Therapeutic effect of Artemia enriched with Escherichia coli expressing double-stranded RNA in the black tiger shrimp Penaeus monodon

Thammasorn, Thitiporn; Somchai, Parinyachat; Laosutthipong, Chaowanee; Jitrakorn, Sarocha; Wongtripop, Somjai; Thitamadee, Siripong; Withyachumnarnkul, Boonsirm; Saksmerprome, Vanvimon

doi:10.1016/j.antiviral.2013.08.005

Cited by 27 publications

(16 citation statements)

References 15 publications

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“…The effect of culture medium on productivity of E. coli expression system has been studied by varying various media to achieve the optimal production of bioactive compounds [ 21 , 22 ], indicating the need for optimization of culture conditions when attempting cultivation of new species. Previous work by our group demonstrated the production of dsRNA against shrimp viruses in RNase-deficient E. coli using LB broth medium [ 18 , 19 , 23 , 24 ]. A single population of dsRNA against an individual shrimp viral gene is produced in the range of a few micrograms per 100 mL E. coli culture under a lab-scale experiment.…”

Section: Resultsmentioning

confidence: 99%

Large-scale production and antiviral efficacy of multi-target double-stranded RNA for the prevention of white spot syndrome virus (WSSV) in shrimp

et al. 2015

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BackgroundRNA interference (RNAi) is a specific and effective approach for inhibiting viral replication by introducing double-stranded (ds)RNA targeting the viral gene. In this study, we employed a combinatorial approach to interfere multiple gene functions of white spot syndrome virus (WSSV), the most lethal shrimp virus, using a single-batch of dsRNA, so-called “multi-WSSV dsRNA.” A co-cultivation of RNase-deficient E. coli was developed to produce dsRNA targeting a major structural protein (VP28) and a hub protein (WSSV051) with high number of interacting protein partners.ResultsFor a co-cultivation of transformed E. coli, use of Terrific broth (TB) medium was shown to improve the growth of the E. coli and multi-WSSV dsRNA yields as compared to the use of Luria Bertani (LB) broth. Co-culture expression was conducted under glycerol feeding fed-batch fermentation. Estimated yield of multi-WSSV dsRNA (μg/mL culture) from the fed-batch process was 30 times higher than that obtained under a lab-scale culture with LB broth. Oral delivery of the resulting multi-WSSV dsRNA reduced % cumulative mortality and delayed average time to death compared to the non-treated group after WSSV challenge.ConclusionThe present study suggests a co-cultivation technique for production of antiviral dsRNA with multiple viral targets. The optimal multi-WSSV dsRNA production was achieved by the use of glycerol feeding fed-batch cultivation with controlled pH and dissolved oxygen. The cultivation technique developed herein should be feasible for industrial-scale RNAi applications in shrimp aquaculture. Interference of multiple viral protein functions by a single-batch dsRNA should also be an ideal approach for RNAi-mediated fighting against viruses, especially the large and complicated WSSV.

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Section: Resultsmentioning

confidence: 99%

Large-scale production and antiviral efficacy of multi-target double-stranded RNA for the prevention of white spot syndrome virus (WSSV) in shrimp

et al. 2015

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“…In the past decade, RNAi-based technology has gained attention for its potential to control shrimp viral diseases that have caused significant losses in aquaculture. Inhibition of shrimp viruses by viral-specific dsRNA were demonstrated in insect cell lines (He et al, 2009;Theerawanitchpan et al, 2012) and in penaeid shrimp (Robalino et al, 2005;Yodmuang et al, 2006;Ongvarrasopone et al, 2008;Saksmerprome et al, 2009Saksmerprome et al, , 2013Thammasorn et al, 2013). Given that sequences of viral genes of interest are available, dsRNA targeting those genes can be synthesized by in vitro transcription and in vivo bacterial system.…”

Section: Introductionmentioning

confidence: 97%

Use of microalgae Chlamydomonas reinhardtii for production of double-stranded RNA against shrimp virus

Somchai

Jitrakorn

Thitamadee

et al. 2016

Aquaculture Reports

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a b s t r a c t RNA interference has been proposed to be a promising tool for combating shrimp viruses. Antiviral double-stranded (ds)RNA has been mostly produced in Escherichia coli-expression system because of its high efficiency and inexpensive operations. However, overusing the bacteria may raise concerns regarding public health and environmental contamination, and seeking for a new dsRNA production platform would be alternative for future molecular farming. In this study, we exploited the green microalgae Chlamydomonas reinhardtii to produce dsRNA targeting the lethal shrimp yellow head virus (YHV). The expression plasmid pSL18 for C. reinhardtii was constructed to contain YHV-specific hairpin RNA expression cassette, and the successful assembly of pSL18-YHV was confirmed by PCR and enzymatic digestions. Glass bead method was employed for transformation of C. reinhardtii nuclear genome with pSL18-YHV. Microalgal expression of dsRNA-YHV, approximately 45 ng from 100-mL culture, was detected by qRT-PCR. Oral feeding experiment on postlarval shrimp revealed that the formulated feed with C. reinhardtii expressing dsRNA-YHV, at the ratio of 1 × 10 8 transformants per gram feed, improved 22% survival rate after YHV challenge. The present study suggests that C. reinhardtii can be bioengineered to produce viralspecific dsRNA for shrimp viral disease control, and the developed qRT-PCR could detect microalgal dsRNA with detection limit of subpicogram.

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“…It proved to be superior to previously reported methods based on nucleic acid amplification techniques (NAAT) such as conventional RT-PCR [1], [8] that are often less sensitive and often do not give a clear indication of severity of infection. Although quantification of LSNV has been reported by real-time RT-PCR assays using SYBR chemistry [25], the method requires sophisticated and expensive equipment that restricts its wide application. Recently, a real-time LAMP method was used to amplify lamda DNA as a model with high specificity and sensitivity by measuring the turbidity that arises from the magnesium pyrophosphate product of the LAMP reaction [10].…”

Section: Resultsmentioning

confidence: 99%

Demonstration of a Very Inexpensive, Turbidimetric, Real-Time, RT-LAMP Detection Platform Using Shrimp Laem-Singh Virus (LSNV) as a Model

et al. 2014

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Rapid and accurate detection of pathogens under field laboratory conditions is necessary for effective control of veterinary pathogens. Here we describe a prototype, portable, pathogen detection device developed for single tube, real-time, reverse transcription, loop-mediated isothermal amplification (RT-LAMP) using Laem-Singh virus (LSNV) as a model. LSNV is an RNA virus and a component cause of growth retardation in black tiger shrimp. We chose its RNA-dependent RNA polymerase (RdRp) gene as the target for our tests. The basis for detection was measurement of turbidity arising from formation of a white, insoluble magnesium pyrophosphate precipitate byproduct upon amplification of the RdRp target sequence from 100 ng template RNA extracted from shrimp. The measurement device consisted of a heating block to maintain constant temperature in the RT-LAMP reaction for 8 Eppindorf sample tubes, a light-emitting diode (LED) light source providing red light emission at 650 nm wavelength to pass through sample tubes, a light dependent resistance (LDR) photo-detector and a software program to report turbidity events and could potentially be marketed for under US$3000. The device was connected to a computer to display real-time results in a variety of formats. The optimized protocol for LSNV detection consisted of incubation of the sample tubes at 65°C for 1 h during which turbidity was continuously measured, and quantitative results could be obtained by reaction time measurement. The sensitivity of detection was comparable to that of conventional nested RT-PCR and there was no cross reaction with other common shrimp viruses. The device was used for quantitative measurement of relative copy numbers of LSNV RdRp in 8 shrimp tissues and they were found to be highest in the gills followed in order by the lymphoid organ and hemolymph (p≤0.05). This platform can be easily adapted for detection of other pathogens under field laboratory settings.

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Therapeutic effect of Artemia enriched with Escherichia coli expressing double-stranded RNA in the black tiger shrimp Penaeus monodon

Cited by 27 publications

References 15 publications

Large-scale production and antiviral efficacy of multi-target double-stranded RNA for the prevention of white spot syndrome virus (WSSV) in shrimp

Large-scale production and antiviral efficacy of multi-target double-stranded RNA for the prevention of white spot syndrome virus (WSSV) in shrimp

Use of microalgae Chlamydomonas reinhardtii for production of double-stranded RNA against shrimp virus

Demonstration of a Very Inexpensive, Turbidimetric, Real-Time, RT-LAMP Detection Platform Using Shrimp Laem-Singh Virus (LSNV) as a Model

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