Procedures and potential pitfalls for constructing a bee-infecting RNA virus clone

Abstract

Viruses are factors that can fluctuate insect populations, including honey bees. Most honey bee infecting viruses are single positive-stranded RNA viruses that may not specifically infect honey bees and can be hazardous to other pollinator insects. In addition, these viruses could synergize with other stressors to worsen the honey bee population decline. To identify the underlying detailed mechanisms, reversed genetic studies with infectious cDNA clones of the viruses are necessary. Moreover, an infectious cDNA clone can be applied to studies as an ideal virus isolate that consists of a single virus species with a uniform genotype. However, only a few infectious cDNA clones have been reported in honey bee studies since the first infectious cDNA clone was published four decades ago. This article discusses steps, rationales, and potential issues in bee-infecting RNA virus cloning. In addition, failed experiences of cloning a Deformed wing virus isolate that was phylogenetically identical to Kakugo virus were addressed. We hope the information provided in this article can facilitate further developments of reverse-genetic studies of bee-infecting viruses to clarify the roles of virus diseases in the current pollinator declines.

A DNA Plasmid-Based Approach for Efficient Synthesis of Sacbrood Virus Infectious Clones within Host Cells

“…A few limitations of using Sf9 cells, not the natural host, as a surrogate host for SBV have been revealed. Previous studies [ 21 , 33 ], which used purified virions and viral genome RNA, respectively, suggested the potential of SBV to infect surrogate cell lines. However, these studies reported failures to inoculate naïve cells upon subculture and similar findings were observed in a study on Deformed wing virus in Sf9 cells [ 34 ].…”

“…However, implementing the standard method for producing infectious cloned viruses can present significant technical challenges [ 21 ]. Firstly, the need to produce full-length viral RNA in 8–10 kb sizes, which is considerably larger than the typical sizes required for gene expression or RNA interference experiments, poses a major obstacle for researchers.…”