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Structure prediction of the 5′ non-translated region (NTR) of four iflavirus RNAs revealed two types of potential internal ribosome entry site (IRES), which are discriminated by size and level of complexity, in this group of viruses. In contrast to the intergenic IRES of dicistroviruses, the potential 5′ IRES structures of iflaviruses do not have pseudoknots. To test the activity of one of these, a bicistronic construct was made in which the 5′ NTR ofVarroa destructorvirus 1 (VDV-1) containing a putative IRES was cloned in between two reporter genes, enhanced green fluorescent protein and firefly luciferase (Fluc). The presence of the 5′ NTR of VDV-1 greatly enhanced the expression levels of the second reporter gene (Fluc) inLymantria disparLd652Y cells. The 5′ NTR was active in a host-specific manner, as it showed lower activity inSpodoptera frugiperdaSf21 cells and no activity inDrosophila melanogasterS2 cells.

Honey bee virus research is an enormously broad area, ranging from subcellular molecular biology through physiology and behaviour, to individual and colony-level symptoms, transmission and epidemiology. The research methods used in virology are therefore equally diverse. This article covers those methods that are very particular to virological research in bees, with numerous cross-referrals to other BEEBOOK papers on more general methods, used in virology as well as other research. At the root of these methods is the realization that viruses at their most primary level inhabit a molecular, subcellular world, which they manipulate and interact with, to produce all higher order phenomena associated with virus infection and disease. Secondly, that viruses operate in an exponential world, while the host operates in a linear world and that much of the understanding and management of viruses hinges on reconciling these fundamental mathematical differences between virus and host. The article concentrates heavily on virus propagation and methods for detection, with minor excursions into surveying, sampling management and background information on the many viruses found in bees.