By Hui Gong*
Among the infectious pathogens, viral epizootics have caused the most catastrophic losses to the whole of the shrimp industry over the years. Although over 20 shrimp viruses have been identified, the most economically significant are white spot syndrome virus (WSSV), yellow head virus (YHV), taura syndrome virus (TSV), infectious myonecrosis virus (IMNV) and infectious hypodermal and haematopoietic necrosis virus, densovirus (DNV), etc. Shrimp relies solely on an innate immune system, and its diverse defense mechanisms against pathogenic infections include RNAi and signaling pathways such as Toll, Imd and Jak-STAT pathways, clotting and melanization (via prophenoloxidase cascade), in addition to some cellular mechanisms such as phagocytosis, apoptosis, nodule formation, encapsulation.
The RNAi response is crucial in controlling virus replication and limiting virus induced pathology and inherently provides specific antiviral response. Also implicated in antiviral responses, various signaling pathways lead to the activation of transcription factors and the subsequent expression of antimicrobial peptides. This article is mainly focusing on the recent research discoveries of the antiviral mechanism of RNAi in penaeid shrimp, whereas the signaling pathways in shrimp immune response will be a separate topic for future discussion.
First discovered in plants, later in invertebrate, RNA interference (RNAi) is the mechanism mediated by small RNAs, with typical lengths among 21-30 ribonucleotides. There are three categories, namely small interfering RNAs (siRNAs), microRNAs (miRNAs) and Piwi-associated interfering RNAs (piRNAs) in invertebrates. Like other invertebrates, the shrimp innate immune system is triggered by the recognition of the invading microbes by the molecules called Pattern Recognition Proteins (PRPs). The PRPs recognize and bind to the microbes and activate various immune responses. For viral pathogens, the shrimp specific PRPs could respond to the ssRNA and dsRNA from viruses in order to initiate the RNAi pathways.
Using WSSV (DNA virus) and TSV (RNA virus) as examples, an RNAi pathway in shrimp is graphically illustrated (Figure 1). In term of the siRNA mediated antiviral responses in shrimp, dsRNAs can be generated from the replication intermediate of DNA or RNA viruses. Shrimp Dicer-2 recognizes and cuts the dsRNA into 22–24 ribonucleotide siRNAs. Then, siRNAs are sent to an RNA-induced silencing complex (RISC) (containing Ago2) and degrade viral RNAs. The miRNAs are first transcribed to long pri-mRNAs from shrimp or viral DNA genome. The pri-miRNA is cut into the pre-miRNA by Drosha in the nucleus. Then pre-miRNA is exported to the cytoplasm for further processing by Dicer-1. The miRNA:miRNA* duplex intermediate is incorporated into RISC (containing Ago1), and one chain stays in the RISC to bind target mRNAs, followed by target mRNAs either being degraded or the translation being inhibited.
Since the discovery of RNAi mechanisms, major proteins involved in RNAi pathways have been identified in penaeid shrimp. Drosha, Dicer 1 and Dicer 2 proteins, different types of Argonaute proteins (the key component of RISC), as well as several members of transactivation response RNA-binding protein (TRBP) have been characterized in various shrimp species. Details are summarized in Table 1. These findings validate the existence of RNAi machinery in penaeid shrimp, but the understanding of RNAi systems in shrimp is still quite limited.
To date, there is a growing body of evidence that administration of synthesized dsRNA or siRNA could protect shrimp against viral infections (Table 2). Administration of dsRNA or siRNA specific to particular viral genes have shown protective effects in suppressing viral replication in vivo and inhibiting the viral disease progression, such as WSSV, YHV, IMNV, TSV, gill associated virus (GAV), and DNV among others. RNAi delivery systems, such as oral delivery vs. injection have been evaluated. From a practical point of view, oral administration of dsRNA or siRNA through feeding is more desirable than individual shrimp injection. However, the issue of insufficient delivery of the designated RNA when passing from the gut to the hemolymph via digestion and absorption processes, especially due to degradation of the RNA constructs by the gut enzymes such as nucleases, should be carefully considered in experimental design and investigation.
There is great potential for adopting RNAi technology against various infectious diseases, and eventually developing effective strategies for controlling the viral diseases. However, the basic research and application of RNAi technology in shrimp aquaculture are still in very early developmental stages, with several serious challenges. Firstly, lack of whole genome sequences and no permanent shrimp line to study RNAi mechanism in shrimp and host-pathogen interaction at the molecular level. This will be needed in order to develop the most suitable strategies against pathogens. Secondly, lack of standardization methods of assays and inconsistent results generated from the various studies is a fundamental issue that needs to be addressed systematically. Thirdly, the development and employment of safe, feasible, and both functionally- and cost-effective delivery methods of RNAi to shrimp at specific stage(s) deserves to be further explored. Nonetheless, the future of applying RNAi in controlling viral diseases in shrimp is highly promising from both preventive and therapeutic perspectives, with more understanding of the integrated immune network of shrimp and its interaction with viral pathogens.
Hui Gong, PhD, is an Associate Professor at the College of Natural and Applied Sciences at the University of Guam. Her expertise in shrimp aquaculture has built on 17 years of experience in applied research in both academic and industrial backgrounds.