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siRNA injection induces sequence independent protection in Penaeus monodon against white spot syndrome virus (WSSV)
Abstract
White spot syndrome virus (WSSV) is a major disease in crustaceans, particularly shrimp, due to the current intensity of aquaculture practices. Novel strategies including vaccination to control this virus would be highly desirable. However, invertebrates lack a true adaptive immune response system and seem to rely on various innate immune responses. An alternative and more specific approach to counteract WSSV infections in shrimp could be by the exploitation of RNA interference. As long dsRNA molecules induce a general, sequence-independent anti-viral immunity in shrimp [Robalino, J., Browdy, C.L., Prior, S., Metz, A., Parnell, P., Gross, P., Warr, G., 2004. J. Virol. 78, 10442-10448], it was investigated whether shorter 21 nt siRNAs with homology to the WSSV vp15 and vp28 genes would give a sequence-specific interference response in the shrimp Penaeus monodon. Vp28 siRNAs as well as nonspecific control gfp siRNAs were able to specifically and efficiently silence their homologous genes in a heterologous baculovirus insect cell expression system. However, in shrimps no such a specific effect was observed. Shrimp injected with vp15 or vp28 siRNAs before WSSV challenge gave a significantly lower mortality rate, but not significantly different when shrimps were injected with gfp siRNA. Thus, large dsRNA molecules as well as siRNAs induce a sequence-independent anti-viral immunity when injected in shrimp.
... The RNAi pathway is thought to be an ancient mechanism for protecting the host and its genome against viruses and rogue genetic elements by the process of mRNA degradation that is induced by double-stranded RNA (dsRNA) in a sequence-specific manner (Jones et al. 1999;Zamore et al. 2000;Bernstein et al. 2001). Several recent papers have reported that siRNAs (Westenberg et al. 2005;Xu, Han & Zhang 2007) or dsRNA synthesized via in vitro methods (Robalino et al. 2004(Robalino et al. , 2005Kim et al. 2007) serve as potential therapeutic agents for treating white spot syndrome disease. In the marine shrimp Litopenaeus vannamei (Boone, 1931), the antiviral response can be induced by sequence-independent or sequence-specific dsRNA, which may activate RNAi-like mechanisms (Robalino et al. 2004(Robalino et al. , 2005. ...
... In the marine shrimp Litopenaeus vannamei (Boone, 1931), the antiviral response can be induced by sequence-independent or sequence-specific dsRNA, which may activate RNAi-like mechanisms (Robalino et al. 2004(Robalino et al. , 2005. Westenberg et al. (2005) have used siR-NAs specific to the VP15 and VP28 genes of WSSV and observed that shrimp injected with VP15 or VP28 siRNASs before WSSV challenge had a significantly lower mortality. Xu et al. (2007) have used a specific 21-bp short-interfering RNA targeting the VP28 gene of WSSV, and the results revealed that the transcription and expression of the VP28 gene were silenced. ...
... Application of RNAi technology against WSSV is a possible solution to control the quick spread of this deadly disease (Robalino et al. 2004(Robalino et al. , 2005Westenberg et al. 2005;Kim et al. 2007;Xu et al. 2007). It is not possible to apply in vitro synthesized dsRNA and siRNA available for RNAi therapy in the shrimp culture ponds, because they cannot be produced in large quantities. ...
... Several researchers have demonstrated various vaccination procedures for shrimp (Rout et al. 2007;Balasubramanian et al. 2008;Rajesh Kumar et al. 2008;Satoh et al. 2008). Recently, RNAi technology was found to be e¡ective against viral infections in shrimp (Robalino et al. 2005;Westenberg, Heinhuis, Zuidema & Vlak 2005;Shekhar & Lu 2009). However, no studies have been reported using dsRNA in M. japonicus for protection against WSSV. ...
... Although RNAi is a commonly used technique for the functional characterization of genes (Agrawal, Dasaradhi, Mohmmed, Malhotra, Bhatnagar & Mukherjee 2003;Vanhecke & Janitz 2005), currently, it is also being evaluated as a therapeutic strategy against viruses in many organisms including shrimp (Ding, Li, Lu, Li & Li 2004;Tirasophon et al. 2007;Xu et al. 2007;Shekhar & Lu 2009). Protection of shrimp using RNAi technology can be achieved by two di¡erent pathways: sequence-independent and sequence-spe-ci¢c (Robalino, Browdy, Prior, Metz, Parnell, Gross & Warr 2004;Westenberg et al. 2005). An important example for sequence-independent antiviral immunity is RNAi using the GFP gene, which can be used to knock down gene expression and inhibit viral replication (Yodmuang, Tirasophon, Roshorm, Chinnirunvong & Panyim 2006;Kim et al. 2007). ...
... RNAi experiments using several WSSV genes have been reported to inhibit viral replication in various shrimp species such as P. monodon, L. vannamei and P. chinensis using a sequence-speci¢c dsRNA pathway. They are VP28, VP281 and protein kinase genes in P. chinensis (Kim et al. 2007); DNA polymerase, ribonuclease reductase, ORF252, VP28 and VP19 in L.vannamei (Robalino et al. 2005); and VP28 and VP15 (Westenberg et al. 2005;Tirasophon et al. 2007;Sarathi et al. 2008) in P. monodon. Protection against WSSV using dsRNA-VP28 in P. monodon and P. chinensis has been reported as 100% survival ob-served up to 30 and 7 days post-WSSV infection, whereas more than 95% survival in L. vannamei up to 9 dpi. ...
Recent applications of RNA interference technology against the white spot syndrome virus (WSSV) were found to be promising and effective antiviral strategies. In the present study, a gene-specific full-length double-stranded RNA (dsRNA) was prepared for the WSSV-VP28 gene using the in vitro transcription method. Challenging experiments were performed by an intramuscular injection of VP28-dsRNA in Marsupenaeus japonicus, followed by WSSV infection after 24 h, expecting 95–100% mortality within in 9 days of duration. The results revealed that the protective efficiency of VP28-dsRNA against WSSV infection is more than 85%, 25 days post infection (dpi). Various assays including bioassay, reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting, immunohistochemistry (IHC), histology and 4′,6-diamidino-2-phenylindole (DAPI) staining were performed to confirm the knockdown of the VP28 gene. No positive signals were observed in the gill and heart tissues of VP28-dsRNA/WSSV-infected shrimp 7 dpi by RT-PCR and Western blotting. No infection was observed in the VP28-dsRNA/WSSV-infected group through histology and IHC. DAPI staining of haemocytes revealed no signs of condensation and fragmentation in the VP28-dsRNA-administered group. The unrelated YHVGP116-dsRNA had no significant effect on the expression of the VP28 gene, confirming that the knockdown is specific to VP28-dsRNA.
... An alternative and effective methodology to prevent this infection in shrimp could be the utilization of an RNA interference. Shorter 21-nucleotide siRNAs with homology were investigated in the WSSV and either vp15, vp28 or gfp genes give a sequence-specific interference and response in the shrimp Penaeus monodon in one study [141]. Vp15 is a basic DNA-binding protein of WSSV [142]; vp28 is a main WSSV cover protein that probably also participates in systemic virus infection [143]; gfp siRNAs are useful for nonspecific control of siRNA effects. ...
... Time-mortality graph of shrimps (Penaeus monodon) injected with siRNAs[141]. ...
Gene editing and gene silencing techniques have the potential to revolutionize our knowledge of biology and diseases of fish and other aquatic animals. By using such techniques, it is feasible to change the phenotype and modify cells, tissues and organs of animals in order to cure abnormalities and dysfunctions in the organisms. Gene editing is currently experimental in wide fields of aquaculture, including growth, controlled reproduction, sterility and disease resistance. Zink finger nucleases, TALENs and CRISPR/Cas9 targeted cleavage of the DNA induce favorable changes to site-specific locations. Moreover, gene silencing can be used to inhibit the translation of RNA, namely, to regulate gene expression. This methodology is widely used by researchers to investigate genes involved in different disorders. It is a promising tool in biotechnology and in medicine for investigating gene function and diseases. The production of food fish has increased markedly, making fish and seafood globally more popular. Consequently, the incidence of associated problems and disease outbreaks has also increased. A greater investment in new technologies is therefore needed to overcome such problems in this industry. To put it concisely, the modification of genomic DNA and gene silencing can comprehensively influence aquatic animal medicine in the future. On the ethical side, these precise genetic modifications make it more complicated to recognize genetically modified organisms in nature and can cause several side effects through created mutations. The aim of this review is to summarize the current state of applications of gene modifications and genome editing in fish medicine.
... Innate antiviral immunity in shrimps was depicted by in vivo RNAi knockdown experiments for the first time in shrimps by employing a non-specific and sequence-independent dsRNA to inhibit TSV or W SSV infections in L. vannamei (Robalino, 2004). Westenberg et al. (2005) suggest the poor uptake of dsRNA/siRNA by the cells as a possible explanation for the sequence-independent reaction. Circulation of this RNA in the hemolymph may induce a general defense response by signaling through a cellular receptor. ...
... Still, innate anti-viral immunity, sequence-dependent antiviral protection and gene silencing could be induced by injection of long dsRNA molecules (Robalino et al., 2005). Another study used short siRNA (21bp) against WSSV vp28 or vp15 genes and reported a significant reduction in shrimp mortality compared to controls (Westenberg et al., 2005). Administration of a sequence-specific vp28-siRNA into infected shrimp every day for three days induce gene silencing in vivo in Penaeus japonicus shrimp with no WSSV DNA found at the end of the experiment, suggesting that vp28-siRNA completely eradicated the virus from infected shrimp . ...
Ribonucleic acid interference (RNAi), a valuable tool for manipulating gene functionality in the laboratory, has also emerged as a powerful tool to suppress infection or replication of many pathogens that cause severe economic losses in fish farming. By taking advantage of the cell’s endogenous RNAi apparatus, small interfering RNA of ~21-22 bp can be introduced into cells to induce target specific mRNA degradation. With the growing appreciation for the potential of RNAi technology, the diversity in vivo relevance to aquaculture is seemingly vast. Studies in the future should address the hurdles like delivery strategy stability and degradation of RNAi therapeutic molecule by nucleases in aquatic animals. In this article, we review the literature in the field of RNAi technology in aquaculture, summarize the status and prospects, which may open doors to its applicability potential as a therapeutic strategy to modulate host-pathogen interactions and inspire further trials.
... Higher rates of survival reported for synchronous injection of long dsRNA encoding VP28 than DNA polymerase-encoding dsRNA of WSSV in L. vannamei challenged with WSSV (Robalino et al. 2004;. Westenberg et al. (2005), on the other hand attempted RNAi strategy by injecting a specific 21bp short-interference RNA (VP28-siRNA) by selectively targeting the VP28 gene of WSSV instead of long dsRNA, a potent antiviral which can induce an immune response (Westenberg et al. 2005). Their result showed significantly lower mortality rate in shrimps injected with VP28 siRNA (1.6 Mol/1g of P. monodon). ...
... Higher rates of survival reported for synchronous injection of long dsRNA encoding VP28 than DNA polymerase-encoding dsRNA of WSSV in L. vannamei challenged with WSSV (Robalino et al. 2004;. Westenberg et al. (2005), on the other hand attempted RNAi strategy by injecting a specific 21bp short-interference RNA (VP28-siRNA) by selectively targeting the VP28 gene of WSSV instead of long dsRNA, a potent antiviral which can induce an immune response (Westenberg et al. 2005). Their result showed significantly lower mortality rate in shrimps injected with VP28 siRNA (1.6 Mol/1g of P. monodon). ...
VP28 is an essential WSSV envelope protein used to develop innate immune recognition in shrimp against WSSV, owing to its remarkable capability to localize on the surface of shrimp epithelial cells. Transgenically engineered viral VP28 protein from several expression systems was used to increase the survival chances of shrimp against WSSV pathogen, and their efficacy was found to last for 2–3 weeks following therapy. Though few reports have shown that DNA vaccination using the VP28 DNA construct is capable of protecting shrimp from WSSV, the exact mechanism behind the immunity is still unknown. Several research groups have provided insights into VP28‐driven dsRNA vaccination to protect shrimp against WSSV infection, but the stability attained by many of these vaccines turned out to be short‐lived. RNAi treatment with VP28dsRNA, shrimp showed better resistance to the WSSV challenge, although multiple doses of dsRNA are required to provide extended protection for up to 28 days. However, no foolproof protection was currently offered by VP28‐based recombinant protein or DNA vaccination against WSSV. In recent years, attention has been directed towards identifying small molecule inhibitors of VP28 through molecular docking and molecular dynamics simulation studies and is an emerging approach in drug development and design against WSSV. This review highlights the opportunities, limits and challenges of VP28‐based control strategies of White Spot Syndrome Virus, by emphasizing the status quo of current methodological approaches and suggesting future research directions of using it as a potential drug discovery target.
... VP28 and VP26 are responsible for virus trafficking into the host cell (Tang et al., 2007;van Hulten et al., 2001a;Youtong et al., 2011;Zhang et al., 2002). Reports also show RNAi towards these structural proteins like VP28 are effective in controlling the WSSV infection or multiplication (Kim et al., 2007;Nilsen et al., 2017;Robalino et al., 2005;Sanjuktha et al., 2012;Sarathi et al., 2008;Sudhakaran et al., 2011;Westenberg et al., 2005;Xu, Han, and Zhang, 2007). ...
... On the other side 3, 6, 12 and 18 h time point failed to give protection/silencing against WSSV (data not shown), and virion copy number were almost similar to control GFP-dsRNA treated group; which also denotes RNAi is sequence specific. Similar results were obtained when dsRNA was injected 24 h or 48 h prior to challenge (Kim et al., 2007;Nilsen et al., 2017;Robalino et al. 2005;Sanjuktha, et al., 2012;Sarathi et al., 2008;Sudhakaran et al., 2011;Westenberg et al., 2005;Xu, Han, and Zhang, 2007). Shrimps do have enzymes required for RNAi machinery, especially Argonaute and Dicer protein components of the RNAi pathway are available in shrimp (Phetrungnapha et al., 2013 andSu et al., 2008). ...
... Oral delivery of bacterially-expressed vp28 dsRNA has also been reported to provide some protection [21]. While, most of these studies involved challenge of treated shrimp with the virus within 72 h post-injection of RNAi elements [20,25,29], only one study tried to evaluate the effectiveness of RNAi treatment in protecting shrimp against WSSV 10 days and 20 days post-treatment [17]. For anRNAi-based treatment to be commercially viable, it should be able to provide protection against WSSV for at least 7 to 10 days post-RNAi treatment. ...
... Only one previous study has evaluated the effectiveness of dsRNA targeting vp26 and vp28 on limiting WSSV infection 10 days and 20 days post treatment, reporting 20 to 37% and 0 to 13% survival when challenged with virus 10 days and 20 days after dsRNA treatment, respectively [17]. The increased protection (in terms of % survival as well as duration of protection) observed in the present study compared to the previous studies (Table 1) may have been due to the fact that four WSSV genes were targeted simultaneously in the present study, as opposed to targeting of a single WSSV genes [20,25,29,17]. While two of the gene targets, vp28 and vp19 encode structural proteins, the other two gene targets rr2 and tk-tmk encode proteins involved in DNA replication. ...
... Double-stranded (ds)RNA-mediated viral inhibition through RNA interference (RNAi) is powerful and efficient to control and prevent shrimp virus diseases (Robalino et al. 2005;Tirasophon et al. 2005;Westenberg et al. 2005;Yodmuang et al. 2006;Shekhar and Lu 2008;Saksmerprome et al. 2009;Theerawanitchpan et al. 2012;Saksmerprome et al. 2013;Thammasorn et al. 2013). Simple and inexpensive production of dsRNA for shrimp aquaculture was developed by introducing a recombinant plasmid with viral-specific hairpin expression cassette into RNase-deficient Escherichia coli (Saksmerprome et al. 2009). ...
... Since the transformed L. plantarum is not genetically modified to be an RNase-deficient strain, the quantity of its expressed dsYHV determined by qRT-PCR would more likely represent the remaining long dsYHV copies not digested by the bacterial RNase enzyme into small RNAs. While most of the previous reports emphasized the use of long dsRNA (>100 bp) to promote shrimp viral sequence-specific interference (Robalino et al. 2005;Kim et al. 2007;Saksmerprome et al. 2009;Mejia-Ruiz et al. 2011), a few reports demonstrated the ability of small interfering RNAs to suppress shrimp viral replication (Westenberg et al. 2005;Wu et al. 2007;Xu et al. 2007). Therefore, the transformed L. plantarum producing a mixture of long and short dsRNA should still be applicable for shrimp viral disease control. ...
We engineered probiotic bacteria, Lactobacillus plantarum, to produce specific double-stranded (ds)RNA for viral inhibition in penaeid shrimp. When supplemented in feed, the developed strain should offer specific viral disease resistance and still remain other beneficial effects on shrimp health against other bacterial pathogens. The pWH1520 expression vector was constructed to encode hairpin RNA targeting shrimp yellow head virus (YHV) in L. plantarum. The quantity of YHV-specific dsRNA (dsYHV) was approximately 1.66 ± 0.25 ng from 7 × 10¹¹ CFU of L. plantarum. Feeding shrimp with the probiotic expressing dsYHV (at 10¹¹ CFU g⁻¹ feed) for 5 days prior to YHV challenge provided shrimp partial protection against YHV. In vitro agar well diffusion analysis revealed that both wild-type and transformed L. plantarum inhibited growth of Vibrio parahaemolyticus, the shrimp pathogen responsible for acute hepatopancreatic necrosis disease (AHPND) and early mortality syndrome (EMS). When tested in shrimp by immersion method, both L. plantarum strains (at 10⁷ CFUs ml⁻¹ seawater) also provided shrimp protection against V. parahaemolyticus. In conclusion, this study suggested the potential of the dsRNA-expressed L. plantarum for promoting shrimp health against the pathogenic virus. In addition, engineering L. plantarum for dsRNA production did not alter the probiotic’s intrinsic antibacterial property, and therefore, the developed strain can be used to protect shrimp against the important bacterial pathogen, V. parahaemolyticus causing AHPND/EMS.
... Oral delivery of bacterially-expressed vp28 dsRNA has also been reported to provide some protection [21]. While, most of these studies involved challenge of treated shrimp with the virus within 72 h post-injection of RNAi elements [20,25,29], only one study tried to evaluate the effectiveness of RNAi treatment in protecting shrimp against WSSV 10 days and 20 days post-treatment [17]. For anRNAi-based treatment to be commercially viable, it should be able to provide protection against WSSV for at least 7 to 10 days post-RNAi treatment. ...
... Only one previous study has evaluated the effectiveness of dsRNA targeting vp26 and vp28 on limiting WSSV infection 10 days and 20 days post treatment, reporting 20 to 37% and 0 to 13% survival when challenged with virus 10 days and 20 days after dsRNA treatment, respectively [17]. The increased protection (in terms of % survival as well as duration of protection) observed in the present study compared to the previous studies (Table 1) may have been due to the fact that four WSSV genes were targeted simultaneously in the present study, as opposed to targeting of a single WSSV genes [20,25,29,17]. While two of the gene targets, vp28 and vp19 encode structural proteins, the other two gene targets rr2 and tk-tmk encode proteins involved in DNA replication. ...
Anil Kumar, et al., (2015) Double Stranded RNA Simultaneously Targeting four White Spot Syndrome Virus (WSSV) genes provides protection against WSSV in Lito-penaeus Vannamei. Int J Marine Sci Ocean Technol. 2(2), 5-10 Citation: Anil Kumar, et al., (2015) Double Stranded RNA Simultaneously Targeting four White Spot Syndrome Virus (WSSV) genes provides protection against WSSV in Litopenaeus Vannamei. Int J Marine Sci Ocean Technol. 2(2), 5-10.
... Therefore, for the pathogens that posed the greatest disease risk to shrimp farming, exclusion through the use of SPF breeding lines has provided the central pillar to mitigate disease risk. Notably, much research since has focused on developing means to reduce, and ideally clear, pathogens from infected shrimp using RNA interference (RNAi) [227][228][229][230][231][232][233][234][235], however, there are no published accounts of such methods being applied routinely to achieve SPF status in commercial breeding lines. Recent research suggests there may be other therapeutics and novel strategies that offer the promise of improved disease outcomes for breeding lines such as selection for the presence of protective EVEs (endogenous viral elements) and "New circular DNA vaccines" [236] or transgenerational immune priming (see review by Roy et al. [237]). ...
Intensification of the shrimp sector, also referred to as vertical expansion, has been predominately driven by consecutive incidences of global disease outbreaks, which have caused enormous economic loss for the main producer countries. A growing segment of the shrimp farming industry has opted to use production systems with higher density, biosecurity, and operating control to mitigate the risks posed by disease. However, successful super-intensive shrimp production is reliant on an advanced understanding of many important biological and economic parameters in the farming system, coupled with effective monitoring, to maintain optimal production. Compared to traditional extensive or semi-intensive systems, super-intensive systems require higher inputs of feed, energy, labor, and supplements. These systems are highly sensitive to the interactions between these different inputs and require that the biological and economical parameters of farming are carefully balanced to ensure success. Advancing nutritional knowledge and tools to support consistent and efficient production of shrimp in these high-cost super-intensive systems is also necessary. Breeding programs developing breeding-lines selected for these challenging super-intensive environments are critical. Understanding synergies between the key areas of production systems, nutrition, and breeding are crucial for super-intensive farming as all three areas coalesce to influence the health of shrimp and commercial farming success. This article reviews current strategies and innovations being used for Litopenaeus vannamei in production systems, nutrition, and breeding, and discusses the synergies across these areas that can support the production of healthy and high-quality shrimp in super-intensive systems. Finally, we briefly discuss some key issues of social license pertinent to the super-intensive shrimp farming industry.
... In contrast to the other control plasmid, pshGFP plasmid showed a large reduction in both PmRab7 and Ef-1α mRNA expressions ( Figure 3). This may suggest the sequence-independent effect of siRNA especially in GFP siRNA that can induce a non-specific innate immune response against WSSV in shrimp as previously reported (Westenberg, Heinhuis, Zuidema, & Vlak, 2005). It may lead to non-specific mRNA degradation as well as global inhibition in protein translation of critical host transcripts (Oates, Bruce, & Ho, 2000;Zhao, Cao, Li, & Meng, 2001). ...
RNA interference (RNAi) was investigated as potential antiviral strategy to mitigate losses in shrimp aquaculture. With this aim, an effective short‐hairpin RNA (shRNA) expressed intracellularly from bacterial vectors incorporating eukaryotic promoters offers an alternative to an injected synthetic small‐interfering RNA (siRNA) or long double‐stranded RNA (dsRNA). The vector‐based RNAi designed to contain a U6 snRNA polymerase III promoter sequence from zebrafish (Danio rerio) for driving shRNA was previously introduced to shrimp cell extract and was able to express the shRNA. Here, four DNA plasmids containing putative zebrafish U6 promoter to drive shRNA against PmRab7‐specific sequence were used to transfect primary hemocyte culture. The cells were subsequently infected by Yellow head virus (YHV). As results, when analyzed by RT‐PCR at 24 hr post‐transfection, Penaeus monodon Rab7 (PmRab7) mRNA transcription was inhibited most significantly by the pshPmRab7‐2 construct. YHV replication in primary shrimp hemocyte cultures was shown to be inhibited substantially by this PmRab7 gene‐specific hpRNA construct. Transfection of pshPmRab7‐2 construct also reduced YHV replication most effectively when analyzed similarly between 24 hr until 72 hr post‐infection. These results demonstrate a potential application of DNA‐based shRNA construct as effective molecule for antiviral therapy in shrimp.
... In particular, silencing of Rab 7, the key protein of endocytotic pathway, was found to protect shrimp against WSSV, yellowhead virus (YHV), Taura syndrome virus (TSV) and Laem-Singh virus (LSNV) (Ongvarrasopone et al. 2008(Ongvarrasopone et al. , 2010(Ongvarrasopone et al. , 2011. In addition to dsRNA, the RNAi mechanism can be activated by administering small siRNAs (Westenberg et al. 2005), short/long hairpin RNA (shRNA/ lhRNA) (Krishnan et al. 2009a) and antisense RNA (Lu & Sun 2005). Similarly, antisense RNA construct targeting VP24 and VP28 of WSSV with the shrimp histone-3 promoter was injected to P. monodon and the immunized shrimp in both VP24 and VP28 groups conferred significant protection of 80% and 90%, respectively against WSSV challenge, without any manifestation of infection (Ahanger et al. 2014). ...
Crustacean aquaculture, dominated by shrimp, is a highly profitable food‐producing sector in the world. However, a variety of biotic and abiotic stressors can have adverse effect on the immune system of shrimp making them susceptible to diseases. Although a vertebrate‐like adaptive immune system is lacking in shrimp, an efficient innate immune system renders protection against invading pathogens. The innate immune system comprises two distinct but overlapping components, the cellular and humoral, and these are regulated through several signal transduction pathways. The signal pathways are initiated by the recognition of pathogen‐associated molecular patterns by germline‐encoded pattern recognition receptors leading to the production of different effector molecules that act against the pathogens. RNAi‐mediated post‐transcriptional gene silencing and microRNA regulation of immune response have also been found to be functional in shrimp. Similarly, apoptosis and apoptosis‐related genes are also reported, besides interferon (IFN) system‐like antiviral regulatory mechanism. Further, some form of immune memory, termed ‘immune priming’ or ‘innate immunity with specificity’ and ‘quasi‐immune response’ is recorded in shrimp and these abilities have been exploited in verifying the immunoprotection against different pathogens. Antigens developed either directly from the pathogens or through recombinant proteins have been tested for immune‐protective ability. RNAi‐mediated protection has also been demonstrated against different shrimp viruses. This review summarizes the available scientific information on immune responses and the immunoprotection trials carried out in crustaceans with a focus on shrimp. The available research evidences indicate the potential of developing effective immunoprophylactic measures in shrimp.
... Cada um desses dsRNAs inespecíficos induziu imunidade antiviral generalizada, sequência-independente. Essa resposta inespecífica ao vírus WSSV foi novamente observada em outro camarão (Penaeus monodon) por Westenberg et al. (2005). Estes pesquisadores injetaram siRNAs de 21 nt contra os genes do vírus que codificam as proteínas VP15 (ligante de DNA) e VP28 (vide página anterior). ...
A RNAi tem sido amplamente utilizada nas mais diversas espécies, principalmente em organismos “não modelos”, nos quais os métodos de manipulação genética são limitados. Nesses organismos, o emprego da técnica de RNAi vem permitindo avanços sem precedentes na investigação da função de um número ilimitado de genes, especialmente aqueles ligados ao desenvolvimento, fisiologia endócrina, reprodução, comportamento e imunologia (Bellés, 2010). Dentre as potenciais aplicações, o silenciamento de genes virais se destaca como estratégia terapêutica promissora na veterinária, particularmente quando confere resistência a doenças em animais de interesse econômico, sobretudo os de grande porte. Essa nova vertente da engenharia genética também emerge como ferramenta viável para aplicação em questões ligadas à produção alimentar sustentável e impacto ambiental (Long et al., 2010). Os avanços técnicos na área de RNAi na última década denotam seu caráter revolucionário no campo da genética, possibilitando resultados robustos, com custo e consumo de tempo menores do que usualmente são gastos com a produção de animais nocaute e transgênicos. Mesmo que os mecanismos moleculares envolvendo RNAi não estejam completamente elucidados, a comunidade científica utiliza-se das vantagens da técnica para desenvolver pesquisas básicas e aplicadas.
... Proteins involved in the RNAi mechanism were found in shrimp confirming the existence of shrimp RNAi machinery (Dechklar et al., 2008;Su et al., 2008;Unajak et al., 2006). Double-stranded RNAs specifically against non-structural gene products including protease, polymerase and helicase mRNAs of shrimp pathogens have been investigated to effectively inhibit viral propagation in cultured cells and shrimps (Saksmerprome et al., 2017;Theerawanitchpan et al., 2012;Tirasophon et al., 2005;Tirasophon et al., 2007;Westenberg et al., 2005;Yodmuang et al., 2006). In particular, RNA-dependent RNA polymerase (RdRp) has become an attractive target (Saksmerprome et al., 2009;Saksmerprome et al., 2013), and the RNAi-based strategy has been investigated as a potential measure for treatment and prevention of viral diseases in cultured shrimps. ...
Double-stranded RNA (dsRNA) is employed to down-regulate the expression of specific genes of shrimp viral pathogens through the RNA interference (RNAi) pathway. The administration of dsRNA into shrimp has been shown to be an effective strategy to block yellow head virus (YHV) progression. In this study, a vector (pLVX-AcGFP1-N1) was developed to introduce a long-hairpin RNA (lhRNA) silencing cassette under a CMV promoter, so-called “pLVX-lhRdRp”, against the RNA-dependent RNA polymerase (RdRp) gene of YHV. A primary culture of hemocytes isolated from Penaeus monodon was transfected with the pLVX-lhRdRp vector, generating transcripts of lhRNAs as early as 12 h post transfection. Twelve hours prior to YHV challenge, the primary hemocyte cell culture was transfected with pLVX-lhRdRp, whereas control groups were transfected with pLVX-AcGFP1-N1 or no transfection. The group treated with pLVX-lhRdRp significantly suppressed YHV replication at 24-72 h after YHV challenge. The results from RT-PCR and immunohistochemistry confirmed that both mRNA and protein expression of YHV were effectively inhibited by the pLVX-lhRdRp vector. Thus, our hemocyte culture and dsRNA expression plasmid with constitutive promoter have potential as a platform to test DNA constructs expressing long-hairpin RNA against pathogenic viral infection and as a RNAi-based DNA vaccine in shrimp.
... The vaccination results by using other vaccine components, such as VP15 (42 percent) (Westenberg et al. 2005) ...
... and a T7 promoter was added to the 5′ end ( Table 1). The dsRNA coding the GFP has been widely used as a non-specific control in a variety of RNA interference studies (Ponprateep et al. 2012;Westenberg et al. 2005). We amplified the Pt-CHH cDNA with a pair of T7 linked primers, and the Pt-CHH DNA fragment with the T7 promoter was used as a template for the synthesis of dsRNA using an in vitro transcription T7 kit (TaKaRa, Japan). ...
The crustacean hyperglycemic hormone (CHH) gene of Portunus trituberculatus (Pt-CHH) consists of four exons and three introns spanning 3849 bp in size and generating two mature mRNA, Pt-CHH1, and Pt-CHH2. The primary gene transcript produces a cDNA encoding for the putative Pt-CHH2 from exons 1, 2, 3, and 4 and an alternative transcript encodes for a putative Pt-CHH1 peptide from exons 1, 2, and 4. A promoter fragment of about 3 kb was obtained by genomic walking. The tissue-specific expression pattern is examined by reverse transcriptase chain reaction, and the results show that Pt-CHH1 is detected in the eyestalk, brain, muscle, and blood. However, Pt-CHH2 is detected in the ganglia thoracalis and gill. The results indicate that the expression of Pt-CHH2 in the gill might suggest a potential role in osmoregulation. The Pt-CHH transcript level in the gill increases when the crab is exposed to low salinity. The injection of dsRNA for Pt-CHH causes a significant reduction in Pt-CHH2 transcript level and the activity of Na⁺/K⁺-ATPase, and carbonic anhydrase (CA) show a serious decrease. In conclusion, this study provides molecular evidence to support the osmoregulatory function of Pt-CHH2.
... where animals treated with dsRNA targeting viral genes vp26 and vp28 gradually lost the antiviral effect 10 days after the initiation of RNAi treatment (Mejía-Ruíz et al., 2011;Nilsen et al., 2017). In P. monodon, protection against WSSV lasts 13 days upon RNAi treatment via viral-specific dsRNA injection (Westenberg et al., 2005). The presence of the RNAi ArCad phenotype in A. franciscana nauplii suggests that RNAi is sustained for at least up two weeks in dsRNA treated females. ...
Artemia and the RNA interference technique were used in research to explore the function of genes in crustaceans. In this study, we annotated and characterized nine putative genes involved in RNA interference in Artemia franciscana including two Dicer, three Argonauts, two dsRNA binding proteins, Drosha and Sid-1 together with evidence of Pasha and Exportin-5. The phylogenetic results indicated the identity of these genes and revealed that genes homologous to those in both the siRNA and miRNA pathway in arthropods are also present in Artemia franciscana. This study provides the first look at the core genes of the RNAi machinery of A. franciscana. Additionally, egg-sac microinjection was used to generate the desired RNAi phenotype. The high numbers of nauplii with the desired phenotypes obtained from injected maternal A. franciscana (both from the first and second brood) demonstrated the reliability of the egg-sac microinjection method, providing a methodology to study gene function on high numbers of individuals be treating the parental generation
... Shrimp infected with WSSV, principally display white spots or patches embedded in the exoskeleton and epidermis [12] and show a cumulative mortality of upto 100% within 10 days in commercial shrimp farms [13]. Previous strategies to prevent and control WSSV, generally included an improvement of environmental conditions, stocking up of specific pathogen-free or pathogenresistant post-larvae, induction of nonspecific antiviral responses to antiviral drugs or immunostimulants [14][15][16], neutralization of antibodies [17][18][19][20], specific immunization by vaccines [21][22][23][24][25][26], and suppression of the virus with RNAi technology [27][28][29][30][31]. ...
... The increase in LDH expression and activity, along with glucose and lactate concentrations induced by the WSSV infection were completely ameliorated by the silencing of HIF-1α demonstrating that the observed effects were mediated by HIF-1. Several studies have demonstrated the effects of silencing of target genes on molecular, cellular, and metabolic responses in shrimp exposed to viral infections such as the WSSV [11,[14][15][16][17][18][19][20][21][22][23][58][59][60][61][62][63][64]. Anaerobic glycolysis in white shrimp exposed to hypoxia up-regulates glycolytic genes, including the LDH subunits, via HIF-1 [3,7,8]. ...
Lactate dehydrogenase (LDH) is key for anaerobic glycolysis. LDH is induced by the hypoxia inducible factor -1 (HIF-1). HIF-1 induces genes involved in glucose metabolism and regulates cellular oxygen homeostasis. HIF-1 is formed by a regulatory α-subunit (HIF-1α) and a constitutive β-subunit (HIF-1β). The white spot syndrome virus (WSSV) induces anaerobic glycolysis in shrimp hemocytes, associated with lactate accumulation. Although infection and lactate production are associated, the LDH role in WSSV-infected shrimp has not been examined. In this work, the effects of HIF-1 silencing on the expression of two LDH subunits (LDHvan-1 and LDHvan-2) in shrimp infected with the WSSV were studied. HIF-1α transcripts increased in gills, hepatopancreas, and muscle after WSSV infection, while tHIF-1β remained constitutively expressed. The expression for both LDH subunits increased in each tissue evaluated during the WSSV infection, translating into increased enzyme activity. Glucose concentration increased in each tissue evaluated, while lactate increased in gills and hepatopancreas, but not in muscle. Silencing of HIF-1α blocked the increase of LDH expression and enzyme activity, along with glucose (all tissues) and lactate (gills and hepatopancreas) concentrations produced by WSSV infection. These results demonstrate that HIF-1 up regulates the expression of LDH subunits during WSSV infection, and that this induction contributes to substrate metabolism in energetically active tissues of infected shrimp.
... According to Westenberg et al. [69], small interfering RNA (siRNA) can inhibit WSSV gene expression and replication in a sequence-independent manner. Based on these observations it could be assumed that siRNA against major envelope proteins could be a potent anti-WSSV mechanism to protect shrimps against infections. ...
The shrimp industries make a remarkable economic development in the aquaculture industries in India. The task behaving the forming is the viral contaminations in the cultural industries. India was means with pre diagnosis carried mostly against WSSV and very less enquiries on IHHNV. This review article deals on major DNA viruses White Spot Syndrome virus (WSSV), Monodon Baculovius (MBV), Infectious Hypodermal and Hematopoietic Necrosis virus (IHHNV) and Hepatopancreatic Parvovirus (HPV) with making an details molecular understanding and diagnostic awareness.
... There have been several reports demonstrating the efficacy of dsRNA and siRNA against shrimp viruses such as WSSV, TSV, and YHV (Krishnan et al., 2009;Sanjuktha et al., 2012). Several recent studies have reported that siRNA (Westenberg et al., 2005;Xu et al., 2007) or in vitro and bacterially expressed dsRNA (Kim et al., 2007;Robalino et al., 2004Robalino et al., , 2005Sanjuktha et al., 2012) show great potential therapeutic agents for treating shrimp viral diseases. ...
In the present study, a suitable carrier system was developed for the delivery of dsRNA into Penaeus monodon (P. monodon) post larvae to silence the Monodon baculovirus (MBV) structural gene of p74. The carrier system was developed by layer by layer adsorption of oppositely charged chitosan-dextran sulfate, on charged silica nanoparticles. The silica template was removedto produce multilayered hollow nanocapsules (CS-DS) that were utilized for dsRNA loading at an alkaline pH. The capsule's surface was modified by conjugating with shrimp feed for enhanced cellular uptake. In vivo cellular uptake of CS-DS/FITC loaded nanocapsules conjugated with feed was studied after oral administration into post-larvae. The results revealed that the encapsulated FITC was effectively delivered and exhibited a sustained release into the cytoplasm of shrimp post-larvae. The MBV challenge study for structural gene p74was conducted after 3-25 days of post infection (dpi) with respective CS-DS/dsRNA coated with feed. The results showed a significant survival rate of 86.63% and effective gene silencing in P. monodon. Our findings indicated that the delivery of dsRNA using shrimp feed coatedCS-DSnanocapsules could be a novel approach to prevent viral infections in shrimp.
... The idea is that RNAi makes the gene not to work, or silencing or knock down the expression, it is called post-transcriptional gene silencing. Several recent studies have reported that siRNAs (Westenberg et al. 2005;Xu et al. 2007) or dsRNA synthesized by in vitro methods (Kim et al. 2007;Robalino et al. 2004Robalino et al. , 2005 serve as potential therapeutic agents for treating white spot syndrome disease. These approaches are costly and laborious for the synthesis of large amounts of siRNA or dsRNA by in vitro methods. ...
Indian aquaculture industry was growing steadily and showed a sixfold growth in production over the last two decades. However, the overall development of aquaculture in India did not reach the levels as projected due to frequent disease outbreaks and related issues. Shrimp and fish are predominantly affected by viruses including white spot syndrome virus, monodon baculovirus, hepatopancreatic parvovirus, viral nervous necrosis, white tail disease, and hypodermal and hematopoietic necrosis baculovirus that cause outbreaks across the countries. Owing to these viral pathogens, the production of aquaculture fishes and crustaceans has dramatically been dropped. There are no specific measures to control these viral infections since it causes mortality at all life stages of cultured aquatic organisms. Early detection of the diseases may be beneficial to prevent the spreading and mass mortality. In India, the Central Institute of Brackish Water Aquaculture has developed a non-invasive diagnostic tool for early and precise detection of monodon baculovirus infection in Indian tiger shrimp Penaeus monodon using SYBR Green-based real-time polymerase chain reaction (PCR) technique. More than twenty-five cell lines from freshwater, brackish water, and marine fish have been developed, characterized, and stored in C. Abdul Hakeem College for research and viral diagnosis. This review reveals the distinctive tools that are being used in aquaculture for the detection of pathogens and preventive measures. Advanced molecular methods such as nested PCR and SYBR Green-based real-time PCR are found to be sensitive and effective for the quantitation. Simple and inexpensive methods such as microscopic evaluation and histopathological analysis detects the progression of the disease. These assays can be used as a diagnostic method for emerging diseases in addition to avoid forthcoming of another.
... There have been several reports demonstrating the efficacy of dsRNA and siRNA against shrimp viruses such as WSSV, TSV, and YHV (Krishnan et al., 2009; Sanjuktha et al., 2012). Several recent studies have reported that siRNA (Westenberg et al., 2005; Xu et al., 2007) or in vitro and bacterially expressed dsRNA (Kim et al., 2007; Robalino et al., 2004 Robalino et al., , 2005 Sanjuktha et al., 2012) show great potential therapeutic agents for treating shrimp viral dis- eases. Often, viral invasion in shrimps occurs via. ...
... In our transcriptome data, three unigenes identified as important components of the RNA interference (RNAi) pathway, Argonaute 1, Argonaute 2 and Dicer 2 were highly expressed. Previous research reported a significant inhibition of virus replication via the dsRNA (Robalino et al., 2005;Yodmuang et al., 2006;Sarathi et al., 2008;Ongvarrasopone et al., 2008) and siRNA (Westenberg et al., 2005;Wu et al., 2007;Xu et al., 2007) of specific genes, suggesting that an RNAi mechanism against virus infection possibly exists in shrimps. Subsequently, the genes encoding Argonaute (Unajak et al., 2006), Dicer (Su et al., 2008) and other members of the RNAi pathway were revealed in shrimps. ...
The world production of shrimp such as the Malaysian giant freshwater prawn, Macrobrachium rosenbergii is seriously affected by the white spot syndrome virus (WSSV). There is an urgent need to understand the host pathogen interaction between M. rosenbergii and WSSV which will be able to provide a solution in controlling the spread of this infectious disease and lastly save the aquaculture industry. Now, using Next Generation Sequencing (NGS), we will be able to capture the response of the M. rosenbergii to the pathogen and have a better understanding of the host defence mechanism. Two cDNA libraries, one of WSSV-challenged M. rosenbergii and a normal control one, were sequenced using the Illumina HiSeqTM 2000 platform. After de novo assembly and clustering of the unigenes from both libraries, 63,584 standard unigenes were generated with a mean size of 698 bp and an N50 of 1137 bp. We successfully annotated 35.31% of all unigenes by using BLASTX program (E-value <10-5) against NCBI non-redundant (Nr), Swiss-Prot, Kyoto Encyclopedia of Genes and Genome pathway (KEGG) and Orthologous Groups of proteins (COG) databases. Gene Ontology (GO) assessment was conducted using BLAST2GO software. Differentially expressed genes (DEGs) by using the FPKM method showed 8,443 host genes were significantly up-regulated whereas 5,973 genes were significantly down-regulated. The differentially expressed immune related genes were grouped into 15 animal immune functions. The present study showed that WSSV infection has a significant impact on the transcriptome profile of M. rosenbergii’s hepatopancreas, and further enhanced the knowledge of this host-virus interaction. Furthermore, the high number of transcripts generated in this study will provide a platform for future genomic research on freshwater prawns.
... RNAi has been denoted as a powerful tool against WSSV when viral genes are silenced. This effect has been documented in several shrimp species such as Marsupenaeus japonicus (Wu et al. 2007;Xu et al. 2007;Sudhakaran et al. 2011), Penaeus chinensis (Kim et al. 2007), Penaeus monodon (Westenberg et al. 2005;Attasart et al. 2009), and Litopenaeus vannamei (Robalino et al. 2005;Wu et al. 2007). The efficiency of RNAi to inhibit the replication of WSSV in shrimp depends on the protein target, dsRNA size, dsRNA dosage applied, and the number of inoculated virions (Robalino et al. 2005;Wu et al. 2007;Alvarez-Ruiz et al. 2013). ...
The white spot syndrome virus (WSSV) remains the most devastating viral pathogen of shrimp culture worldwide. Gene silencing by RNA interference (RNAi) using double stranded RNA (dsRNA) has been considered a powerful tool for conferring protection against WSSV when viral genes are silenced, as documented in several shrimp species. However, this effect is not long lasting. Our results provide the first evidence that long-term silencing of the LvRab7 endogen produced antiviral effect against WSSV, which endured at least 21 d after dsRNA treatment (dat). Until now, the most efficient way to implement RNAi with dsRNA into the shrimp is by injection. Consequently, its application to broodstock in hatcheries is possible, minimizing the risk of vertical transmission of the virus. We show that the expression of Rab7 in hemocytes is lowest at 2 dat and finally recovers to basal status. In contrast, in gills and pleopods, gene expression silencing continued for at least 21 d. We challenged Litopenaeus vannamei broodstock with WSSV at 7, 14, or 21 dat reaching mortality rates of 0, 40, and 27%, respectively. In conclusion, the LvRab7 gene silencing is progressive and effective against WSSV. However, further studies are necessary to elucidate the functions of Rab7 in shrimp cells before applying this methodology at a commercial level.
... Resultados similares han sido reportados en Fenneropenaeus chinensis administrando ARN de doble cadena dirigido contra las proteínas VP281, VP28 y la PK (Kim et al. 2007). En el camarón tigre Penaeus monodon se redujo la infección silenciando los genes vp15 y vp28 (Westenberg et al. 2005). Otro estudio reporta la completa inhibición de la infección por WSSV mediante tres inyecciones consecutivas de RNAi corto dirigido contra VP28 en Marsupenaeus japonicus (Xu et al. 2007). ...
White spot syndrome virus (WSSV) causes high mortality to farmed shrimp and serious economic losses. Its unique sequence and genome structure has placed WSSV in its own new family Nimaviridae. Recently, high performance molecular techniques have made it possible to identify and characterize several WSSV structural proteins. These include 'shotgun' sequencing and isobaric tags for relative and absolute quantification (iTRAQ). Such techniques have made it possible to characterize 14 new WSSV proteins. Location and characterization of structural proteins can help to understand WSSV morphogenesis and pathogenesis. Both processes are essential to understand the mechanism of infection and to develop novel control methods. At present no effective treatments exist to fight WSSV in the field. WSSV structural proteins such as VP28 and VP19 have been evaluated to reduce the impact of WSSV. These molecules are essential early in the infection. Neutralization assays using specific antibodies against WSSV structural proteins have shown an increased survival of treated shrimp. Recently, RNA interference (RNAi) constructs directed against structural proteins have been used as a new tool to reduce/inhibit WSSV replication. A better comprehension of the host-pathogen interaction would allow the development of new methods to control WSSV. The use of high throughput techniques to determine the location and function of structural proteins will contribute to develop new strategies against infection. Intervention strategies aimed to block virus entry into the host cells may be a valuable output from these studies.
... summer mortality in C. gigas: Huvet et al. 2004; Martelia sydneyi in Saccostrea glomerata: Newton et al. 2004). Another pathogen of shrimp is the white spot syndrome virus, which can be controlled by the anti-viral immunity of injected double stranded (ds)RNA molecules and single stranded (si)RNA (Westenberg et al. 2005). ...
The development and application of genomics has been facilitated in a number of fields by the availability of new methodologies and tools, such as high throughput DNA sequencing and complementary DNA (cDNA) microar-rays. Genomic tools are already used in research on commercially important fish and shellfish species. Thousands of expressed sequence tags (EST) are now available for some of these species, and the sequencing of complete genomes of tilapia, cod, salmonids, flatfishes, sea bass and Pacific oyster has been proposed. Microarray technology through simultaneous analysis of the expression of thousands of genes allows the identification of candidate genes involved in the function of multiple physiological, morphological and behavioural traits of interests in organisms and populations from different environments. This paper reviews the current development of genomic technologies, and pinpoints their potential beneficial applications as well as implications for fisheries management and aquaculture. Résumé – Quel rôle pour la génomique dans la gestion des pêches et de l'aquaculture ? Le développement et l'application de la génomique ont été facilités dans un certain nombre de domaines par la disponibilité de nouveaux outils et de nouvelles méthodes, tels que le séquençage d'ADN à haut débit et les puces à ADN (« microarrays »). Les outils de la génomique sont déjà développés sur les espèces de poissons et d'invertébrés d'importance commerciale. Des milliers d'étiquettes, marqueurs de séquence exprimée (EST) sont désormais disponibles pour quelques unes de ces espèces et le séquençage du génome complet de tilapia, de la morue, de salmonidés, de poissons plats, du bar, et de l'huître creuse sont demandés. La technologie des puces à ADN, au travers d'analyse simultanée de l'expression de milliers de gènes, permet l'identification de gènes candidats impliqués dans les multiples fonctions physiologiques, morphologiques et comportementales chez les organismes et les populations d'environnements variés. Cet article pré-sente la synthèse de récents développements de ces technologies du génome, et met en évidence leurs applications potentielles ainsi que les implications dans la gestion des pêches et de l'aquaculture.
... The dsCHH RNA probe was designed to silence the two P. clarkii CHH genes CHH1 and CHH2 (Manfrin et al., 2014), both involved in the hyperglycemic activity stimulation. The double-stranded RNA codifying the Green Fluorescent Protein (dsRNA-GFP) has been widely used as non-specific control in a variety of RNA interference studies (RNAi) (Westenberg et al., 2005; Ponprateep et al., 2012). Injections and hemolymph withdrawal were performed through the abdominal hemolymph sinuses. ...
RNA interference has frequently been applied to modulate gene function in organisms. With the aim of creating new autocidal methods based on neuro-endocrine disruptors for invasive populations of Procambarus clarkii, we silenced the Crustacean Hyperglycemic Hormone (CHH) by injecting the corresponding dsRNA. CHH is a pleiotropic hormone that primarily regulates the mobilization of energy reserves and plays a pivotal role in stress responses. Here, we describe two experiments aimed at testing whether CHH silencing significantly alters important physiological aspects. The first experiment investigates the effects of CHH silencing at the glycemic and transcriptomic level in the eyestalk. The second experiment explores the long-term effects of CHH silencing and the effects on mortality and moulting rates. Osmotic deficits and mortality were recorded in specimens injected with CHH dsRNA, whilst controls were injected with GFP dsRNA. After 20 days, despite still silenced for CHH, individuals that survived recovered a strong hyperglycemic response after serotonin injection due to the compensatory effect of two peptides belonging to the crustacean neurohormone CHH protein family. © 2015, Universita degli Studi di Modena e Reggio Emilia. All rights reserved.
... Previous studies have successfully demonstrated the use of RNA-based vaccines to protect shrimp from WSSV infection (Kim et al., 2007;Xu et al., 2007). An antiviral effect of dsRNA or silencing RNA (siRNA) synthesized from the VP28 gene of WSSV has been reported and administered to shrimp before WSSV challenge (Sarathi et al., 2008;Westenberg et al., 2005). In this study, we explore the possibility of MrNv-VLP as a nanocarrier to deliver VP28 dsRNA to prevent viral infection in shrimp. ...
... Inhibition of YHV replication by cognate dsRNA significantly resulted in lower mortality in the black tiger shrimp (Tirasophon et al. 2007;Yodmuang et al. 2006). Meanwhile, recent studies revealed the existence of both innate (non-sequence specific) and RNAi related (sequence specific) antiviral phenomena in a crustacean model (Robalino et al. 2005;Robalino et al. 2007b;Robalino et al. 2004;Westenberg et al. 2005). However, the protection induced by dsRNA could be overwhelmed by a higher dose (8-fold) of infectious virus. ...
White spot syndrome virus (WSSV) is marked as one of the most economically devastating pathogens in shrimp aquaculture worldwide. Infection of cultured shrimp can lead to mass mortality (up to 100%). Although progress has been made, our understanding of WSSV’s infection process and the virus–host–environment interaction is far from complete. This in turn hinders the development of effective mitigation strategies against WSSV. Infection models occupy a crucial first step in the research flow that tries to elucidate the infectious disease process to develop new antiviral treatments. Moreover, since the establishment of continuous shrimp cell lines is a work in progress, the development and use of standardized in vivo infection models that reflect the host–pathogen interaction in shrimp is a necessity. This review critically examines key aspects of in vivo WSSV infection model development that are often overlooked, such as standardization, (post)larval quality, inoculum type and choice of inoculation procedure, housing conditions, and shrimp welfare considerations. Furthermore, the usefulness of experimental infection models for different lines of WSSV research will be discussed with the aim to aid researchers when choosing a suitable model for their research needs.
Viral disease pandemics are a major cause of economic losses in crustacean farming worldwide. While RNA interference (RNAi)-based therapeutics have shown promise at a laboratory scale, without an effective oral delivery platform, RNA-based therapy will not reach its potential against controlling viral diseases in crustaceans. Using a reverse-engineered shrimp RNA virus, Macrobrachium rosenbergii nodavirus (MrNV), we have developed a shrimp viral vector for delivering an engineered RNA cargo. By replacing the RNA-dependent RNA polymerase (RdRp) protein-coding region of MrNV with a cargo RNA encoding green fluorescent protein (GFP) as a proof-of-concept, we generated a replication-incompetent mutant MrNV(ΔRdRp) carrying the GFP RNA cargo resulting in MrNV(ΔRdRp)-GFP. Upon incorporating MrNV(ΔRdRp)-GFP in the diet of the marine Pacific white shrimp (Penaeus vannamei), MrNV(ΔRdRp) particles were visualized in hemocytes demonstrating successful vector internalization. Fluorescence imaging of hemocytes showed the expression of GFP protein and the MrNV capsid RNA (RNA 2) as well as the incorporated GFP RNA cargo. Detection of cargo RNA in hepatopancreas and pleopods indicated the systemic spread of the viral vector. The quantitative load of both the MrNV RNA2 and GFP RNA progressively diminished within eight days post-administration of the viral vector, which indicated a lack of MrNV(ΔRdRp)-GFP replication in shrimp. In addition, no pathological hallmarks of the wild-type MrNV infection were detected using histopathology in the target tissue of treated shrimp. The data unequivocally demonstrated the successful engineering of a replication-incompetent viral vector for RNA delivery, paving the way for the oral delivery of antiviral therapeutics in farmed crustaceans.
The wide exploration of interferon (IFN)s in vertebrates for medical purposes has attracted researchers to investigate the existence of a similar role of interferon in other organisms such as invertebrates, including insects, and crustacea. A review of the literature indicates that there is no evidence of interferon existing either in insects such as D. melanogaster and A. gambiae which have had their genomes fully sequenced or in crustacea. However, a nonspecific antiviral state in crustacean, such as P. monodon can be efficiently triggered by both dsRNA and siRNA. The evidence suggests that anonymous cytokines, similar to interferon and not identical to any vertebrate IFNs, related to antiviral protection, do exist in crustacea. However, how widely spread of interferon immune response inducer or interferon-like molecules in this group is an important issue that remains to be explored.
Circular RNAs (circRNAs) are non-coding RNAs (ncRNAs) originating from a post-transcriptional modification process called back-splicing. Despite circRNAs being traditionally considered by-products rather than independently functional, circRNAs play many vital roles, such as in host immunity during viral infection. However, in shrimp, these remain largely unexplored. Therefore, this study aims to identify circRNAs in Litopenaeus vannamei in the context of WSSV infection, one of the most eradicative pathogens threatening shrimp populations worldwide. We identified 290 differentially expressed circRNAs (DECs) in L. vannamei upon WSSV infection. Eight DECs were expressed from their parental genes, including alpha-1-inhibitor-3, calpain-B, integrin-V, hemicentin-2, hemocytin, mucin-17, proPO2, and rab11-FIP4. These were examined quantitatively by qRT-PCR, which revealed the relevant expression profiles to those obtained from circRNA-Seq. Furthermore, the structural and chemical validation of the DECs conformed to the characteristics of circRNAs. One of the functional properties of circRNAs as a miRNA sponge was examined via the interaction network between DECs and WSSV-responsive miRNAs, which highlighted the targets of miRNA sponges. Our discovery could provide insight into the participation of these ncRNAs in shrimp antiviral responses.
Fisheries play an important role in the world economy, and it contributes to the nations’ income, exports, food, nutritional security, and employment opportunities. In recent years, the capture fisheries decline due to over exploitation and illegal fishing. Aquaculture is one of the lucrative foods producing industries due to its delicious taste and high-income generation in the international market; thus, aquaculture production has increased lately with various culturing methods of culture practices. Unfortunately, in the past two decades, the aquaculture industry faces serious infectious disease problems, and it affects the production level. The traditional practices are also not effective and create so many environmental hazards for disease control. Considering the potential threat and environmental issues, it should be focused on developing alternative applications for the solution of the problems. The alternative practices have versatile characteristics like safety, efficiency, and affordability and are highly recommended in aquaculture practices to achieve higher production. In the present review, we have discussed the alternative approaches for shrimp health management for improving food production, including antimicrobial and non-specific immune-stimulating herbal and algal compounds; in silico approach for designing suitable antimicrobial drugs; antimicrobial biosurfactants from microbial sources; pre and probiotics; different generation of vaccines and its production in different expression system; chicken edible antibody production and its influences in shrimp aquaculture system; Specific Pathogen Free (SPF) shrimp; RNA interference (RNAi); Phage Therapy and Gene editing technology (CRISPR). The above-said approaches help to improve the aquatic cultivable shrimps’ health leading to improve food production.
Since proteins play an important role in the life of an organism, many researchers are now looking at how genes and proteins interact to form different proteins. It is anticipated that the creation of adequate tools for rapid analysis of proteins will accelerate the determination of functional aspects of these biomolecules and develop new biomarkers and therapeutic targets for the diagnosis and treatment of various diseases. Though shrimp contains high-quality marine proteins, there are reports about the heavy losses to the shrimp industry due to the poor quality of shrimp production and many times due to mass mortality also. Frequent outbreaks of diseases, water pollution, and quality of feed are some of the most recognized reasons for such losses. In the seafood export market, shrimp occupies the top position in currency earnings and strengthens the economy of many developing nations. Therefore, it is vital for shrimp-producing companies they produce healthy shrimp with high-quality protein. Though aquaculture is a very competitive market, global awareness regarding the use of scientific knowledge and emerging technologies to obtain better-farmed organisms through sustainable production has enhanced the importance of proteomics in seafood biology research. Proteomics, as a powerful tool, has therefore been increasingly used to address several issues in shrimp aquaculture. In the present paper, efforts have been made to address some of them, particularly the role of proteomics in reproduction, breeding and spawning, immunological responses and disease resistance capacity, nutrition and health, microbiome and probiotics, quality and safety of shrimp production, bioinformatics applications in proteomics, the discovery of protein biomarkers, and mitigating biotic and abiotic stresses. Future challenges and research directions on proteomics in shrimp aquaculture have also been discussed.
Prevention and control of diseases is a significant factor contributing to sustainable aquaculture. Discovery of RNA interference (RNAi) mechanism has opened new vistas in developing effective vaccines against viral diseases in aquaculture species, especially shrimps. Since the mechanism of action does not involve the host immune system, these are not vaccines in the true sense, but the RNAi molecules being developed globally to control diseases have both prophylactic and therapeutic effects. In general, a crucial viral gene essential for replication/pathogenesis is targeted for degradation by RNAi through post-transcriptional gene silencing. Several double stranded RNA (dsRNA) substrates like siRNA, shRNA and lhRNA can be utilized for this purpose. Single stranded antisense RNA (asRNA) molecules are also shown to exert gene silencing by RNAi mechanism. Several software are available for designing RNAi molecules and care should be taken to avoid off-target effects. Although the dsRNA / asRNA molecules can be delivered transiently for short-term silencing, more effective gene silencing can be achieved by employing plasmid vector-based dsRNA / asRNA expression constructs. Further, DNA vector-based RNAi molecules are more stable and can be delivered to aquaculture species through the immersion route that is least stressful, effective and economical. However, plasmid-based RNAi vaccines need to be designed in accordance with the regulatory guidelines.
Among different categories of management options available to combat infectious diseases in shrimp, vaccination is one of the most promising ones. Several vaccination methods such as inactivated (using temperature, gamma irradiation, binary ethylenimine (BEI) and formalin), recombinant and DNA vaccine and RNAi technology have been employed over the years. For white spot syndrome virus (WSSV), many viral genes, especially structural protein VP28, have been targeted for producing different types of vaccines. Many prokaryotic and eukaryotic expression systems, with their own advantages and limitations, have been used for generating recombinant vaccines. DNA vaccine has been suggested to be an ideal approach, considering many advantages including the ability of plasmid DNA to be vertically transmitted from mother to progeny. However, the efficacy of vaccine depends on many host- and pathogen-associated factors. Among various delivery methods, oral vaccination has been suggested as the most appropriate method. Further, the stability of oral vaccines in the gastro-intestinal tract of the animal has been enhanced by encapsulation with chitosan, glucan, liposomes and Artemia sp. Use of polyvalent vaccine, vaccine supplemented with nutritional additives, adjuvants and immunostimulants has also been reported to enhance the protective efficacy of vaccine in shrimp.
In the past two decades, with the discovery of RNA interference (RNAi) or post-transcriptional gene silencing (PTGS), the modern molecular biology field has been boosted with its immense applications. This rapid and powerful silencing method is useful in studying the gene function as well as in therapeutic applications for disease treatment. The RNAi or PTGS is a biological process of mRNA degradation induced by complementary double-stranded (ds) small interfering RNA (siRNA) sequences that mediate suppression of target protein-coding gene expression and provide resistance to both exogenous and endogenous microbial nucleic acids. This sequence-specific natural gene silencing mechanism has revolutionized experimental biology. RNAi technology has important practical applications in aquatic animal health, including functional genomics, therapeutic intervention, and other areas. Here in this chapter, we introduced the RNAi or PTGS mechanisms and the current understanding of gene silencing in aquatic animals in both fish and shellfish and will propose key areas of aquaculture fields where gene silencing could be applied.
Growing demands for aquatic sources of animal proteins have attracted significant investments in aquaculture research in recent years. The crustacean aquaculture industry has undergone substantial growth to accommodate a rising global demand, however such large-scale production is susceptible to pathogen-mediated destruction. It is clear that a thorough understanding of the crustacean innate immune system is imperative for future research into combating current and future pathogens of the main food crop species. Through a comparative genomics approach utilising extant data from 55 species, we describe the innate immune system of crustaceans from the Malacostraca class. We identify 7407 malacostracan genes from 39 gene families implicated in different aspects of host defence and demonstrate dynamic evolution of innate immunity components within this group. Malacostracans have achieved flexibility in recognising infectious agents through divergent evolution and expansion of pathogen recognition receptors genes. Antiviral RNAi, Toll and JAK-STAT signal transduction pathways have remained conserved within Malacostraca, although the Imd pathway appears to lack several key components. Immune effectors such as the antimicrobial peptides (AMPs) have unique evolutionary profiles, with many malacostracan AMPs not found in other arthropod groups. Lastly, we describe four putative novel immune gene families, characterised by distinct protein domains, potentially representing important evolutionary novelties of the malacostracan immune system.
VP28 is an essential WSSV envelope protein used to develop innate immune
recognition in shrimp against WSSV, owing to its remarkable capability to localize
on the surface of shrimp epithelial cells. Transgenically engineered viral VP28
protein from several expression systems was used to increase the survival chances
of shrimp against WSSV pathogen, and their efficacy was found to last for 2–
3 weeks following therapy. Though few reports have shown that DNA vaccination
using the VP28 DNA construct is capable of protecting shrimp from WSSV, the
exact mechanism behind the immunity is still unknown. Several research groups
have provided insights into VP28-driven dsRNA vaccination to protect shrimp
against WSSV infection, but the stability attained by many of these vaccines
turned out to be short-lived. RNAi treatment with VP28dsRNA, shrimp showed
better resistance to the WSSV challenge, although multiple doses of dsRNA are
required to provide extended protection for up to 28 days. However, no foolproof
protection was currently offered by VP28-based recombinant protein or DNA
vaccination against WSSV. In recent years, attention has been directed towards
identifying small molecule inhibitors of VP28 through molecular docking and
molecular dynamics simulation studies and is an emerging approach in drug
development and design against WSSV. This review highlights the opportunities,
limits and challenges of VP28-based control strategies of White Spot Syndrome
Virus, by emphasizing the status quo of current methodological approaches and
suggesting future research directions of using it as a potential drug discovery target
In decapod crustaceans, the insulin-like peptides such as insulin-like receptor (IR) are involved in the key regulatory processes in sexual differentiation and sexual characteristic maintenance. Here, we studied the localization of MrIR (M. rosenbergii insulin-like receptor) in Macrobrachium rosenbergii and found that MrIR was mainly expressed in the spermatocytes, androgenic gland cells, and secretory epithelial cells of terminal ampullae. The co-localization between MrIR and MrIAG (M. rosenbergii insulin-like androgenic gland gene) confirmed that MrIR functions as a receptor for MrIAG. This is the first time the siRNA method was used to knock-down MrIR in M. rosenbergii. The optimal injection dose of siRNA-MrIR knockdown was 0.5 μg/g body weight and long-term knockdown experiment yielded neofemales. Through histological observations, we inferred that the injection dose of 0.5 μg/g body weight can efficiently retard the spermatogenesis in testes. The seminiferous lobules were loosely arranged, and only spermatocytes could be observed. Morphologically, neofemales developed as female prawns that contained brood chamber (sperm-receiving pouch), setal buds, ovipositing setae and ovigerous setae. However, the second pereiopod was longer than in female control, but shorter than in male control. The current study provides insight into the MrIR knockdown-induced sex reversal in M. rosenbergii.
Nanotechnology a multidisciplinary field involves the design and production of functional systems at the molecular level. In aquaculture, the application of nanotechnology it’s still at infant stage and it potent enough to solve many issues related to nutrition, animal production, reproduction, disease diagnosis, prevention and treatment. Worldwide, during the last decade though shrimp culture has been one of the most prevalent practices in marine industry and it has been threatened by viral diseases frequently. Among various shrimp viral pathogens, white spot syndrome virus (WSSV) is exceedingly pathogenic and conscientious for huge economic loss in shrimp culture industry. In this review, the application of nanotechnology in diagnosis and management of WSSV in aquaculture is discussed in detail.
Aquatic animal diseases are one of the most significant constraints to the development and management of aquaculture worldwide. As a result, measures to combat diseases of fish and shellfish have assumed a high priority in many aquaculture producing countries. RNA interference (RNAi) has emerged as a powerful tool to manipulate gene expression in the laboratory. The discovery of RNA interference in mid ninety's added a new dimension in the regulation of gene expression by different types of RNA. It is a phenomenon in which double-stranded RNA (dsRNA) is the initiating factor in post-transcriptional gene silencing. It is a process in which the introduction of a double-stranded RNA (dsRNA) in the cells causes the specific degradation of a mRNA containing the same sequence. The double-stranded RNA (dsRNA) and short interfering RNA (siRNAs) alone cannot degrade mRNA but require the assistance of two enzymes namely Dicer and RNA-induced silencing complex (RISC). Among its many potential biomedical applications, silencing of viral genes stands out as a promising therapeutic strategy. The specific nature of the inhibition of protein production of cells has resulted in an explosion of research to understand and exploit RNAi. The technique is now established in vitro systems, and much work is focused on adapting RNAi for in vivo application. The field of RNA interference (RNAi) is moving at an impressive pace and generating excitingresults. A better understanding of post-transcriptional gene silencing (PTGS) shouldallow a more efficient response to viral infection and the development of transgene/host associations that can override silencing to allow the expression of interested proteins. The potential of the technology in understanding physiological and pathological processes is significant, while its development as a therapeutic agent holds much promise as targeted agents.
Background
Growing global demands for crustacean food crop species have driven large investments in aquaculture research worldwide. However, large-scale production is susceptible to pathogen-mediated destruction particularly in developing economies. Thus, a thorough understanding of the immune system components of food crop species is imperative for research to combat pathogens. ResultsThrough a comparative genomics approach utilising extant data from 55 species, we describe the innate immune system of the class Malacostraca, which includes all food crop species. We identify 7407 malacostracan genes from 39 gene families implicated in different aspects of host defence and demonstrate dynamic evolution of innate immunity components within this group. Malacostracans have achieved flexibility in recognising infectious agents through divergent evolution and expansion of pathogen recognition receptors genes. Antiviral RNAi, Toll and JAK-STAT signal transduction pathways have remained conserved within Malacostraca, although the Imd pathway appears to lack several key components. Immune effectors such as the antimicrobial peptides (AMPs) have unique evolutionary profiles, with many malacostracan AMPs not found in other arthropods. Lastly, we describe four putative novel immune gene families, potentially representing important evolutionary novelties of the malacostracan immune system. Conclusion
Our analyses across the broader Malacostraca have allowed us to not only draw analogies with other arthropods but also to identify evolutionary novelties in immune modulation components and form strong hypotheses as to when key pathways have evolved or diverged. This will serve as a key resource for future immunology research in crustacean food crops.
DNA vaccines present the aquaculture industry with an effective and economically viable method of controlling viral pathogens that drastically affect productivity. Since specific immune response is rudimentary in invertebrates, the presence of RNA interference (RNAi) pathway in shrimps provides a promising new approach to vaccination. Plasmid DNA vaccines that express short or long double stranded RNA in vivo have shown protection against viral diseases. The design, construction and considerations for preparing such vaccines are discussed.
Background and objective: Providing healthy food and required for increasing population of the country, that their health is important for future physical and mental health, Including responsibilities that it should be considered by wise strategy. Among these, marines have multiple capabilities for preventing diseases and providing health and in comparison with other proteins are more completely nutrients. In this study pathogenicity of virus of white spot disease that is the cause of the death and destruction of shrimps, in the Litopenaeus vannamei have been studied experimentally. Materials and methods: In this research, 200 Litopenaeus vannamei weighing approximately 10-12 gr were randomly selected,and Infected animals being tested, and by using diagnostic kits IQ2000 PCR have been confirmed. From the frozen body of Indian white shrimps that previously have shown symptoms of white spot disease, with using Nested Polymerase chain reaction (Nested-PCR) method, the virus existence was proven, that was used as virus source, and during a day in several meals along with food was given to Litopenaeus vannamei. After 3 days, clinical symptoms including: rapid separation of the cuticle from the epidermis, empty stomach and intestines, yellow and brittle of hepatopancreas, and red moving limbs was observed. Samples of tissues from treatment groups for study PCR and next steps were selected. Results: samples with Nested-PCR method, according to instructions of diagnostic special kit of white spot disease, multiple reaction steps passed and Finally, by ultraviolet rays were observed and recorded, of which 90 samples, in 62 cases positive reactions were observed.
The 97-megabase genomic sequence of the nematode Caenorhabditis elegans reveals over 19,000 genes. More than 40 percent of the predicted protein products find significant matches in other organisms.
There is a variety of repeated sequences, both local and dispersed. The distinctive distribution of some repeats and highly
conserved genes provides evidence for a regional organization of the chromosomes.
White spot syndrome baculovirus (WSBV) has been found across different shrimp species and in different Asian countries. The detection of WSBV in shrimp with white spot syndrome has already been achieved by means of 1-step polymerase chain reaction (PCR). In an attempt to establish a more sensitive assay, we evaluated the effect of 2-step amplification with nested primers on the sensitivity of WSBV diagnostic PCR. The sensitivity of the 2-step amplification was 10(3) to 10(4) times higher than that of 1-step amplification. Using both techniques, we successfully detected WSBV DNA in cultured and captured shrimp, crabs and other arthropods. Cultured Penaeus monodon (black tiger shrimp), P. japonicus (kuruma shrimp), P. penicillatus (red tail shrimp), and Metapenaeus ensis (sand shrimp) displaying white spot syndrome were collected from farms at different localities. One-step amplification of the DNA extracted from these shrimps consistently yielded an expected 1447 bp PCR product. Some of the tested specimens of cultured Scylla serrata (mud crab) that exhibited white spot syndrome were positive in 1-step WSBV diagnostic PCR, while others were positive only in 2-step WSBV diagnostic PCR. Use of the 2-step amplification protocol also detected a WSBV-specific DNA fragment in Macrobrachium rosenbergii (the giant freshwater prawn) exhibiting white spot syndrome. We also confirmed that WSBV exists in wild-caught shrimp (P. monodon, P. japonicus, P. semisulcatus and P. penicillatus) and crabs (Charybdis feriatus, Portunus pelagicus and P. sanguinolentus) collected from the natural environment in coastal waters around southern Taiwan. Detection of WSBV in non-cultured arthropods collected from WSBV-affected shrimp farms revealed that copepods, the pest crab Helice tridens, small pest Palaemonidae prawn and the larvae of an Ephydridae insect were reservoir hosts of WSBV. The relatedness between WSBV and Thailand's systemic ectodermal and mesodermal baculovirus (SEMBV) is discussed in this paper.
White spot syndrome virus (WSSV) is at present a major scourge to worldwide shrimp cultivation. We have determined the entire sequence of the double-stranded, circular DNA genome of WSSV, which contains 292,967 nucleotides encompassing 184 major open reading frames (ORFs). Only 6% of the WSSV ORFs have putative homologues in databases, mainly representing genes encoding enzymes for nucleotide metabolism, DNA replication, and protein modification. The remaining ORFs are mostly unassigned, except for five, which encode structural virion proteins. Unique features of WSSV are the presence of a very long ORF of 18,234 nucleotides, with unknown function, a collagen-like ORF, and nine regions, dispersed along the genome, each containing a variable number of 250-bp tandem repeats. The collective information on WSSV and the phylogenetic analysis on the viral DNA polymerase suggest that WSSV differs profoundly from all presently known viruses and that it is a representative of a new virus family.
White spot syndrome virus (WSSV) is a large DNA virus infecting shrimp and other crustaceans. The virus particles contain at least five major virion proteins, of which three (VP26, VP24, and VP15) are present in the rod-shaped nucleocapsid and two (VP28 and VP19) reside in the envelope. The mode of entry and systemic infection of WSSV in the black tiger shrimp, Penaeus monodon, and the role of these proteins in these processes are not known. A specific polyclonal antibody was generated against the major envelope protein VP28 using a baculovirus expression vector system. The VP28 antiserum was able to neutralize WSSV infection of P. monodon in a concentration-dependent manner upon intramuscular injection. This result suggests that VP28 is located on the surface of the virus particle and is likely to play a key role in the initial steps of the systemic WSSV infection in shrimp.
The fly Drosophila melanogaster is one of the most intensively studied organisms in biology and serves as a model system for the investigation of many developmental and cellular processes common to higher eukaryotes, including humans. We have determined the nucleotide sequence of nearly all of the approximately 120-megabase euchromatic portion of the Drosophila genome using a whole-genome shotgun sequencing strategy supported by extensive clone-based sequence and a high-quality bacterial artificial chromosome physical map. Efforts are under way to close the remaining gaps; however, the sequence is of sufficient accuracy and contiguity to be declared substantially complete and to support an initial analysis of genome structure and preliminary gene annotation and interpretation. The genome encodes approximately 13,600 genes, somewhat fewer than the smaller Caenorhabditis elegans genome, but with comparable functional diversity.
The fly Drosophila melanogaster is one of the most intensively studied organisms in biology and serves as a model system for the investigation of many developmental
and cellular processes common to higher eukaryotes, including humans. We have determined the nucleotide sequence of nearly
all of the ∼120-megabase euchromatic portion of theDrosophila genome using a whole-genome shotgun sequencing strategy supported by extensive clone-based sequence and a high-quality bacterial
artificial chromosome physical map. Efforts are under way to close the remaining gaps; however, the sequence is of sufficient
accuracy and contiguity to be declared substantially complete and to support an initial analysis of genome structure and preliminary
gene annotation and interpretation. The genome encodes ∼13,600 genes, somewhat fewer than the smaller Caenorhabditis elegansgenome, but with comparable functional diversity.
The fly Drosophila melanogaster is one of the most intensively studied organisms in biology and serves as a model system for the investigation of many developmental and cellular processes common to higher eukaryotes, including humans. We have determined the nucleotide sequence of nearly all of the approximately 120-megabase euchromatic portion of the Drosophila genome using a whole-genome shotgun sequencing strategy supported by extensive clone-based sequence and a high-quality bacterial artificial chromosome physical map. Efforts are under way to close the remaining gaps; however, the sequence is of sufficient accuracy and contiguity to be declared substantially complete and to support an initial analysis of genome structure and preliminary gene annotation and interpretation. The genome encodes approximately 13,600 genes, somewhat fewer than the smaller Caenorhabditis elegans genome, but with comparable functional diversity.
In a diverse group of organisms that includes Caenorhabditis elegans, Drosophila, planaria, hydra, trypanosomes, fungi and plants, the introduction of double-stranded RNAs inhibits gene expression in a sequence-specific manner. These responses, called RNA interference or post-transcriptional gene silencing, may provide anti-viral defence, modulate transposition or regulate gene expression. We have taken a biochemical approach towards elucidating the mechanisms underlying this genetic phenomenon. Here we show that 'loss-of-function' phenotypes can be created in cultured Drosophila cells by transfection with specific double-stranded RNAs. This coincides with a marked reduction in the level of cognate cellular messenger RNAs. Extracts of transfected cells contain a nuclease activity that specifically degrades exogenous transcripts homologous to transfected double-stranded RNA. This enzyme contains an essential RNA component. After partial purification, the sequence-specific nuclease co-fractionates with a discrete, approximately 25-nucleotide RNA species which may confer specificity to the enzyme through homology to the substrate mRNAs.
White spot syndrome virus (WSSV) is an invertebrate virus causing considerable mortality in penaeid shrimp. The oval-to-bacilliform shaped virions, isolated from infected Penaeus monodon, contain four major proteins: VP28, VP26, VP24 and VP19 (28, 26, 24 and 19 kDa, respectively). VP26 and VP24 are associated with the nucleocapsid and the remaining two with the envelope. Forty-one N-terminal amino acids of VP24 were determined biochemically allowing the identification of its gene (vp24) in the WSSV genome. Computer-assisted analysis revealed a striking similarity between WSSV VP24, VP26 and VP28 at the amino acid and nucleotide sequence level. This strongly suggests that these structural protein genes may have evolved by gene duplication and subsequently diverged into proteins with different functions in the WSSV virion, i.e. envelope and nucleocapsid. None of these three structural WSSV proteins showed homology to proteins of other viruses including baculoviruses, underscoring the distinct taxonomic position of WSSV among invertebrate viruses.
The immune system provides protection from a wide range of pathogens. One
component of immunity, the phylogenetically ancient innate immune response,
fights infections from the moment of first contact and is the fundamental
defensive weapon of multicellular organisms. The Toll family of receptors
has a crucial role in immune defence. Studies in fruitflies and in mammals
reveal that the defensive strategies of invertebrates and vertebrates are
highly conserved at the molecular level, which raises the exciting prospects
of an increased understanding of innate immunity.
Double-stranded RNA induces potent and specific gene silencing through a process referred to as RNA interference (RNAi) or
posttranscriptional gene silencing (PTGS). RNAi is mediated by RNA-induced silencing complex (RISC), a sequence-specific,
multicomponent nuclease that destroys messenger RNAs homologous to the silencing trigger. RISC is known to contain short RNAs
(∼22 nucleotides) derived from the double-stranded RNA trigger, but the protein components of this activity are unknown. Here,
we report the biochemical purification of the RNAi effector nuclease from culturedDrosophila cells. The active fraction contains a ribonucleoprotein complex of ∼500 kilodaltons. Protein microsequencing reveals that
one constituent of this complex is a member of the Argonaute family of proteins, which are essential for gene silencing in
Caenorhabditis elegans,Neurospora, and Arabidopsis. This observation begins the process of forging links between genetic analysis of RNAi from diverse organisms and the biochemical
model of RNAi that is emerging from Drosophila in vitro systems.
Toll-like receptors (TLRs) are a family of innate immune-recognition receptors that recognize molecular patterns associated with microbial pathogens, and induce antimicrobial immune responses. Double-stranded RNA (dsRNA) is a molecular pattern associated with viral infection, because it is produced by most viruses at some point during their replication. Here we show that mammalian TLR3 recognizes dsRNA, and that activation of the receptor induces the activation of NF-kappaB and the production of type I interferons (IFNs). TLR3-deficient (TLR3-/-) mice showed reduced responses to polyinosine-polycytidylic acid (poly(I:C)), resistance to the lethal effect of poly(I:C) when sensitized with d-galactosamine (d-GalN), and reduced production of inflammatory cytokines. MyD88 is an adaptor protein that is shared by all the known TLRs. When activated by poly(I:C), TLR3 induces cytokine production through a signalling pathway dependent on MyD88. Moreover, poly(I:C) can induce activation of NF-kappaB and mitogen-activated protein (MAP) kinases independently of MyD88, and cause dendritic cells to mature.
We report the first complete genome sequence of a marine invertebrate virus. White spot bacilliform virus (WSBV; or white spot syndrome virus) is a major shrimp pathogen with a high mortality rate and a wide host range. Its double-stranded circular DNA genome of 305,107 bp contains 181 open reading frames (ORFs). Nine homologous regions containing 47 repeated minifragments that include direct repeats, atypical inverted repeat sequences, and imperfect palindromes were identified. This is the largest animal virus that has been completely sequenced. Although WSBV is morphologically similar to insect baculovirus, the two viruses are not detectably related at the amino acid level. Rather, some WSBV genes are more homologous to eukaryotic genes than viral genes. In fact, sequence analysis indicates that WSBV differs from all known viruses, although a few genes display a weak homology to herpesvirus genes. Most of the ORFs encode proteins that bear no homology to any known proteins, either suggesting that WSBV represents a novel class of viruses or perhaps implying a significant evolutionary distance between marine and terrestrial viruses. The most unique feature of WSBV is the presence of an intact collagen gene, a gene encoding an extracellular matrix protein of animal cells that has never been found in any viruses. Determination of the genome of WSBV will facilitate a better understanding of the molecular mechanism underlying the pathogenesis of the WSBV virus and will also provide useful information concerning the evolution and divergence of marine and terrestrial animal viruses at the molecular level.
The Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) Se8 gene was recently shown to encode the viral envelope fusion (F) protein. A 60-kDa C-terminal subunit (F1) of the 76-kDa primary translation product of this gene was found to be the major envelope protein of SeMNPV budded virus (BV) (W. F. J. IJkel, M. Westenberg, R. W. Goldbach, G. W. Blissard, J. M. Vlak, and D. Zuidema, Virology
275:30–41, 2000). A specific inhibitor was used to show that furin is involved in cleavage of the precursor envelope fusion
(F0) protein. BV produced in the presence of the inhibitor possesses the uncleaved F0 protein, while an F protein with a mutation in the furin cleavage site was translocated to the plasma membrane but lost its
fusogenic activity. These results indicate that cleavage of F0 is required to activate the SeMNPV F protein and is necessary for BV infectivity. Specific antibodies against F1 and against the putative N terminus (F2) of the primary translation product were used to show that the F protein is BV specific and that BVs contain both the 60-
(F1) and 21-kDa (F2) cleavage products. In nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis both subunits migrate as a single
80-kDa protein, indicating that the subunits remain associated by a disulfide linkage. In addition, the presence of the F
protein predominately as a monomer suggests that disulfide links are not involved in oligomerization. Thus, the envelope fusion
protein from group II nucleopolyhedroviruses of baculoviruses has properties similar to those of proteins from a number of
vertebrate viruses.
White spot syndrome virus (WSSV) infects penaeid shrimp and other crustaceans. The WSSV virion consists of an enveloped rod-shaped nucleocapsid enclosing a large circular double-stranded DNA genome of 293 kbp. The virion envelope contains two major proteins of 28 (VP28) and 19 kDa (VP19) and the nucleocapsid consists of three major proteins of 26 (VP26), 24 (VP24) and 15 kDa (VP15). Study on the morphogenesis of the WSSV particle requires the genomic identification and chemical characterization of these WSSV virion proteins. An internal amino acid sequence of envelope protein VP19 was obtained by amino acid sequencing and used to locate the VP19 open reading frame of this protein on the genome, as WSSV ORF182. VP19 contained two putative transmembrane domains, which may anchor this protein in the WSSV envelope. Similarly, the gene for VP15 was located on the WSSV genome as ORF109. N-terminal amino acid sequencing on VP15 suggested that this protein was expressed from the second ATG of its ORF and the first methionine is lost by N-terminal protein processing. The 15 kDa protein is very basic and is a candidate DNA-binding protein in the WSSV nucleocapsid. None of the five major structural WSSV proteins appear to be glycosylated, which is an unusual feature among enveloped animal viruses.
Pattern recognition proteins such as lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) play an important role in the innate immune response of crustaceans and insects. Random sequencing of cDNA clones from a hepatopancreas cDNA library of white spot virus (WSV)-infected shrimp provided a partial cDNA (PsEST-289) that showed similarity to the LGBP gene of crayfish and insects. Subsequently full-length cDNA was cloned by the 5'-RACE (rapid amplification of cDNA ends) technique and sequenced. The shrimp LGBP gene is 1,352 bases in length and is capable of encoding a polypeptide of 376 amino acids that showed significant similarity to homologous genes from crayfish, insects, earthworms, and sea urchins. Analysis of the shrimp LGBP deduced amino acid sequence identified conserved features of this gene family including a potential recognition motif for beta-(1-->3) linkage of polysaccharides and putative RGD cell adhesion sites. It is known that LGBP gene expression is upregulated in bacterial and fungal infection and that the binding of lipopolysaccharide and beta-1,3-glucan to LGBP activates the prophenoloxidase (proPO) cascade. The temporal expression of LGBP and proPO genes in healthy and WSV-challenged Penaeus stylirostris shrimp was measured by real-time quantitative reverse transcription-PCR, and we showed that LGBP gene expression in shrimp was upregulated as the WSV infection progressed. Interestingly, the proPO expression was upregulated initially after infection followed by a downregulation as the viral infection progressed. The downward trend in the expression of proPO coincided with the detection of WSV in the infected shrimp. Our data suggest that shrimp LGBP is an inducible acute-phase protein that may play a critical role in shrimp-WSV interaction and that the WSV infection regulates the activation and/or activity of the proPO cascade in a novel way.
White spot syndrome virus (WSSV) is at present one of the major pathogens in shrimp culture worldwide. The complete genome of this virus has been sequenced recently. To identify the structural and functional proteins of WSSV, the purified virions were separated by SDS-PAGE. Twenty-four protein bands were excised, in-gel digested with trypsin, and subjected to matrix-assisted laser desorption ionization-time of flight mass spectrometry and electrospray ionization tandem mass spectrometry, respectively. Eighteen proteins matching the open reading frames of WSSV genome were identified. Except for three known structural proteins and collagen, the functions of the remaining 14 proteins were unknown. Temporal analysis revealed that all the genes were transcribed in the late stage of WSSV infection except for vp121. Of the newly identified proteins, VP466 (derived from band 16) was further characterized. The cDNA encoding VP466 was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. Specific antibody was generated with the purified GST-VP466 fusion protein. Western blot showed that the mouse anti-GST-VP466 antibody bound specifically to a 51-kDa protein of WSSV. Immunogold labeling revealed that VP466 protein is a component of the viral envelope. Results in this investigation thus proved the effectiveness of proteomic approaches for discovering new proteins of WSSV.
Anopheles gambiae is the principal vector of malaria, a disease that afflicts more than 500 million people and causes more than 1 million deaths each year. Tenfold shotgun sequence coverage was obtained from the PEST strain of A. gambiae and assembled into scaffolds that span 278 million base pairs. A total of 91% of the genome was organized in 303 scaffolds; the largest scaffold was 23.1 million base pairs. There was substantial genetic variation within this strain, and the apparent existence of two haplotypes of approximately equal frequency ("dual haplotypes") in a substantial fraction of the genome likely reflects the outbred nature of the PEST strain. The sequence produced a conservative inference of more than 400,000 single-nucleotide polymorphisms that showed a markedly bimodal density distribution. Analysis of the genome sequence revealed strong evidence for about 14,000 protein-encoding transcripts. Prominent expansions in specific families of proteins likely involved in cell adhesion and immunity were noted. An expressed sequence tag analysis of genes regulated by blood feeding provided insights into the physiological adaptations of a hematophagous insect.
Introduction of double-stranded RNA (dsRNA) into a wide variety of cells and organisms results in post-transcriptional depletion
of the homologue endogenous mRNA. This well-preserved phenomenon known as RNA interference (RNAi) is present in evolutionarily
diverse organisms such as plants, fungi, insects, metazoans, and mammals. Because the identification of the targeted mRNA
by the RNAi machinery depends upon Watson-Crick base-pairing interactions, RNAi can be exquisitely specific. We took advantage
of this powerful and flexible technique to demonstrate that selective silencing of genes essential for viral propagation
prevents in vitro and in vivo viral infection. Using the baculovirus Autographa californica, a rapidly replicating and highly cytolytic double-stranded DNA virus that infects many different insect species, we show
for the first time that introduction of dsRNA from gp64 and ie1, two genes essential for baculovirus propagation, results in prevention of viral infection in vitro and in vivo. This is the first report demonstrating the use of RNAi to inhibit a viral infection in animals. This inhibition was specific,
because dsRNA from the polyhedrin promoter (used as control) or unrelated dsRNAs did not affect the time course of viral
infection. The most relevant consequences from the present study are: 1) RNAi offers a rapid and efficient way to interfere
with viral genes to assess the role of specific proteins in viral function and 2) using RNAi to interfere with viral genes
essential for cell infection may provide a powerful therapeutic tool for the treatment of viral infections.
White spot syndrome virus (WSSV) iis an invertebrate virus causing considerable mortality in penaeid shrimp. The oval-to-bacilliform shaped virions, isolated from infected Penaeus monodon, contain four major proteins: VP28, VP26, VP24 and VP19 (28, 26, 24 and 19 kDa, respectively), VP26 and VP24 are associated with the nucleocapsid and the remaining two with the envelope. Forty-one N-terminal amino acids of VP24 were determined biochemically allowing the identification of its gene (vp24) in the WSSV genome. Computer-assisted analysis revealed a striking similarity between WSSV VP24, VP26 and VP28 at the amino acid and nucleotide sequence level, This strongly suggests that these structural protein genes may have evolved by gene duplication and subsequently diverged into proteins with different functions in the WSSV virion, i.e. envelope and nucleocapsid, None of these three structural WSSV proteins showed homology to proteins of other viruses including baculoviruses, underscoring the distinct taxonomic position of WSSV among invertebrate viruses.
Multi-centre databases are making an increasing contribution to medical understanding. While the statistical handling of randomized experimental studies is well documented in the medical literature, the analysis of observational studies requires the addressing of additional important issues relating to the timing of entry to the study and the effect of potential explanatory variables not introduced until after that time. A series of analyses is illustrated on a small data set. The influence of single and multiple explanatory variables on the outcome after a fixed time interval and on survival time until a specific event are examined. The analysis of the effect on survival of factors that only come into play during follow-up is then considered. The aim of each analysis, the choice of data used, the essentials of the methodology, the interpretation of the results and the limitations and underlying assumptions are discussed. It is emphasized that, in contrast to randomized studies, the basis for selection and timing of interventions in observational studies is not precisely specified so that attribution of a survival effect to an intervention must be tentative. A glossary of terms is provided.
Simultaneous capillary-gravity solitary waves (simultons or quadratic solitons) are shown to be possible in a rectangular liquid channel of arbitrary finite depth bounded below by a solid plate and above with a free deformable surface with constant surface tension. A second-harmonic resonance between two waveguide modes (fundamental and second-harmonic waves) is studied with the inclusion of dispersion in the system. The nonlinearly coupled amplitude equations for the two slowly varying envelopes of the fundamental and the second-harmonic wave components are derived using the method of multiple scales. Two types of capillary-gravity simulton solutions are explicitly obtained and an experiment for observing such hydrodynamic simultons is suggested.
The history and characteristics of two cells lines developed from primary explants of pupal tissue from the insect, Spodoptera frugiperda (J.E. Smith), are described. One cell line, IPLB-SF 21, was developed with hemolymph-supplemented medium and has been maintained continuously on the medium. The second cell line, IPLB-SF-1254, was developed with a medium containing a combination of vertebrate sera plus hemolymph and was adapted to hemolyphn-free medium at the 6th passage. The IPLB-SF-21 cell line has a population doubling time of 26 to 30 hr; the doubling time of the IPLB-SF-1254 line is 36 hr. The chromosomal morphology and distribution was typical of other lepidopteran cell lines. Serological studies showed that both cell lines have at least one antigen which also is common is tissue antigens from pupae of Spodoptera frugiperda.
The first chordates appear in the fossil record at the time of the Cambrian explosion, nearly 550 million years ago. The modern ascidian tadpole represents a plausible approximation to these ancestral chordates. To illuminate the origins of chordate and vertebrates, we generated a draft of the protein-coding portion of the genome of the most studied ascidian, Ciona intestinalis. TheCiona genome contains ∼16,000 protein-coding genes, similar to the number in other invertebrates, but only half that found in vertebrates. Vertebrate gene families are typically found in simplified form in Ciona, suggesting that ascidians contain the basic ancestral complement of genes involved in cell signaling and development. The ascidian genome has also acquired a number of lineage-specific innovations, including a group of genes engaged in cellulose metabolism that are related to those in bacteria and fungi.
About 25 years ago, researchers first demonstrated that a short synthetic oligodeoxynucleotide, referred to as antisense, can inhibit replication of Rous sarcoma virus through hybridization to viral RNA. Since then, several hybridization-based oligonucleotide approaches have been developed to elucidate the functions of genes and their potential as therapeutic agents. Short-interfering (si) RNA is the most recent example. To effectively inhibit gene expression, an antisense or siRNA must be resistant to nucleases, be taken up efficiently by cells, hybridize efficiently with the target mRNA and activate selective degradation of the target mRNA or block its translation without causing undesirable side effects. However, both antisense and siRNA agents have been shown to exert non-target-related biological effects including immune stimulation. Do antisense and siRNA agents work as ligands for Toll-like receptors (TLRs), a family of pathogen-associated, molecular pattern recognition receptors?
Nature is the international weekly journal of science: a magazine style journal that publishes full-length research papers in all disciplines of science, as well as News and Views, reviews, news, features, commentaries, web focuses and more, covering all branches of science and how science impacts upon all aspects of society and life.
There are five different viruses which are currently being studied for their impact on commercial farming of the black tiger prawn (Penaeus monodon) in Thailand. Some of these viruses cause disease in other penaeid shrimp species and even other crustacean species. Some occur not only in cultivated shrimp in other Asian countries, but also in those from Australia and the western hemisphere. In descending order from greatest to least economic impact on the Thai shrimp industry, the five viruses are: white-spot baculovirus, yellow-head virus, hepatopancreatic parvo-like virus, infectious hypodermal and hematopoeitic necrosis virus and monodon baculovirus. The purpose of this review is to summarize recent work on these viruses and to suggest future directions of research that may be useful in the effort to develop a sustainable shrimp industry.
Techniques for the detection of white spot baculovirus virus (WSBV) by polymerase chain reaction are well established. In this study, two primer sets designed from an isolate of WSBV from Penaeus monodon, PmNOB III, were used to detect WSBV infection in cultured and wild decapods in Taiwan. WSBV positive cases were found in all of four major marine cultured shrimp, P. monodon, P. japonicus, P. penicillatus and Metapenaeus ensis. Wild P. semisulcatus was also found to be naturally infected by WSBV. On the other hand, no cases of naturally occurring WSBV infection have yet been found in the wild shrimp Exopalaemen orientalis (from a milkfish culture farm), Trachypenaeus curvirostris, M. ensis (from the coast of Taiwan), Macrobrachium sp. and Procambarus clarkii (from rivers in Taiwan). Furthermore, neither the wild crabs, Calappa lophos, Portunus sanguinolentus, Charybdis granulata and C. feriata, nor the wild lobsters Panulirus ornatus, P. versicolor, P. longipes and P. penicillatus, collected from the coast of Taiwan showed any evidence of being naturally infected by WSBV. When captured specimens of these decapods were artificially infected by feeding them with tissues from severely PmNOB III infected P. monodon, wild shrimp mortality reached moderate to high levels at 18 days post infection. Using PCR analysis, WSBV DNA could be detected in the moribund specimens during the experimental period and in the survivors on the final day of the experiment. The mortalities in wild crabs and lobsters, however, were not significantly different from control groups. Nevertheless, WSBV DNA was also detectable in these specimens. WSBV was thus shown to have a wide host range and to exhibit different infectivity in the various decapods investigated in the present study.
White Spot Syndrome Virus (WSSV) is an invertebrate virus, causing considerable mortality in shrimp. Two structural proteins of WSSV were identified. WSSV virions are enveloped nucleocapsids with a bacilliform morphology with an approximate size of 275 × 120 nm, and a tail-like extension at one end. The double-stranded viral DNA has an approximate size 290 kb. WSSV virions, isolated from infected shrimps, contained four major proteins: 28 kDa (VP28), 26 kDa (VP26), 24 kDa (VP24), and 19 kDa (VP19) in size, respectively. VP26 and VP24 were found associated with nucleocapsids; the others were associated with the envelope. N-terminal amino acid sequences of nucleocapsid protein VP26 and the envelope protein VP28 were obtained by protein sequencing and used to identify the respective genes (vp26 and vp28) in the WSSV genome. To confirm that the open reading frames of WSSV vp26 (612) and vp28 (612) are coding for the putative major virion proteins, they were expressed in insect cells using baculovirus vectors and analyzed by Western analysis. A polyclonal antiserum against total WSSV virions confirmed the virion origin of VP26 and VP28. Both proteins contained a putative transmembrane domain at their N terminus and many putative N- and O-glycosylation sites. These major viral proteins showed no homology to baculovirus structural proteins, suggesting, together with the lack of DNA sequence homology to other viruses, that WSSV may be a representative of a new virus family, Whispoviridae.
The 97-megabase genomic sequence of the nematode Caenorhabditis elegans reveals over 19,000 genes. More than 40 percent of the predicted protein products find significant matches in other organisms. There is a variety of repeated sequences, both local and dispersed. The distinctive distribution of some repeats and highly conserved genes provides evidence for a regional organization of the chromosomes.
The history and characteristics of two cells lines developed from primary explants of pupal tissue from the insect, Spodoptera frugiperda (J.E. Smith), are described. One cell line, IPLB-SF 21, was developed with hemolymph-supplemented medium and has been maintained continuously on the medium. The second cell line, IPLB-SF-1254, was developed with a medium containing a combination of vertebrate sera plus hemolymph and was adapted to hemolyphn-free medium at the 6th passage. The IPLB-SF-21 cell line has a population doubling time of 26 to 30 hr; the doubling time of the IPLB-SF-1254 line is 36 hr. The chromosomal morphology and distribution was typical of other lepidopteran cell lines. Serological studies showed that both cell lines have at least one antigen which also is common is tissue antigens from pupae of Spodoptera frugiperda.
The interferon-induced protein kinase DAI, the double-stranded RNA (dsRNA)-activated inhibitor of translation, plays a key role in regulating protein synthesis in higher cells. Once activated, in a process that involves autophosphorylation, it phosphorylates the initiation factor eIF-2, leading to inhibition of polypeptide chain initiation. The activity of DAI is controlled by RNA regulators, including dsRNA activators and highly structured single-stranded RNAs which block activation by dsRNA. To elucidate the mechanism of activation, we studied the interaction of DAI with RNA duplexes of discrete sizes. Molecules shorter than 30 bp fail to bind stably and do not activate the enzyme, but at high concentrations they prevent activation by long dsRNA. Molecules longer than 30 bp bind and activate the enzyme, with an efficiency that increases with increasing chain length, reaching a maximum at about 85 bp. These dsRNAs fail to activate at high concentrations and also prevent activation by long dsRNA. Analysis of complexes between dsRNA and DAI suggests that at maximal packing the enzyme interacts with as little as a single helical turn of dsRNA (11 bp) but under conditions that allow activation the binding site protects about 80 bp of duplex. When the RNA-binding site is fully occupied with an RNA activator, the complex appears to undergo a conformational change.
Conventional mouse hybridoma technology was utilized to produce a panel of monoclonal antibodies which reacted with baculovirus proteins. Using an enzyme-linked immunosorbent assay (ELISA), the hybridomas which were raised against polyhedrin from Autographa californica nuclear polyhedrosis virus (AcNPV) and Choristoeura fumiferana nuclear polyhedrosis virus (CfNPV) were found to cross-react differentially with polyhedrins and granulins from several species of baculoviruses. Hybridoma antibodies which reacted against the nonoccluded form (NOV) of AcNPV in an ELISA test expressed different specificities for the occluded form of the virus (OV), a mutant strain of AcNPV, and CfNPV. Four hybridoma clones produced antibody which neutralized the infectivity of AcNPV NOV. One hybridoma antibody reacted strongly with the uninfected Spodoptera frugiperda host cell line. Using Western blot analysis, it was shown that hybridoma antibodies against polyhedrin reacted differentially with the complete polypeptide and protease-generated fragments of polyhedrin. The polypeptide specificity of 19 of 28 hybridoma antibodies which reacted with OV and NOV of AcNPV was assigned using Western blot analysis.
Multi-centre databases are making an increasing contribution to medical understanding. While the statistical handling of randomized experimental studies is well documented in the medical literature, the analysis of observational studies requires the addressing of additional important issues relating to the timing of entry to the study and the effect of potential explanatory variables not introduced until after that time. A series of analyses is illustrated on a small data set. The influence of single and multiple explanatory variables on the outcome after a fixed time interval and on survival time until a specific event are examined. The analysis of the effect on survival of factors that only come into play during follow-up is then considered. The aim of each analysis, the choice of data used, the essentials of the methodology, the interpretation of the results and the limitations and underlying assumptions are discussed. It is emphasized that, in contrast to randomized studies, the basis for selection and timing of interventions in observational studies is not precisely specified so that attribution of a survival effect to an intervention must be tentative. A glossary of terms is provided.
Experimental introduction of RNA into cells can be used in certain biological systems to interfere with the function of an endogenous gene. Such effects have been proposed to result from a simple antisense mechanism that depends on hybridization between the injected RNA and endogenous messenger RNA transcripts. RNA interference has been used in the nematode Caenorhabditis elegans to manipulate gene expression. Here we investigate the requirements for structure and delivery of the interfering RNA. To our surprise, we found that double-stranded RNA was substantially more effective at producing interference than was either strand individually. After injection into adult animals, purified single strands had at most a modest effect, whereas double-stranded mixtures caused potent and specific interference. The effects of this interference were evident in both the injected animals and their progeny. Only a few molecules of injected double-stranded RNA were required per affected cell, arguing against stochiometric interference with endogenous mRNA and suggesting that there could be a catalytic or amplification component in the interference process.
White spot syndrome virus (WSSV), the causative agent of white spot syndrome in shrimp, has a wide host range which extends to crabs, copepods and other arthropods. In this study, benthic larvae of the mud crab Scylla serrata were captured from Taiwan's coastal waters and screened for the presence of WSSV by polymerase chain reaction (PCR) and in situ hybridization. WSSV was detected in around 60% of the larvae, and this prevalence rate remained fairly constant when the captured larvae were subsequently maintained in an aerated system in the laboratory. WSSV-free larvae obtained from a hatchery were challenged by immersion in a WSSV inoculum. Fifteen days after challenge, cumulative mortality in the experimental group reached 43% compared to 20% in the control group. PCR detection of WSSV in both moribund and surviving specimens clearly implicated the virus as the cause of death in most cases. Histological and in situ hybridization data confirmed that WSSV tissue tropism in Scylla serrata crab larvae is similar to that found in shrimp.
RNA interference (RNAi) is a phenomenon in which introduced double-stranded RNAs (dsRNAs) silence gene expression through specific degradation of their cognate mRNAs. Recent analyses in vitro suggest that dsRNAs may be copied, or converted, into 21-23 nucleotide (nt) guide RNAs that direct the nucleases responsible for RNAi to their homologous mRNA targets. Such small RNAs are also associated with gene silencing in plants.
We developed a quantitative single-embryo assay to examine the mechanism of RNAi in vivo. We found that dsRNA rapidly induced mRNA degradation. A fraction of dsRNAs were converted into 21-23 nt RNAs, and their time of appearance and persistence correlated precisely with inhibition of expression. The strength of RNAi increased disproportionately with increasing dsRNA length, but an 80bp dsRNA was capable of effective gene silencing. RNAi was saturated at low dsRNA concentration and inhibited by excess unrelated dsRNA. The antisense strand of the dsRNA determined target specificity, and excess complementary sense or antisense single-stranded RNAs (ssRNAs) competed with the RNAi reaction.
Processed dsRNAs can act directly to mediate RNAi, with the antisense strand determining mRNA target specificity. The involvement of 21-23 nt RNAs is supported by the kinetics of the processing reaction and the observed size dependence. RNAi depends on a limiting factor, possibly the nuclease that generates the 21-23 mer species. The active moiety appears to contain both sense and antisense RNA strands.
Innate antiviral substances occur in vertebrates and may function as host defenses. Virus infections are common among invertebrates, but little is known about the ability of invertebrates to control viral infections. Pre-existing antiviral substances may be particularly important, since invertebrates lack the antiviral defense conferred by specific immunity. In our study, we found that tissue extracts of blue crab (Callinectes sapidus), shrimp (Penaeus setiferus), and crayfish (Procambarus clarkii) contained antiviral activities that inhibit a variety of DNA and RNA viruses, i.e. Sindbis virus (SB), vaccinia virus (VAC), vesicular stomatitis virus (VS), mengo virus (MENGO), banzi virus (BANZI) and poliomyelitis (POLIO). The concentration of inhibitory activity was relatively high, ranging from 102 to 216 U/g tissue for Sindbis virus, using the various tissue extracts. The other viruses were somewhat less sensitive to the inhibitor. The main antiviral activity in the inhibitor preparation from blue crab resided in an approximately 440 kDa fraction. It was inactivated significantly by lipid extraction, but not by proteinase K or glycosidases. The antiviral mechanism of the inhibitor from the blue crab was inhibition of virus attachment to eukaryotic cells, as evidenced by inhibitory activity at 4 degrees C. These studies are among the first to show the existence of broadly active antiviral activities in aquatic crustaceans. These antiviral substances may function as innate host defenses in these species that lack specific antibody immunity and, therefore, merit further study.
Genetic interference mediated by double-stranded RNA (RNAi) has been a valuable tool in the analysis of gene function in Caenorhabditis elegans. Here we report an efficient induction of RNAi using bacteria to deliver double-stranded RNA. This method makes use of bacteria that are deficient in RNaseIII, an enzyme that normally degrades a majority of dsRNAs in the bacterial cell. Bacteria deficient for RNaseIII were engineered to produce high quantities of specific dsRNA segments. When fed to C. elegans, such engineered bacteria were found to produce populations of RNAi-affected animals with phenotypes that were comparable in expressivity to the corresponding loss-of-function mutants. We found the method to be most effective in inducing RNAi for non-neuronal tissue of late larval and adult hermaphrodites, with decreased effectiveness in the nervous system, in early larval stages, and in males. Bacteria-induced RNAi phenotypes could be maintained over the course of several generations with continuous feeding, allowing for convenient assessments of the biological consequences of specific genetic interference and of continuous exposure to dsRNAs.
The pathogenicity of white spot syndrome virus (WSSV) for the red swamp crawfish (Procambarus clarkii) was investigated after infection by intramuscular (i.m.) injection and oral route. The cumulative mortality of crawfish injected i.m. with WSSV reached 100% in 5 days. After oral feeding WSSV-infected kuruma shrimp (Penaeus japonicus) muscle tissues to the crawfish the cumulative mortality of this host reached 100% in 11 days. On reinfection trials, all the crawfish fed WSSV-infected crawfish muscle tissues died in 9 days. All the shrimp injected with a filtrate of infected crawfish heart tissues died in 12 days with typical signs of white spot syndrome (WSS). Electron microscopy clearly demonstrated that WSSV propagated in the cells of the crawfish midgut. This study showed that the red swamp crawfish can be used as alternative experimental host in the study of WSSV.
Gene silencing mediated by double-stranded RNA (dsRNA) is a sequence-specific, highly conserved mechanism in eukaryotes. In plants, it serves as an antiviral defence mechanism. Animal cells also possess this machinery but its specific function is unclear. Here we demonstrate that dsRNA can effectively protect human cells against infection by a rapidly replicating and highly cytolytic RNA virus. Pre-treatment of human and mouse cells with double-stranded, short interfering RNAs (siRNAs) to the poliovirus genome markedly reduces the titre of virus progeny and promotes clearance of the virus from most of the infected cells. The antiviral effect is sequence-specific and is not attributable to either classical antisense mechanisms or to interferon and the interferon response effectors protein kinase R (PKR) and RNaseL. Protection is the result of direct targeting of the viral genome by siRNA, as sequence analysis of escape virus (resistant to siRNAs) reveals one nucleotide substitution in the middle of the targeted sequence. Thus, siRNAs elicit specific intracellular antiviral resistance that may provide a therapeutic strategy against human viruses.
Gene expression in haemocytes of the kuruma prawn (Penaeus japonicus) was investigated using an expressed sequence tag (EST) approach. Partial nucleotide sequences of cDNA library clones constructed from normal and white spot syndrome virus (WSSV)--infected P. japonicus haemocytes were determined. Of 635 clones obtained from the normal library, 284 (44.7%) significantly matched sequences in GenBank, and of 370 clones obtained from WSSV-infected library, 174 (47.0%) significantly matched sequences in the database. One hundred fifty-two deduced proteins were newly identified. Of these, 28 types were involved in biodefence. For the prophenoloxidase system, there are prophenoloxidase, coagulation factor G-beta chain precursor, factor D, Masquarade-like protease, transglutaminase (TGase), clottable protein and eight types of protease inhibitors (two types of antileukoproteinase, alpha-2-macroglobulin, chelonianin, elastase inhibitor, two types of Kazal inhibitor and Kunitz-type inhibitor). For antibacterial peptides, there are bactinecin 11, penaeidin-2 precursor and lysozyme c type. The others defence-related proteins are basophil leukocyte interleukin-3-regulated protein, natural killer enhancing factor (NK-EF), integral membrane protein (CD34+), ESM-1, Notch homologue and Drac homologue. For the adhesion proteins, there are beta-integrin, cell adhesion molecule (CAM) and three types of collagens. All ESTs representing protease inhibitors and tumour-related proteins were found only in the WSSV-infected library. Those encoding for apoptotic peptides were expressed at high levels in infected library. The putative defence proteins accounted for 2.7% of total ESTs in a normal shrimp library and 15.7% of the total ESTs in an infected library.
Small interfering RNAs (siRNAs) are the mediators of mRNA degradation in the process of RNA interference (RNAi). Here, we describe a human biochemical system that recapitulates siRNA-mediated target RNA degradation. By using affinity-tagged siRNAs, we demonstrate that a single-stranded siRNA resides in the RNA-induced silencing complex (RISC) together with eIF2C1 and/or eIF2C2 (human GERp95) Argonaute proteins. RISC is rapidly formed in HeLa cell cytoplasmic extract supplemented with 21 nt siRNA duplexes, but also by adding single-stranded antisense RNAs, which range in size between 19 and 29 nucleotides. Single-stranded antisense siRNAs are also effectively silencing genes in HeLa cells, especially when 5'-phosphorylated, and expand the repertoire of RNA reagents suitable for gene targeting.
The onset and duration of resistance in experimental survivors of Penaeus japonicus produced by an intramuscular injection with white spot syndrome virus (WSSV) were surveyed by re-challenge tests with the virus conducted at weeks 1-4 and months 1-3 post initial exposure (PIE) to the virus. Virus neutralising activity in the survivors' plasma was also examined. Plasma-treated WSSV was separated from the plasma by centrifugation and then injected into naïve shrimp, in parallel with each re-challenge test. Re-challenge tests of the survivors conducted at weeks 1-4 PIE revealed that the resistance commenced at week 3 (relative percent survival, RPS: 39%) and almost fully developed at week 4 (RPS: 58%), because statistically significant differences in survival rates were observed between the test (previously virus exposed) and control groups at weeks 3 and 4. Re-challenge at months 1-3 PIE resulted in RPS values of 67, 54 and 6%, respectively, indicating the resistance persisted until month 2. RPS values in neutralisation tests performed at weeks 1-4 and months 1-3 PIE were -5, 14, 36, 50, 100, 38 and 6%, respectively, which coincided with the RPS values in each re-challenge test conducted in parallel. The present results demonstrated that resistance of P. japonicus against the viral pathogen developed 3 or 4 weeks after an exposure to the virus, and it persisted for another month at 24 degrees C. The resistance was paralleled by a humoral neutralising factor(s) in the plasma of shrimp.
The first chordates appear in the fossil record at the time of the Cambrian explosion, nearly 550 million years ago. The modern
ascidian tadpole represents a plausible approximation to these ancestral chordates. To illuminate the origins of chordate
and vertebrates, we generated a draft of the protein-coding portion of the genome of the most studied ascidian, Ciona intestinalis. TheCiona genome contains ∼16,000 protein-coding genes, similar to the number in other invertebrates, but only half that found in vertebrates.
Vertebrate gene families are typically found in simplified form in Ciona, suggesting that ascidians contain the basic ancestral complement of genes involved in cell signaling and development. The
ascidian genome has also acquired a number of lineage-specific innovations, including a group of genes engaged in cellulose
metabolism that are related to those in bacteria and fungi.