Simultaneous Production of a Virus-Like Particle Linked to dsRNA to Enhance dsRNA Delivery for Yellow Head Virus Inhibition.

A co-expressed Penaeus stylirostris densovirus ( Pst DNV) capsid and dsRNA specific to the yellow head virus (YHV) protease (CoEx cp Pst DNV/ds pro ) has been shown to suppress YHV replication in the Pacific white-legged shrimp ( Litopenaeus vannamei ). However, maintaining two plasmids in a single bacterial cell is not desirable; therefore, a single plasmid harboring both the Pst DNV capsid and the dsRNA-YHV- pro gene was constructed under the regulation of a single T7 promoter, designated pET28a-Linked cp Pst DNV-ds pro . Following induction, this novel construct expressed an approximately 37-kDa recombinant protein associated with a roughly 400-bp dsRNA (Linked cp Pst DNV-ds pro ). Under a transmission electron microscope, the virus-like particles (VLP; Linked Pst DNV VLPs-ds pro ) obtained were seen to be monodispersed, similar to the native Pst DNV virion. A nuclease digestion assay indicated dsRNA molecules were both encapsulated and present outside the Linked Pst DNV VLPs-ds pro . In addition, the amount of dsRNA produced from this strategy was higher than that obtained with a co-expression strategy. In a YHV infection challenge, the Linked Pst DNV VLPs-ds pro was more effective in delaying and reducing mortality than other constructs tested. Lastly, the linked construct provides protection for the dsRNA cargo from nucleolytic enzymes present in the shrimp hemolymph. This is the first report of a VLP carrying virus-inhibiting dsRNA that could be produced without disassembly and reassembly to control virus infection in shrimp.