Double-Stranded RNA Induces Sequence-Specific Antiviral Silencing in Addition to Nonspecific Immunity in a Marine Shrimp: Convergence of RNAInterference and Innate Immunity in the Invertebrate Antiviral Response?

Abstract

Double-stranded RNA (dsRNA) is a common by-product of viral infections and a potent inducer of innate antiviral immune responses in vertebrates.In the marine shrimp Litopenaeus vannamei, innate antiviral immunity is also induced by dsRNA in a sequence-independent manner. In this study, the hypothesis that dsRNA can evoke not only innate antiviral immunity but also a sequence-specific antiviral response in shrimp was tested. It was found that viral sequence-specific dsRNA affords potent antiviral immunity in vivo, implying the involvement of RNA interference (RNAi)-like mechanisms in the antiviral response of the shrimp. Consistent with the activation of RNAi by virus-specific dsRNA, endogenous shrimp genes could be silenced in a systemic fashion by the administration of cognate long dsRNA. While innate antiviral immunity, sequencedependent antiviral protection, and gene silencing could all be induced by injection of long dsRNA molecules, injection of short interfering RNAs failed to induce similar responses, suggesting a size requirement for extracellular dsRNA to engage antiviral mechanisms and gene silencing. We propose a model of antiviral immunity in shrimp by which viral dsRNA engages not only innate immune pathways but also an RNAi-like mechanism to induce potent antiviral responses in vivo.Double-stranded RNA (dsRNA) is a hallmark of viral infections, and thus, it is not surprising that the immune system has evolved the capacity to recognize dsRNA and respond to it by mounting antiviral responses. In vertebrates, these innate antiviral responses rely in part on the recognition of dsRNA by Toll-like receptor 3 and by RNA-dependent protein kinase (32, 47). The consequences of dsRNA recognition include activation of the interferon system, initiation of apoptosis, and inhibition of cellular protein synthesis. From an evolutionary perspective, innate immune activation by dsRNA has long been thought to be exclusive to vertebrates. This view has been encouraged by the fact that genes encoding homologues of interferons, their receptors, and most of the prominent interferon-regulated genes are absent in fully sequenced invertebrate genomes (1, 7, 10, 11). Nevertheless, it is a reasonable expectation that invertebrates should have an innate immune system capable of recognizing dsRNA as a signature of viral infection. A previous study suggested such a capability by demonstrating that exposure of a marine shrimp to dsRNA induced innate antiviral immunity in a sequence-independent manner (36). The mechanisms underlying this phenomenon as well as its occurrence in other invertebrate taxa remain unknown, but it is clear that the recognition of dsRNA by another pathway, RNA interference (RNAi), is widely distributed among invertebrates and likely an important component of the invertebrate antiviral response.RNAi comprises a set of related cellular processes by which dsRNA molecules direct the suppression of gene expression based on sequence homology between the dsRNA trigger and the target gene. The specific mechanisms u...

Diversity, evolution, and therapeutic applications of small RNAs in prokaryotic and eukaryotic immune systems

“…Sequence-dependent antiviral responses and gene silencing were triggered by the injection of long dsRNAs in vivo. This supports that marine shrimp possess not only innate immunity to viruses, but also an adaptive siRNA-mediated mechanism of antiviral immunity [112].…”

RNA Interference with Special Reference to Combating Viruses of Crustacea

“…One observation that has occurred a few times in the literature is the fact that non-target, non-specific dsRNA used as negative controls in viral knockdown experiments has had a protective effect against some viruses including WSSV [154], YHV [207] and even totally unrelated, nonspecific dsRNA (green fluorescent protein) down regulated YHV genes in Penaeus monodon [207]. Similarly, La Fauce and Owens [93] found non-specific dsRNA knocked down against PmergDNV (see above).…”

“…In almost all cases reported, survival increased following the administration of dsRNA, triggering of the RNAi pathway. RNAi has down regulated White Spot Syndrome Virus and allowed a 85 % reduction in mortality when targeting the VP28 envelope gene, suggested to play a role in WSSV infection [114,148]; 78 % reduction in mortality when targeting RR2 ribonucleotide reductase; 70 % reduction in mortality against VP19 envelope gene [154]; 47 % reduction in mortality against viral ubiquitin ligase [62]; 44 % reduction in mortality when antagonistic to the DNA polymerase. Similarly, when yellow head virus was down regulated by attacking its protease gene and a host host Rab7 GTPase, mortality was reduced by 90 % [143].…”

Biometrical genetic analysis of luteovirus transmission in the aphid Schizaphis graminum

“…Other responses to viral pathogens could include apoptosis (Narayanan, 1998) or RNA interference (RNAi). RNAi is a sequence-specific targeting of pathogen RNA, which is initially induced by a double-stranded RNA, and has been found to be active against viruses in mosquitoes ( Keene et al, 2004), Drosophila (Li et al, 2002) and shrimp (Robalino et al, 2004(Robalino et al, , 2005). An RNAi response to luteoviruses in aphids is considered unlikely since luteoviruses do not uncoat or replicate in the aphid host.…”