RESUMEN
Purpose: Retinoblastoma (RB) is a retinal tumor that most commonly occurs in children. Approximately 40% of RB patients carry germline mutations in the RB1 gene. RB survivors with germline mutations are at increased risk of passing on the disease to future offspring and of secondary cancer in adulthood. This highlights the importance of genetic testing in disease management and counseling. This study aimed to identify germline RB1 mutations and to correlate the mutations with clinical phenotypes of RB patients. Methods: Genomic DNA was extracted from peripheral blood mononuclear cells isolated from 52 RB patients (27 unilaterally and 25 bilaterally affected probands). Mutations in the RB1 gene, including the promoter and exons 1-27 with flanking intronic sequences, were identified by direct sequencing. The samples with negative test results were subjected to multiplex ligation-dependent probe amplification (MLPA) to detect any gross mutations. A correlation of germline RB1 mutations with tumor laterality or age at diagnosis was determined for RB patients. Age at diagnosis was examined in regard to genetic test results and the presence of extraocular tumor extension. Results: Germline RB1 mutations were detected in 60% (31/52) of patients. RB1 mutations were identified in 92% (22/25) of bilateral RB patients, and a high rate of germline RB1 mutations was found in unilateral RB cases (33% or 9/27). Whole gene and exon deletions were reported in five patients. Twenty-three distinct mutations as a result of base substitutions and small deletions were identified in 26 patients; seven mutations were novel. Nonsense and splicing mutations were commonly identified in RB patients. Furthermore, a synonymous mutation was detected in a patient with familial RB; affected mutation carriers in this family exhibited differences in disease severity. The types of germline RB1 mutations were not associated with age at diagnosis or laterality. In addition, patients with positive and negative test results for germline RB1 mutations were similar in age at diagnosis. The incidence of extraocular tumors was high in patients with heritable RB (83% or 5/6), particularly in unilateral cases (33% or 3/9); the mean age at diagnosis of these patients was not different from that of patients with intraocular tumors. Conclusions: This study provides a data set of an RB1 genotypic spectrum of germline mutations and clinical phenotypes and reports the distribution of disease-associated germline mutations in Thai RB patients who attended our center. Our data and the detection methods could assist in identifying a patient with heritable RB, establishing management plans, and informing proper counseling for patients and their families.
Asunto(s)
Mutación de Línea Germinal , Fenotipo , Neoplasias de la Retina/genética , Proteínas de Unión a Retinoblastoma/genética , Retinoblastoma/genética , Ubiquitina-Proteína Ligasas/genética , Adulto , Secuencia de Bases , Niño , Preescolar , Exones , Femenino , Expresión Génica , Genotipo , Humanos , Lactante , Patrón de Herencia , Masculino , Reacción en Cadena de la Polimerasa Multiplex , Linaje , Regiones Promotoras Genéticas , Neoplasias de la Retina/diagnóstico , Neoplasias de la Retina/patología , Retinoblastoma/diagnóstico , Retinoblastoma/patología , Índice de Severidad de la Enfermedad , TailandiaRESUMEN
The derivation of three-dimensional (3D) stratified neural retina from pluripotent stem cells has permitted investigations of human photoreceptors. We have generated a H9 human embryonic stem cell subclone that carries a green fluorescent protein (GFP) reporter under the control of the promoter of cone-rod homeobox (CRX), an established marker of postmitotic photoreceptor precursors. The CRXp-GFP reporter replicates endogenous CRX expression in vitro when the H9 subclone is induced to form self-organizing 3D retina-like tissue. At day 37, CRX+ photoreceptors appear in the basal or middle part of neural retina and migrate to apical side by day 67. Temporal and spatial patterns of retinal cell type markers recapitulate the predicted sequence of development. Cone gene expression is concomitant with CRX, whereas rod differentiation factor neural retina leucine zipper protein (NRL) is first observed at day 67. At day 90, robust expression of NRL and its target nuclear receptor NR2E3 is evident in many CRX+ cells, while minimal S-opsin and no rhodopsin or L/M-opsin is present. The transcriptome profile, by RNA-seq, of developing human photoreceptors is remarkably concordant with mRNA and immunohistochemistry data available for human fetal retina although many targets of CRX, including phototransduction genes, exhibit a significant delay in expression. We report on temporal changes in gene signatures, including expression of cell surface markers and transcription factors; these expression changes should assist in isolation of photoreceptors at distinct stages of differentiation and in delineating coexpression networks. Our studies establish the first global expression database of developing human photoreceptors, providing a reference map for functional studies in retinal cultures.
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Antígenos de Diferenciación/biosíntesis , Diferenciación Celular , Proteínas del Ojo/biosíntesis , Células Madre Embrionarias Humanas/metabolismo , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Transcriptoma , Línea Celular , Células Madre Embrionarias Humanas/citología , Humanos , Células Fotorreceptoras Retinianas Conos/citología , Células Fotorreceptoras Retinianas Bastones/citologíaRESUMEN
MYCN proto-oncogene, bHLH transcription factor (MYCN) amplification is associated with aggressive retinoblastoma (RB) and neuroblastoma (NB) cancer recurrence that is resistant to chemotherapies. Therefore, there is an urgent need to identify new therapeutic tools. This study aimed to evaluate the potential repurposing of ceftriaxone for the treatment of MYCN-amplified RB and NB, based on the clinical observations that the drug was serendipitously found to decrease the volume of the MYCN-driven RB subtype. Using patient-derived tumor organoids and tumor cell lines, we demonstrated that ceftriaxone is a potent and selective growth inhibitor targeting MYCN-driven RB and NB cells. Profiling of drug-induced transcriptomic changes, cell-cycle progression, and apoptotic death indicated cell-cycle arrest and death of drug-treated MYCN-amplified tumor cells. Drug target identification, using an affinity-based proteomic and molecular docking approach, and functional studies of the target proteins revealed that ceftriaxone targeted DEAD-box helicase 3 X-linked (DDX3X), thereby inhibiting translation in MYCN-amplified tumors but not in MYCN-nonamplified cells. The data suggest the feasibility of repurposing ceftriaxone as an anticancer drug and provide insights into the mechanism of drug action, highlighting DDX3X as a potential target for treating MYCN-driven tumors.
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Neuroblastoma , Neoplasias de la Retina , Retinoblastoma , Humanos , Proteína Proto-Oncogénica N-Myc/genética , Proteína Proto-Oncogénica N-Myc/metabolismo , Ceftriaxona , Retinoblastoma/tratamiento farmacológico , Retinoblastoma/genética , Simulación del Acoplamiento Molecular , Proteómica , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/genética , Neuroblastoma/metabolismo , Línea Celular Tumoral , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Regulación Neoplásica de la Expresión GénicaRESUMEN
BACKGROUND: Recurrence of retinoblastoma (RB) following chemoreduction is common and is often managed with local (intra-arterial/intravitreal) chemotherapy. However, some tumors are resistant to even local administration of maximum feasible drug dosages, or effective tumor control and globe preservation may be achieved at the cost of vision loss due to drug-induced retinal toxicity. The aim of this study was to identify drugs with improved antitumor activity and more favorable retinal toxicity profiles via screening of potentially repurposable FDA-approved drugs in patient-derived tumor organoids. METHODS: Genomic profiling of five RB organoids and the corresponding parental tissues was performed. RB organoids were screened with 133 FDA-approved drugs, and candidate drugs were selected based on cytotoxicity and potency. RNA sequencing was conducted to generate a drug signature from RB organoids, and the effects of drugs on cell cycle progression and proliferative tumor cone restriction were examined. Drug toxicity was assessed with human embryonic stem cell-derived normal retinal organoids. The efficacy/toxicity profiles of candidate drugs were compared with those of drugs in clinical use. RESULTS: RB organoids maintained the genomic features of the parental tumors. Sunitinib was identified as highly cytotoxic against both classical RB1-deficient and novel MYCN-amplified RB organoids and inhibited proliferation while inducing differentiation in RB. Sunitinib was a more effective suppressor of proliferative tumor cones in RB organoids and had lower toxicity in normal retinal organoids than either melphalan or topotecan. CONCLUSION: The efficacy and retinal toxicity profiles of sunitinib suggest that it could potentially be repurposed for local chemotherapy of RB.
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Antineoplásicos , Neoplasias de la Retina , Retinoblastoma , Humanos , Retinoblastoma/tratamiento farmacológico , Retinoblastoma/genética , Retinoblastoma/patología , Sunitinib/farmacología , Sunitinib/uso terapéutico , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Neoplasias de la Retina/tratamiento farmacológico , Neoplasias de la Retina/genética , Neoplasias de la Retina/patología , Organoides/metabolismoRESUMEN
Purpose: The study aimed to generate a stepwise method to reduce the workload of full-scale RB1 sequencing for germline mutation screening in retinoblastoma (RB) patients. The implication of germline mutation in tumor focality was also determined in this study. Methods: A stepwise method was created on the basis of "hotspot" exons analyzed using data on germline RB1 mutation in the RB1-Leiden Open Variation Database and then tested for mutation screening in the blood DNA of 42 patients with RB. The method was compared with the clinical next-generation sequencing (NGS) panel in terms of sequencing outcomes. The germline RB1 mutation was examined in association with multifocality in RB. Results: Germline RB1 mutation was identified in 61% of all bilateral cases in the first step of the 3 stepwise method and in 78% and 89% for the two and three steps combined, respectively. NGS detected a mosaic variant of RB1 that was not detected by the first two steps and increased the sensitivity from 78% to 83%. Analysis of the relationship between mutation status and tumor focality indicated that multifocality in RB was dependent on germline RB1 mutation, confirming a higher tendency to have a germline RB1 mutation in patients with multifocal RB. Conclusions: A 3 stepwise method reduces the workload needed for sequencing of the RB1 for bilateral cases. NGS outweighs conventional sequencing in terms of the identification of germline mosaic variants. Multifocal tumors in RB may be used to presume germline mutation. Translational Relevance: The presence of "hotspot" exons of germline RB1 mutation in bilateral cases facilitates a mutation screening. However, when genetic testing is not available, multifocality in RB regardless of tumor laterality is predictive of germline RB1 mutation.
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Neoplasias de la Retina , Retinoblastoma , ADN , Análisis Mutacional de ADN , Mutación de Línea Germinal , Humanos , Mutación/genética , Neoplasias de la Retina/diagnóstico , Neoplasias de la Retina/genética , Neoplasias de la Retina/patología , Retinoblastoma/diagnóstico , Retinoblastoma/genética , Retinoblastoma/patología , Proteínas de Unión a Retinoblastoma/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Considerable attention has recently been given to adipose-derived stem cells (ASCs) as an important source for differentiation to Schwann cells in the treatment of peripheral nerve injury, with considerable clinical advantages over the use of mesenchymal stem cells derived from bone marrow or autologous Schwann cells. However, the relationship between adipose donor site and differentiated ASC phenotype and function is presently unknown. This work systematically studied the differentiation of ASCs harvested from three anatomical sites: (i) subcutaneous; (ii) perinephric; and (iii) epididymal adipose tissue. We show that ASC source is a major determining factor of immunophenotype, multilineage differentiation, Schwann-cell protein expression, and paracrine ability to stimulate neuronal growth. Upregulation of S100ß, glial fibrillary acidic protein (GFAP), and p75NGFR was observed in differentiated ASCs from perinephric fat tissue, while only the expression of S100ß or GFAP and p75NGFR was elevated in differentiated ASCs from subcutaneous or epididymal fat tissue. Although the co-culture of differentiated ASCs with NG108-15 neuronal cells demonstrated that ASCs from each source could stimulate neurite outgrowth and number, differentiated ASCs from subcutaneous and perinephric fat versus epididymal fat were most effective, which was attributed to high-brain-derived neurotropic factor/nerve growth factor and low-neurotrophin-3 levels. Thus, ASCs can be obtained from different anatomical locations, and this determines Schwann-cell phenotype upon differentiation and extent of function. This work is therefore of relevance in local therapeutic delivery of ASCs for the repair of peripheral nerve injury, but also in the broader context of ASC use in related stem-cell therapies.
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Tejido Adiposo/citología , Células Madre Adultas/citología , Diferenciación Celular/fisiología , Células Madre Mesenquimatosas/citología , Células de Schwann/citología , Tejido Adiposo/fisiología , Células Madre Adultas/fisiología , Animales , Western Blotting , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Proteína Ácida Fibrilar de la Glía/metabolismo , Inmunofenotipificación , Masculino , Células Madre Mesenquimatosas/fisiología , Factor de Crecimiento Nervioso/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Neurotrofina 3/metabolismo , Especificidad de Órganos/fisiología , Ratas , Ratas Wistar , Receptor de Factor de Crecimiento Nervioso/metabolismo , Subunidad beta de la Proteína de Unión al Calcio S100 , Proteínas S100/metabolismo , Células de Schwann/fisiologíaRESUMEN
This study aimed to examine the pharmacological profiles of multiple chemo drug candidates in systematic circulation to enhance their specific interactions with five human cancer cell lines. ZnO nanoparticles were successfully bound with chemo drugs via physical adsorption. The drug loading capacity was confirmed by FTIR, whereas the loading efficiency was determined via UV-vis spectrometry. The mean hydrodynamic size increased to 69-82 nm after chemo-drug immobilization via non-covalent interaction with ZnO. Among the nine formulated chemo drugs, the carboplatin (CP)-doxorubicin (DOX)-ZnO complex under UV light irradiation exhibited high sensitivity towards human breast adenocarcinoma cells without affecting human keratinocyte immortal cells with an IC50 of 0.137 µg/mL, whereas the loading capacity and efficiency of CP-DOX-ZnO were 77.81% and 99.05%, respectively. Fluorescence images confirmed that CP-DOX-ZnO using DOX served as a fluorescence enhancer specifically bound onto the cell membranes, which became almost saturated after 24 h incubation. Carboplatin-DOX-ZnO was possibly endocytosed by cancer cells and was selectively internalized into the target cells; thus, free chemo drug was released in the cytoplasm, which induced acute apoptosis. This resulted in complete inhabitation of growth signal of target cancer cells.
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Antineoplásicos/química , Antineoplásicos/farmacología , Carboplatino/química , Doxorrubicina/química , Rayos Ultravioleta , Óxido de Zinc/química , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cuello del Útero/patología , Neoplasias del Colon/patología , Femenino , Humanos , Neoplasias Hepáticas/patología , Neoplasias de la Boca/patologíaRESUMEN
Stunted shrimp caused by Penaeus monodon densovirus (PmDNV) infection is one of the main problems leading to a significant economic loss in Thailand. To control this pandemic disease, a double-stranded-RNA-mediated virus-specific gene silencing approach was applied to inhibit viral replication. In this study, two dsRNAs corresponding to the non-structural protein (ns1) and the structural protein (vp) genes of PmDNV were synthesized and introduced into shrimp haemolymph prior to viral challenge. After allowing viral replication for two weeks, the suppression effect by each dsRNA was evaluated by semi-quantitative PCR and compared with the control. A reduction of PmDNV in shrimp treated with each dsRNA was observed. In contrast, a high level of viral infection was detected in the control group (NaCl). Based on a limited sample number, we reached the tentative conclusion that virus-specific dsRNA can inhibit PmDNV replication, in which the dsRNA-ns1 was more effective than the dsRNA-vp.
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Densovirus/fisiología , Silenciador del Gen , Penaeidae/virología , ARN Bicatenario/metabolismo , Replicación Viral , Animales , Densovirus/genética , Genes Virales , ARN Bicatenario/genética , Tailandia , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Proteínas Estructurales Virales/genética , Proteínas Estructurales Virales/metabolismoRESUMEN
Retinoblastoma is a retinal cancer that is initiated in response to biallelic loss of RB1 in almost all cases, together with other genetic/epigenetic changes culminating in the development of cancer. RB1 deficiency makes the retinoblastoma cell-of-origin extremely susceptible to cancerous transformation, and the tumor cell-of-origin appears to depend on the developmental stage and species. These are important to establish reliable preclinical models to study the disease and develop therapies. Although retinoblastoma is the most curable pediatric cancer with a high survival rate, advanced tumors limit globe salvage and are often associated with high-risk histopathological features predictive of dissemination. The advent of chemotherapy has improved treatment outcomes, which is effective for globe preservation with new routes of targeted drug delivery. However, molecularly targeted therapeutics with more effectiveness and less toxicity are needed. Here, we review the current knowledge concerning retinoblastoma genesis with particular attention to the genomic and transcriptomic landscapes with correlations to clinicopathological characteristics, as well as the retinoblastoma cell-of-origin and current disease models. We further discuss current treatments, clinicopathological correlations, which assist in guiding treatment and may facilitate globe preservation, and finally we discuss targeted therapeutics for future treatments.
RESUMEN
Purpose: MYCNOS (MYCN opposite strand) is co-amplified with MYCN in pediatric cancers, including retinoblastoma. MYCNOS encodes several RNA variants whose functions have not been elucidated in retinoblastoma. Thus, we attempted to identify MYCNOS variants in retinoblastoma and aimed to decipher the role of MYCNOS variant 1 (MYCNOS1) on the activity of MYCN-amplified retinoblastoma. Methods: The profiles of MYCNOS variants and MYCN status were determined in 17 retinoblastoma tissues, cell lines, retinas, and retinal organoids. A functional study of MYCNOS1 expression was conducted in patient-derived tumor cells and in retinoblastoma cell lines via short hairpin RNA-mediated gene silencing. We carried out MYCN expression, cell viability, cell cycle, apoptosis, soft agar colony formation, and transwell assays to examine the role of MYCNOS1 in MYCN and cell behaviors. We analyzed a transcriptome of MYCN-amplified retinoblastoma cells deficient for MYCNOS1 and, finally, tested the responses of these cells to chemotherapeutic agents. Results: Expression of MYCNOS1 was associated with the expression and copy number of MYCN. Knockdown of MYCNOS1 caused instability of the MYCN protein, leading to cell cycle arrest and impaired proliferation and chemotaxis-directed migration in MYCN-amplified retinoblastoma cells in which RB1 was intact. MYCNOS1 expression was associated with gene signatures of photoreceptor cells and epithelial-mesenchymal transition. MYCNOS1 silencing enhanced the response of retinoblastoma cells to topotecan but not carboplatin. Conclusions: MYCNOS1 supports progression of retinoblastoma. Inhibition of MYCNOS1 expression may be necessary to suppress MYCN activity when treating MYCN-amplified cancers without RB1 mutation.
Asunto(s)
Genes de Retinoblastoma/genética , Proteína Proto-Oncogénica N-Myc/genética , Proteínas de Neoplasias/genética , ARN Largo no Codificante/genética , Neoplasias de la Retina/genética , Retinoblastoma/genética , Línea Celular , Niño , Femenino , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Humanos , Masculino , Mutación/genética , ARN Interferente Pequeño/genéticaRESUMEN
Persistent or recurrent retinoblastoma (RB) is associated with the presence of vitreous or/and subretinal seeds in advanced RB and represents a major cause of therapeutic failure. This necessitates the development of novel therapies and thus requires a model of advanced RB for testing candidate therapeutics. To this aim, we established and characterized a three-dimensional, self-organizing organoid model derived from chemotherapy-naïve tumors. The responses of organoids to drugs were determined and compared to relate organoid model to advanced RB, in terms of drug sensitivities. We found that organoids had histological features resembling retinal tumors and seeds and retained DNA copy-number alterations as well as gene and protein expression of the parental tissue. Cone signal circuitry (M/L+ cells) and glial tumor microenvironment (GFAP+ cells) were primarily present in organoids. Topotecan alone or the combined drug regimen of topotecan and melphalan effectively targeted proliferative tumor cones (RXRγ+ Ki67+) in organoids after 24-h drug exposure, blocking mitotic entry. In contrast, methotrexate showed the least efficacy against tumor cells. The drug responses of organoids were consistent with those of tumor cells in advanced disease. Patient-derived organoids enable the creation of a faithful model to use in examining novel therapeutics for RB.
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Carcinogénesis/patología , Modelos Biológicos , Organoides/patología , Retinoblastoma/tratamiento farmacológico , Retinoblastoma/patología , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Carcinogénesis/efectos de los fármacos , Preescolar , Variaciones en el Número de Copia de ADN/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Lactante , Melfalán/farmacología , Melfalán/uso terapéutico , Estadificación de Neoplasias , Organoides/efectos de los fármacos , Neoplasias de la Retina/tratamiento farmacológico , Neoplasias de la Retina/patología , Retinoblastoma/genética , Topotecan/farmacología , Topotecan/uso terapéuticoRESUMEN
We discuss the use of pluripotent stem cell lines carrying fluorescent reporters driven by retinal promoters to derive three-dimensional (3-D) retina in culture and how this system can be exploited for elucidating human retinal biology, creating disease models in a dish, and designing targeted drug screens for retinal and macular degeneration. Furthermore, we realize that stem cell investigations are labor-intensive and require extensive resources. To expedite scientific discovery by sharing of resources and to avoid duplication of efforts, we propose the formation of a Retinal Stem Cell Consortium. In the field of vision, such collaborative approaches have been enormously successful in elucidating genetic susceptibility associated with age-related macular degeneration.
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Células Madre Pluripotentes , Retina/citología , Degeneración Retiniana , Técnicas de Cultivo de Tejidos/métodos , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Línea Celular , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Genes Reporteros/fisiología , Humanos , Degeneración Macular/metabolismo , Degeneración Macular/terapia , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Retina/metabolismo , Degeneración Retiniana/metabolismo , Degeneración Retiniana/terapia , Neuronas Retinianas/citología , Epitelio Pigmentado de la Retina/metabolismoRESUMEN
We describe a simple, rapid and highly selective protocol for the primary culture of Schwann cells in vitro from freshly dissociated adult rat nerve. The protocol is based on a selective culture medium comprising both mitogens (forskolin and optionally N2 supplement plus bovine pituitary extract), to stimulate growth of Schwann cells, plus an inhibitory substrate to simultaneously restrict fibroblast overgrowth (D-valine), contained in DMEM. This protocol differs from other available methods in that it uses the preferential capacity of Schwann cells to metabolize D-valine because of the difference in expression of a D-amino acid oxidase (DAAO) enzyme between Schwann cells and fibroblasts plus the presence of a selective mitogen to stimulate growth of Schwann cells. This permits derivation of highly pure Schwann cells directly from fresh adult nerve. Average Schwann cell purities of 97% can be achieved after 19 d without pre-degeneration, purification or antimitotic steps.
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Técnicas de Cultivo de Célula , Células de Schwann/citología , Animales , Medios de Cultivo , Masculino , Ratas , Ratas Wistar , Células de Schwann/metabolismo , Nervio Ciático/citología , Valina/metabolismoRESUMEN
Penaeus monodon densovirus (PmDNV) is one of the major causes of stunted shrimp in Thailand and leads to considerable economic losses in overall shrimp production. Present study shows that the double-stranded RNA corresponding to the non-structural protein gene (ns1) and structural protein gene (vp) of PmDNV effectively inhibit viral propagation in naturally pre-infected shrimp. Multiple application of dsRNA was performed by injection into the haemolymph. The total amount of virus in the hepatopancreas of treated shrimp was measured by semi-quantitative PCR and histological methods. Observations indicated that PmDNV was almost eradicated in comparison to the high viral propagation in the control groups (no dsRNA and non-related dsRNA-gfp). For heavily infected shrimp, simultaneously knock down of ns1 and vp genes exhibited greater potency for viral depletion than dsRNA-ns1 alone. Furthermore, typical hypertrophic nuclei were also reduced in treated shrimp. This study therefore demonstrates the first result of an effective anti-PmDNV therapy in naturally pre-infected shrimp.
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Densovirus/crecimiento & desarrollo , Penaeidae/virología , Animales , Antivirales/farmacología , Productos Biológicos/farmacología , Densovirus/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Histocitoquímica , ARN Bicatenario/farmacología , ARN Viral/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tailandia , Carga Viral , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/farmacología , Replicación Viral/efectos de los fármacosRESUMEN
Although a significant progress has been achieved on dsRNA mediated anti-virus strategy development, there is still no effective means to control the virulent white spot syndrome virus (WSSV). Six double-stranded RNAs specific to different essential genes of WSSV (ie1, ie3, pol (DNA polymerase), rr2 (ribonucleotide reductase small subunit), vp26, and vp28) were employed to suppress viral replication in shrimp. At the condition that non-specific inhibitory effect was overwhelmed, the relative protective degree of these dsRNAs against WSSV infection (rr2>ie3>vp26, vp28>ie1>pol) was observed by semi-quantitative PCR. Besides, more than one injection of dsRNA was needed for an efficient viral inhibition. To improve viral protection in Penaeus monodon, synchronized blocking of viral cellular transport (by dsRNA-PmRab7) and viral essential gene synthesis (by dsRNA-rr2) was first performed in this study. The suppression effects of shrimp mortality by either combined dsRNAs of rr2 and PmRab7 or dsRNA-rr2 alone was monitored for 8 days after viral challenge. Approximately 95% of shrimp survivals were detected from both combined dsRNAs and dsRNA-rr2 alone whereas all shrimp without dsRNA were dead. It revealed that there was no additive inhibitory effect of the combined dsRNAs over dsRNA-rr2 alone.