhibidores , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Transducción de Señal , Regulación hacia Arriba

RESUMEN

Antigen cross-presentation is a crucial step in the assembly of an antitumor immune response leading to activation of naïve CD8 T cells. This process has been extensively used in clinical trials, in which dendritic cells generated in vitro are loaded with tumor antigens and then autotransplanted to the patients. Recently, the use of autologous transplant of dendritic cells fused with dying tumor cells has demonstrated good results in clinical studies. In this work, we generated a similar process in vivo by treating mice with dead tumor cells [cell bodies (CBs)] expressing the fusogenic protein of the infectious salmon anemia virus (ISAV). ISAV fusion protein retains its fusogenic capability when is expressed on mammalian cells in vitro and the CBs expressing it facilitates DCs maturation, antigen transfer by antigen-presenting cells, and increase cross-presentation by DCs in vitro. Additionally, we observed in the melanoma model that CBs with or without ISAV fusion protein reduce tumor growth in prophylactic treatment; however, only ISAV expressing CBs showed an increase CD4 and CD8 cells in spleen. Overall, our results suggest that CBs could be used as a complement with other type of strategies to amplify antitumor immune response.

RESUMEN

A slightly beige-white pigmented, Gram-staining-negative, rod-shaped bacterium, strain M1A1T, was isolated from seawater samples obtained in Fildes Bay, Antarctica (62°12' S 58° 57' W). Phylogenetic analysis based on nearly full-length 16S rRNA gene sequences showed that the isolate shared 98.4 % 16S rRNA gene sequence identity to the type strain of Psychromonas arctica, but less than 97 % 16S rRNA gene sequence similarities to all other species of the genus Psychromonas. DNA-DNA hybridization with Psychromonas arctica DSM 14288T showed low values (21 %, reciprocal 27 %). The main cellular fatty acid of strain M1A1T was summed feature 3 fatty acids (C16 : 1ω7c/C16 : 1ω8c), followed by C16 : 0. Based on phylogenetic, chemotaxonomic, genomic and phenotypic analyses, we propose a novel species of the genus Psychromonas with the name Psychromonas aquatilis sp. nov. and the strain M1A1T (=CIP 111183T=CCM 8710T=LMG 29766T) as type strain.

Asunto(s)

Gammaproteobacteria/clasificación , Filogenia , Agua de Mar/microbiología , Regiones Antárticas , Técnicas de Tipificación Bacteriana , Composición de Base , Chile , ADN Bacteriano/genética , Ácidos Grasos/química , Gammaproteobacteria/genética , Gammaproteobacteria/aislamiento & purificación , Hibridación de Ácido Nucleico , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN

RESUMEN

BACKGROUND: ISAV is a member of the Orthomyxoviridae family that affects salmonids with disastrous results. It was first detected in 1984 in Norway and from then on it has been reported in Canada, United States, Scotland and the Faroe Islands. Recently, an outbreak was recorded in Chile with negative consequences for the local fishing industry. However, few studies have examined available data to test hypotheses associated with the phylogeographic partitioning of the infecting viral population, the population dynamics, or the evolutionary rates and demographic history of ISAV. To explore these issues, we collected relevant sequences of genes coding for both surface proteins from Chile, Canada, and Norway. We addressed questions regarding their phylogenetic relationships, evolutionary rates, and demographic history using modern phylogenetic methods. RESULTS: A recombination breakpoint was consistently detected in the Hemagglutinin-Esterase (he) gene at either side of the Highly Polymorphic Region (HPR), whereas no recombination breakpoints were detected in Fusion protein (f) gene. Evolutionary relationships of ISAV revealed the 2007 Chilean outbreak group as a monophyletic clade for f that has a sister relationship to the Norwegian isolates. Their tMRCA is consistent with epidemiological data and demographic history was successfully recovered showing a profound bottleneck with further population expansion. Finally, selection analyses detected ongoing diversifying selection in f and he codons associated with protease processing and the HPR region, respectively. CONCLUSIONS: Our results are consistent with the Norwegian origin hypothesis for the Chilean outbreak clade. In particular, ISAV HPR0 genotype is not the ancestor of all ISAV strains, although SK779/06 (HPR0) shares a common ancestor with the Chilean outbreak clade. Our analyses suggest that ISAV shows hallmarks typical of RNA viruses that can be exploited in epidemiological and surveillance settings. In addition, we hypothesized that genetic diversity of the HPR region is governed by recombination, probably due to template switching and that novel fusion gene proteolytic sites confer a selective advantage for the isolates that carry them. Additionally, protein modeling allowed us to relate the results of phylogenetic studies with the predicted structures. This study demonstrates that phylogenetic methods are important tools to predict future outbreaks of ISAV and other salmon pathogens.

Asunto(s)

Evolución Molecular , Isavirus/genética , Filogeografía , Animales , Canadá/epidemiología , Chile/epidemiología , Variación Genética , Isavirus/clasificación , Isavirus/aislamiento & purificación , Modelos Genéticos , Noruega/epidemiología , Infecciones por Orthomyxoviridae/epidemiología , Infecciones por Orthomyxoviridae/veterinaria , Infecciones por Orthomyxoviridae/virología , Filogenia , ARN Viral/genética , Recombinación Genética , Salmón/virología , Análisis de Secuencia de ARN

RESUMEN

The infectious salmon anemia virus (ISAV), which belongs to the Orthomyxoviridae family, has been responsible for major losses in the salmon industry, with mortalities close to 100% in areas where Atlantic salmon (Salmo salar) is grown. This work studied the effect of ribavirin (1-ß-d-ribofuranosyl-1,2,3-triazole-3-carbaxaide), a broad-spectrum antiviral compound with proven ability to inhibit the replicative cycle of the DNA and RNA viruses. The results show that ribavirin was able to inhibit the infectivity of ISAV in in vitro assays. In these assays, a significant inhibition of the replicative viral cycle was observed with a 50% inhibitory concentration (IC50) of 0.02 µg/ml and an IC90 of 0.4 µg/ml of ribavirin. After ribavirin treatment, viral proteins were not detectable and a reduction of viral mRNA association with ribosomes was observed. Ribavirin does not affect the levels of EF1a, nor its association with polysomes, suggesting that the inhibition of RNA synthesis occurs specifically for the virus mRNAs and not for cellular mRNAs. Moreover, ribavirin caused a significant reduction in genomic and viral RNA messenger levels. The study of the inhibitory mechanism showed that it was not reversed by the addition of guanosine. Furthermore, in vivo assays showed a reduction in the mortality of Salmo salar by more than 90% in fish infected with ISAV and treated with ribavirin without adverse effects. In fact, these results show that ribavirin is an antiviral that could be used to prevent ISAV replication either in vitro or in vivo.

Asunto(s)

Antivirales/farmacología , Enfermedades de los Peces/tratamiento farmacológico , Isavirus/efectos de los fármacos , Infecciones por Orthomyxoviridae/veterinaria , Ribavirina/farmacología , Salmo salar/virología , Animales , Células Cultivadas , Enfermedades de los Peces/virología , Técnica del Anticuerpo Fluorescente , Guanosina/farmacología , Concentración 50 Inhibidora , Isavirus/fisiología , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Infecciones por Orthomyxoviridae/virología , Reacción en Cadena de la Polimerasa , Polirribosomas/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/metabolismo , ARN Viral/biosíntesis , ARN Viral/metabolismo , Replicación Viral/efectos de los fármacos

RESUMEN

The infectious salmon anemia virus (ISAV) is the causative agent of the ISA syndrome that affects mainly Atlantic salmon (Salmo salar) and has caused high mortality epidemics in Norway, Scotland, Canada, the United States and Chile. It is classified as an Orthomyxoviridae, its genome is composed of 8 single-strand RNA segments with negative polarity that code for 11 polypeptides. Through functional studies of the coded proteins it has been established that RNA segments 5 and 6 code for a fusion protein and hemagglutinin, respectively, while two polypeptides coded by segments 7 and 8 inhibit interferon induction. The functions of the rest of the possible proteins coded by the viral genome have been assigned by comparison with the corresponding ones of the influenza virus genome. As to its pathogenicity, some growth parameters such as incubation period, resistance to chemical and physical factors, establishment of the infection in other marine species, and dissemination ability among the different organs have been evaluated in several salmonids. Genomic analysis has shown (i) the existence of a high polymorphism region (HPR) in segment 6, and (ii) sequence insertion in segment 5. More than 20 HPR variants have been determined, all originating from HPR0, which is associated with low pathogenicity, while 4 different sequence insertions in segment 5 have not been related with some characteristic of the virus infection. Much progress has been made in the characterization of the virus in 20 years of study, but more detailed knowledge of the specific function of the proteins coded by all the viral genes is still missing, including the pathogenicity mechanism at the molecular level.

Asunto(s)

Enfermedades de los Peces/virología , Isavirus/genética , Isavirus/patogenicidad , Infecciones por Orthomyxoviridae/veterinaria , Salmo salar/virología , Animales , Canadá , Chile , Brotes de Enfermedades , Noruega , Infecciones por Orthomyxoviridae/epidemiología , Infecciones por Orthomyxoviridae/virología , Polimorfismo Genético , ARN Viral/genética , Escocia , Estados Unidos , Proteínas Virales/genética , Virulencia

RESUMEN

Infectious Pancreatic Necrosis Virus (IPNV) is a bisegmented, double-stranded RNA virus, which belongs to the Birnaviridae family. In the current study, we have analyzed the RNA replication intermediates (RI) purified throughout the viral replication cycle in cultured cells. Equilibrium ultracentrifugation of infected cellular lysates resulted in two major peaks of viral components. The first peak, at a buoyant density of 1.33 g/cm(3), contained assembled IPNV viral particles A and B, whereas the second peak, located at buoyant densities >1.4 g/cm(3), contained a higher molecular weight viral ribonucleoprotein complex composed of, at least, VPg/VP1 and a heterogeneous population of single- and double-stranded viral RNA species. Interestingly, analyses of these dsRNA RI indicated that they contain single-stranded segments of incompletely synthesized positive-strands of RNA. Northern blot experiments of total RNA isolated from infected cells confirmed our proposed configuration of the RNA RI, where the full-length negative-strand of RNA is used as the template for the synthesis of several 3'-truncated forms of the positive-strand of the viral RNA. Together, our results indicate that IPNV utilizes the negative-strand of RNA as template for genome replication.

Asunto(s)

Virus de la Necrosis Pancreática Infecciosa/fisiología , ARN Viral/aislamiento & purificación , ARN Viral/metabolismo , Replicación Viral , Animales , Línea Celular , Centrifugación Isopicnica , Sustancias Macromoleculares/aislamiento & purificación , Salmón