Shrimp is one of the major aquaculture species that is farmed at an industrial scale in Asia and the Americas. The growth and profitability of shrimp farming is hampered due to viral disease outbreaks and currently, there is no commercially available therapeutic to control these diseases. Despite the advancement of gene-therapy technology such as RNAi, the lack of a cost-effective oral delivery method impedes their applicability to shrimp aquaculture. We present here an orally administered shrimp viral vector capable of delivering a gene payload. As a proof-of-concept study, we reverse-engineered Macrobrachium rosenbergii nodavirus (MrNV) making it replication-deficient by replacing its RNA-dependent RNA-polymerase (RdRp) gene with a marker gene, green fluorescent protein (GFP). The recombinant MrNV was produced using a baculovirus (BV) expression system in insect cell, Sf9. The assembly of mature virions of MrNV carrying GFP gene in Sf9 cells was confirmed by transmission electron microscopy and by measuring GFP expression by fluorescence microscopy and flow cytometry.
An MrNV viral vector should meet two criteria: First, the recombinant MrNV (MrNV-GFP_rBV) should be able to successfully deliver and express GFP gene into shrimp cells. Second, the viral vector should be able to deliver the payload via oral route in shrimp. For this, we delivered MrNV-GFP_rBV by injection and oral route by mixing the MrNV-GFP_rBV with in shrimp feeds. Three groups comprising five individual SPF Penaeus vannamei shrimp (2.5-3g) were treated with: Group 1- was injected once with ~1 x 107 pfu/mL of MrNV-GFP_rBV; Group 2- was fed with commercial feeds soaked with SF9 cells infected with MrNV-GFP_rBV at 5% of the biomass for 5 days; Group 3 - was fed with the same commercial feed (without the virus) and served as the negative/naïve control. Hemolymph were drawn from shrimp in all three groups after 5 days, hemocytes were separated by centrifugation and seeded in 24-well plate with L-15 media, then viewed under fluorescent microscope.
Results show that hemocytes from both Groups 1 and 2, i.e. rBV-MrNV-GFP delivered by injection and oral feeding expressed GFP (Fig. 1). These results provide a strong evidence that a baculovirus based shrimp noda viral vector can effectively deliver a foreign (GFP) gene in vivo by injection and via oral route.