Abstract
BackgroundFor 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 in vitro 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, Varroa Destructor.ResultsThe 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.ConclusionThe 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.
Highlights
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
Recent advances in molecular technology have greatly expanded our ability to detect and elucidate the molecular events associated with virus infections and pathogenesis
As with all single-strand positive sense RNA viruses, replication of honey bee viruses proceeds via the production of a negative-strand intermediate and its presence is indicative of active viral replication
Summary
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. It is very imperative to develop new methods that would permit the in vitro pathogenesis study of honey bee viruses. 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, Varroa Destructor. Observations of virus cytopathic effect (CPE) in cultured cells, a standard method used for unraveling the mechanisms of viral replication and the specific host responses to viral infections, are not possible. It is to develop new methods that would permit the study of virus replication in vitro
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