A simple one-step method for producing dsRNA from E. coli to inhibit shrimp virus replication

Posiri, Pratsaneeyaporn; Ongvarrasopone, Chalermporn; Panyim, Sakol

doi:10.1016/j.jviromet.2012.11.033

Cited by 60 publications

(68 citation statements)

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“…The amount of dsRNA-ORF89 in feed and the quantity of ingested feed will be important factors for success in a WSSV treatment (6). The amount of specific dsRNA recommended for the antiviral effect to take effect from the feed should be 5-10 times greater than that injected (11,15,33). Feed containing dsRNA-ORF89 must be capable of bringing an optimal concentration to the target organs and cells; i.e., the sites of primary WSSV replication, such as the epithelial cells in the anterior intestine in the digestive tract, and in the gills (34).…”

Section: Discussionmentioning

confidence: 99%

“…La cantidad de dsRNA-ORF89 incorporado en el alimento y la cantidad ingerida puede ser un factor importante en el éxito del tratamiento para WSSV (6). La cantidad de dsRNA específico recomendada para que el efecto antiviral tenga efecto desde el alimento deberá ser 5-10 veces mayor que la inyectada (11,15,33).…”

Section: Discussionunclassified

“…Los métodos más comunes de administrar el dsRNA son por inyección, inmersión, o vía oral (per os) (10). Cada uno de estos métodos posee diversas ventajas y desventajas; por ejemplo, por inyección directa se ha visto un importante efecto antiviral (11) no obstante, este método de aplicación es costoso, requiere mucho trabajo y es considerado impráctico por la alta cantidad de organismos que se requiere proteger en los estanques de cultivo (12). El método de…”

Section: Introductionunclassified

“…The most common methods of administering dsRNA are by injection, immersion, or oral route (per os) (10). Each of these methods has several advantages and disadvantages; For example, direct injection has been shown to have an important antiviral effect (11). However, this method of application is costly, labor intensive and considered impractical because of the large number of organisms that need to be protected in culture ponds (12).…”

Section: Introductionmentioning

confidence: 99%

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Digestibilidad in vitro del dsRNA específico por enzimas del tracto digestivo del camarón Litopenaeus vannamei

2017

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Objective. The digestibility of specific dsRNA by action of the enzymes of digestive tract of the whiteleg shrimp Litopenaeus vannamei was determined in vitro. Materials and methods. Digestive enzyme activity (amylase, lipase, protease, DNase and RNase) was measured in the stomach, digestive gland, and anterior, middle, and posterior intestine of juvenile shrimp, and the digestibility of DNA, RNA and the dsRNA-ORF89, specific to WSSV, was determined by in vitro assays, as well as electrophoretic and densitometric analyses. Results. The highest enzymatic activity was found in the digestive gland: amylase (81.41%), lipase (92.60%), protease (78.20%), DNase (90.85%), and RNase (93.14%). The highest digestive capacity against DNA, RNA, and dsRNA was found in the digestive gland (5.11 ng of DNA per minute, 8.55 ng of RNA per minute, and 1.48 ng dsRNA per minute). Conclusions. The highest digestibility of dsRNA-ORF89, specific to WSSV, was found in the digestive gland, whereas the lowest digestibility was observed in the posterior intestine. This is the first report regarding the digestibility of dsRNA-ORF89 by whiteleg shrimp digestive tract enzymes, with potential therapeutic importance in shrimp culture to prevent WSSV disease through balanced feed.Keywords: Aquaculture, balanced feed, digestive gland, nucleic acids, WSSV (Sources: MeSH, FAO). RESUMENObjetivo. La digestibilidad del dsRNA específico para el virus de la mancha blanca (WSSV) por acción de las enzimas del tracto digestivo del camarón Litopenaeus vannamei fue analizada in vitro. Material y métodos. Se midió la actividad de enzimas digestivas (proteasa, amilasa, lipasa, ADNasa y ARNasa) en el estómago, la glándula digestiva, el intestino anterior, medio y posterior en juveniles de camarón patiblanco y se evaluó la digestibilidad de ácidos nucleicos ADN, ARN y dsRNA-ORF89 especifico contra el virus WSSV, por análisis electroforéticos y densitometría. Resultados. La actividad enzimática más alta se encontró en la glándula digestiva del camarón: amilasa (81.41%), lipasa (92.60%), proteasa (78.20%), ADNasa (90.85%) y ARNasa (93.14%). Se evidenció la capacidad digestiva del camarón patiblanco contra el ADN, ARN y dsRNA-ORF89 encontrando en la glándula digestiva la mayor digestión (5.11 ng de ADN por minuto, 8.55 ng de ARN por minuto y 1.48 ng de dsRNA por minuto). Conclusiones. La mayor digestibilidad del dsRNA-ORF89, específico contra el virus WSSV, se encontró en la glándula digestiva y la menor en el intestino posterior. Este es el primer informe relacionado con la digestibilidad del dsRNA-ORF89 por las enzimas del camarón patiblanco con potencial importancia terapéutica en el cultivo de camarón para prevenir la enfermedad del WSSV a través del alimento balanceado. INTRODUCCIÓNUna de las herramientas más poderosas utilizadas en la acuicultura frente a patógenos virales como el virus de la mancha blanca (WSSV) es el mecanismo de ARN de interferencia (ARNi) inducido por ARN de doble cadena (dsRNA, por sus siglas en ingles double-stranded RNA) (1). P...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionunclassified

Section: Introductionunclassified

Section: Introductionmentioning

confidence: 99%

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Digestibilidad in vitro del dsRNA específico por enzimas del tracto digestivo del camarón Litopenaeus vannamei

2017

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“…The transformed HT115 (DE3) strain can eventually produce up to 45 μ g dsRNA/1‐OD600/mL E. coli culture (Posiri et al . ), and large‐scale in vivo dsRNA production is approximately one‐fourth the cost of production using a commercial in vitro transcription kit (Saksmerprome et al . ).…”

Section: Methodsmentioning

confidence: 97%

RNA interference in shrimp and potential applications in aquaculture

Nguyen

Christiaens

Bossier

et al. 2016

Reviews in Aquaculture

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As the RNA interference mechanism was discovered, researchers have made huge strides in unravelling biological mechanisms at the gene level in a wide range of organisms. RNAi is a simple and rapid method that allows silencing gene expression at the post-transcriptional level and is triggered by the introduction of double-stranded RNA into the cell, leading to the degradation of the corresponding mRNA. In this review, we will provide an overview of successful RNAi experiments to date in Crustacea, and shrimp in particular, with special focus on various potential applications of RNA interference in research and aquaculture. An important aspect of RNAi in any species is the delivery of the double-stranded RNA and the barriers that have to be overcome to get the dsRNA into the target cells. Different options, experimental procedures, success rates and challenges in using RNAi are discussed in this review

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Optimization of dietary RNA interference delivery to western flower thrips Frankliniella occidentalis and onion thrips Thrips tabaci

Andongma

Greig

Dyson

et al. 2019

Arch Insect Biochem Physiol

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In insect reverse genetics, dietary delivery of interfering RNAs is a practical approach in nonmodel species, such as thrips, whose small size, and feeding behavior restricts the use of other delivery methods. In a laboratory context, an unsuitable diet could confound the interpretation of an RNA interference (RNAi) phenotype, however well-formulated artificial diets can minimize experimental variability, reduce the need for insect handling, and can further be used for roles, such as delivering double-strand RNA (dsRNA)-expressing recombinant bacteria.In this study, artificial diets for oral delivery of dsRNA were developed for two important pest thrips species, western flower thrips (Frankliniella occidentalis) and onion thrips (Thrips tabaci), with the goal of (a) stimulating feeding behavior, (b) supporting optimal growth rates of dsRNA-expressing symbiotic bacteria, and (c) nutritionally supporting the thrips for sufficient periods to observe RNAi phenotypes. The efficacy of artificial diets for ingesting "naked" dsRNA or dsRNA-expressing symbionts and dsRNA delivery via host plant uptake was evaluated. Compared with previously published diet formulations, new combinations based on tryptone, yeast, and soy were superior for enhancing feeding and longevity. However, simply adding "naked" dsRNA to an ---

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A simple one-step method for producing dsRNA from E. coli to inhibit shrimp virus replication

Cited by 60 publications

References 23 publications

Digestibilidad in vitro del dsRNA específico por enzimas del tracto digestivo del camarón Litopenaeus vannamei

Digestibilidad in vitro del dsRNA específico por enzimas del tracto digestivo del camarón Litopenaeus vannamei

RNA interference in shrimp and potential applications in aquaculture

Optimization of dietary RNA interference delivery to western flower thrips Frankliniella occidentalis and onion thrips Thrips tabaci

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