Construction and Rescue of a Molecular Clone of Deformed Wing Virus (DWV).
ABSTRACT: European honey bees are highly important in crop pollination, increasing the value of global agricultural production by billions of dollars. Current knowledge about virulence and pathogenicity of Deformed wing virus (DWV), a major factor in honey bee colony mortality, is limited. With this study, we close the gap between field research and laboratory investigations by establishing a complete in vitro model for DWV pathogenesis. Infectious DWV was rescued from a molecular clone of a DWV-A genome that induces DWV symptoms such as crippled wings and discoloration. The expression of DWV proteins, production of infectious virus progeny, and DWV host cell tropism could be confirmed using newly generated anti-DWV monoclonal antibodies. The recombinant RNA fulfills Koch's postulates circumventing the need of virus isolation and propagation of pure virus cultures. In conclusion, we describe the development and application of a reverse genetics system for the study of DWV pathogenesis.
Project description:Deformed wing virus (DWV) is considered one of the most damaging pests in honey bees since the spread of its vector, Varroa destructor. In this study, we sequenced the whole genomes of two virus isolates and studied the evolutionary forces that act on DWV genomes. The isolate from a Varroa-tolerant bee colony was characterized by three recombination breakpoints between DWV and the closely related Varroa destructor virus-1 (VDV-1), whereas the variant from the colony using conventional Varroa management was similar to the originally described DWV. From the complete sequence dataset, nine independent DWV-VDV-1 recombination breakpoints were detected, and recombination hotspots were found in the 5' untranslated region (5' UTR) and the conserved region encoding the helicase. Partial sequencing of the 5' UTR and helicase-encoding region in 41 virus isolates suggested that most of the French isolates were recombinants. By applying different methods based on the ratio between non-synonymous (dN) and synonymous (dS) substitution rates, we identified four positions that showed evidence of positive selection. Three of these positions were in the putative leader protein (Lp), and one was in the polymerase. These findings raise the question of the putative role of the Lp in viral evolution.
Project description:Background:Deformed wing virus (DWV) is a serious threat to honey bees (Apis mellifera) and is considered a major cause of elevated losses of honey bee colonies. However, lack of information on the immunogenicity of DWV structural proteins has hindered the development of effective biocontrol drugs. Methods:We optimized the VP1, VP2 and VP3 codons of DWV surface capsid protein genes on the basis of an Escherichia coli codon bias, and the optimized genes of roVP1, roVP2 and roVP3 were separately expressed in E. coli and purified. Next, the three recombinant proteins of roVP1, roVP2 and roVP3 were intramuscularly injected into BALB/c and the immunogenicity was evaluated by the levels of specific IgG and cytokines. Furthermore, anti-roVP-antisera (roVP1 or roVP2 or roVP3) from the immunized mice was incubated with DWV for injecting healthy white-eyed pupae for the viral challenge test, respectively. Results:The optimized genes roVP1, roVP2 and roVP3 achieved the expression in E. coli using SDS-PAGE and Western blotting. Post-immunization, roVP2 and roVP3 exhibited higher immunogenicity than roVP1 and stimulated a stronger humoral immune response in the mice, which showed that the recombinant proteins of roVP3 and roVP2 induced a specific immune response in the mice. In the challenge test, data regarding quantitative real-time RT-PCR (qRT-PCR) from challenged pupae showed that the level of virus copies in the recombinant protein groups was significantly lower than that of the virus-only group at 96 h post-inoculation (P < 0.05). Among them, the degree of neutralization using antibodies raised to the recombinant proteins are between approximately 2-fold and 4-fold and the virus copies of the roVP3 group are the lowest in the three recombinant protein groups, which indicated that specific antibodies against recombinant proteins roVP1, roVP2 and roVP3 of DWV could neutralize DWV to reduce the virus titer in the pupae. Collectively, these results demonstrated that the surface capsid protein of DWV acted as candidates for the development of therapeutic antibodies against the virus.
Project description:Recent honey bee colony losses, particularly during the winter, have been shown to be associated with the presence of both ectoparasitic mites and Deformed Wing Virus (DWV). Whilst the role of Varroa destructor mites as a viral vector is well established, the role of Tropilaelaps mercedesae mites in viral transmission has not been fully investigated. In this study, we tested the effects that V. destructor and T. mercedesae infestation have on fluctuation of the DWV copy number and alteration of the virus variants in honey bees by characterizing individual pupae and their infesting mites. We observed that both mite species were associated with increased viral copy number in honey bee pupae. We found a positive correlation between DWV copy number in pupae and copy number in infesting mites, and the same DWV type A variant was present in either low or high copy number in both honey bee pupae and infesting V. destructor. These data also suggest that variant diversity is similar between honey bee pupae and the mites that infest them. These results support a previously proposed hypothesis that DWV suppresses the honey bee immune system when virus copy number reaches a specific threshold, promoting greater replication.
Project description:We developed a honey bee RNA-virus vector based on the genome of a picorna-like Deformed wing virus (DWV), the main viral pathogen of the honey bee (Apis mellifera). To test the potential of DWV to be utilized as a vector, the 717 nt sequence coding for the enhanced green fluorescent protein (eGFP), flanked by the peptides targeted by viral protease, was inserted into an infectious cDNA clone of DWV in-frame between the leader protein and the virus structural protein VP2 genes. The in vitro RNA transcripts from egfp-tagged DWV cDNA clones were infectious when injected into honey bee pupae. Stable DWV particles containing genomic RNA of the recovered DWV with egfp inserts were produced, as evidenced by cesium chloride density gradient centrifugation. These particles were infectious to honey bee pupae when injected intra-abdominally. Fluorescent microscopy showed GFP expression in the infected cells and Western blot analysis demonstrated accumulation of free eGFP rather than its fusions with DWV leader protein (LP) and/or viral protein (VP) 2. Analysis of the progeny egfp-tagged DWV showed gradual accumulation of genome deletions for egfp, providing estimates for the rate of loss of a non-essential gene an insect RNA virus genome during natural infection.
Project description:Many attempts to develop a reliable cell cultured-based system to study honey bee virus infections have encountered substantial difficulties. We investigated the ability of a cell line from a heterologous insect to sustain infection by a honey bee virus. For this purpose, we infected the Lepidopteran hemocytic cell line (P1) with Deformed wing virus (DWV). The genomic copies of DWV increased upon infection, as monitored by quantitative RT-PCR. Moreover, a tagged-primer-based RT-PCR analysis showed the presence of DWV negative-sense RNA in the cells, indicating virus replication. However, the DWV from infected cells was mildly infectious to P1 cells. Similar results were obtained when the virus was injected into Apis mellifera pupae. Thus, though the virus yields from the infected cells appeared to be very low, we show for the first time that DWV can replicate in a heterologous cell line. Given the availability of many other insect cell lines, our study paves the way for future exploration in this direction. In the absence of adequate A. mellifera cell lines, exploring the ability of alternative cell lines to enable honey bee virus infections could provide the means to study and understand the viral infectious cycle at the cellular level and facilitate obtaining purified isolates of these viruses.
Project description:Transcriptional profiling of honey bee pupae challenging a DWV infection by comparing the gene expression profiles of untreated honey bee pupae with DWV-infected pupae. Goal was to determine the gene expression profile of DWV-infected honey bee pupae.
Project description:Honey bees are agriculturally important, both as pollinators and by providing products such as honey. The sustainability of beekeeping is at risk through factors of global change such as habitat loss, as well as through the spread of infectious diseases. In China and other parts of Asia, beekeepers rely both on native Apis cerana and non-native Apis mellifera, putting bee populations at particular risk of disease emergence from multi-host pathogens. Indeed, two important honey bee parasites have emerged from East Asian honey bees, the mite Varroa destructor and the microsporidian Nosema ceranae. As V. destructor vectors viral bee diseases, we investigated whether another key bee pathogen, Deformed Wing Virus (DWV), may also have originated in East Asian honey bee populations. We use a large-scale survey of apiaries across China to investigate the prevalence and seasonality of DWV in managed A. mellifera and A. cerana colonies, showing that DWV-A prevalence was higher in A. mellifera, with a seasonal spike in prevalence in autumn and winter. Using phylogenetic and population genetic approaches, we show that while China and East Asian DWV isolates show comparatively high levels of genetic diversity, these bee populations are not a source for the current global DWV epidemic.
Project description:Deformed wing virus (DWV) is an emerging infectious disease of the honey bee (Apis mellifera) that is considered a major cause of elevated losses of honey bee colonies. DWV comprises two widespread genotypes: the originally described genotype A, and genotype B. In adult honey bees, DWV-B has been shown to be more virulent than DWV-A. However, their comparative effects on earlier host developmental stages are unknown. Here, we experimentally inoculated honey bee pupae and tested for the relative impact of DWV-A versus DWV-B on mortality and wing deformities in eclosing adults. DWV-A and DWV-B caused similar, and only slightly elevated, pupal mortality (mean 18% greater mortality than control). Both genotypes caused similarly high wing deformities in eclosing adults (mean 60% greater wing deformities than control). Viral titer was high in all of the experimentally inoculated eclosing adults, and was independent of wing deformities, suggesting that the phenotype 'deformed wings' is not directly related to viral titer or viral genotype. These viral traits favor the emergence of both genotypes of DWV by not limiting the reproduction of its vector, the ectoparasitic Varroa destructor mite, in infected pupae, and thereby facilitating the spread of DWV in honey bees infested by the mite.
Project description:The impacts of invertebrate RNA virus population dynamics on virulence and infection outcomes are poorly understood. Deformed wing virus (DWV), the main viral pathogen of honey bees, negatively impacts bee health, which can lead to colony death. Despite previous reports on the reduction of DWV diversity following the arrival of the parasitic mite Varroa destructor, the key DWV vector, we found high genetic diversity of DWV in infested United States honey bee colonies. Phylogenetic analysis showed that divergent US DWV genotypes are of monophyletic origin and were likely generated as a result of diversification after a genetic bottleneck. To investigate the population dynamics of this divergent DWV, we designed a series of novel infectious cDNA clones corresponding to coexisting DWV genotypes, thereby devising a reverse-genetics system for an invertebrate RNA virus quasispecies. Equal replication rates were observed for all clone-derived DWV variants in single infections. Surprisingly, individual clones replicated to the same high levels as their mixtures and even the parental highly diverse natural DWV population, suggesting that complementation between genotypes was not required to replicate to high levels. Mixed clone-derived infections showed a lack of strong competitive exclusion, suggesting that the DWV genotypes were adapted to coexist. Mutational and recombination events were observed across clone progeny, providing new insights into the forces that drive and constrain virus diversification. Accordingly, our results suggest that Varroa influences DWV dynamics by causing an initial selective sweep, which is followed by virus diversification fueled by negative frequency-dependent selection for new genotypes. We suggest that this selection might reflect the ability of rare lineages to evade host defenses, specifically antiviral RNA interference (RNAi). In support of this hypothesis, we show that RNAi induced against one DWV strain is less effective against an alternate strain from the same population.
Project description:Deformed wing virus (DWV) is an important pathogen in a broad range of insects, including honey bees. Concordant with the spread of Varroa, DWV is present in the majority of honey bee colonies and can result in either low-level infections with asymptomatic bees that nonetheless exhibit increased colony loss under stress, or high-level infections with acute effects on bee health and viability. DWV can be transmitted vertically or horizontally and evidence suggests that horizontal transmission via Varroa is associated with acute symptomatic infections. Vertical transmission also occurs and is presumably important for the maintenance of DWV in honey bee populations. To further our understanding the vertical transmission of DWV through queens, we performed three experiments: we studied the quantitative effectiveness of vertical transmission, surveyed the prevalence of successful egg infection under commercial conditions, and distinguished among three possible mechanisms of transmission. We find that queen-infection level predicts the DWV titers in their eggs, although the transmission is not very efficient. Our quantitative assessment of DWV demonstrates that eggs in 1/3 of the colonies are infected with DWV and highly infected eggs are rare in newly-installed spring colonies. Additionally, our results indicate that DWV transmission occurs predominantly by virus adhering to the surface of eggs (transovum) rather than intracellularly. Our combined results suggest that the queens' DWV vectoring capacity in practice is not as high as its theoretical potential. Thus, DWV transmission by honey bee queens is part of the DWV epidemic with relevant practical implications, which should be further studied.