Establishment and characterization of a new fibroblast-like cell line from the skin of a vertebrate model, zebrafish (Danio rerio).

BACKGROUND: The available fully sequenced genome and genetic similarities compared to humans make zebrafish a prominent in vitro vertebrate model for drug discovery & screening, toxicology, and radiation biology. Zebrafish also possess well developed immune systems which is ideal for studying infectious diseases. Fish skin confers immunity by serving as a physical barrier against the invading pathogens in the aquatic habitat. Therefore in vitro models from the skin tissue of zebrafish help to study the physiology, functional genes in vitro, wound healing, and pathogenicity of microbes. Hence the study aimed to develop and characterize a skin cell line from the wild-type zebrafish Danio rerio. METHODS AND RESULTS: A novel cell line designated as DRS (D. rerio skin) was established and characterized from the skin tissue of wild-type zebrafish, D. rerio, by the explant technique. The cells thrived well in the Leibovitz's -15 medium supplemented with 15% FBS and routinely passaged at regular intervals. The DRS cells mainly feature fibroblast-like morphology. The culture conditions of the cells were determined by incubating the cells at varying concentrations of FBS and temperature; the optimum was 15% FBS and 28 °C, respectively. Cells were cryopreserved and revived with 70-75% viability at different passage levels. Two extracellular products from bacterial species Aeromonas hydrophila and Edwardsiella tarda were tested and found toxic to the DRS cells. Mitochondrial genes, namely COI and 16S rRNA PCR amplification and partial sequencing authenticated the species of origin of cells. The modal diploid (2n) chromosome number of the cells was 50. The cell line DRS was found to be free from mycoplasma. The cells were transfected with pMaxGFP plasmid and tested positive for green fluorescence at 24-48 h post-transfection. CONCLUSION: The findings from this study thus confirm the usefulness of the developed cell line in bacterial susceptibility and transgene expression studies.

WSSV susceptibility in the early life stages of Penaeus vannamei shows relationship with bodyweight.

White Spot Syndrome Virus (WSSV) continues to cause considerable loss to shrimp farmers globally with frequent outbreaks even in specific pathogen free Peneaus vannamei. Our studies showed that the bodyweight (BW) of PL has a bearing on their susceptibility to the virus. To test this hypothesis, PL of the same age group and family were grouped according to BW (10-20, 30-40, and 50-60 mg) and challenged through immersion route with two viral doses (106 and 107 virus copies/L of water). It was observed that the PL became susceptible to WSSV at ≥50 mg BW. In the 50-60 mg PL group, the higher challenge dose shows a sharp mortality curve with 100% mortality at 10 days post immersion, while the lower dose shows a steady increase in cumulative mortality that reaches 100% on the 13th day post immersion. The study also brings out that an in vivo viral load of approximately 3.5 to 4.5 × 107WSSV copies/100 ng shrimp DNA results in mortality. This is the first report on the relationship between BW and WSSV susceptibility in shrimp PL. Also reported here is a quantitative assessment of WSSV infection in P. vannamei PL and an optimized challenge protocol.

Gain of function studies on predicted host receptors for white spot virus.

Three decades after its first outbreak, the shrimp white spot virus (WSV) is still a global cause of concern due to considerable losses and lack of effective control measures. Several candidate host receptor proteins have been identified, but the pathogenesis is not clearly understood, although the key role of the WSV envelope protein VP28 in virus internalization is established. Here, protein-protein docking is applied to evaluate the interaction of VP28 trimeric extracellular region with four host (Penaeus monodon) receptors reported earlier, Rab7 GTPase (PmRab7), glucose transporter 1 (PmGLUT1), C-type lectin (PmCTL) and calreticulin (PmCRT). The stability of predicted complexes evaluated in terms of binding energy per unit buried surface area ranged from -8.46 to -11.82 cal mol-1/Å2, which is not sufficient for functional interaction. Nevertheless, each of these host proteins was tested by a gain-of-function approach by observing their ability to make a fish cell line permissive to the shrimp WSV. Full-length expression constructs of the four receptors were transfected into SSN1 snakehead fish cells that are non-permissive to WSV. Transfected SSN1 cells and WSV permissive insect Sf9 cells were challenged with purified WSV. After 24 h, the presence of receptor transcripts was confirmed in the treated SSN1 cells, and not in the non-transfected SSN1 cells. Further, vp28 transcript was detected in Sf9 cells, but not in any of the treated SSN1 cells, indicating that none of the receptors were singly sufficient to make SSN1 cells permissive to WSV, even though PmRab7 was a strong candidate that alone showed >85% protection in virus neutralization experiments. For the other 3 candidates, previous reports predicted the involvement of co-receptors, which is confirmed here by their inability to act singly.

Transcriptome analysis of Clarias magur brain and gonads suggests neuro-endocrine inhibition of milt release from captive GnRH-induced males.

Transcriptome analysis of Clarias magur brain and gonads at preparatory, mature, 6 and 16 h post-GnRH injection (hpi) stages yielded 9.5 GB data with 39,738 contigs. Sequences of 45 reproductive genes were identified for the first time in C. magur along with unique and differentially expressed genes. The expression of 20 genes was validated by qRT-PCR. Upregulation of Cyp11A1, Cyp17A1 and FTZF1 genes in the 16hpi testis accompanied by the 17ß-HSD3 expression indicates testosterone (T) synthesis in response to LH surge, while reduced expression of CYP11B1 suggests a high T: 11-KT ratio. It is evident by the gene expression analysis that the inhibitory neurotransmitter GABA, altered T: 11-KT, increased testicular bile acids, and oxytocin-like neuropeptide in the male brain, appear to be involved in arresting the pulsatile motion of testicular smooth muscles. The work generates important leads for an effective induced breeding strategy for silurid catfish.

Molecular characterization and expression analysis of secretory immunoglobulin M (IgM) heavy chain gene in rohu, Labeo rohita.

Immunoglobulin M (IgM) is the major isotype among teleost immunoglobulins. The present study was aimed to explore IgM heavy chain gene and its expression profile in rohu. Full-length IgM heavy chain cDNA of rohu consisted of 1994 bp encoding a polypeptide of 576 amino acid residues including a leader peptide, variable (VH) and constant (CH1-CH2-CH3-CH4) domains confirming the secretory form of IgM. The sequence carries conserved residues such as cysteine, tryptophan and amino acid motifs like 'YYCAR' and 'FDYWGKGT-VTV-S'. The predicted 3 D model confirmed various domains of rohu IgM heavy chain. Phylogenetic tree analysis revealed that IgM heavy chain gene of rohu shared the same cluster with that of other cyprinid fishes. Tissue distribution analysis showed the predominant level of IgM heavy chain gene expression in kidney, spleen and intestine. IgM heavy chain gene expression in rohu kidney was found to be up-regulated and reached a maximum at 7 days post-challenge with Aeromonas hydrophila. These findings demonstrate the first report of full-length secretory IgM heavy chain gene in rohu. Besides, IgM heavy chain gene was highly expressed in major lymphoid tissues and bacterial challenge influenced its expression which further confirmed its role in the adaptive humoral immune response.

Development and characterization of a new DRCF cell line from Indian wild strain zebrafish Danio rerio (Hamilton 1822).

Danio rerio, zebrafish, has been widely used as a non-mammalian vertebrate model organism in various studies. The present research describes to develop and characterize a new cell line from a wild strain Indian zebrafish native to Brahmaputra River, Assam, India. The new cell line designated as DRCF was developed from the caudal fin of D. rerio. The cell line was successfully subcultured up to 31 passages. Growth studies revealed that cell growth of DRCF was optimal at 28 °C in L-15 medium supplemented with 20% FBS. Molecular characterization of the DRCF cell line using mitochondrial genes namely cytochrome oxidase subunit I gene (COI) and 16S rRNA authenticated the true origin of the cell line. The chromosome analysis of the DRCF cell line expressed its 50 diploid chromosome number of D. rerio. The immunocytochemical characterization of the cell line exhibited its fibroblastic morphology. The expression of the green fluorescent protein (GFP) following transfection revealed the suitability of the cell line for transfection studies.

A combined approach of 16S rRNA and a functional marker gene, soxB to reveal the diversity of sulphur-oxidising bacteria in thermal springs.

With the advent of new molecular tools, new taxa of sulphur-oxidising bacteria (SOB) in diverse environments are being discovered. However, there is a significant gap of knowledge about the ecology and diversity of SOB in thermal springs. Here, the species diversity and phylogenetic affiliations of SOB were investigated using 16S rRNA and functional gene marker, soxB in thermal springs of Thane district of Maharashtra, India. Most SOB detected by 16S rDNA sequences belong to different operational taxonomic units (OTU's): Firmicutes, α-, ß-, γ-Proteobacteria and Actinobacteria with the dominance of first class. However, the soxB gene clone library sequences had shown affiliation with the ß-, γ- and α-Proteobacteria. ß-Proteobacteria-related sequences were dominant, with 53.3% clones belonging to genus Hydrogenophaga. The thiosulphate oxidation assay carried out for different isolates having distinct identity showed the mean sulphate-sulphur production from 117.86 ± 0.50 to 218.82 ± 2.56 mg SO4-S l-1 after 9 days of incubation. Also, sulphur oxidation by the genus Nitratireductor, Caldimonas, Geobacillus, Paenibacillus, Brevibacillus, Tristrella and Chelatococcus has been reported for the first time that reveals ecological widening over which thiotrophs are distributed.

Regulation of compensatory growth by molecular mechanism in Labeo rohita juveniles under different feeding regimes.

A study was carried out to assess the regulation of compensatory growth under different restriction feeding regimes in Labeo rohita juveniles by the interaction of various feed intake and growth regulating genes. A 60 day feeding trial was conducted with five treatment groups, Control (3% body weight, bw), T1 (alternate days), T2 (0.5% bw), T3 (1% bw) and T4 (2% bw) and feeding was done for first 30 days of the trial. For next 30 days, all the treatment groups were fed at a rate of 3% bw as in the control group. There was significant (p < 0.05) difference in the weight gain among the treatment groups with lowest FCR and highest PER was found in T2 group. Ghrelin gene mRNA levels were upregulated during first 30th days of the trial with highest expression levels in the T2 group. The expression levels of leptin gene mRNA were found significantly different (p < 0.05) among the treatments, which was down-regulated during initial 30 days and upregulated as the experiment progress towards 60th day. The IGF-1 mRNA expression levels were upregulated more in liver compared to the muscle tissue. The results of the study suggest that increased ghrelin levels and decreased leptin levels lead to hyperphagia during the onset of refeeding, which further triggers the compensatory growth in L. rohita. The present study describes the molecular mechanism behind the compensatory growth following a different feed restriction regime in L. rohita which is regulated due to the interaction of different energy homeostasis and growth regulating genes.

Development of a fluorescent transgenic zebrafish biosensor for sensing aquatic heavy metal pollution.

We report a transgenic zebrafish (Danio rerio) designed to respond to heavy metals using a metal-responsive promoter linked to a fluorescent reporter gene (DsRed2). The metallothionein MT-Ia1 promoter containing metal-responsive elements was derived from the Asian green mussel, Perna viridis. The promoter is known to be induced by a broad spectrum of heavy metals. The promoter-reporter cassette cloned into the Tol2 transposon vector was microinjected into zebrafish embryos that were then reared to maturity. A transgene integration rate of 28 % was observed. The confirmed transgenics were mated with wild-type counterparts, and pools of F1 embryos were exposed to sub-lethal doses of Cd(2+), Cu(2+), Hg(2+), Pb(2+) and Zn(2+). The red fluorescence response of zebrafish embryos was observed 8 h post- exposure to these sub-lethal doses of heavy metals using a fluorescence microscope. Reporter expression estimated by real-time PCR revealed eightfold, sixfold and twofold increase on exposure to highest concentrations of Hg(2+), Cd(2+) and Cu(2+), while Pb(2+) and Zn(2+) had no effect. This biosensor could be a first-level screening method for confirming aquatic heavy metal bio-toxicity to eukaryotes.

Expression studies on NA+/K(+)-ATPase in gills of Penaeus monodon (Fabricius) acclimated to different salinities.

The decapod crustacean Penaeus monodon survives large fluctuations in salinity through osmoregulation in which Na+/K(+)-ATPase (NKA) activity in the gills plays a central role. Adult P. monodon specimens were gradually acclimatized to 5, 25 and 35 per thousand salinities and maintained for 20 days to observe long-term alterations in NKA expression. Specific NKA activity assayed in gill tissues was found to be 3 folds higher at 5 per thousand compared to 25 per thousand (isosmotic salinity) and 0.48 folds lower at 35 per thousand. The enzyme was immunolocalized in gills using mouse α-5 monoclonal antibody that cross reacts with P. monodon NKA α-subunit. At 5 per thousand the immunopositive cells were distributed on lamellar tips and basal lamellar epithelium of the secondary gill filaments and their number was visibly higher. At both 25 per thousand and 35 per thousand NKA positive cells were observed in the inter-lamellar region but the expression was more pronounced at 25 per thousand. Gill architecture was normal at all salinities. However, the 1.5 fold increase in NKA α-subunit mRNA at 5 per thousand measured by quantitative RT-PCR (qRT-PCR) using EF1α as reference gene was not statistically significant. The study confirms the osmoregulating ability of P. monodon like other crustaceans at lower salinities. It is likely that significant increase in NKA transcript level happens at an earlier time point. At higher salinities all three methods record only marginal or no change from isosmotic controls confirming the hypothesis that the animal largely osmoconforms in hyperosmotic environment.

Molecular cloning, sequencing and tissue-level expression of complement C3 of Labeo rohita (Hamilton, 1822).

Complement component C3 plays a central role in all known complement activation pathways. In the present study, we cloned, sequenced and analyzed the full-length cDNA sequence of Labeo rohita complement C3 (LRC3). The expression pattern of complement C3 mRNA in different tissues of healthy rohu and after challenge with Aeromonas hydrophila were evaluated using real-time PCR. The LRC3 cDNA sequence of rohu comprised of 5081 bp encoding a predicted protein of 1645 amino acids. The deduced amino acid sequence had the characteristic domain architecture. About eight domains specific to complement C3 are present in the sequence starting from signal peptide to netrin C345C (NTR) domain. The post-translational processing signal sequence (RKRR), the C3-convertase cleavage site sequence (LAR) and the canonical thiol-ester motif (GCGEQ) were found to be conserved in the LRC3. Real-time PCR analysis revealed the highest expression of C3 in liver and extra-hepatic expression of C3 was also observed in all the tissues studied. A. hydrophila challenge resulted in significant up-regulated expression of C3 transcripts in both liver and kidney at 6, 12, 24, 48 and 72 h post-infection.

Molecular phylogeny of elasmobranchs inferred from mitochondrial and nuclear markers.

The elasmobranchs (sharks, rays and skates) being the extant survivors of one of the earliest offshoots of the vertebrate evolutionary tree are good model organisms to study the primitive vertebrate conditions. They play a significant role in maintaining the ecological balance and have high economic value. Due to over-exploitation and illegal fishing worldwide, the elasmobranch stocks are being decimated at an alarming rate. Appropriate management measures are necessary for restoring depleted elasmobranch stocks. One approach for restoring stocks is implementation of conservation measures and these measures can be formulated effectively by knowing the evolutionary relationship among the elasmobranchs. In this study, a total of 30 species were chosen for molecular phylogeny studies using mitochondrial cytochrome c oxidase subunit I, 12S ribosomal RNA gene and nuclear Internal Transcribed Spacer 2. Among different genes, the combined dataset of COI and 12S rRNA resulted in a well resolved tree topology with significant bootstrap/posterior probabilities values. The results supported the reciprocal monophyly of sharks and batoids. Within Galeomorphii, Heterodontiformes (bullhead sharks) formed as a sister group to Lamniformes (mackerel sharks): Orectolobiformes (carpet sharks) and to Carcharhiniformes (ground sharks). Within batoids, the Myliobatiformes formed a monophyly group while Pristiformes (sawfishes) and Rhinobatiformes (guitar fishes) formed a sister group to all other batoids.

Toll-like receptor of mud crab, Scylla serrata: molecular characterisation, ontogeny and functional expression analysis following ligand exposure, and bacterial and viral infections.

Toll-like receptors are sentinels of innate immune system, which recognise pathogen-associated molecular patterns, and subsequently activate production of antimicrobial peptides to contain the infection. In the present study, we cloned and characterised a Toll gene from Scylla serrata (SsToll) encoding 1005 amino acids with typical Toll-like receptor domain topology. Phylogenetic analysis revealed that it belongs to insect-type invertebrate Toll family showing 100 % identity with Scylla paramamosain (SpToll). The expression pattern of mRNA in different tissues indicated that SsToll is constitutively expressed in all the tissues examined, with varying expression levels. The expression was also detected in all the life-stages (egg, zoea stages 1-5, megalopa and crab instar) with the highest level observed in zoea 2. In-vitro studies using crab haemocyte culture demonstrated that SsToll transcripts are distinctly modulated in response to ligands such as peptidoglycan and lipopolysaccharide at all time-points. A significant change in SsToll expression was also noticed in haemocytes exposed to poly I:C (3-9 h). On the contrary, the transcription level of SsToll in response to white spot syndrome virus (WSSV) challenge was noticeably different. The change in expression in vitro was not significant at early time-points until 3 h; the transcripts showed a significant up-regulation commencing from 6 h, but not beyond 12 h. However, in vivo expression was unaffected at early time-points of WSSV challenge (until 12 h) and a gradual up-regulation was detected at 24 h. In-vivo challenge with Vibrio parahaemolyticus resulted in delayed up-regulation of the gene. The results obtained in the present study suggest that SsToll might be involved in the innate immunity of mud crab.

First report on vertical transmission of a plasmid DNA in freshwater prawn, Macrobrachium rosenbergii.

Outbreak of WSSV disease is one of the major stumbling blocks in shrimp aquaculture. DNA vaccines have shown potential for mass scale vaccination owing to their stability, cost effectiveness and easy maintenance. Development of economically feasible delivery strategies remains to be a major challenge. This study demonstrates vertical transmission of a plasmid DNA in a decapod Macrobrachium rosenbergii for the first time. Females at three different maturation stages (immature, matured and berried) and mature males were injected with a plasmid DNA and allowed to spawn with untreated counterparts. Using specific primers the plasmid DNA could be amplified from the offspring of all groups except that of berried females. For this confirmation genomic DNA was isolated from 3 pools of 10 post larvae in each group. This presents an ideal strategy to protect young ones at zero stress.

Development of chitosan conjugated DNA vaccine against nodavirus in Macrobrachium rosenbergii (De Man, 1879).

The protective efficacy of a DNA construct containing extra small virus antisense (XSVAS) gene of nodavirus encapsulated with chitosan nanoparticles (NPs) was investigated in giant freshwater prawn Macrobrachium rosenbergii (De Man, 1879). The delivery was carried out using oral and immersion methods. A plasmid concentration of 100 ng µL(-1) when conjugated with chitosan NPs was found to be more effective in increasing the survivability of the infected prawn. The particle mean size, zeta potential and loading efficiency percentage were 297 nm, 27 mV and 85%, respectively. The ability of the chitosan to form a complex with the plasmid was studied by agarose gel electrophoresis. The NPs were characterized by atomic force microscopy (AFM). Persistence study showed the presence of the DNA construct up to 30th day post-treatment. The oral treatment was found to be better than the immersion treatment for delivery of the chitosan-conjugated DNA construct. This is probably the first report on the delivery of nanoconjugated DNA construct in M. rosenbergii, against nodavirus.

Short peptidoglycan recognition protein 5 modulates immune response to bacteria in Indian major carp, Cirrhinusmrigala.

Peptidoglycan recognition proteins (PGRPs) function in host antibacterial responses by recognizing bacterial peptidoglycan (PGN). In the present study, a short pgrp5 (named mpgrp5) was identified in Cirrhinus mrigala (mrigal). The full-length cDNA of the mpgrp5 gene was 1255 bp, containing an open reading frame of 746 bp encoding a protein of 248 amino acids. The predicted protein contained the typical Pgrp/amidase domain, conserved Zn2+, and PGN binding residues. The phylogenetic analysis revealed that the mpgrp5 is closely related to Pgrps reported in Labeo rohita, Cyrinus carpio, and Ctenopharyngodon idella. The ontogenetic expression of mpgrp5 was highest at 7 days post-hatching (dph) and its possible maternal transfer. mpgrp5 was constitutively expressed in all tissues examined, with the highest expression observed in the intestine. Furthermore, mpgrp5 was found upregulated in mrigal post-challenge in a time-dependent manner at 6hpi in the liver (3.16 folds, p < 0.05) and kidney (2.79 folds, p < 0.05) and at 12hpi in gill (1.90 folds, p < 0.01), skin (1.93 folds, p < 0.01), and intestine, (2.71 folds, p < 0.05) whereas at 24hpi in spleen (4.0 folds, p < 0.01). Our results suggest that mpgrp5 may play an important role in antibacterial immune response from early life stages in mrigal.

Development of a broad-spectrum fluorescent heavy metal bacterial biosensor.

Bacterial biosensors can measure pollution in terms of their actual toxicity to living organisms. A recombinant bacterial biosensor has been constructed that is known to respond to toxic levels of Zn(2+), Cd(2+) and Hg(2+). The zinc regulatory gene zntR and zntA promoter from znt operon of E. coli have been used to trigger the expression of GFP reporter protein at toxic levels of these ions. The sensor was induced with 3-800 ppm of Zn(2+), 0.005-4 ppm of Cd(2+) and 0.001-0.12 ppm of Hg(2+) ions. Induction studies were also performed in liquid media to quantify GFP fluorescence using fluorimeter. To determine the optimum culture conditions three different incubation periods (16, 20 and 24 h) were followed. Results showed an increased and consistent fluorescence in cells incubated for 16 h. Maximum induction for Zn(2+), Cd(2+) and Hg(2+) was observed at 20, 0.005 and 0.002 ppm, respectively. The pPROBE-zntR-zntA biosensor reported here can be employed as a primary screening technique for aquatic heavy metal pollution.

A GFP-based bacterial biosensor with chromosomally integrated sensing cassette for quantitative detection of Hg(II) in environment.

A mercury biosensor was constructed by integrating biosensor genetic elements into E. coli JM109 chromosome in a single copy number, using the attP/attB recombination mechanism of lambda phage. The genetic elements used include a regulatory protein gene (merR) along with operator/promoter (O/P) derived from the mercury resistance operon from pDU1358 plasmid of Serratia marcescens. The expression of reporter gene gfp is also controlled by merR/O/P. Integration of the construct into the chromosome was done to increase the stability and precision of the biosensor. This biosensor could detect Hg(II) ions in the concentration range of 100-1700 nmol/L, and manifest the result as the expression of GFP. The GFP expression was significantly different (P < or = 0.05) for each concentration of inducing Hg(II) ions in the detection range, which reduces the chances of misinterpretation of results. A model using regression method was also derived for the quantification of the concentration of Hg(II) in water samples.

Characterization of mitochondrial genome of Indian Ocean blue-spotted maskray, Neotrygon indica and its phylogenetic relationship within Dasyatidae Family.

The present study characterized complete mitochondrial genome of Blue-spotted maskray, Neotrygon indica and studied the evolutionary relationship of the species within the Dasyatidae family. The total length of the mitogenome was 17,974 bp including 37 genes and a non-coding control region. The average frequency of nucleotides in protein-coding genes was A: 29.1 %, T: 30.2 %, G: 13.0 % and C: 27.7 % with AT content of 59.3 %. The values of AT and GC skewness were -0.018 and -0.338, respectively. Comparative analyses showed a large number of average synonymous substitutions per synonymous site (Ks) in gene NADH4 (5.07) followed by NADH5 (4.72). High values of average number of non-synonymous substitutions per non-synonymous site (Ka) were observed in genes ATPase8 (0.54) and NADH2 (0.44). Genes NADH4L and NADH2 showed high interspecific genetic distance values of 0.224 ± 0.001 and 0.213 ± 0.002, respectively. Heat map analysis showed variation in codon usage among different species of the Dasyatidae family. The phylogenetic tree showed a sister relationship between the Dasyatinae and the Neotrygoninae subfamilies. Neotrygon indica formed as a sister species to the clade consisting of N. varidens and N. orientalis. Based on the present results, Neotrygon indica could have diverged from the common ancestor of the two latter in the Plio-Pleistocene. The present study showed distinct characteristics of N. indica from its congeners through comparative mitogenomics.

RNA interference-based therapeutics for shrimp viral diseases.

RNA interference (RNAi) has emerged as a powerful tool to manipulate gene expression in the laboratory. The presence of a double-stranded RNA (dsRNA) in eukaryotic cells triggers this post-transcriptional gene-silencing mechanism, leading to a sequence-specific degradation of the target mRNA. Among its many potential biomedical applications, silencing of viral genes stands out as a promising therapeutic strategy. Marine shrimp viral diseases, especially white spot disease (WSD), represents one of the most attractive targets for the development of therapeutic RNAi owing to its widespread economic impact. This review summarizes the current knowledge in the therapeutic application of RNAi for combating viral diseases in shrimp. The basic principles of RNAi are described, focusing on features important for its therapeutic manipulation. Subsequently, a stepwise strategy for the development of therapeutic RNAi is presented.