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. Honeybees are the most important managed pollinating insect, contributing billions of dollars to annual global agricultural production. Over the last century a parasitic mite, Varroa, has spread worldwide, with significant impacts on honeybee colony health as a consequence of its transmission of a cocktail of viruses while feeding on honeybee ‘blood’. The most important virus for colony health is deformed wing virus (DWV), high levels of which cause developmental deformities and premature ageing resulting in high overwintering colony losses. In experiments on individual Varroa-exposed pupae we demonstrate that a single type of virulent DWV is amplified 1,000–10,000 times in the recipient pupae, despite the mite containing a high diversity of replicating DWV strains. We could recapitulate this by direct injection of pupae with mixed virus populations, showing the virulent strain is advantaged by the route of transmission. In parallel, we detected changes in the immune response and developmental gene expression of the honeybee and propose that these contribute to the characteristic pathogenesis of DWV. Identification of a virulent strain of DWV has implications for therapeutic or prophylactic interventions to improve honeybee colony health, as well as contributing to our understanding of the biology of this important honeybee viral pathogen.

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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-1VVD and VDV-1DVD, exhibit crossovers between the 5′-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-1DVD levels in honeybee pupae and associated mites, suggesting that this recombinant, with a DWV-derived 5′-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 5′-UTR (VDV-1VVD).

Iflaviridae is a family of small non-enveloped viruses with monopartite, positive-stranded RNA genomes of approximately 9–11 kilobases. Viruses of all classified species infect arthropod hosts, with the majority infecting insects. Both beneficial and pest insects serve as hosts, and infections can be symptomless (Nilaparvatalugens honeydew virus 1) or cause developmental abnormalities (deformed wing virus), behavioural changes (sacbrood virus) and premature mortality (infectious flacherie virus). The host range has not been examined for most members. The most common route of infection for iflaviruses is the ingestion of virus-contaminated food sources. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Iflaviridae, which is available at www.ictv.global/report/iflaviridae.

The nucleolus and specific nucleolar proteins are involved in the life cycles of some plant and animal viruses, but the functions of these proteins and of nucleolar trafficking in virus infections are largely unknown. The ORF3 protein of the plant virus, groundnut rosette virus (an umbravirus), has been shown to cycle through the nucleus, passing through Cajal bodies to the nucleolus and then exiting back into the cytoplasm. This journey is absolutely required for the formation of viral ribonucleoprotein particles (RNPs) that, themselves, are essential for the spread of the virus to noninoculated leaves of the shoot tip. Here, we show that these processes rely on the interaction of the ORF3 protein with fibrillarin, a major nucleolar protein. Silencing of the fibrillarin gene prevents long-distance movement of groundnut rosette virus but does not affect viral replication or cell-to-cell movement. Repressing fibrillarin production also localizes the ORF3 protein to multiple Cajal body-like aggregates that fail to fuse with the nucleolus. Umbraviral ORF3 protein and fibrillarin interact in vitro and, when mixed with umbravirus RNA, form an RNP complex. This complex has a filamentous structure with some regular helical features, resembling the RNP complex formed in vivo during umbravirus infection. The filaments formed in vitro are infectious when inoculated to plants, and their infectivity is resistant to RNase. These results demonstrate previously undescribed functions for fibrillarin as an essential component of translocatable viral RNPs and may have implications for other plant and animal viruses that interact with the nucleolus.

The nucleolus and Cajal bodies (CBs) are prominent interacting subnuclear domains involved in a number of crucial aspects of cell function. Certain viruses interact with these compartments but the functions of such interactions are largely uncharacterized. Here, we show that the ability of the groundnut rosette virus open reading frame (ORF) 3 protein to move viral RNA long distances through the phloem strictly depends on its interaction with CBs and the nucleolus. The ORF3 protein targets and reorganizes CBs into multiple CB‐like structures and then enters the nucleolus by causing fusion of these structures with the nucleolus. The nucleolar localization of the ORF3 protein is essential for subsequent formation of viral ribonucleoprotein (RNP) particles capable of virus long‐distance movement and systemic infection. We provide a model whereby the ORF3 protein utilizes trafficking pathways involving CBs to enter the nucleolus and, along with fibrillarin, exit the nucleus to form viral ‘transport‐competent’ RNP particles in the cytoplasm.