Viral diseases remain one of the major bottlenecks in the growth and profitability of shrimp farming worldwide. Despite enormous progress on developing vial disease diagnostics and preventative measures like developing biosecurity protocols, probiotics, prebiotics, immunostimulants among other things, there is no commercially available antiviral therapeutics to control viral diseases in shrimp. RNAi-based therapeutics have shown promise at experimental levels, but unavailability of an oral delivery platform that can be scalable and cost-effects appear to thwart the commercial development of antiviral therapeutics. We developed an oral delivery platform based on a replication-deficient shrimp virus that can deliver RNAi-based anti-viral therapeutics through commercial diets.
Recently, we engineered an infectious cDNA clone of Macrobrachium rosenbergii nodavirus (MrNV) using a baculovirus expression system and Sf9 insect cells. MrNV is a non-enveloped RNA virus and consists of two positive sense, single-stranded RNA, RNA-1 that encodes an RNA-dependent RNA polymerase (RdRp) and a B2 protein, and RNA-2 that encodes the viral capsid protein. MrNV RNA1 and RNA2 were cloned downstream of polyhedrin and P10 promoters in a baculovirus pFastBacDUAL vector and upon infection of Sf9 cells with recombinant baculovirus (rBV), both MrNV and rBV were produced. The infectivity of Sf9 cell derived MrNV was demonstrated via an immersion challenge that resulted in clinical signs and histopathology similar to wild type MrNV. Subsequently, the MrNV RdRp gene was replaced with a GFP marker and the engineered virus, MrNVΔRdRp-GFP was found to successfully deliver GFP via oral administration through diet.
To determine the feasibility of using the MrNV-based viral vector as an oral delivery platform for RNAi molecules, we cloned hairpin RNA targeting structural and non-structural genes of white spot syndrome virus (WSSV). The engineered viruses, MrNVΔRdRp[hRNA] were produced in Sf9 cells and the cell culture biomass was mixed with a commercial diet for feeding SPF Penaeus vannamei before WSSV challenge. Three MrNVΔRdRp[hRNA] constructs, including two that targets a WSSV structural gene and one targeting a non-structural gene, were tested for their efficacy in reducing WSSV load. MrNVΔRdRp[GFP] mixed with commercial diet and commercial diet alone were used as controls. Among three viral constructs tested, MrNVΔRdRp[hRNA] targeting a non-structural gene significantly reduced WSSV load compared to the two other constructs and control treatments, and delayed WSSV-induced mortality compared to controls. This has opened up an avenue for an oral delivery of anti-viral therapeutics in shrimp.
Key words: RNAi, WSSV vaccine, oral delivery, MrNV, anti-viral therapy.