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

Jaruwan Worawittayatada; Kitipong Angsujinda; Rapee Sinnuengnong; Pongsopee Attasart; Duncan R Smith; Wanchai Assavalapsakul

doi:10.3390/v14122594

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

Viruses. 2022 Nov 22;14(12):2594. doi: 10.3390/v14122594.

Authors

Jaruwan Worawittayatada¹, Kitipong Angsujinda², Rapee Sinnuengnong^{1

3}, Pongsopee Attasart⁴, Duncan R Smith⁵, Wanchai Assavalapsakul¹

Affiliations

¹ Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
² Aquatic Resources Research Institute, Chulalongkorn University, Bangkok 10330, Thailand.
³ Department of Research and Development, Queen Saovabha Memorial Institute, The Thai Red Cross Society, Bangkok 10330, Thailand.
⁴ Center of Applied Shrimp Research and Innovation, Institute of Molecular Biosciences, Mahidol University, Nakorn Pathom 73170, Thailand.
⁵ Institute of Molecular Biosciences, Mahidol University, Nakorn Pathom 73170, Thailand.

Abstract

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.

Keywords: Penaeus stylirostris densovirus; dspro; inhibition; shrimp; virus-like particle; yellow head virus.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Capsid Proteins / genetics
Densovirinae* / genetics
Densovirus* / genetics
Penaeidae*
RNA, Double-Stranded / genetics
RNA, Double-Stranded / metabolism
Recombinant Proteins / genetics
Roniviridae* / genetics
Roniviridae* / metabolism

Substances

Capsid Proteins
Recombinant Proteins
RNA, Double-Stranded

Grants and funding

This research was funded by the 90th Anniversary of Chulalongkorn University (Rachadapisek Sompote Fund) Grant No. GCUGR1125622065M to J.W. and W.A., and Grants for Development of New Faculty Staff, Ratchadaphiseksomphot Endowment Fund, Chulalongkorn University Grant No. DNS_64_062_65_003_1 to K.A.