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 (PstDNV) capsid and dsRNA specific to the yellow head virus (YHV) protease (CoEx cpPstDNV/dspro) 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 PstDNV capsid and the dsRNA-YHV-pro gene was constructed under the regulation of a single T7 promoter, designated pET28a-Linked cpPstDNV-dspro. Following induction, this novel construct expressed an approximately 37-kDa recombinant protein associated with a roughly 400-bp dsRNA (Linked cpPstDNV-dspro). Under a transmission electron microscope, the virus-like particles (VLP; Linked PstDNV VLPs-dspro) obtained were seen to be monodispersed, similar to the native PstDNV virion. A nuclease digestion assay indicated dsRNA molecules were both encapsulated and present outside the Linked PstDNV VLPs-dspro. 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 PstDNV VLPs-dspro 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.

Administration of co-expressed Penaeus stylirostris densovirus-like particles and dsRNA-YHV-Pro provide protection against yellow head virus in shrimp.

The activation of the innate RNA interference pathway through double-stranded RNAs (dsRNAs) is one of the approaches to protecting shrimp from viruses. Previous studies have shown that injection of specific dsRNAs can successfully inhibit viral infection in shrimp. However, inhibition requires high levels of dsRNA and dsRNA stability in shrimp is limited. Virus-like particles (VLPs) have been applied to deliver nucleic acids into host cells because of the protection of dsRNAs from host endonucleases as well as the target specificity provided by VLPs. Therefore, this study aimed to develop Penaeus stylirostris densovirus (PstDNV) VLPs for dsRNA deliver to shrimp. The PstDNV capsid protein was expressed and can be self-assembled to form PstDNV VLPs. Co-expression of dsRNA-YHV-Pro and PstDNV capsid protein was achieved in the same bacterial cells, whose structure was displayed as the aggregation of VLPs by TEM. Tested for their inhibiting yellow head virus (YHV) from infecting shrimp, the dsRNA-YHV-Pro-PstDNV VLPs gave higher levels of YHV suppression and a greater reduction in shrimp mortality than the delivery of naked dsRNA-YHV-Pro. Therefore, PstDNV-VLPs are a promising vehicle for dsRNA delivery that maintains the anti-virus activity of dsRNA in shrimp over a longer period of time as compared to native dsRNAs.