Sacbrood virus (SBV) of honey bees is a picornavirus in the genus Iflavirus. Given its relatively small and simple genome structure, single positive-strand RNA with only one ORF, cloning the full genomic sequence is not difficult. However, adding nonsynonymous mutations to the bee iflavirus clone is difficult because of the lack of information about the viral protein processes. Furthermore, the addition of a reporter gene to the clones has never been accomplished. In preliminary trials, we found that the site between 3′ untranslated region (UTR) and poly(A) can retain added sequences. We added enhanced green fluorescent protein (EGFP) expression at this site, creating a SBV clone with an expression tag that does not affect virus genes. An intergenic region internal ribosome entry site (IRES) from Black queen cell virus (BQCV) was inserted to initiate EGFP expression. The SBV-IRES-EGFP clone successfully infected Apis cerana and Apis mellifera, and in A. cerana larvae, it was isolated and passaged using oral inoculation. The inoculated larvae had higher mortality and the dead larvae showed sacbrood symptoms. The added IRES-EGFP remained in the clone through multiple passages and expressed the expected EGFP in all infected bees. We demonstrated the ability to add gene sequences in the site between 3′-UTR and poly(A) in SBV and the potential to do so in other bee iflaviruses; however, further investigations of the mechanisms are needed. A clone with a desired protein expression reporter will be a valuable tool in bee virus studies.

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.