Aquatic invertebrates farmed at an industrial scale continue to face the risk of viral outbreaks due to a lack of antiviral therapies. While RNA interference (RNAi) by sequence-specific dsRNA has been identified as a promising antiviral tool, its application to farm-scale bottlenecks with intramuscular (IM) injection being the only delivery method. Only through an oral delivery platform with a vector capable of delivering therapeutic RNA molecules into shrimp cells shall RNAi reach its potential against viral diseases of aquatic organisms. We present here an oral delivery platform using a shrimp virus capsid protein as the viral vector. The reverse-engineered Macrobrachium rosenbergii nodavirus (rMrNV) was made replication-deficient by replacing its RNA-dependent RNA-polymerase (RdRp) with a cargo RNA represented by green fluorescent protein (GFP). Efficient production of the viral vector was achieved using baculovirus dual-expression system capable of simultaneous production of both capsid protein and GFP RNA cargo facilitating assembly. To ensure its applicability to farm-scale, we demonstrate here the oral delivery and detection of GFP RNA into shrimp cells as well as proving MrNVcp is indeed replication-deficient by testing its viability and screening for any pathological effects. We went on to explore its applicability against white spot syndrome virus (WSSV) by replacing GFP with WSSV VP28 hairpin RNA.
Here we demonstrate that our viral vector is non-replicating and can deliver the cargo GFP RNA through the diet. For this, detection of the cargo RNAs (MrNVcp and GFP) were done in shrimp fed with commercial diets incorporated with MrNVcp-GFP. Results show that RNA copy number of MrNVcp gradually declined to 10 copies after 8 days post-feeding (Fig.1A) as also seen for GFP RNA detection (Fig. 2B). These results are proof that MrNVcp is effective in delivering RNA cargo via oral route comparable to injection method. Histological analyses ruled out any pathological damages post-feeding that may be indicative of a viral infection. Combined with the detection results (Fig. 1A), the generated MrNVcp viral vector is non-replicating and non-infectious. These results show a promising shrimp viral vector-based oral delivery platform for RNAi therapeutics.