29 results on '"Toro-Ascuy D"'
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2. Development of plaque assay for Chilean Infectious Salmon Anaemia Virus, application for virus purification and titration in salmon ASK cells
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Castillo‐Cerda, M T, primary, Cottet, L, additional, Toro‐Ascuy, D, additional, Spencer, E, additional, and Cortez‐San Martín, M, additional
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- 2013
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3. Development of plaque assay for Chilean Infectious Salmon Anaemia Virus, application for virus purification and titration in salmon ASK cells.
- Author
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Castillo‐Cerda, M T, Cottet, L, Toro‐Ascuy, D, Spencer, E, and Cortez‐San Martín, M
- Subjects
ATLANTIC salmon ,FISH viruses ,ORTHOMYXOVIRUSES ,CELL culture ,CELL lines ,PLAQUE assay technique ,IN situ hybridization ,IMMUNOFLUORESCENCE - Abstract
The article presents a study which investigates the development of plaque assay for Chilean infectious salmon anaemia virus (ISAV) belonging to the Orthomyxoviridae family and the Isavirus genus. The study involves various methods in detecting ISAV including in situ hybridization, indirect immunofluorescence and isolation in cell cultures of Atlantic salmon kidney (ASK) cells. Results found that ASK cell line is susceptible to ISAV infection and cytopathic effects post-infection.
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- 2014
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4. Salmon-IgM Functionalized-PLGA Nanosystem for Florfenicol Delivery as an Antimicrobial Strategy against Piscirickettsia salmonis .
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Velásquez F, Frazao M, Diez A, Villegas F, Álvarez-Bidwell M, Rivas-Pardo JA, Vallejos-Vidal E, Reyes-López F, Toro-Ascuy D, Ahumada M, and Reyes-Cerpa S
- Abstract
Salmonid rickettsial septicemia (SRS), caused by Piscirickettsia salmonis , has been the most severe health concern for the Chilean salmon industry. The efforts to control P. salmonis infections have focused on using antibiotics and vaccines. However, infected salmonids exhibit limited responses to the treatments. Here, we developed a poly (D, L-lactide-glycolic acid) (PLGA)-nanosystem functionalized with Atlantic salmon IgM (PLGA-IgM) to specifically deliver florfenicol into infected cells. Polymeric nanoparticles (NPs) were prepared via the double emulsion solvent-evaporation method in the presence of florfenicol. Later, the PLGA-NPs were functionalized with Atlantic salmon IgM through carbodiimide chemistry. The nanosystem showed an average size of ~380-410 nm and a negative surface charge. Further, florfenicol encapsulation efficiency was close to 10%. We evaluated the internalization of the nanosystem and its impact on bacterial load in SHK-1 cells by using confocal microscopy and qPCR. The results suggest that stimulation with the nanosystem elicits a decrease in the bacterial load of P. salmonis when it infects Atlantic salmon macrophages. Overall, the IgM-functionalized PLGA-based nanosystem represents an alternative to the administration of antibiotics in salmon farming, complementing the delivery of antibiotics with the stimulation of the immune response of infected macrophages.
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- 2024
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5. Rescue of Infectious Salmon Anemia Virus (ISAV) from Cloned cDNA.
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Toro-Ascuy D, Cárdenas M, Vásquez-Martínez Y, and Cortez-San Martín M
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- Animals, DNA, Complementary genetics, Cell Line, RNA, Viral genetics, Salmon genetics, Mammals genetics, Isavirus genetics, Orthomyxoviridae genetics, Orthomyxoviridae Infections veterinary, Fish Diseases
- Abstract
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., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
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- 2024
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6. KLF17 is an important regulatory component of the transcriptomic response of Atlantic salmon macrophages to Piscirickettsia salmonis infection.
- Author
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Pérez-Stuardo D, Frazão M, Ibaceta V, Brianson B, Sánchez E, Rivas-Pardo JA, Vallejos-Vidal E, Reyes-López FE, Toro-Ascuy D, Vidal EA, and Reyes-Cerpa S
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- Animals, Gene Expression Profiling, Macrophages metabolism, Transcription Factors metabolism, RNA metabolism, Mammals, Salmo salar genetics
- Abstract
Piscirickettsia salmonis is the most important health problem facing Chilean Aquaculture. Previous reports suggest that P. salmonis can survive in salmonid macrophages by interfering with the host immune response. However, the relevant aspects of the molecular pathogenesis of P. salmonis have been poorly characterized. In this work, we evaluated the transcriptomic changes in macrophage-like cell line SHK-1 infected with P. salmonis at 24- and 48-hours post-infection (hpi) and generated network models of the macrophage response to the infection using co-expression analysis and regulatory transcription factor-target gene information. Transcriptomic analysis showed that 635 genes were differentially expressed after 24- and/or 48-hpi. The pattern of expression of these genes was analyzed by weighted co-expression network analysis (WGCNA), which classified genes into 4 modules of expression, comprising early responses to the bacterium. Induced genes included genes involved in metabolism and cell differentiation, intracellular transportation, and cytoskeleton reorganization, while repressed genes included genes involved in extracellular matrix organization and RNA metabolism. To understand how these expression changes are orchestrated and to pinpoint relevant transcription factors (TFs) controlling the response, we established a curated database of TF-target gene regulatory interactions in Salmo salar , SalSaDB. Using this resource, together with co-expression module data, we generated infection context-specific networks that were analyzed to determine highly connected TF nodes. We found that the most connected TF of the 24- and 48-hpi response networks is KLF17, an ortholog of the KLF4 TF involved in the polarization of macrophages to an M2-phenotype in mammals. Interestingly, while KLF17 is induced by P. salmonis infection, other TFs, such as NOTCH3 and NFATC1, whose orthologs in mammals are related to M1-like macrophages, are repressed. In sum, our results suggest the induction of early regulatory events associated with an M2-like phenotype of macrophages that drives effectors related to the lysosome, RNA metabolism, cytoskeleton organization, and extracellular matrix remodeling. Moreover, the M1-like response seems delayed in generating an effective response, suggesting a polarization towards M2-like macrophages that allows the survival of P. salmonis . This work also contributes to SalSaDB, a curated database of TF-target gene interactions that is freely available for the Atlantic salmon community., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Pérez-Stuardo, Frazão, Ibaceta, Brianson, Sánchez, Rivas-Pardo, Vallejos-Vidal, Reyes-López, Toro-Ascuy, Vidal and Reyes-Cerpa.)
- Published
- 2023
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7. Overnutrition as a risk factor for more serious respiratory viral infections in children: A retrospective study in hospitalized patients.
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Cáceres C, Castillo M, Carrillo K, Tapia CV, Valderrama G, Maquilón C, Toro-Ascuy D, Zorondo-Rodríguez F, and Fuenzalida LF
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- Humans, Child, Child, Preschool, Adolescent, Retrospective Studies, Overweight epidemiology, Obesity epidemiology, Risk Factors, Viruses, Virus Diseases epidemiology, Respiratory Tract Infections epidemiology
- Abstract
Background: The prevalence of obesity has increased dramatically in children worldwide. Obesity has been recognized as a risk factor for more serious viral respiratory infections, mainly in adults., Objective: To study the relationship between overnutrition (obesity and overweight) and clinical severity in children hospitalized with acute respiratory infections of viral origin., Methods: One hundred and forty-three clinical records of children between 2 and 18 years old hospitalized for acute respiratory infection at Clínica Dávila (2014-2018) were analyzed, recording the respiratory viruses detected at the time of hospitalization, weight, and height. Nutritional status was estimated using Z score or body mass index, according to age., Results: Eighty-tree3 children (58%) were positive for more than one respiratory virus. The main virus detected in monoinfection was adenovirus (9.8%), followed by respiratory syncytial virus (7.7%) and parainfluenza virus (7.7%). There were no deaths. Patients with obesity presented more days of hospitalization (P = .04), oxygen therapy (P = .03) and mechanical ventilation (P < .001), as well as a higher probability of requiring mechanical ventilation (P = .001) and of ICU admission (P = .003) compared with children with normal weight. Patients with overweight presented more days of mechanical ventilation (P < .001) than patients with normal weight. No significant differences were found between the presence of viral coinfection and nutritional status., Conclusion: Overnutrition is associated with greater severity of viral respiratory infection in hospitalized children., (Copyright © 2023 SEEN and SED. Published by Elsevier España, S.L.U. All rights reserved.)
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- 2023
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8. Microbiota Profile of the Nasal Cavity According to Lifestyles in Healthy Adults in Santiago, Chile.
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Toro-Ascuy D, Cárdenas JP, Zorondo-Rodríguez F, González D, Silva-Moreno E, Puebla C, Nunez-Parra A, Reyes-Cerpa S, and Fuenzalida LF
- Abstract
Background: The respiratory microbiome is dynamic, varying between anatomical niches, and it is affected by various host and environmental factors, one of which is lifestyle. Few studies have characterized the upper respiratory tract microbiome profile according to lifestyle. We explored the association between lifestyles and microbiota profiles in the upper respiratory tract of healthy adults., Methods: We analyzed nasal samples from 110 healthy adults who were living in Santiago, Chile, using 16S ribosomal RNA gene-sequencing methods. Volunteers completed a structured questionnaire about lifestyle., Results: The composition and abundance of taxonomic groups varied across lifestyle attributes. Additionally, multivariate models suggested that alpha diversity varied in the function of physical activity, nutritional status, smoking, and the interaction between nutritional status and smoking, although the significant impact of those variables varied between women and men. Although physical activity and nutritional status were significantly associated with all indexes of alpha diversity among women, the diversity of microbiota among men was associated with smoking and the interaction between nutritional status and smoking., Conclusions: The alpha diversity of nasal microbiota is associated with lifestyle attributes, but these associations depend on sex and nutritional status. Our results suggest that future studies of the airway microbiome may provide a better resolution if data are stratified for differences in sex and nutritional status.
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- 2023
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9. Overnutrition, Nasopharyngeal Pathogenic Bacteria and Proinflammatory Cytokines in Infants with Viral Lower Respiratory Tract Infections.
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Arias-Bravo G, Valderrama G, Inostroza J, Tapia C, Toro-Ascuy D, Ramilo O, Orellana P, Cifuentes-Muñoz N, Zorondo-Rodríguez F, Mejias A, and Fuenzalida LF
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- Bacteria, Child, Child, Preschool, Cytokines, Haemophilus influenzae, Humans, Infant, Moraxella catarrhalis, Nasopharynx, Staphylococcus aureus, Streptococcus pneumoniae, Overnutrition, Respiratory Tract Infections microbiology
- Abstract
Background: Little is known about the interaction between the nasopharyngeal bacterial profile and the nutritional status in children. In this study, our main goal was to evaluate the associations between overnutrition and the presence of four potentially pathogenic bacteria in the nasopharynx of infants with viral lower respiratory tract infections (LRTI). In addition, we determined whether changes in the nasopharyngeal bacterial profile were associated with mucosal and serum proinflammatory cytokines and with clinical disease severity. Methods: We enrolled 116 children less than 2 years old hospitalized for viral LRTI during two consecutive respiratory seasons (May 2016 to August 2017); their nutritional status was assessed, and nasopharyngeal and blood samples were obtained. S. aureus, S. pneumoniae, H. influenzae, M. catarrhalis, and respiratory viruses were identified in nasopharyngeal samples by qPCR. Cytokine concentrations were measured in nasopharyngeal and blood samples. Disease severity was assessed by the length of hospitalization and oxygen therapy. Results: Nasopharyngeal pathogenic bacteria were identified in 96.6% of the enrolled children, and 80% of them tested positive for two or more bacteria. The presence and loads of M. catarrhalis was higher (p = 0.001 and p = 0.022, respectively) in children with overnutrition (n = 47) compared with those with normal weights (n = 69). In addition, the detection of >2 bacteria was more frequent in children with overnutrition compared to those with normal weight (p = 0.02). Multivariate regression models showed that the presence and loads of S. pneumoniae and M. catarrhalis were associated with higher concentrations of IL-6 in plasma and TNF-α in mucosal samples in children with overnutrition. Conclusions: The nasopharyngeal profile of young children with overnutrition was characterized by an over representation of pathogenic bacteria and proinflammatory cytokines.
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- 2022
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10. Comparison of the First and Second Wave of Infections by SARS-CoV-2: A Retrospective and Longitudinal Study From a Primary Health Care Center in Santiago of Chile.
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Acuña-Castillo C, Inostroza-Molina A, Castro SA, Molina-Cabrera S, Leiva-Salcedo E, Riquelme D, Luraschi R, Barrera-Avalos C, Vallejos-Vidal E, Mella-Torres A, Valdés D, Torres C, Maisey K, Escobar A, Reyes-Cerpa S, Toro-Ascuy D, Imarai M, Reyes-López FE, and Sandino AM
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- Chile epidemiology, Humans, Longitudinal Studies, Pandemics, Primary Health Care, Retrospective Studies, COVID-19 epidemiology, SARS-CoV-2
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The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Many countries have reported the experience of at least two contagion waves, describing associated mortality rates and population behavior. The analysis of the effect of this pandemic in different localities can provide valuable information on the key factors to consider in the face of future massive infectious diseases. This work describes the first retrospective and comparative study about behavior during the first and second waves of the COVID-19 pandemic in Chile from a primary Healthcare Center. From 19,313 real-time quantitative PCR (RT-qPCR) tests assessed, the selected 1,694 positive diagnostics showed a decrease in mortality rate in the second wave (0.6%) compared with the first (4.6%). In addition, we observed that infections in the second wave were mainly in young patients with reduced comorbidities. The population with a complete vaccination schedule shows a decrease in the duration of symptoms related to the disease, and patients with more comorbidities tend to develop severe illness. This report provides evidence to partially understand the behavior and critical factors in the severity of the COVID-19 pandemic in the population of Santiago of Chile., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Acuña-Castillo, Inostroza-Molina, Castro, Molina-Cabrera, Leiva-Salcedo, Riquelme, Luraschi, Barrera-Avalos, Vallejos-Vidal, Mella-Torres, Valdés, Torres, Maisey, Escobar, Reyes-Cerpa, Toro-Ascuy, Imarai, Reyes-López and Sandino.)
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- 2022
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11. Factors Influencing the Acceptance of COVID-19 Vaccines in a Country with a High Vaccination Rate.
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Toro-Ascuy D, Cifuentes-Muñoz N, Avaria A, Pereira-Montecinos C, Cruzat G, Peralta-Arancibia K, Zorondo-Rodríguez F, and Fuenzalida LF
- Abstract
Control of the COVID-19 pandemic largely depends on the effectiveness of the vaccination process. An understanding of the factors that underlie the willingness to accept vaccination contributes pivotal information to controlling the pandemic. We analyzed the association between the willingness to accept the available COVID-19 vaccines and vaccine determinants amidst the Chilean vaccination process. Individual-level survey data was collected from 744 nationally representative respondents and multivariate regression models were used to estimate the association between outcome and explanatory variables. We found that trust in COVID-19 vaccines, scientists, and medical professionals significantly increased the willingness to: accept the vaccines and booster doses, as well as annual vaccinations and the vaccination of children. Our results are critical to understanding the acceptance of COVID-19 vaccines in the context of a country with one of the world's highest vaccination rates. We provide useful information for decision-making and policy design, in addition to establishing guidelines regarding how to effectively explain vaccination programs to citizens.
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- 2022
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12. Epitranscriptomic regulation of HIV-1 full-length RNA packaging.
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Pereira-Montecinos C, Toro-Ascuy D, Ananías-Sáez C, Gaete-Argel A, Rojas-Fuentes C, Riquelme-Barrios S, Rojas-Araya B, García-de-Gracia F, Aguilera-Cortés P, Chnaiderman J, Acevedo ML, Valiente-Echeverría F, and Soto-Rifo R
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- 5' Untranslated Regions, Adenosine genetics, Adenosine metabolism, Gene Products, gag genetics, Methylation, RNA, Viral genetics, RNA, Viral metabolism, Virion metabolism, HIV-1 metabolism
- Abstract
During retroviral replication, the full-length RNA serves both as mRNA and genomic RNA. However, the mechanisms by which the HIV-1 Gag protein selects the two RNA molecules that will be packaged into nascent virions remain poorly understood. Here, we demonstrate that deposition of N6-methyladenosine (m6A) regulates full-length RNA packaging. While m6A deposition by METTL3/METTL14 onto the full-length RNA was associated with increased Gag synthesis and reduced packaging, FTO-mediated demethylation promoted the incorporation of the full-length RNA into viral particles. Interestingly, HIV-1 Gag associates with the RNA demethylase FTO in the nucleus and contributes to full-length RNA demethylation. We further identified two highly conserved adenosines within the 5'-UTR that have a crucial functional role in m6A methylation and packaging of the full-length RNA. Together, our data propose a novel epitranscriptomic mechanism allowing the selection of the HIV-1 full-length RNA molecules that will be used as viral genomes., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)
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- 2022
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13. The Landscape of IFN/ISG Signaling in HIV-1-Infected Macrophages and Its Possible Role in the HIV-1 Latency.
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Rojas M, Luz-Crawford P, Soto-Rifo R, Reyes-Cerpa S, and Toro-Ascuy D
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- Animals, HIV Infections virology, Humans, Signal Transduction physiology, HIV Infections metabolism, Interferon Type I metabolism, Interferons metabolism, Macrophages metabolism, Virus Latency physiology
- Abstract
A key characteristic of Human immunodeficiency virus type 1 (HIV-1) infection is the generation of latent viral reservoirs, which have been associated with chronic immune activation and sustained inflammation. Macrophages play a protagonist role in this context since they are persistently infected while being a major effector of the innate immune response through the generation of type-I interferons (type I IFN) and IFN-stimulated genes (ISGs). The balance in the IFN signaling and the ISG induction is critical to promote a successful HIV-1 infection. Classically, the IFNs response is fine-tuned by opposing promotive and suppressive signals. In this context, it was described that HIV-1-infected macrophages can also synthesize some antiviral effector ISGs and, positive and negative regulators of the IFN/ISG signaling. Recently, epitranscriptomic regulatory mechanisms were described, being the N6-methylation (m6A) modification on mRNAs one of the most relevant. The epitranscriptomic regulation can affect not only IFN/ISG signaling, but also type I IFN expression, and viral fitness through modifications to HIV-1 RNA. Thus, the establishment of replication-competent latent HIV-1 infected macrophages may be due to non-classical mechanisms of type I IFN that modulate the activation of the IFN/ISG signaling network.
- Published
- 2021
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14. CBP80/20-dependent translation initiation factor (CTIF) inhibits HIV-1 Gag synthesis by targeting the function of the viral protein Rev.
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García-de-Gracia F, Gaete-Argel A, Riquelme-Barrios S, Pereira-Montecinos C, Rojas-Araya B, Aguilera P, Oyarzún-Arrau A, Rojas-Fuentes C, Acevedo ML, Chnaiderman J, Valiente-Echeverría F, Toro-Ascuy D, and Soto-Rifo R
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- Cells, Cultured, Down-Regulation, HEK293 Cells, HIV-1 genetics, HIV-1 metabolism, HeLa Cells, Humans, Jurkat Cells, Protein Biosynthesis genetics, rev Gene Products, Human Immunodeficiency Virus physiology, Eukaryotic Initiation Factors physiology, gag Gene Products, Human Immunodeficiency Virus biosynthesis, rev Gene Products, Human Immunodeficiency Virus antagonists & inhibitors
- Abstract
Translation initiation of the human immunodeficiency virus type-1 (HIV-1) full-length RNA has been shown to occur through cap-dependent and IRES-driven mechanisms. Previous studies suggested that the nuclear cap-binding complex (CBC) rather than eIF4E drives cap-dependent translation of the full-length RNA and we have recently reported that the CBC subunit CBP80 supports the function of the viral protein Rev during nuclear export and translation of this viral transcript. Ribosome recruitment during CBC-dependent translation of cellular mRNAs relies on the activity CBP80/20 translation initiation factor (CTIF), which bridges CBP80 and the 40S ribosomal subunit through interactions with eIF3g. Here, we report that CTIF inhibits HIV-1 and HIV-2 Gag synthesis from the full-length RNA. Our results indicate that CTIF associates with HIV-1 Rev through its N-terminal domain and is recruited onto the full-length RNA ribonucleoprotein complex in order to interfere with Gag synthesis. We also demonstrate that CTIF induces the cytoplasmic accumulation of Rev impeding the association of the viral protein with CBP80. We finally show that Rev interferes with the association of CTIF with CBP80 indicating that CTIF and Rev compete for the CBC subunit.
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- 2021
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15. In Situ Hybridization-Proximity Ligation Assay (ISH-PLA) to Study the Interaction of HIV-1 RNA and Remodeling Proteins.
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Toro-Ascuy D, Gaete-Argel A, Rojas-Celis V, and Valiente-Echeverria F
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- HeLa Cells, Humans, Protein Binding, HIV-1 genetics, In Situ Hybridization methods, RNA, Viral metabolism, RNA-Binding Proteins metabolism
- Abstract
The mechanisms involved in the posttranscriptional control of the replicative cycle of the human immunodeficiency virus (HIV), specifically the molecular events which allow the interaction between the viral genomic RNA (gRNA) and the cellular machinery for the transport, translation, or intracellular packaging, have not been yet elucidated. In this chapter, we describe the in situ hybridization-proximity ligation assay (ISH-PLA) to characterize interactions between the genomic RNA (gRNA) of HIV-1 and viral proteins or host proteins involved in nuclear export and translation initiation. We also present data that validate the ISH-PLA as a simple and useful tool to study HIV-1 gRNA-protein interactions within cells.
- Published
- 2021
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16. Non-Specific Antibodies Induce Lysosomal Activation in Atlantic Salmon Macrophages Infected by Piscirickettsia salmonis .
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Pérez-Stuardo D, Espinoza A, Tapia S, Morales-Reyes J, Barrientos C, Vallejos-Vidal E, Sandino AM, Spencer E, Toro-Ascuy D, Rivas-Pardo JA, Reyes-López FE, and Reyes-Cerpa S
- Subjects
- Animals, Fish Diseases microbiology, Lysosomes microbiology, Macrophages microbiology, Piscirickettsiaceae Infections veterinary, Salmon microbiology, Antibodies, Bacterial immunology, Fish Diseases immunology, Lysosomes immunology, Macrophages immunology, Piscirickettsia immunology, Piscirickettsiaceae Infections immunology, Salmon immunology
- Abstract
Piscirickettsia salmonis , an aggressive intracellular pathogen, is the etiological agent of salmonid rickettsial septicemia (SRS). This is a chronic multisystemic disease that generates high mortalities and large losses in Chilean salmon farming, threatening the sustainability of the salmon industry. Previous reports suggest that P. salmonis is able to survive and replicate in salmonid macrophages, inducing an anti-inflammatory environment and a limited lysosomal response that may be associated with host immune evasion mechanisms favoring bacterial survival. Current control and prophylaxis strategies against P. salmonis (based on the use of antibiotics and vaccines) have not had the expected success against infection. This makes it urgent to unravel the host-pathogen interaction to develop more effective therapeutic strategies. In this study, we evaluated the effect of treatment with IgM-beads on lysosomal activity in Atlantic salmon macrophage-enriched cell cultures infected with P. salmonis by analyzing the lysosomal pH and proteolytic ability through confocal microscopy. The impact of IgM-beads on cytotoxicity induced by P. salmonis in infected cells was evaluated by quantification of cell lysis through release of Lactate Dehydrogenase (LDH) activity. Bacterial load was determined by quantification of 16S rDNA copy number by qPCR, and counting of colony-forming units (CFU) present in the extracellular and intracellular environment. Our results suggest that stimulation with antibodies promotes lysosomal activity by lowering lysosomal pH and increasing the proteolytic activity within this organelle. Additionally, incubation with IgM-beads elicits a decrease in bacterial-induced cytotoxicity in infected Atlantic salmon macrophages and reduces the bacterial load. Overall, our results suggest that stimulation of cells infected by P. salmonis with IgM-beads reverses the modulation of the lysosomal activity induced by bacterial infection, promoting macrophage survival and bacterial elimination. This work represents a new important evidence to understand the bacterial evasion mechanisms established by P. salmonis and contribute to the development of new effective therapeutic strategies against SRS., (Copyright © 2020 Pérez-Stuardo, Espinoza, Tapia, Morales-Reyes, Barrientos, Vallejos-Vidal, Sandino, Spencer, Toro-Ascuy, Rivas-Pardo, Reyes-López and Reyes-Cerpa.)
- Published
- 2020
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17. Bacterial Synthesis of Ternary CdSAg Quantum Dots through Cation Exchange: Tuning the Composition and Properties of Biological Nanoparticles for Bioimaging and Photovoltaic Applications.
- Author
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Órdenes-Aenishanslins N, Anziani-Ostuni G, Monrás JP, Tello A, Bravo D, Toro-Ascuy D, Soto-Rifo R, Prasad PN, and Pérez-Donoso JM
- Abstract
In this study, we introduce a biological method for the production of ternary Quantum Dots (QDs): complex nanostructures with tunable optical and structural properties that utilizes post-synthesis modifications through cation exchange. This versatile in-situ cation exchange method being reported for the first time shows great potential for extending the scope of microbial synthesis. By using this bacterial-based method, we easily synthesize and purify CdS, CdSAg, and Ag
2 S nanocrystals of a size below 15 nm and with variable morphologies that exhibit fluorescence emissions covering a broad spectral range (from 400 to 800 nm). Energy-dispersive X-ray spectroscopy (EDS) results indicate the partial replacement of Cd2+ by Ag+ when AgNO3 concentration is increased. This replacement produces CdSAg ternary QDs hetero-structures with high stability, fluorescence in the NIR-I (700 - 800 nm), and 36.13% quantum yield. Furthermore, this reaction can be extended for the production of soluble Ag2 S nanoparticles (NPs) without any traces of Cd. QDs biosynthesized through this cation exchange process display very low toxicity when tested in bacterial or human cell lines. Biosynthesized ternary hetero-structures were used as red fluorescent dyes to label HeLa cells in confocal microscopy studies, which validates its use in bioimaging applications in the near infrared region. In addition, the application of biologically-produced cadmium NPs in solar cells is reported for the first time. The three biosynthesized QDs were successfully used as photosensitizers, where the CdSAg QDs show the best photovoltaic parameters. Altogether, obtained results validate the use of bacterial cells for the controlled production of nanomaterials with properties that allow their application in diverse technologies. We developed a simple biological process for obtaining tunable Quantum Dots (QDs) with different metal compositions through a cation exchange process. Nanoparticles (NPs) are produced in the extracellular space of bacterial cells exposed to cysteine and CdCl2 in a reaction that depends on S2- generation mediated by cysteine desulfhydrase enzymes and uses cellular biomolecules to stabilize the nanoparticle. Using this extracellular approach, water-soluble fluorescent CdS, CdSAg, and Ag2 S Quantum Dots with a tunable emission ranging from 400 to 800 nm were generated. This is the first study reporting the use of microorganisms to produce tunable ternary QDs and the first time that a cation exchange process mediated by cells is described. Obtained results validate the use of biological synthesis to produce NPs with new characteristics and opens a completely new research field related to the use of microorganisms to synthesize complex NPs that are difficult to obtain with regular chemical methods.- Published
- 2020
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18. Development of an Isavirus minigenome system to study the function of the pocket RNA-binding domain of the viral nucleoprotein (NP) in salmon cells.
- Author
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Toro-Ascuy D, Santibañez A, Peña V, Beltran-Pavez C, Cottet L, Molina C, Montoya M, Sandoval N, Vásquez-Martínez Y, Mascayano C, and Cortez-San Martín M
- Subjects
- Animals, Genomics, Genome, Viral, Isavirus genetics, RNA-Binding Motifs genetics, Ribonucleoproteins genetics, Salmo salar virology, Viral Proteins genetics
- Abstract
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., (© 2019 John Wiley & Sons Ltd.)
- Published
- 2020
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19. New Challenges of HIV-1 Infection: How HIV-1 Attacks and Resides in the Central Nervous System.
- Author
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Rojas-Celis V, Valiente-Echeverría F, Soto-Rifo R, and Toro-Ascuy D
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- Central Nervous System metabolism, HIV Infections drug therapy, HIV Infections metabolism, HIV-1 pathogenicity, Humans, Quality of Life, Central Nervous System virology, HIV Infections physiopathology, HIV-1 metabolism, Neurocognitive Disorders virology
- Abstract
Acquired immunodeficiency syndrome (AIDS) has become one of the most devastating pandemics in recorded history. The main causal agent of AIDS is the human immunodeficiency virus (HIV), which infects various cell types of the immune system that express the CD4 receptor on their surfaces. Today, combined antiretroviral therapy (cART) is the standard treatment for all people with HIV; although it has improved the quality of life of people living with HIV (PLWH), it cannot eliminate the latent reservoir of the virus. Therefore HIV/AIDS has turned from a fatal disease to a chronic disease requiring lifelong treatment. Despite significant viral load suppression, it has been observed that at least half of patients under cART present HIV-associated neurocognitive disorders (HAND), which have been related to HIV-1 infection and replication in the central nervous system (CNS). Several studies have focused on elucidating the mechanism by which HIV-1 can invade the CNS and how it can generate the effects seen in HAND. This review summarizes the research on HIV-1 and its interaction with the CNS with an emphasis on the generation of HAND, how the virus enters the CNS, the relationship between HIV-1 and cells of the CNS, and the effect of cART on these cells.
- Published
- 2019
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20. A Rev-CBP80-eIF4AI complex drives Gag synthesis from the HIV-1 unspliced mRNA.
- Author
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Toro-Ascuy D, Rojas-Araya B, García-de-Gracia F, Rojas-Fuentes C, Pereira-Montecinos C, Gaete-Argel A, Valiente-Echeverría F, Ohlmann T, and Soto-Rifo R
- Subjects
- Cell Line, Eukaryotic Initiation Factor-4A metabolism, HIV-1 metabolism, HeLa Cells, Humans, Multiprotein Complexes genetics, Multiprotein Complexes metabolism, Nuclear Cap-Binding Protein Complex metabolism, Protein Binding, RNA Splicing, RNA, Messenger metabolism, RNA, Viral genetics, RNA, Viral metabolism, Virus Replication genetics, gag Gene Products, Human Immunodeficiency Virus biosynthesis, rev Gene Products, Human Immunodeficiency Virus metabolism, Eukaryotic Initiation Factor-4A genetics, HIV-1 genetics, Nuclear Cap-Binding Protein Complex genetics, RNA, Messenger genetics, gag Gene Products, Human Immunodeficiency Virus genetics, rev Gene Products, Human Immunodeficiency Virus genetics
- Abstract
Gag synthesis from the full-length unspliced mRNA is critical for the production of the viral progeny during human immunodeficiency virus type-1 (HIV-1) replication. While most spliced mRNAs follow the canonical gene expression pathway in which the recruitment of the nuclear cap-binding complex (CBC) and the exon junction complex (EJC) largely stimulates the rates of nuclear export and translation, the unspliced mRNA relies on the viral protein Rev to reach the cytoplasm and recruit the host translational machinery. Here, we confirm that Rev ensures high levels of Gag synthesis by driving nuclear export and translation of the unspliced mRNA. These functions of Rev are supported by the CBC subunit CBP80, which binds Rev and the unspliced mRNA in the nucleus and the cytoplasm. We also demonstrate that Rev interacts with the DEAD-box RNA helicase eIF4AI, which translocates to the nucleus and cooperates with the viral protein to promote Gag synthesis. Finally, we show that the Rev/RRE axis is important for the assembly of a CBP80-eIF4AI complex onto the unspliced mRNA. Together, our results provide further evidence towards the understanding of the molecular mechanisms by which Rev drives Gag synthesis from the unspliced mRNA during HIV-1 replication.
- Published
- 2018
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21. Rescue of Infectious Salmon Anemia Virus (ISAV) from Cloned cDNA.
- Author
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Toro-Ascuy D and Martín MC
- Subjects
- Animals, Cell Line, Cloning, Molecular, Gene Expression, Gene Order, Genome, Viral, Plasmids genetics, Reverse Genetics, Transfection, DNA, Complementary, Isavirus genetics, RNA, Viral, Virus Replication
- Abstract
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 fish 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 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.
- Published
- 2017
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22. Avian Influenza Virus H5 Strain with North American and Eurasian Lineage Genes in an Antarctic Penguin.
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Barriga GP, Boric-Bargetto D, San Martin MC, Neira V, van Bakel H, Thompsom M, Tapia R, Toro-Ascuy D, Moreno L, Vasquez Y, Sallaberry M, Torres-Pérez F, González-Acuña D, and Medina RA
- Subjects
- Animals, Antarctic Regions epidemiology, Genes, Viral, Influenza in Birds epidemiology, Phylogeny, Influenza A virus classification, Influenza A virus genetics, Influenza in Birds virology, Spheniscidae virology
- Published
- 2016
- Full Text
- View/download PDF
23. Interactions between the HIV-1 Unspliced mRNA and Host mRNA Decay Machineries.
- Author
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Toro-Ascuy D, Rojas-Araya B, Valiente-Echeverría F, and Soto-Rifo R
- Subjects
- Humans, HIV-1 genetics, Host-Pathogen Interactions, RNA Stability, RNA, Viral metabolism
- Abstract
The human immunodeficiency virus type-1 (HIV-1) unspliced transcript is used both as mRNA for the synthesis of structural proteins and as the packaged genome. Given the presence of retained introns and instability AU-rich sequences, this viral transcript is normally retained and degraded in the nucleus of host cells unless the viral protein REV is present. As such, the stability of the HIV-1 unspliced mRNA must be particularly controlled in the nucleus and the cytoplasm in order to ensure proper levels of this viral mRNA for translation and viral particle formation. During its journey, the HIV-1 unspliced mRNA assembles into highly specific messenger ribonucleoproteins (mRNPs) containing many different host proteins, amongst which are well-known regulators of cytoplasmic mRNA decay pathways such as up-frameshift suppressor 1 homolog (UPF1), Staufen double-stranded RNA binding protein 1/2 (STAU1/2), or components of miRNA-induced silencing complex (miRISC) and processing bodies (PBs). More recently, the HIV-1 unspliced mRNA was shown to contain N ⁶-methyladenosine (m⁶A), allowing the recruitment of YTH N ⁶-methyladenosine RNA binding protein 2 (YTHDF2), an m⁶A reader host protein involved in mRNA decay. Interestingly, these host proteins involved in mRNA decay were shown to play positive roles in viral gene expression and viral particle assembly, suggesting that HIV-1 interacts with mRNA decay components to successfully accomplish viral replication. This review summarizes the state of the art in terms of the interactions between HIV-1 unspliced mRNA and components of different host mRNA decay machineries., Competing Interests: The authors declare no conflict of interest.
- Published
- 2016
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24. Isolation and Characterization of Salmonid CD4+ T Cells.
- Author
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Maisey K, Montero R, Corripio-Miyar Y, Toro-Ascuy D, Valenzuela B, Reyes-Cerpa S, Sandino AM, Zou J, Wang T, Secombes CJ, and Imarai M
- Subjects
- Animals, CD3 Complex immunology, CD3 Complex metabolism, Cell Culture Techniques, Cell Line, Cell Proliferation, Fish Proteins genetics, Fish Proteins metabolism, GATA3 Transcription Factor genetics, GATA3 Transcription Factor metabolism, Interleukin-15 metabolism, Lymphocyte Activation, Mammals immunology, Phylogeny, Receptors, Antigen, T-Cell metabolism, STAT1 Transcription Factor genetics, STAT1 Transcription Factor metabolism, T-Box Domain Proteins genetics, T-Box Domain Proteins metabolism, CD4-Positive T-Lymphocytes physiology, Head Kidney immunology, Oncorhynchus mykiss immunology, Spleen immunology
- Abstract
This study reports the isolation and functional characterization of rainbow trout (Oncorhynchus mykiss) CD4-1(+) T cells and the establishment of an IL-15-dependent CD4-1(+) T cell line. By using Abs specific for CD4-1 and CD3ε it was possible to isolate the double-positive T cells in spleen and head kidney. The morphology and the presence of transcripts for T cell markers in the sorted CD4-1(+)CD3ε(+) cells were studied next. Cells were found to express TCRα, TCRβ, CD152 (CTLA-4), CD154 (CD40L), T-bet, GATA-3, and STAT-1. The sorted CD4-1(+) T cells also had a distinctive functional attribute of mammalian T lymphocytes, namely they could undergo Ag-specific proliferation, using OVA as a model Ag. The OVA-stimulated cells showed increased expression of several cytokines, including IFN-γ1, IL-4/13A, IL-15, IL-17D, IL-10, and TGF-β1, perhaps indicating that T cell proliferation led to differentiation into distinct effector phenotypes. Using IL-15 as a growth factor, we have selected a lymphoid cell line derived from rainbow trout head kidney cells. The morphology, cell surface expression of CD4-1, and the presence of transcripts of T cell cytokines and transcription factors indicated that this is a CD4-1(+) T cell line. To our knowledge, this is the first demonstration of the presence of CD4-1(+)CD3ε(+) T cells in salmonids. As in mammals, CD4-1(+) T cells may be the master regulators of immune responses in fish, and therefore these findings and the new model T cell line developed will contribute to a greater understanding of T cell function and immune responses in teleost fish., (Copyright © 2016 by The American Association of Immunologists, Inc.)
- Published
- 2016
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25. DEAD-box RNA helicase DDX3 connects CRM1-dependent nuclear export and translation of the HIV-1 unspliced mRNA through its N-terminal domain.
- Author
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Fröhlich A, Rojas-Araya B, Pereira-Montecinos C, Dellarossa A, Toro-Ascuy D, Prades-Pérez Y, García-de-Gracia F, Garcés-Alday A, Rubilar PS, Valiente-Echeverría F, Ohlmann T, and Soto-Rifo R
- Subjects
- Active Transport, Cell Nucleus genetics, Gene Expression Regulation, Viral, HIV Infections therapy, HIV Infections virology, HIV-1 pathogenicity, Host-Pathogen Interactions genetics, Humans, Protein Biosynthesis, Protein Structure, Tertiary, RNA, Messenger biosynthesis, RNA, Messenger genetics, RNA, Viral genetics, Virus Replication genetics, Exportin 1 Protein, DEAD-box RNA Helicases genetics, HIV Infections genetics, HIV-1 genetics, Karyopherins genetics, Receptors, Cytoplasmic and Nuclear genetics
- Abstract
DEAD-box RNA helicase DDX3 is a host factor essential for HIV-1 replication and thus, a potential target for novel therapies aimed to overcome viral resistance. Previous studies have shown that DDX3 promotes nuclear export and translation of the HIV-1 unspliced mRNA. Although the function of DDX3 during both processes requires its catalytic activity, it is unknown whether other domains surrounding the helicase core are involved. Here, we show the involvement of the N- and C-terminal domains of DDX3 in the regulation of HIV-1 unspliced mRNA translation. Our results suggest that the intrinsically disordered N-terminal domain of DDX3 regulates its functions in translation by acting prior to the recruitment of the 43S pre-initiation complex onto the viral 5'-UTR. Interestingly, this regulation was conserved in HIV-2 and was dependent on the CRM1-dependent nuclear export pathway suggesting a role of the RNA helicase in interconnecting nuclear export with ribosome recruitment of the viral unspliced mRNA. This specific function of DDX3 during HIV gene expression could be exploited as an alternative target for pharmaceutical intervention., (Copyright © 2016 Elsevier B.V. All rights reserved.)
- Published
- 2016
- Full Text
- View/download PDF
26. Development of a reverse genetic system for infectious salmon anemia virus: rescue of recombinant fluorescent virus by using salmon internal transcribed spacer region 1 as a novel promoter.
- Author
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Toro-Ascuy D, Tambley C, Beltran C, Mascayano C, Sandoval N, Olivares E, Medina RA, Spencer E, and Cortez-San Martín M
- Subjects
- Animals, Fish Proteins metabolism, Fluorescence, Genes, Reporter, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Isavirus chemistry, Isavirus physiology, Orthomyxoviridae Infections virology, Recombinant Proteins genetics, Recombinant Proteins metabolism, Salmo salar virology, Virus Replication, Fish Diseases virology, Fish Proteins genetics, Isavirus genetics, Orthomyxoviridae Infections veterinary, Promoter Regions, Genetic, Reverse Genetics methods
- Abstract
Infectious salmon anemia (ISA) is a serious disease of marine-farmed Atlantic salmon (Salmo salar) caused by ISA virus (ISAV), belonging to the genus Isavirus, family Orthomyxoviridae. There is an urgent need to understand the virulence factors and pathogenic mechanisms of ISAV and to develop new vaccine approaches. Using a recombinant molecular biology approach, we report the development of a plasmid-based reverse genetic system for ISAV, which includes the use of a novel fish promoter, the Atlantic salmon internal transcribed spacer region 1 (ITS-1). Salmon cells cotransfected with pSS-URG-based vectors expressing the eight viral RNA segments and four cytomegalovirus (CMV)-based vectors that express the four proteins of the ISAV ribonucleoprotein complex allowed the generation of infectious recombinant ISAV (rISAV). We generated three recombinant viruses, wild-type rISAV(901_09) and rISAVr(S6-NotI-HPR) containing a NotI restriction site and rISAV(S6/EGFP-HPR) harboring the open reading frame of enhanced green fluorescent protein (EGFP), both within the highly polymorphic region (HPR) of segment 6. All rescued viruses showed replication activity and cytopathic effect in Atlantic salmon kidney-infected cells. The fluorescent recombinant viruses also showed a characteristic cytopathic effect in salmon cells, and the viruses replicated to a titer of 6.5105 PFU/ml,similar to that of the wild-type virus. This novel reverse genetics system offers a powerful tool to study the molecular biology of ISAV and to develop a new generation of ISAV vaccines to prevent and mitigate ISAV infection, which has had a profound effect on the salmon industry.
- Published
- 2015
- Full Text
- View/download PDF
27. Induction of anti-inflammatory cytokine expression by IPNV in persistent infection.
- Author
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Reyes-Cerpa S, Reyes-López F, Toro-Ascuy D, Montero R, Maisey K, Acuña-Castillo C, Sunyer JO, Parra D, Sandino AM, and Imarai M
- Subjects
- Animals, Birnaviridae Infections immunology, DNA Primers genetics, Enzyme-Linked Immunosorbent Assay, Kaplan-Meier Estimate, Kidney metabolism, Neutralization Tests, Real-Time Polymerase Chain Reaction, Spleen metabolism, Birnaviridae Infections veterinary, Cytokines immunology, Fish Diseases immunology, Fish Diseases virology, Infectious pancreatic necrosis virus immunology, Inflammation Mediators metabolism, Oncorhynchus mykiss
- Abstract
Infectious Pancreatic Necrosis Virus (IPNV) is the agent of a well-characterized acute disease that produces a systemic infection and high mortality in farmed fish species but also persistent infection in surviving fish after outbreaks. Because viral persistence of susceptible mammal hosts appears to be associated with the modulation of anti-inflammatory cytokine expression, in this study we examined the expression levels of key pro- and anti-inflammatory cytokines in kidney and spleen of trout, as well as humoral immune response (IgM and IgT) during experimental persistent viral infection and in the acute phase of infection as a comparison. IPNV infection in rainbow trout resulted in a distinct profile of cytokine expression depending on the type of infection, acute or persistent. Levels of early pro-inflammatory cytokines, IL-1β and IL-8, did not increase in the head kidney of the fish with persistent asymptomatic infection but increased in some of the symptomatic infected fish. The antiviral cytokine IFNα was not significantly induced in any of the infected fish groups. The level of expression of the Th1-related cytokine IL-12 was significantly higher in trout with persistent asymptomatic infection than in symptomatic fish. This was also accompanied by an increase in IFNγ. The anti-inflammatory cytokines IL-10 and TGF-β1 had distinct expression profiles. While IL-10 expression increased in all infected fish, TGF-β1 was only up-regulated in fish with persistent infection. All infected fish had significantly lower total IgM levels than the non-infected fish whereas IgT levels did not change. Specific and neutralizing antibodies against IPNV were not observed in acute and persistent infection except in the group of fish with the lowest degree of clinical signs. Interestingly, the lack of humoral immune response could be associated with the high expression of anti-inflammatory cytokines, which might inhibit antibody production. The balance between pro-inflammatory Th1 type cytokines and the regulatory cytokines could explain the high percentage of survival and the resolution of the inflammatory response in the IPNV-infected fish but also the establishment of viral persistence., (Copyright © 2014 Elsevier Ltd. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
28. IPNV modulation of pro and anti-inflammatory cytokine expression in Atlantic salmon might help the establishment of infection and persistence.
- Author
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Reyes-Cerpa S, Reyes-López FE, Toro-Ascuy D, Ibañez J, Maisey K, Sandino AM, and Imarai M
- Subjects
- Animals, Birnaviridae Infections immunology, Cell Line, Fish Diseases virology, Gene Expression Profiling, Gills immunology, Head Kidney immunology, Birnaviridae Infections veterinary, Cytokines metabolism, Fish Diseases immunology, Gene Expression Regulation, Infectious pancreatic necrosis virus physiology, Salmo salar
- Abstract
IPNV is the agent of a well-characterized acute disease that produces a systemic infection and high mortality in farmed fish species and persistent infection in surviving fish after outbreaks. Because modulation of the host expression of pro and anti-inflammatory cytokines can help establish persistence, in this study, we examined the expression of IL-1β, IL-8, IFNα1 and IL-10 during acute and persistent IPNV infection of Atlantic salmon. Results showed that IPNV infection induces an increase of the IFNα1 and IL-10 mRNA levels in the spleen and head kidney (HK) of fish after acute experimental infection. Levels of the pro-inflammatory cytokines IL-1β and IL-8 did not rise in the spleen although an increase of IL-1β, but not of IL-8, was observed in head kidney. In carrier asymptomatic salmon, cytokine gene expression of IFNα1 in the spleen and IL-10 in head kidney were also significantly higher than expression in non-carrier fish. Interestingly, a decrease of IL-8 expression was also observed. IPNV infection of SHK-1, which is a macrophage-like cell line of salmon, also induced an increase of expression of the anti-inflammatory cytokine IL-10 with no effects on the expression of IL-1β and IL-8. The effects are induced by an unknown mechanism during viral infection because poly I:C and the viral genomic dsRNA showed the opposite effects on cytokine expression in SHK-1 cells. In summary, IPNV always induces up-regulation of the anti-inflammatory cytokine IL-10 in Atlantic salmon. As this is accompanied by a lack of induction of the pro-inflammatory cytokines IL-1β and IL-8, the anti-inflammatory milieu may explain the high frequency, prevalence and persistence of IPNV in salmon. Effects might be part of the viral mechanisms of immune evasion., (Copyright © 2011 Elsevier Ltd. All rights reserved.)
- Published
- 2012
- Full Text
- View/download PDF
29. Identification of CD3ε, CD4, CD8β splice variants of Atlantic salmon.
- Author
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Maisey K, Toro-Ascuy D, Montero R, Reyes-López FE, and Imarai M
- Subjects
- Animals, Base Sequence, Birnaviridae Infections immunology, Birnaviridae Infections metabolism, CD3 Complex immunology, CD4 Antigens immunology, CD8 Antigens immunology, Cloning, Molecular, DNA Primers genetics, DNA, Complementary genetics, Fish Diseases metabolism, Gills metabolism, Head Kidney metabolism, Infectious pancreatic necrosis virus, Molecular Sequence Data, Protein Isoforms immunology, RNA, Messenger genetics, Real-Time Polymerase Chain Reaction veterinary, Sequence Analysis, DNA veterinary, Spleen metabolism, Thymus Gland metabolism, Birnaviridae Infections veterinary, CD3 Complex genetics, CD4 Antigens genetics, CD8 Antigens genetics, Fish Diseases immunology, Protein Isoforms genetics, RNA, Messenger metabolism, Salmo salar genetics
- Abstract
In vertebrates, CD3 complex and CD4 and CD8 co-receptors are essential for signal transduction during T cell activation. In the present study, we report the mRNA spliced variants of the Atlantic salmon CD3ε, CD4 and CD8β and the effect of pathogen encounter on the expression of these variants. CD3ε is alternatively spliced in thymus, head kidney, spleen and gills to give rise to the complete mRNA sequence and to an alternative product that lacks the transmembrane exon. CD4 is also alternatively spliced in the thymus, head kidney, spleen and gills to form two variants, although the alternative product is barely detectable. The alternative product lacks the exon 1B encoding the D1 domain, which is essential for binding to MHC class II proteins. Two amplicons were also found for the CD8β gene; sequencing analysis revealed that the main PCR product corresponds to the previously reported CD8β sequence, whereas the variant sequence encodes a potential protein that lacks the Ig-like domain. The expression of CD3, CD4, CD8β genes also analyzed in head kidney of LPS-treated and IPNV infected salmon and different patterns of expression were observed. The presence and balance of the different variants of T cell co-receptors could be related to the ability of fish to induce a particular type of immune response, as well as, the ability of the pathogen to modify the fish immune response., (Copyright © 2011 Elsevier Ltd. All rights reserved.)
- Published
- 2011
- Full Text
- View/download PDF
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