Development of a Honey Bee RNA Virus Vector Based on the Genome of 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 LP and/or VP2 proteins. 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.

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

“…The levels of DWV in these pupae, 10 10 − 10 11 GE ( Fig. 2c), were similar to those observed in the pupae infected with DWV-GFP by injection 40 . Therefore, such injection-infected pupae could be used as an adequate surrogate for Varroa-infected pupae in cannibalism experiments.…”

“…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.…”

“…Honey bee pupae at the white eye developmental stage collected from Varroa-free brood cells were injected with 8 mL of a ltered extract containing 10 7 of DWV-GFP virus particles. This extract was generated using individual pupae infected with in vitro RNA transcript from the construct pDWV-L-GFP carrying an enhanced-GFP coding sequence 40 , which gave an equimolar ratio of DWV to GFP in qRT-PCR tests, indicating that it contained mainly intact recombinant virus without GFP deletions. The extractinjected pupae were incubated in the dark for 48 hr at +33 o C, relative humidity 85% prior to development of GFP uorescence visible when illuminated with long wave, 395 nm, ultraviolet light illumination ( Fig.…”

“…Extraction of total RNA from pools of frozen worker bees started with lysis in guanidine isothiocyanate buffer as described previously 49 , followed by further disruption using QIAShredder (QIAGEN) and puri cation using RNeasy kits (QIAGEN). Quanti cation of DWV and GFP RNA in these RNA extracts was carried out by RT-qPCR as previously 40 and included cDNA synthesis using Superscript III (Invitrogen) and random hexanucleotides as primers, and qPCR using SYBR green (BioRad) and the primers speci c to DWV genomic RNA (5'-GAGATCGAAGCGCATGAACA-3' and 5'-TGAATTCAGTGTCGCCCATA-3', positions 6497 -6626 nt of DWV, positions 7268 -7397 of DWV-L-GFP), to the region spanning the eGFP -structural VP2 interface (GFP-speci c primer 5'-GCATGGACGAGCTGTACAAG-3', and DWV-speci c 5'-CCTTTTCTAATTCAACTTCACC -3', positions 2526 -2624 of DWV-L-GFP genome), and to the honey bee b-actin mRNA (5'-AGGAATGGAAGCTTGCGGTA-3' and 5'-AATTTTCATGGTGGATGGTGC-3'). The plasmid pDWV-L-GFP 40 was used as a standard for quanti cation of DWV and GFP copy numbers, which were log-transformed prior to statistical analyses.…”

“…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.…”