Distribution of deformed wing virus within honey bee (Apis mellifera) brood cells infested with the ectoparasitic mite Varroa destructor

The distribution of deformed wing virus (DWV) in adult female Varroa destructor and in their progeny in relation to the pupal host bee was investigated to evaluate acquisition and transfer of DWV by the mites. The results clearly show that adult female mites regularly act as competent vectors of DWV, however, they do not acquire or transfer virus on all possible occasions. Mother mites may contain DWV while the pupal host remains free from overt infection and both mother mites and mite progeny may not acquire detectable amounts of DWV from an infected host bee. However, a majority of mites feeding on pupae that emerge with deformed wings will contain DWV. The data also demonstrates that both adult and immature mite progeny most likely acquire DWV from DWV-infected host bees and not from their mother mites. Possible explanations for the obtained results are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Acaricide treatment affects viral dynamics in Varroa destructor-infested honey bee colonies via both host physiology and mite control

Honey bee (Apis mellifera) colonies are declining, and a number of stressors have been identified that affect, alone or in combination, the health of honey bees. The ectoparasitic mite Varroa destructor, honey bee viruses that are often closely associated with the mite, and pesticides used to control the mite population form a complex system of stressors that may affect honey bee health in different ways. During an acaricide treatment using Apistan (plastic strips coated with tau-fluvalinate), we analyzed the infection dynamics of deformed wing virus (DWV), sacbrood virus (SBV), and black queen cell virus (BQCV) in adult bees, mite-infested pupae, their associated Varroa mites, and uninfested pupae, comparing these to similar samples from untreated control colonies. Titers of DWV increased initially with the onset of the acaricide application and then slightly decreased progressively coinciding with the removal of the Varroa mite infestation. This initial increase in DWV titers suggests a physiological effect of tau-fluvalinate on the host's susceptibility to viral infection. DWV titers in adult bees and uninfested pupae remained higher in treated colonies than in untreated colonies. The titers of SBV and BQCV did not show any direct relationship with mite infestation and showed a variety of possible effects of the acaricide treatment. The results indicate that other factors besides Varroa mite infestation may be important to the development and maintenance of damaging DWV titers in colonies. Possible biochemical explanations for the observed synergistic effects between tau-fluvalinate and virus infections are discussed.     

Deformed wing virus in western honey bees (Apis mellifera) from Atlantic Canada and the first description of an overtly-infected emerging queen

Deformed wing virus (DWV) in western honey bees (Apis mellifera) often remains asymptomatic in workers and drones, and symptoms have never been described from queens. However, intense infections linked to parasitism by the mite Varroa destructor can cause worker wing deformity and death within 67 h of emergence. Ten workers (eight with deformed wings and two with normal wings) and three drones (two with deformed wings and one with normal wings) from two colonies infected with V. destructor from Nova Scotia, Canada, and two newly-emerged queens (one with deformed wings and one with normal wings) from two colonies infected with V. destructor from Prince Edward Island, Canada, were genetically analyzed for DWV. We detected DWV in all workers and drones, regardless of wing morphology, but only in the deformed-winged queen. This is the first report of DWV from Atlantic Canada and the first detection of a symptomatic queen with DWV from anywhere.     

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.     

Deformed wing virus

Deformed wing virus (DWV; Iflaviridae) is one of many viruses infecting honeybees and one of the most heavily investigated due to its close association with honeybee colony collapse induced by Varroa destructor. In the absence of V. destructor DWV infection does not result in visible symptoms or any apparent negative impact on host fitness. However, for reasons that are still not fully understood, the transmission of DWV by V. destructor to the developing pupae causes clinical symptoms, including pupal death and adult bees emerging with deformed wings, a bloated, shortened abdomen and discolouration. These bees are not viable and die soon after emergence. In this review we will summarize the historical and recent data on DWV and its relatives, covering the genetics, pathobiology, and transmission of this important viral honeybee pathogen, and discuss these within the wider theoretical concepts relating to the genetic variability and population structure of RNA viruses, the evolution of virulence and the development of disease symptoms.     

Dead or alive: deformed wing virus and Varroa destructor reduce the life span of winter honeybees

Elevated winter losses of managed honeybee colonies are a major concern, but the underlying mechanisms remain controversial. Among the suspects are the parasitic mite Varroa destructor, the microsporidian Nosema ceranae, and associated viruses. Here we hypothesize that pathogens reduce the life expectancy of winter bees, thereby constituting a proximate mechanism for colony losses. A monitoring of colonies was performed over 6 months in Switzerland from summer 2007 to winter 2007/2008. Individual dead workers were collected daily and quantitatively analyzed for deformed wing virus (DWV), acute bee paralysis virus (ABPV), N. ceranae, and expression levels of the vitellogenin gene as a biomarker for honeybee longevity. Workers from colonies that failed to survive winter had a reduced life span beginning in late fall, were more likely to be infected with DWV, and had higher DWV loads. Colony levels of infection with the parasitic mite Varroa destructor and individual infections with DWV were also associated with reduced honeybee life expectancy. In sharp contrast, the level of N. ceranae infection was not correlated with longevity. In addition, vitellogenin gene expression was significantly positively correlated with ABPV and N. ceranae loads. The findings strongly suggest that V. destructor and DWV (but neither N. ceranae nor ABPV) reduce the life span of winter bees, thereby constituting a parsimonious possible mechanism for honeybee colony losses.     

Lower Virus Infections in Varroa destructor-Infested and Uninfested Brood and Adult Honey Bees (Apis mellifera) of a Low Mite Population Growth Colony Compared to a High Mite Population Growth Colony

A comparison was made of the prevalence and relative quantification of deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), black queen cell virus (BQCV), Kashmir bee virus (KBV), acute bee paralysis virus (ABPV) and sac brood virus (SBV) in brood and adult honey bees (Apis mellifera) from colonies selected for high (HMP) and low (LMP) Varroa destructor mite population growth. Two viruses, ABPV and SBV, were never detected. For adults without mite infestation, DWV, IAPV, BQCV and KBV were detected in the HMP colony; however, only BQCV was detected in the LMP colony but at similar levels as in the HMP colony. With mite infestation, the four viruses were detected in adults of the HMP colony but all at higher amounts than in the LMP colony. For brood without mite infestation, DWV and IAPV were detected in the HMP colony, but no viruses were detected in the LMP colony. With mite infestation of brood, the four viruses were detected in the HMP colony, but only DWV and IAPV were detected and at lower amounts in the LMP colony. An epidemiological explanation for these results is that pre-experiment differences in virus presence and levels existed between the HMP and LMP colonies. It is also possible that low V. destructor population growth in the LMP colony resulted in the bees being less exposed to the mite and thus less likely to have virus infections. LMP and HMP bees may have also differed in susceptibility to virus infection.     

Deformed wing virus associated with <Emphasis Type="Italic">Tropilaelaps mercedesae</Emphasis> infesting European honey bees (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

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.     

Infectivity of DWV Associated to Flower Pollen: Experimental Evidence of a Horizontal Transmission Route

Deformed wing virus (DWV) is a honeybee pathogen whose presence is generally associated with infestation of the colony by the mite Varroa destructor, leading to the onset of infections responsible for the collapse of the bee colony. DWV contaminates bee products such as royal jelly, bee-bread and honey stored within the infected hive. Outside the hive, DWV has been found in pollen loads collected directly from infected as well as uninfected forager bees. It has been shown that the introduction of virus-contaminated pollen into a DWV-free hive results in the production of virus-contaminated food, whose role in the development of infected bees from virus-free eggs has been experimentally demonstrated. The aim of this study was twofold: (i) to ascertain the presence of DWV on pollen collected directly from flowers visited by honeybees and then quantify the viral load and (ii) determine whether the virus associated with pollen is infective. The results of our investigation provide evidence that DWV is present on pollen sampled directly from visited flowers and that, following injection in individuals belonging to the pollinator species Apis mellifera, it is able to establish an active infection, as indicated by the presence of replicating virus in the head of the injected bees. We also provide the first indication that the pollinator species Osmia cornuta is susceptible to DWV infection.     

Virus infection causes specific learning deficits in honeybee foragers

In both mammals and invertebrates, virus infections can impair a broad spectrum of physiological functions including learning and memory formation. In contrast to the knowledge on the conserved mechanisms underlying learning, the effects of virus infection on different aspects of learning are barely known. We use the honeybee (Apis mellifera), a well-established model system for studying learning, to investigate the impact of deformed wing virus (DWV) on learning. Injection of DWV into the haemolymph of forager leads to a RT-PCR detectable DWV signal after 3 days. The detailed behavioural analysis of DWV-infected honeybees shows an increased responsiveness to water and low sucrose concentrations, an impaired associative learning and memory formation, but intact non-associative learning like sensitization and habituation. This contradicts all present studies in non-infected bees, where increased sucrose responsiveness is linked to improved associative learning and to changes in non-associative learning. Thus, DWV seems to interfere with molecular mechanism of learning by yet unknown processes that may include viral effects on the immune system and on gene expression.     

Knock‐on community impacts of a novel vector: spillover of emerging DWV‐B from Varroa‐infested honeybees to wild bumblebees

Novel transmission routes can directly impact the evolutionary ecology of infectious diseases, with potentially dramatic effect on host populations and knock‐on effects on the wider host community. The invasion of Varroa destructor, an ectoparasitic viral vector in Western honeybees, provides a unique opportunity to examine how a novel vector affects disease epidemiology in a host community. This specialist honeybee mite vectors deformed wing virus (DWV), an important re‐emerging honeybee pathogen that also infects wild bumblebees. Comparing island honeybee and wild bumblebee populations with and without V. destructor, we show that V. destructor drives DWV prevalence and titre in honeybees and sympatric bumblebees. Viral genotypes are shared across hosts, with the potentially more virulent DWV‐B overtaking DWV‐A in prevalence in a current epidemic. This demonstrates disease emergence across a host community driven by the acquisition of a specialist novel transmission route in one host, with dramatic community level knock‐on effects.     

Detection of deformed wing virus in the greenhouse for possible horizontal transmission of virus in honey bee colony

The honey bee Apis mellifera L. is a crucial insect in the agricultural industry and natural ecosystem by being a major pollinator. Nevertheless, honey bee population has been recently facing a decline. Among the several factors responsible for this decline, deformed wing virus (DWV) is considered a primary cause that negatively affects honey bee health. DWV is a cosmopolitan honey bee pathogen and causes morphological disadvantages in individual honey bees and colony collapse. Regarding the horizontal transmission of DWV, in addition to Varroa destructor, a well-known major vector of DWV, flowers have recently been implied as a transmission route. Therefore, in this study, we detected DWV from various substances, including flowers, honey bee feces, pupa, larva, nurse bee, surface of nurse bee, pollen collected by forager bee, and forager bee samples in four strawberry greenhouses, which could suggest the potential for the horizontal transmission of DWV in the semi-field condition. We also detected DWV in pollen collected by DWV-negative forager bees, implying that flowers can serve as a potential source of virus infection. These findings suggest that the surrounding environment such as shared floral sources affects the spread of DWV.     

On the Front Line: Quantitative Virus Dynamics in Honeybee (Apis mellifera L.) Colonies along a New Expansion Front of the Parasite Varroa destructor

Over the past fifty years, annual honeybee (Apis mellifera) colony losses have been steadily increasing worldwide. These losses have occurred in parallel with the global spread of the honeybee parasite Varroa destructor. Indeed, Varroa mite infestations are considered to be a key explanatory factor for the widespread increase in annual honeybee colony mortality. The host-parasite relationship between honeybees and Varroa is complicated by the mite''s close association with a range of honeybee viral pathogens. The 10-year history of the expanding front of Varroa infestation in New Zealand offered a rare opportunity to assess the dynamic quantitative and qualitative changes in honeybee viral landscapes in response to the arrival, spread and level of Varroa infestation. We studied the impact of de novo infestation of bee colonies by Varroa on the prevalence and titres of seven well-characterised honeybee viruses in both bees and mites, using a large-scale molecular ecology approach. We also examined the effect of the number of years since Varroa arrival on honeybee and mite viral titres. The dynamic shifts in the viral titres of black queen cell virus and Kashmir bee virus mirrored the patterns of change in Varroa infestation rates along the Varroa expansion front. The deformed wing virus (DWV) titres in bees continued to increase with Varroa infestation history, despite dropping infestation rates, which could be linked to increasing DWV titres in the mites. This suggests that the DWV titres in mites, perhaps boosted by virus replication, may be a major factor in maintaining the DWV epidemic after initial establishment. Both positive and negative associations were identified for several pairs of viruses, in response to the arrival of Varroa. These findings provide important new insights into the role of the parasitic mite Varroa destructor in influencing the viral landscape that affects honeybee colonies.     

Deformed Wing Virus Implicated in Overwintering Honeybee Colony Losses

The worldwide decline in honeybee colonies during the past 50 years has often been linked to the spread of the parasitic mite Varroa destructor and its interaction with certain honeybee viruses. Recently in the United States, dramatic honeybee losses (colony collapse disorder) have been reported; however, there remains no clear explanation for these colony losses, with parasitic mites, viruses, bacteria, and fungal diseases all being proposed as possible candidates. Common characteristics that most failing colonies share is a lack of overt disease symptoms and the disappearance of workers from what appears to be normally functioning colonies. In this study, we used quantitative PCR to monitor the presence of three honeybee viruses, deformed wing virus (DWV), acute bee paralysis virus (ABPV), and black queen cell virus (BQCV), during a 1-year period in 15 asymptomatic, varroa mite-positive honeybee colonies in Southern England, and 3 asymptomatic colonies confirmed to be varroa mite free. All colonies with varroa mites underwent control treatments to ensure that mite populations remained low throughout the study. Despite this, multiple virus infections were detected, yet a significant correlation was observed only between DWV viral load and overwintering colony losses. The long-held view has been that DWV is relatively harmless to the overall health status of honeybee colonies unless it is in association with severe varroa mite infestations. Our findings suggest that DWV can potentially act independently of varroa mites to bring about colony losses. Therefore, DWV may be a major factor in overwintering colony losses.Deformed wing virus (DWV), acute bee paralysis virus (ABPV), and black queen cell virus (BQCV) are single-stranded positive-sense RNA viruses of the order Picornavirales and are regularly detected in honeybee populations in the United Kingdom (1). ABPV has been assigned to the family Dicistroviridae and is known to follow a classic acute-type infection strategy since relatively low loads (10³ to 10⁶ viruses per honeybee) can rapidly translate into overt symptoms of paralysis and ultimately death for the honeybee, depending on the mode of transmission (6, 33). ABPV shares >92% sequence homology with other members of the family Dicistroviridae, Kashmir bee virus and Israeli acute paralysis virus, across the eight conserved domains of the RNA-dependent RNA polymerase gene, and it has been proposed that these viruses have recently diverged and are variants of each other (7). Advances in the study of this proposed ABPV complex is revealing the significant impact these viruses may have on honeybee colonies on a global scale. For example, a recent study in the United States has observed a correlation between Israeli acute paralysis virus and colony collapse disorder (17). That said, other agents, including bacteria and microsporidia, have also been proposed as important factors in the onset of colony loss (25, 27).BQCV is similar to ABPV in that it, too, follows a typical acute infection strategy. This virus is known to infect honeybee queen cell larvae, causing the larvae to discolor and die (5). It has been shown to be associated with the microsporidian Nosema apis (4) although whether N. apis has a direct role in the transmission of this virus still needs to be determined. Both ABPV and BQCV have been detected in worker honeybees and pupae (38), and the viruses are transmitted orally, via food and feeding activities (14). BQCV has also been detected in queen honeybees (13), suggesting that vertical transmission is also important for this virus. Both BQCV (12) and ABPV (38) have been detected within the varroa mite; however, only ABPV (9) has been shown to be vectored by varroa mites and has been found associated with dead colonies infested with varroa mites in Germany, Russia, and the United States (1). Later modeling work (33) indicated that very large (10,000+) mite populations are required to kill a colony since it is difficult for ABPV to become established among the bee population due to its high virulence.DWV is currently designated as a member of the unassigned genus Iflavirus within the order Picornavirales. It is generally considered as less virulent than ABPV or Kashmir bee virus, but it is known to cause overt symptoms of wing deformities in developing honeybees, resulting in emerging honeybees that are unable to fly and die shortly (5). It is also speculated that a cloud of DWV sequence variants exists that have evolved from a common ancestor. This is due to the high sequence similarities DWV isolates share with Kakugo virus and Varroa destructor virus within the RNA-dependent RNA polymerase gene, yet differences in virus epidemiology and pathological effects distinguish them from each other (29). DWV has been detected in worker honeybees, pupae, larvae, drones, and queens (15, 18, 20) as well as within the varroa mite (38, 43) and more recently the mite Tropilaelaps mercedesae (21), implying a range of horizontal and vertical transmission routes. Despite their global occurrence, it is generally accepted that DWVs play a secondary role in the causes of honeybee disease compared to their parasitic and bacterial counterparts as the viruses routinely reside at low levels in colonies, with symptomatic infections being rare (5). Moreover, multiple variants with differing infection strategies can account for a lack of discernible symptoms.Whether these viruses follow a persistent, latent, inapparent, or progressive infection strategy still remains unclear. Persistent (often called chronic) infections imply that the rate of infection within a host is in balance with the reproduction rate of the infected cell type or host itself. This is achieved through a combination of changing virus replication and host immune responses. Latent infections occur when the virus lies dormant within the host (replication inactive) until activation by defined stimuli. Progressive infections are caused by viruses that enter the host cell and replicate undetected for many cellular generations over many years before manifesting overt or acute symptoms. These three infection strategies all evade the host immune system, which results in the inability of the host to fully expel the virus, and this inability is often lethal. Inapparent (often referred to as covert) infections are indicative of a highly evolved relationship between the virus and natural host. Moreover, these infections are distinct in that the natural host can eventually clear itself from this short-term infection (19). Infections of DWV are often described as inapparent (15); however, Yue et al. (44) have suggested that a distinction should be made between “true inapparent” and their newly defined “covert infection” based on the long-term nature of DWV infection in honeybee colonies and on the nature of its transmission. This conclusion is congruent with current knowledge that traditional serological screening methods for DWV have limitations in their sensitivity (20). Therefore, the presence and duration of DWV within colonies have often been underestimated using serological assays as the overt symptoms of the deformed wing phenotype (>10¹¹ virions per honeybee) are short-lived. Advances in virus detection methodologies have enabled the development of more sensitive techniques, such as PCR, and this has demonstrated that DWV persists for longer periods within colonies (38). However, based on the current research evidence, a case could be made that DWV actually follows the classic persistent infection strategy.DWV is thought to have an intricate relationship with varroa mites such that immunosuppression of the honeybee pupae by the mites results in increased DWV amplification when the honeybees are exposed to other pathogens (42). It has additionally been shown that the number of mites parasitizing honeybee pupae is positively correlated with the probability of their developing malformed wings (10). Other findings indicate that DWV replication within the mite and subsequent transmission to developing honeybees lead to the increased likelihood of the bees'' emerging with wing deformities (24, 43). Taken together, the expectation is that DWV-associated colony collapse would typically occur in the presence of a large (>2,000) varroa mite infestation carrying high levels of DWV and with a high proportion of deformed honeybees. While the effect of varroa mite-induced DWV disease is well recognized, i.e., wing deformities coupled with downregulation of immunity-related genes and antimicrobial peptides (36, 42) and impaired learning behavior (28), the impact of non-varroa mite-vectored DWV within asymptomatic honeybees still needs to be realized. Moreover, it was recently reported that varroa mite-free bumblebees that tested positive for DWV actually showed symptoms of DWV infection (23). Even though these bumblebees were in close proximity to DWV-infected and varroa mite-infested honeybee colonies, it is evidence that the dependency of DWV on varroa mite vectoring for a symptomatic infection (manifested as classic wing deformities or other symptoms) may not be as critical as previously thought.The purpose of this study was to investigate asymptomatic viral dynamics within husbanded honeybee colonies over an annual cycle. We set out to observe the relationship, if any, between virus infections, varroa mite parasitism and vectoring, honeybee colony health, and colony longevity. For the first time, a quantitative analysis of three picorna-like honeybee viruses over the course of a year was undertaken for DWV, ABPV, and BQCV.     

Genetic variability and phylogenetic analysis among strains of deformed wing virus infesting honey bees and other organisms

Various viruses can infect honey bees, but deformed wing virus (DWV) is considered the most dangerous virus to them and has role in the sudden decline of bee colonies. This virus has different strains; however, there are no available studies to compare the characteristics of these strains utilizing bioinformatics. In this study, 27 strains of deformed wing virus were analyzed based on their sequences and their genetic relationships. Also, some primers were designed and tested to identify their ability to separate DWV strains. The percentages range from 28.99% to 29.63%, 22.28% to 22.78%, 15.73% to 16.28%, and 31.71% to 32.86% for nucleotides A, G, C, and T, respectively in all strains. The numbers of polymorphic sites as well as nucleotide diversity were highly similar in all strains. Statistical analyses generally showed the absence of high variations between sequences. Also, the phylogenetic tree classified strains into three groups. The network between strains of each group was established and discussed based on their geographical locations. Two groups contained strains from USA and Europe while one group contained strains from Asia. Rapid variations and mutations in the sequences of DWV were suggested. Notably, genetic studies on DWV are lacking in some geographical regions. The variations between strains detected in honey bees and other organisms were discussed. Four primers were designed and tested beside two reference primers. One of the designed primers showed the best results in binding with all DWV strains except one.     

Virus infections in Brazilian honey bees

This work describes the first molecular-genetic evidence for viruses in Brazilian honey bee samples. Three different bee viruses, Acute bee paralysis virus (ABPV), Black queen cell virus (BQCV), and Deformed wing virus (DWV) were identified during a screening of RNAs from 1920 individual adult bees collected in a region of southeastern Brazil that has recently shown unusual bee declines. ABPV was detected in 27.1% of colony samples, while BQCV and DWV were found in 37% and 20.3%, respectively. These levels are substantially lower than the frequencies found for these viruses in surveys from other parts of the world. We also developed and validated a multiplex RT-PCR assay for the simultaneous detection of ABPV, BQCV, and DWV in Brazil.     

Effect of 1,3-1,6 β-Glucan on Natural and Experimental Deformed Wing Virus Infection in Newly Emerged Honeybees (Apis mellifera ligustica)

The Western Honeybee is a key pollinator for natural as well as agricultural ecosystems. In the last decade massive honeybee colony losses have been observed worldwide, the result of a complex syndrome triggered by multiple stress factors, with the RNA virus Deformed Wing Virus (DWV) and the mite Varroa destructor playing crucial roles. The mite supports replication of DWV to high titers, which exert an immunosuppressive action and correlate with the onset of the disease.The aim of this study was to investigate the effect of 1,3–1,6 β-glucan, a natural innate immune system modulator, on honeybee response to low-titer natural and high-titer experimental DWV infection. As the effects exerted by ß-glucans can be remarkably different, depending on the target organism and the dose administered, two parallel experiments were performed, where 1,3–1,6 ß-glucan at a concentration of 0.5% and 2% respectively, was added to the diet of three cohorts of newly emerged honeybees, which were sampled from a Varroa-free apiary and harboured a low endogenous DWV viral titer. Each cohort was subjected to one of the following experimental treatments: no injection, injection of a high-copy number DWV suspension into the haemocel (experimental DWV infection) or injection of PBS into the haemocoel (physical injury). Control bees fed a ß-glucan-free diet were subjected to the same treatments. Viral load, survival rate, haemocyte populations and phenoloxidase activity of each experimental group were measured and compared. The results indicated that oral administration of 0.5% ß-glucan to naturally infected honeybees was associated with a significantly decrease of the number of infected bees and viral load they carried, and with a significant increase of the survival rate, suggesting that this natural immune modulator molecule might contribute to increase honeybee resistance to viral infection.     

Venereal and vertical transmission of deformed wing virus in honeybees (Apis mellifera L.)

Deformed wing virus (DWV) infected semen was used for artificial insemination of DWV-free virgin queens. High titres of DWV could subsequently be detected not only in the spermatheca, but also in the ovaries, demonstrating venereal transmission of DWV in honey bees. Subsequent vertical transmission of the virus to the progeny of DWV infected queens was also demonstrated. Neither transmission route is 100% effective. Whether venereal transmission of DWV occurs during natural mating remains to be determined. The implications for the use, sale and transport of semen samples for artificial insemination are discussed.     

Molecular and biological characterization of deformed wing virus of honeybees (Apis mellifera L.)

Deformed wing virus (DWV) of honeybees (Apis mellifera) is closely associated with characteristic wing deformities, abdominal bloating, paralysis, and rapid mortality of emerging adult bees. The virus was purified from diseased insects, and its genome was cloned and sequenced. The genomic RNA of DWV is 10,140 nucleotides in length and contains a single large open reading frame encoding a 328-kDa polyprotein. The coding sequence is flanked by a 1,144-nucleotide 5' nontranslated leader sequence and a 317-nucleotide 3' nontranslated region, followed by a poly(A) tail. The three major structural proteins, VP1 (44 kDa), VP2 (32 kDa), and VP3 (28 kDa), were identified, and their genes were mapped to the N-terminal section of the polyprotein. The C-terminal part of the polyprotein contains sequence motifs typical of well-characterized picornavirus nonstructural proteins: an RNA helicase, a chymotrypsin-like 3C protease, and an RNA-dependent RNA polymerase. The genome organization, capsid morphology, and sequence comparison data indicate that DWV is a member of the recently established genus Iflavirus.     

Changes of RNA virus infection rates and gut microbiota in young worker Apis mellifera (Hymenoptera: Apidae) of a chalkbrood-infected colony after a pollination task in a greenhouse

Western honey bee (Apis mellifera Linnaeus) populations recently have been in decline worldwide owing not only to colony collapse disorder but also other infectious diseases. The problem is neither decreasing nor has it been resolved. Chalkbrood (Ascosphaera apis, Maassen ex Claussen) is a well-known fungal brood disease that is now found throughout the world, and there are indications that the incidence of chalkbrood may be on the rise. Here, we conducted comparative studies to analyze infection rates of pathogenic RNA viruses and gut microbiota of young worker honey bees in two colonies: a healthy control colony raised in an open field and a test colony showing the chalkbrood symptom after a 2-month pollination task in a strawberry greenhouse. We found that the number of young worker bees with deformed wing virus (DWV) RNA was significantly elevated in the chalkbrood-infected colony, and two kinds of gut γ-proteobacteria, Frischella perrara gen. nov. and Pasteurellaceae bacterium Trm1, were especially increased in DWV-infected animals. These results showed that the DWV infection rates of worker honey bees were enhanced when their brood was infected with chalkbrood disease and that the gut microbiota in worker bees was significantly affected by the virus infection.