Deformed Wing Virus Infection Research Articles

Reduction in deformed wing virus infection in larval and adult honey bees (Apis mellifera L.) by double‐stranded RNA ingestion

Deformed wing virus (DWV) is a serious pathogen of the honey bee, Apis mellifera L., vectored by the parasitic mite Varroa destructor. The virus is associated with wing deformity in symptomatic bees, and premature death and reduced colony performance in asymptomatic bees. In the present study we reduced DWV infection by feeding both first instar larvae and adult A. mellifera with a double-stranded (ds) RNA construct, DWV-dsRNA, which is specific to DWV in DWV-inoculated bees, by mixing it with their food. We showed that feeding DWV to larvae causes wing deformity in adult bees in the absence of varroa mites and decreases survival rates of adult bees relative to bees not fed DWV. Feeding larvae with DWV-dsRNA in advance of inoculation with virus reduced the DWV viral level and reduced wing deformity relative to larvae fed DWV or DWV with green fluorescent protein-dsRNA (probably a result of RNA silencing), but did not affect survival to the adult stage. Feeding DWV-dsRNA did not affect larval survival rates, which suggests that dsRNA is non-toxic to larvae. Feeding adult workers with DWV-dsRNA in advance of inoculation with virus increased their longevity and reduced DWV concentration relative to controls.

Evidence for damage-dependent hygienic behaviour towards Varroa destructor-parasitised brood in the western honey bee, Apis mellifera

The ectoparasitic mite Varroa destructor and honey bee pathogenic viruses have been implicated in the recent demise of honey bee colonies. Several studies have shown that the combination of V. destructor and deformed wing virus (DWV) poses an especially serious threat to honey bee health. Mites transmitting virulent forms of DWV may cause fatal DWV infections in the developing bee, while pupae parasitised by mites not inducing or activating overt DWV infections may develop normally. Adult bees respond to brood diseases by removing affected brood. This hygienic behaviour is an essential part of the bees' immune response repertoire and is also shown towards mite-parasitised brood. However, it is still unclear whether the bees react towards the mite in the brood cell or rather towards the damage done to the brood. We hypothesised that the extent of mite-associated damage rather than the mere presence of parasitising mites triggers hygienic behaviour. Hygienic behaviour assays performed with mites differing in their potential to transmit overt DWV infections revealed that brood parasitised by 'virulent' mites (i.e. mites with a high potential to induce fatal DWV infections in parasitised pupae) were removed significantly more often than brood parasitised by 'less virulent' mites (i.e. mites with a very low potential to induce overt DWV infections) or non-parasitised brood. Chemical analyses of brood odour profiles suggested that the bees recognise severely affected brood by olfactory cues. Our results suggest that bees show selective, damage-dependent hygienic behaviour, which may be an economic way for colonies to cope with mite infestation.

Dynamics of persistent and acute deformed wing virus infections in honey bees, Apis mellifera.

The dynamics of viruses are critical to our understanding of disease pathogenesis. Using honey bee Deformed wing virus (DWV) as a model, we conducted field and laboratory studies to investigate the roles of abiotic and biotic stress factors as well as host health conditions in dynamics of virus replication in honey bees. The results showed that temperature decline could lead to not only significant decrease in the rate for pupae to emerge as adult bees, but also an increased severity of the virus infection in emerged bees, partly explaining the high levels of winter losses of managed honey bees, Apis mellifera, around the world. By experimentally exposing adult bees with variable levels of parasitic mite Varroa destructor, we showed that the severity of DWV infection was positively correlated with the density and time period of Varroa mite infestation, confirming the role of Varroa mites in virus transmission and activation in honey bees. Further, we showed that host conditions have a significant impact on the outcome of DWV infection as bees that originate from strong colonies resist DWV infection and replication significantly better than bee originating from weak colonies. The information obtained from this study has important implications for enhancing our understanding of host‑pathogen interactions and can be used to develop effective disease control strategies for honey bees.

Negative correlation between Nosema ceranae spore loads and deformed wing virus infection levels in adult honey bee workers

Interactions between pathogens might contribute to honey bee colony losses. Here we investigated if there is an association between the microsporidian Nosema ceranae and the deformed wing virus (DWV) in different body sections of individual honey bee workers (Apis mellifera ligustica) under exclusion of the vector Varroa destructor. Our data provide correlational evidence for antagonistic interactions between the two pathogens in the midgut of the bees.

Deformed wing virus and drone mating flights in the honey bee (Apis mellifera): implications for sexual transmission of a major honey bee virus

Deformed wing virus (DWV) represents an ideal model to study the interaction between mode of transmission and virulence in honey bees since it exhibits both horizontal and vertical transmissions. However, it is not yet clear if venereal–vertical transmission represents a regular mode of transmission for this virus in natural honey bee populations. Here, we provide clear evidence for the occurrence of high DWV titres in the endophallus of sexually mature drones collected from drone congregation areas (DCAs). Furthermore, the endophallus DWV titres of drones collected at their maternal hives were no different from drones collected at nearby DCAs, suggesting that high-titre DWV infection of the endophallus does not hinder the ability of drones to reach the mating area. The results are discussed within the context of the dispersal of DWV between colonies and the definition of DWV virulence with respect to the transmission route and the types of tissues infected.

Horizontal transmission of deformed wing virus: pathological consequences in adult bees (Apis mellifera) depend on the transmission route

Recent reports on a steady decline of honeybee colonies in several parts of the world caused great concern. There is a consensus that pathogens are among the key players in this alarming demise of the most important commercial pollinator. One of the pathogens heavily implicated in colony losses is deformed wing virus (DWV). Overt DWV infections manifested as deformed-wing syndrome started to become a threat to honeybees only in the wake of the ectoparasitic mite Varroa destructor, which horizontally transmits DWV. However, a direct causal link between the virus and the symptom 'wing deformity' has not been established yet. To evaluate the impact of different horizontal transmission routes, and especially the role of the mite in the development of overt DWV infections, we performed laboratory infection assays with pupae and adult bees. We could demonstrate that pupae injected with DWV dose-dependently developed overt infections characterized by deformed wings in adult bees, suggesting that DWV, if transmitted to pupae by the mite, is the causative agent of the deformed-wing syndrome. The OID(50) (overt infection dosage) was approximately 2500 genome equivalents. Injecting more than 1×10(7) DWV genome equivalents into adult bees also resulted in overt infections while the same viral dosage fed to adult bees only resulted in covert infections. Therefore, both infection of adult bees through DWV-transmitting phoretic mites and infection of nurse bees through their cannibalizing DWV-infected pupae might represent possible horizontal transmission routes of DWV.

Emerging and re-emerging viruses of the honey bee (Apis melliferaL.)

Until the late 1980s, specific viral infections of the honey bee were generally considered harmless in all countries. Then, with the worldwide introduction of the ectoparasite mite Varroa destructor, beekeepers encountered increasing difficulties in maintaining their colonies. Epidemiological surveys and laboratory experiments have demonstrated that the newly acquired virulence of several viruses belonging to the family Dicistroviridae (acute bee paralysis virus, Kashmir bee virus and Israeli acute paralysis virus) in Europe and the USA had been observed in relation with V. destructor acting as a disseminator of these viruses between and within bee colonies and as an activator of virus multiplication in the infected individuals: bee larvae and adults. Equal emphasis is given to deformed wing virus (DWV) belonging to the Iflaviridae. Overt outbreaks of DWV infections have been shown to be linked to the ability of V. destructor to act not only as a mechanical vector of DWV but also as a biological vector. Its replication in mites prior to its vectoring into pupae seemed to be necessary and sufficient for the induction of a overt infection in pupae developing in non-viable bees with deformed wings. DWV in V. destructor infested colonies is now considered as one of the key players of the final collapse. Various approaches for combating bee viral diseases are described: they include selection of tolerant bees, RNA interference and prevention of new pathogen introduction. None of these approaches are expected to lead to enhanced bee-health in the short term.

Virus infections and winter losses of honey bee colonies (Apis mellifera)

SummaryApiculturists have recently been confronted with drastic and inexplicable winter losses of colonies, and virus infections may be involved. Here, we surveyed 337 Swiss honey bee colonies in the winter of 2005 and 2006 and categorized their health status as: 1. dead (= no or few live bees left); 2. weak (= dwindling, high mortality of adult bees); or 3. healthy (= normal overwintering colony). From each colony, pooled adult workers were analyzed for deformed wing virus (DWV), acute bee paralysis virus (ABPV), chronic bee paralysis virus (CBPV) and Kashmir bee virus (KBV). Neither KBV nor CBPV were found, but significantly higher ABPV and DWV infections were found in dead vs. weak vs. healthy colonies (except DWV in 2006 between weak and healthy). Moreover, ABPV and DWV loads were positively correlated with each other. This is the first report demonstrating statistically significant correlations between viruses associated with Varroa destructor and winter mortality.

Molecular approaches to the analysis of deformed wing virus replication and pathogenesis in the honey bee, Apis mellifera

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.

Localization of deformed wing virus (DWV) in the brains of the honeybee, Apis mellifera Linnaeus

BackgroundDeformed wing virus (DWV) is a positive-strand RNA virus that infects European honeybees (Apis mellifera L.) and has been isolated from the brains of aggressive bees in Japan. DWV is known to be transmitted both vertically and horizontally between bees in a colony and can lead to both symptomatic and asymptomatic infections in bees. In environmentally stressful conditions, DWV can contribute to the demise of a honeybee colony. The purpose of the current study is to identify regions within the brains of honeybees where DWV replicates using in-situ hybridization.ResultsIn-situ hybridizations were conducted with both sense and antisense probes on the brains of honeybees that were positive for DWV as measured by real-time RT-PCR. The visual neuropils demonstrated detectable levels of the DWV positive-strand genome. The mushroom bodies and antenna lobe neuropils also showed the presence of the viral genome. Weaker staining with the sense probe in the same regions demonstrates that the antigenome is also present and that the virus is actively replicating in these regions of the brain.ConclusionThese results demonstrate that in bees infected with DWV the virus is replicating in critical regions of the brain, including the neuropils responsible for vision and olfaction. Therefore DWV infection of the brain could adversely affect critical sensory functions and alter normal bee behavior.

Deformed wing virus: replication and viral load in mites (Varroa destructor)

Deformed wing virus (DWV) normally causes covert infections but can have devastating effects on bees by inducing morphological deformity or even death when transmitted by the ectoparasitic mite Varroa destructor. In order to determine the role of V. destructor in the development of crippled wings, we analysed individual mites for the presence and replication of DWV. The results supported the correlation between viral replication in mites and morphologically deformed bees. Quantification of viral genome equivalents revealed that mites capable of inducing an overt DWV infection contained 10(10)-10(12) genome equivalents per mite. In contrast, mites which could not induce crippled wings contained a maximum of only 10(8) viral genome equivalents per mite. We conclude that the development of crippled wings not only depends on DWV transmission by V. destructor but also on viral replication in V. destructor and on the DWV titre in the parasitizing mites.

Deformed wing virus associated with Tropilaelaps mercedesae infesting European honey bees (Apis mellifera)

Mites in the genus Tropilaelaps (Acari: Laelapidae) are ectoparasites of the brood of honey bees (Apis spp.). Different Tropilaelaps subspecies were originally described from Apis dorsata, but a host switch occurred to the Western honey bee, Apis mellifera, for which infestations can rapidly lead to colony death. Tropilaelaps is hence considered more dangerous to A. mellifera than the parasitic mite Varroa destructor. Honey bees are also infected by many different viruses, some of them associated with and vectored by V. destructor. In recent years, deformed wing virus (DWV) has become the most prevalent virus infection in honey bees associated with V. destructor. DWV is distributed world-wide, and found wherever the Varroa mite is found, although low levels of the virus can also be found in Varroa free colonies. The Varroa mite transmits viral particles when feeding on the haemolymph of pupae or adult bees. Both the Tropilaelaps mite and the Varroa mite feed on honey bee brood, but no observations of DWV in Tropilaelaps have so far been reported. In this study, quantitative real-time RT-PCR was used to show the presence of DWV in infested brood and Tropilaelaps mercedesae mites collected in China, and to demonstrate a close quantitative association between mite-infested pupae of A. mellifera and DWV infections. Phylogenetic analysis of the DWV sequences recovered from matching pupae and mites revealed considerable DWV sequence heterogeneity and polymorphism. These polymorphisms appeared to be associated with the individual brood cell, rather than with a particular host.

First molecular confirmation of deformed wing virus infections of honeybees from a Belgian apiary reveals the presence of black queen cell virus and Varroa destructor virus 1

First molecular confirmation of deformed wing virus infections of honeybees from a Belgian apiary reveals the presence of black queen cell virus and Varroa destructor virus 1

Vertical-transmission routes for deformed wing virus of honeybees (Apis mellifera)

Deformed wing virus (DWV) is a viral pathogen of the European honeybee (Apis mellifera), associated with clinical symptoms and colony collapse when transmitted by the ectoparasitic mite Varroa destructor. In the absence of V. destructor, DWV infection does not result in visible symptoms, suggesting that mite-independent transmission results in covert infections. True covert infections are a known infection strategy for insect viruses, resulting in long-term persistence of the virus in the population. They are characterized by the absence of disease symptoms in the presence of the virus and by vertical transmission of the virus. To demonstrate vertical transmission and, hence, true covert infections for DWV, a detailed study was performed on the vertical-transmission routes of DWV. In total, 192 unfertilized eggs originating from eight virgin queens, and the same number of fertilized eggs from the same queens after artificial insemination with DWV-negative (three queens) or DWV-positive (five queens) semen, were analysed individually. The F0 queens and drones and F1 drones and workers were also analysed for viral RNA. By in situ hybridization, viral sequences were detected in the ovary of an F0 queen that had laid DWV-positive unfertilized eggs and was inseminated with DWV-positive semen. In conclusion, vertical transmission of DWV from queens and drones to drone and worker offspring through unfertilized and fertilized eggs, respectively, was demonstrated. Viral sequences in fertilized eggs can originate from the queen, as well as from drones via DWV-positive semen.

Localization of deformed wing virus infection in queen and drone Apis mellifera L

The distribution of deformed wing virus infection within the honey bee reproductive castes (queens, drones) was investigated by in situ hybridization and immunohistology from paraffin embedded sections. Digoxygenin or CY5.5 fluorochrome end-labelled nucleotide probes hybridizing to the 3' portion of the DWV genome were used to identify DWV RNA, while a monospecific antibody to the DWV-VP1 structural protein was used to identify viral proteins and particles. The histological data were confirmed by quantitative RT-PCR of dissected organs. Results showed that DWV infection is not restricted to the digestive tract of the bee but spread in the whole body, including queen ovaries, queen fat body and drone seminal vesicles.

Detection of Deformed wing virus, a honey bee viral pathogen, in bumble bees ( Bombus terrestris and Bombus pascuorum) with wing deformities

Honey bees ( Apis mellifera) productively infected with Deformed wing virus (DWV) through Varroa destructor ( V. destructor) during pupal stages develop into adults showing wing and other morphological deformities. Here, we report for the first time the occurrence of bumble bees ( Bombus terrestris, Bombus pascuorum) exhibiting wing deformities resembling those seen in clinically DWV-infected honey bees. Using specific RT-PCR protocols for the detection of DWV followed by sequencing of the PCR products we could demonstrate that the bumble bees were indeed infected with DWV. Since such deformed bumble bees are not viable DWV infection may pose a serious threat to bumble bee populations.

Clinical pathologic conference.

<h3>Abstract</h3> The ectoparasitic mite, <i>Varroa destructor</i> and the viruses it vectors, including types A and B of Deformed wing virus (DWV), pose a major threat to honey bees, <i>Apis mellifera</i>. Analysis of 256 mites collected from the same set of colonies on five occasions from May to October 2021 showed that less than a half of them, 39.8% (95% confidence interval (CI): 34.0 - 46.0%), were able to induce an overt-level DWV infection with more than 10⁹ viral genomes per bee in the pupa after 6 days of feeding, with both DWV-A and DWV-B being vectored at similar rates. To investigate the effect of the phoretic stage on the mites’ ability to vector DWV, the mites from two field collection events were divided into two groups, one of which was tested immediately for their infectiveness, and the other was kept with adult worker bees in cages for 12 days prior to testing their infectiveness. We found that while 39.2 % (95% CI: 30.0 – 49.1%) of the immediately tested mites induced overt-level DWV infections, 12-day phoretic passage significantly increased the infectiousness of mites to 89.8% (95% CI: 79.2 – 95.6%). It is likely that Varroa mites that survive brood interruptions in field colonies are increasingly infectious. We found that mite lifespan was significantly affected by the type of DWV it transmitted to pupae. The mites, which induced overt DWV-B but not DWV-A infection had an average lifespan of 15.5 days (95% CI: 11.8 - 19.2 days), which was significantly shorter than those of the mites which induced overt DWV-A but not DWV-B infection, with an average lifespan of 24.3 days (95% CI: 20.2 - 28.5), or the mites which did not induce high levels of DWV-A or DWV-B, with an average survival of 21.2 days (95% CI: 19.0 - 23.5 days). The mites which transmitted high levels of both DWV-A and DWV-B had an intermediate average survival of 20.5 days (95% CI: 15.1 - 25.9 days). The negative impact of DWV-B on mite survival could be a consequence of the ability of DWV-B, but not DWV-A to replicate in Varroa mites.