Penaeus monodon is protected against gill- associated virus by muscle injection but not oral delivery of bacterially expressed dsRNAs

Sellars, Melony J.; Rao, Min; Arnold, Stuart; Wade, Nicholas M.; Cowley, Jeff A.

doi:10.3354/dao02343

Cited by 34 publications

(24 citation statements)

References 30 publications

(47 reference statements)

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“…monodon is consistent with its ability to protect P. monodon against disease following challenge with GAV (Sellars et al 2011). The finding that the single dsGAV1 dsRNA targeted to the extreme 5'-terminus of the GAV genome reduced infection loads slightly more rapidly and substantially was interesting, and in a practical context would reduce complexity and cost of dsRNA production in any commercial application of technology.…”

Section: Accepted M Manuscriptsupporting

confidence: 51%

“…Target site positions for dsRNAs in the GAV ORF1a/1b gene, thus restricting their action to the genomic-length ssRNA transcribed in low abundance and critical for translating enzymes essential for generating genome replication machinery, were as reported previously (Sellars et al 2011), except that the dsGAV5 target region overlapping the helicase motif in ORF1b was substituted for another region (dsGAV6; 17237-18014 nt; GenBank Acc. AF227196.2) positioned slightly further downstream ( Table 1).…”

Section: Accepted Manuscriptmentioning

confidence: 99%

“…To obtain initial evidence that such a strategy is feasible and might provide a means of reducing vertical transmission of GAV from broodstock to farmed progeny, here juvenile P. monodon with subclinical GAV infections collected from a farm in North Queensland were injected with either single dsRNAs or a cocktail of 5 dsRNAs targeted to sequences across the GAV ORF1a/1b replicase gene found previously to be highly effective at protecting shrimp against disease and mortality following GAV challenge (Sellars et al, 2011). By tracking GAV infection loads using real-time quantitative RT-PCR over a 2 week period following dsRNA injection, these were identified to be reduced significantly within 6 to 10 days among shrimp injected with the GAV dsRNA cocktail and 1 of the 2 single GAV dsRNAs examined.…”

Section: Accepted M Manuscriptmentioning

confidence: 99%

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Reduced loads of pre-existing Gill-associated virus (GAV) infection in juvenile Penaeus monodon injected with single or multiple GAV-specific dsRNAs

et al. 2014

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The ability of RNA interference (RNAi) based on injected dsRNA was investigated here for its ability to reduce the severity of pre-existing subclinical Gill-associated virus (GAV) infections in farm stocks of juvenile Black Tiger shrimp (Penaeus monodon). Following tail muscle injection of single or multiple long dsRNAs targeted to sequences positioned across the GAV ORF1a/1b replicase genes, pleopods were sampled sequentially from individuals at regular intervals over a 2 week period to track changes in GAV RNA loads by quantitative real-time RT-PCR. Mean GAV RNA amounts showed statistically significant (P <0.05) declines from Day 6 post-injection in shrimp injected with a cocktail of 5 GAV dsRNAs or a single dsRNA (dsGAV1) that targeted a ~1.2 kb sequence at the extreme 5'-terminus of the GAV genome. GAV RNA amounts were not reduced significantly among shrimp injected with another single dsRNA (dsGAV6) targeted to a sequence just downstream of the helicase motif in the ORF1b gene. Based on these encouraging data, it will be useful to determine whether the RNAi approach can reduce GAV infection loads sufficiently in P. monodon broodstock used in commercial hatcheries in Australia to restrict vertical transmission of GAV to seedstock.

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Section: Accepted M Manuscriptsupporting

confidence: 51%

Section: Accepted Manuscriptmentioning

confidence: 99%

Section: Accepted M Manuscriptmentioning

confidence: 99%

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Reduced loads of pre-existing Gill-associated virus (GAV) infection in juvenile Penaeus monodon injected with single or multiple GAV-specific dsRNAs

et al. 2014

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“…For example, dsRNA directed to the endogenous Gen β-actin in the giant tiger prawn P. monodon caused death (100%) when applied by injection (25 µL shrimp saline solution containing the various dsRNA purified from bacteria). In contrast, no deaths were reported when dsRNA was orally administered in food with bacteria expressing specific dsRNA (6.0 × 10 10 bacteria/g, extruded feed with β-actindsRNA) (35). Similar results were observed when silencing the viral gene of the associated virus of the gills (GAV) of the giant tiger prawn with bacteria expressing specific dsRNA (4.9 × 10 10 bacteria/g extruded feed of GAV-dsRNA) where survival was 97% by injection and 15-35% by oral administration (36).…”

Section: Discussionmentioning

confidence: 95%

“…Por ejemplo, el dsRNA dirigido al gen endógeno β-actina del camarón P. monodon fue capaz de causar la muerte (al 100%) cuando se aplicaba por inyección (25 µl de solución salina de camarón, que contiene los diversos dsRNA purificados a partir de bacterias). Mientras que, no se reportaron mortalidades cuando se administró el dsRNA por vía oral en alimento administrado con bacterias que expresaba dsRNA especifico (6.0 × 10 10 bacterias/g de alimento extruido con β-actin-dsRNA) (35). Resultados similares fueron observados al silenciar el gen viral del virus asociado a las branquias (GAV) del camarón P. monodon con bacterias que expresaban dsRNA especifico (4.9 × 10 10 bacterias/g de alimento extruido con GAV-dsRNA) donde la supervivencia fue del 97% por inyección, y del 15-35% por administración oral (36).…”

Section: Discussionunclassified

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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Biotechnological Approaches in Fish Health Management

Khan

Choudhury

2021

Biotechnological Advances in Aquaculture Health Management

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Penaeus monodon is protected against gill- associated virus by muscle injection but not oral delivery of bacterially expressed dsRNAs

Cited by 34 publications

References 30 publications

Reduced loads of pre-existing Gill-associated virus (GAV) infection in juvenile Penaeus monodon injected with single or multiple GAV-specific dsRNAs

Reduced loads of pre-existing Gill-associated virus (GAV) infection in juvenile Penaeus monodon injected with single or multiple GAV-specific dsRNAs

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

Biotechnological Approaches in Fish Health Management

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