Molecular approaches to the analysis of deformed wing virus replication and pathogenesis in the honey bee, Apis mellifera

<p>Abstract</p> <p>Background</p> <p>For years, the understanding of the pathogenetic mechanisms that underlie honey bee viral diseases has been severely hindered because of the lack of a cell culture system for virus propagation. As a result, it is very imperative to develop new methods that would permit the <it>in vitro </it>pathogenesis study of honey bee viruses. The identification of virus replication is an important step towards the understanding of the pathogenesis process of viruses in their respective hosts. In the present study, we developed a strand-specific RT-PCR-based method for analysis of Deformed Wing Virus (DWV) replication in honey bees and in honey bee parasitic mites, <it>Varroa Destructor</it>.</p> <p>Results</p> <p>The results shows that the method developed in our study allows reliable identification of the virus replication and solves the problem of falsely-primed cDNA amplifications that commonly exists in the current system. Using TaqMan real-time quantitative RT-PCR incorporated with biotinylated primers and magnetic beads purification step, we characterized the replication and tissue tropism of DWV infection in honey bees. We provide evidence for DWV replication in the tissues of wings, head, thorax, legs, hemolymph, and gut of honey bees and also in Varroa mites.</p> <p>Conclusion</p> <p>The strategy reported in the present study forms a model system for studying bee virus replication, pathogenesis and immunity. This study should be a significant contribution to the goal of achieving a better understanding of virus pathogenesis in honey bees and to the design of appropriate control measures for bee populations at risk to virus infections.</p

RNA virus spillover from managed honeybees (Apis mellifera) to wild bumblebees (Bombus spp.).

The decline of many bumblebee species (Bombus spp.) has been linked to an increased prevalence of pathogens caused by spillover from managed bees. Although poorly understood, RNA viruses are suspected of moving from managed honeybees (Apis mellifera) into wild bumblebees through shared floral resources. We examined if RNA viruses spillover from managed honeybees, the extent to which viruses are replicating within bumblebees, and the role of flowers in transmission. Prevalence and active infections of deformed wing virus (DWV) were higher in bumblebees collected near apiaries and when neighboring honeybees had high infection levels. We found no DWV in bumblebees where honeybee foragers and honeybee apiaries were absent. The prevalence of black queen cell virus (BQCV) was also higher in bumblebees collected near apiaries. Furthermore, we detected viruses on 19% of flowers, all of which were collected within apiaries. Our results corroborate the hypothesis that viruses are spilling over from managed honeybees to wild bumblebees and that flowers may be an important route for transmission

Experimental study system to follow virus pathogenicity during honey bee pupal development in the laboratory.

<p>[A] Development of IAPV-inoculated (IAPV), PBS-injected (PBS), and negative control (W/O) individuals. The IAPV group shows progressive symptoms of disease, compared to the normally developing PBS and control group. [B] Close-up of the variable symptoms of IAPV replication in white eye honey bee pupae: Complete cessation of development with no visual evidence of disease (1), Apparently normal development (2), Rapid darkening of different body parts (3,4), Darkening and hindered development combined (5). [C] Control bees are completing metamorphosis.</p

To enrich for IAPV from honey bees of typically mixed infections, repeated cycles of pupal inoculations and later virus purification from the inoculated pupae were performed.

<p>The preferential replication of IAPV during this procedure resulted in virus purifications with negligible levels of contamination from other viruses. [A] Inoculation of honey bee pupae with IAPV. Even though the injection apparatus varied among experiments (see main text), the basic injection site and methodology shown were identical. [B] Electron microscope image of purified IAPV sample, showing clean and uniform virus particles (full and empty particles) around 27 nm.</p