Development of a Honey Bee RNA Virus Vector Based on the Genome of a Deformed Wing Virus

Abstract

We developed a honey bee RNA-virus vector based on the genome of a picorna-like Deformed wing virus (DWV), the main viral pathogen of the honey bee (Apis mellifera). To test the potential of DWV to be utilized as a vector, the 717 nt sequence coding for the enhanced green fluorescent protein (eGFP), flanked by the peptides targeted by viral protease, was inserted into an infectious cDNA clone of DWV in-frame between the leader protein and the virus structural protein VP2 genes. The in vitro RNA transcripts from egfp-tagged DWV cDNA clones were infectious when injected into honey bee pupae. Stable DWV particles containing genomic RNA of the recovered DWV with egfp inserts were produced, as evidenced by cesium chloride density gradient centrifugation. These particles were infectious to honey bee pupae when injected intra-abdominally. Fluorescent microscopy showed GFP expression in the infected cells and Western blot analysis demonstrated accumulation of free eGFP rather than its fusions with DWV leader protein (LP) and/or viral protein (VP) 2. Analysis of the progeny egfp-tagged DWV showed gradual accumulation of genome deletions for egfp, providing estimates for the rate of loss of a non-essential gene an insect RNA virus genome during natural infection.

Bee Viruses: Routes of Infection in Hymenoptera

“…Recently, a molecular clone of CBPV was shown to cause typical clinical symptoms mimicking naturally CBPV-infected honey bees (Seitz et al, 2019). Similarly, SBV and DWV clones have been synthetized to express the enhanced green fluorescent protein (EGFP;Jin et al, 2020;Ryabov et al, 2020). Besides creating a clone that produces typical symptoms, it adds the advantage of a reporter gene for protein expression studies (Jin et al, 2020).…”

Infection of a Lepidopteran Cell Line with Deformed Wing Virus

“…Understanding the course of the virus infection is crucial to develop the means to alleviate and\or suppress its damage to individual honey bees and colonies. To achieve this goal, experimental approaches were developed, including the extensive monitoring of virus infections in honey bee colonies [ 10 , 11 , 12 , 13 , 14 , 15 ], virus infectivity assays, the artificial infections of honey bees in cages [ 7 , 16 , 17 ], experimental colonies [ 8 , 18 ], the development of infectious viral clones [ 19 , 20 ], and fluorescent-labelled tagged viruses [ 21 , 22 ].…”

Pupal Cannibalism byWworker Honey Bees Contributes to the Spread of Deformed Wing Virus.

“…Experiment A tested if cannibalization of the pupae infected with DWV by Varroa mites could result in development of the virus infection in worker bees. It included injecting honey bee pupae with a ltered tissue extract containing DWV-GFP particles 40 , or by a phosphate buffer saline (PBS) control. After 48 hours (hr), when GFP uorescence had developed in the DWV-GFP-injected pupae indicating virus infection ( Fig.…”

“…One reason complicating the study of the impact of cannibalism on DWV circulation is the di culty in distinguishing between DWV infection initiated by cannibalization and by other routes. To solve this problem we used genetically-tagged DWV carrying unique genetic markers, the green uorescent protein (GFP) gene and an introduced unique restriction site 40 , allowing us to trace transmission of the virus. We also carried out pupal cannibalism experiments in controlled laboratory conditions rather than hives, thereby minimizing virus transmission from other sources.…”