The globally distributed ectoparasite Varroa destructor is a vector for viral pathogens of the Western honeybee (Apis mellifera), in particular the Iflavirus Deformed Wing Virus (DWV). In the absence of Varroa low levels DWV occur, generally causing asymptomatic infections. Conversely, Varroa-infested colonies show markedly elevated virus levels, increased overwintering colony losses, with impairment of pupal development and symptomatic workers. To determine whether changes in the virus population were due Varroa amplifying and introducing virulent virus strains and/or suppressing the host immune responses, we exposed Varroa-naïve larvae to oral and Varroa-transmitted DWV. We monitored virus levels and diversity in developing pupae and associated Varroa, the resulting RNAi response and transcriptome changes in the host. Exposed pupae were stratified by Varroa association (presence/absence) and virus levels (low/high) into three groups. Varroa-free pupae all exhibited low levels of a highly diverse DWV population, with those exposed per os (group NV) exhibiting changes in the population composition. Varroa-associated pupae exhibited either low levels of a diverse DWV population (group VL) or high levels of a near-clonal virulent variant of DWV (group VH). These groups and unexposed controls (C) could be also discriminated by principal component analysis of the transcriptome changes observed, which included several genes involved in development and the immune response. All Varroa tested contained a diverse replicating DWV population implying the virulent variant present in group VH, and predominating in RNA-seq analysis of temporally and geographically separate Varroa-infested colonies, was selected upon transmission from Varroa, a conclusion supported by direct injection of pupae in vitro with mixed virus populations. Identification of a virulent variant of DWV, the role of Varroa in its transmission and the resulting host transcriptome changes furthers our understanding of this important viral pathogen of honeybees.

SummaryHoney 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. Métodos estándar para la investigación de virus en Apis mellifera ResumenLa investigación de los virus de la abeja de la miel es un área sumamente amplia, que abarca desde la biología molecular subcelular hasta la fisiología y el comportamiento, desde síntomas al nivel de individuo hasta al nivel de la colmena, transmisión y epidemiología. Los métodos de investigación en virología son, por tanto, diversos. Este artículo incluye aquellos métodos específicos de la investigación virológica en las abejas, con numerosas referencias cruzadas con otros artículos del BEEBOOK y otros más generales, usados tanto en virología como en otras disciplinas. La base de estos métodos es la comprensión de los virus en su nivel primario de hábitat molecular, ambiente subcelular, que manipulan y con el que interactúan, para producir otros fenómenos de orden superior asociados a la infección del virus y la enfermedad. En segundo lugar, estos virus actúan en un mundo exponencial, mientras que los hospedadores actúan en un mundo lineal y gran parte del entendimiento y manejo de los virus depende de los fundamentos matemáticos de las diferencias entre el virus y el hospedador. El artículo se centra principalmente en la propagación de virus y en los métodos para su detección, con inclusiones menores en su estudio, el manejo del muestreo y la información general sobre los numerosos virus que se encuentran en las abejas.

We have used high-throughput Illumina sequencing to identify novel recombinants between Deformed wing virus (DWV) and Varroa destructor virus-1 (VDV-1), which accumulate to higher levels than DWV in both honeybees and Varroa destructor mites. The recombinants, VDV-1 VVD and VDV-1 DVD , exhibit crossovers between the 59-UTR and the regions encoding the structural (capsid) and non-structural viral proteins. This implies that the genomes are modular and that each region may evolve independently, as demonstrated in human enteroviruses. Individual honeybee pupae were infected with a mixture of observed recombinants and DWV. A strong correlation was observed between VDV-1 DVD levels in honeybee pupae and associated mites, suggesting that this recombinant, with a DWV-derived 59-UTR and non-structural protein region flanking a VDV-1-derived capsid-encoding region, is better adapted to transmission between V. destructor and honeybees than the parental DWV or a recombinant bearing the VDV-1-derived 59-UTR (VDV-1 VVD ).