Abstract
Several studies have demonstrated that injection of double-stranded RNAs (dsRNA) homologous to mRNA for the white spot syndrome virus (WSSV) viral protein 28 (VP28) can induce protection in shrimp against WSSV through RNA interference (RNAi). In comparison to shrimp injected with either PBS or a green fluorescent protein (GFP) nonspecific dsRNA, we obtained nearly complete protection against WSSV infection in shrimp injected with VP28 dsRNA. Upregulation of host genes associated with small RNA silencing was measured 48 hours post treatment in groups injected with dsRNA, and although the VP28-treated group remained moderately upregulated after challenge with WSSV, many-fold higher induction was observed in both control groups reflecting the ongoing viral infection. RNA sequencing of VP28-treated shrimp demonstrated a siRNA population dominated by high levels of 22 nt long molecules narrowly targeting the VP28 mRNA both before and after challenge with WSSV. Conversely, while no siRNAs targeting WSSV were detected before challenge, a broad response of 22 nt siRNAs mapping across the entire WSSV genome were found in both control groups after challenge. These results give detailed insight to how dsRNA targeting VP28 function to induce protection against WSSV, by generating a highly focused population of 22 nt long siRNA molecules.
Highlights
As aquaculture production of shrimp has grown into the most valuable seafood commodity over the last 30 years[1], disease outbreaks have become an increasing challenge for the industry
Groups consisting of 5 separate tanks each holding 20 shrimp were injected intramuscularly (IM) with either PBS or double-stranded RNAs (dsRNA) homologous to the green fluorescent protein (GFP)-specific or viral protein 28 (VP28)-coding sequence
RT-qPCR was performed on shrimp stomach tissue in order to verify that both the delivered dsRNA targeting GFP and VP28 were present in the treated shrimp during the sampling period
Summary
As aquaculture production of shrimp has grown into the most valuable seafood commodity over the last 30 years[1], disease outbreaks have become an increasing challenge for the industry. Shrimp can be effectively protected against WSSV and other viruses by injecting specific dsRNA targeting viral genes[5]. The concept of small RNA silencing involves Dicer (Dcr) endonuclease enzymes that recognize and cleave different variants of RNAs with double-stranded motifs, feeding various regulatory pathways with short duplex RNAs. The siRNA pathways play an important role in somatic cells detecting long dsRNA molecules from viral- or transposable element replication. Cleavage of mRNA transcripts by the action of siRNA and RISC inhibits translation of proteins encoded by viruses and mobile genetic elements, and is referred to as RNA interference[11,12,13]. Associated with the miRNA pathway in Drosophila[14, 20], a possible involvement in the antiviral response has been shown for homologue genes in white leg shrimp[21,22,23]. An RNA sequencing study on WSSV infected shrimp (Penaeus chinensis) suggested a siRNA population to be within the 21–23 nucleotide range with a peak at 22 nucleotides[28], and a similar study on the bumble bee (Bombus terrestris) emphasizes on a 22 nucleotide siRNA peak[29]
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