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Varroa mite, Varroa destructor Anderson and Trueman (Acari: Varroidae), causes severe damage to honeybee colonies and facilitates the transmission of several bee viruses, such as deformed wing virus (DWV), which causes wing deformity in newly molted bees. In Japan, western honeybees Apis mellifera Linnaeus (Hymenoptera: Apidae) are reared for both honey production and pollination and are infested with Varroa mites. Detailed surveillance of bee viruses in both A. melliferra and V. destructor has not been conducted in Japan. We surveyed eight major bee viruses in A. mellifera and Varroa mites from Japanese apiaries. DWV is the predominant virus in both A. mellifera and V. destructor in Japan. Two strains of the virus, DWV-A and DWV-B, are considered virulent in honeybees, but only DWV-A was detected in this study. The DWV levels in honeybees were strongly correlated with the Varroa infestation rate. We also detected other viruses in both the bees and mites, such as black queen cell and Lake Sinai viruses, but at much lower infection rates than DWV. Infection rates of these viruses were higher in colonies that did not contain Varroa mites than in Varroa-infested bee colonies. Therefore, Varroa mites in Japanese apiaries could be associated with DWV transmission, but they may rarely be associated with the transmission of other viruses. [ABSTRACT FROM AUTHOR]

Honeybees harbor a distinct core microbiota that plays a crucial role in stress tolerance and disease resistance. However, infections can significantly alter the composition and diversity of their gut microbiota, impacting overall bee health. This study investigates the effects of Deformed Wing Virus (DWV) infection on the gut microbiota of Apis cerana honeybee. The samples were collected from six colonies in Hanoi, Vietnam, and analyzed using high-throughput sequencing of the 16S rRNA gene. Our results show that while the overall diversity of gut microbiota in DWV-infected and uninfected bees did not significantly differ, notable changes were observed in the relative abundances of specific bacterial taxa. In DWV-infected bees, the relative abundance of Proteobacteria significantly decreased, whereas Firmicutes increased compared to uninfected bees. At the genus level, a significant decrease in Gilliamella and an increase in Lactobacillus were observed in infected bees. Functional gene predictions indicated that pathways related to carbohydrate, fatty acid, and lipid metabolism, as well as the biosynthesis of cofactors, vitamins, and amino acids, were upregulated in DWV-infected bees. These findings highlight the impact of DWV infection on the gut microbiota of A. cerana and suggest potential avenues for using probiotics to restore gut microbial balance and improve honeybee health. This research provides a foundation for developing strategies to enhance the resilience of honeybee colonies against viral infections. [ABSTRACT FROM AUTHOR]

Deformed wing virus (DWV) is known as one of the main viruses that affect honeybees' health all around the world. The virus has two widespread genotypes, DWV-A and DWV-B (VDV-1), transmitted mainly by V. destructor mites. In this study, we collected honeycombs with covered broods from 73 apiaries in eight Lithuanian regions and initially investigated the prevalence of V. destructor mites. Mites were collected from May to the end of July in 2021 from 124 hives. The prevalence of V. destructor infestations in beehives reached 30% and 63% in investigated apiaries. The presence of DWV-A and DWV-B pathogens in mites and broods was examined by RT-qPCR targeting the CRPV-capsid region. The molecular characterization of the virus in mite samples was based on sequence analysis of the RNA-dependent RNA polymerase (RdRp) region. In addition, leader polypeptide (LP), structural protein (Vp3), Helicase, and RdRp genes were used for phylogenetic characterization of dual infection. The prevalences of DWV-B in mites and broods were 56.5% and 31.5%, respectively, while DWV-A was detected in 12.9% of mite samples and 24.7% of brood samples. Some of the examined mite samples harboured dual virus infections. Our findings showed that bee colonies from the same apiary were not always infected by the same viruses. Some bee colonies were virus-free, while others were highly infected. Phylogenetic analysis of 21 sequences demonstrated the presence of highly variable DWV-B and DWV-A genotypes in Lithuania and possible recombinant variants of the virus. This study represents the first molecular characterization of mite-borne pathogens hosted by honeybees (Apis mellifera) in Lithuania. [ABSTRACT FROM AUTHOR]

Honey bees (Apis mellifera) play a crucial role in agriculture through their pollination activities. However, they have faced significant health challenges over the past decades that can limit colony performance and even lead to collapse. A primary culprit is the parasitic mite Varroa destructor, known for transmitting harmful bee viruses. Among these viruses is deformed wing virus (DWV), which impacts bee pupae during their development, resulting in either pupal demise or in the emergence of crippled adult bees. In this study, we focused on DWV master variant B. DWV-B prevalence has risen sharply in recent decades and appears to be outcompeting variant A of DWV. We generated a molecular clone of a typical DWV-B strain to compare it with our established DWV-A clone, examining RNA replication, protein expression, and virulence. Initially, we analyzed the genome using RACE-PCR and RT-PCR techniques. Subsequently, we conducted full-genome RT-PCR and inserted the complete viral cDNA into a bacterial plasmid backbone. Phylogenetic comparisons with available full-length sequences were performed, followed by functional analyses using a live bee pupae model. Upon the transfection of in vitro-transcribed RNA, bee pupae exhibited symptoms of DWV infection, with detectable viral protein expression and stable RNA replication observed in subsequent virus passages. The DWV-B clone displayed a lower virulence compared to the DWV-A clone after the transfection of synthetic RNA, as evidenced by a reduced pupal mortality rate of only 20% compared to 80% in the case of DWV-A and a lack of malformations in 50% of the emerging bees. Comparable results were observed in experiments with low infection doses of the passaged virus clones. In these tests, 90% of bees infected with DWV-B showed no clinical symptoms, while 100% of pupae infected with DWV-A died. However, at high infection doses, both DWV-A and DWV-B caused mortality rates exceeding 90%. Taken together, we have generated an authentic virus clone of DWV-B and characterized it in animal experiments. [ABSTRACT FROM AUTHOR]

Identifying traits for adaptation to different management and environmental regimes is key to maintaining robust honey bee populations under global climate change. We compared mite-resistant (Pol-line and Russian) and Italian honey bee stocks in variable-temperature cage experiments (200 bees per cage) with respect to food consumption, thermoregulation, gene expression, and lifespan, in 3 experiments over 2 years. The Italian stock bees consumed more syrup and pollen on average than the mite-resistant stocks, but the mite-resistant stocks maintained higher cluster temperatures and had median lifespans 8 days longer, consistent with the increased expression of vitellogenin relative to Italian stock. Model results indicated that, to maintain the same colony size as the mite-resistant stocks, Italian stock colonies would need about 13% more sealed brood to offset reduced worker lifespans. These differences among bee stocks likely influence colony-level productivity and health, and showed the importance of experimental replication. [ABSTRACT FROM AUTHOR]

Abstract The health of honey bee queens is crucial for colony success, particularly during stressful periods like overwintering. To accompany a previous longitudinal study of colony and worker health, we explored niche-specific gut microbiota, host gene expression, and pathogen prevalence in honey bee queens overwintering in a warm southern climate. We found differential gene expression and bacterial abundance with respect to various pathogens throughout the season. Biologically older queens had larger microbiotas, particularly enriched in Bombella and Bifidobacterium. Both Deformed Wing Virus A and B subtypes were highest in the fat body tissue in January, correlating with colony Varroa levels, and Deformed Wing Virus titers in workers. High viral titers in queens were associated with decreased vitellogenin expression, suggesting a potential trade-off between immune function and reproductive capacity. Additionally, we found a complex and dynamic relationship between these viral loads and immune gene expression, indicating a possible breakdown in the coordinated immune response as the season progressed. Our study also revealed a potential link between Nosema and Melissococcus plutonius infections in queens, demonstrating that seasonal opportunism is not confined to just workers. Overall, our findings highlight the intricate interplay between pathogens, metabolic state, and immune response in honey bee queens. Combined with worker and colony-level metrics from the same colonies, our findings illustrate the social aspect of queen health and resilience over the winter dearth.

The health of honey bee queens is crucial for colony success, particularly during stressful periods like overwintering. To accompany a previous longitudinal study of colony and worker health, we explored niche-specific gut microbiota, host gene expression, and pathogen prevalence in honey bee queens overwintering in a warm southern climate. We found differential gene expression and bacterial abundance with respect to various pathogens throughout the season. Biologically older queens had larger microbiotas, particularly enriched in Bombella and Bifidobacterium. Both Deformed Wing Virus A and B subtypes were highest in the fat body tissue in January, correlating with colony Varroa levels, and Deformed Wing Virus titers in workers. High viral titers in queens were associated with decreased vitellogenin expression, suggesting a potential trade-off between immune function and reproductive capacity. Additionally, we found a complex and dynamic relationship between these viral loads and immune gene expression, indicating a possible breakdown in the coordinated immune response as the season progressed. Our study also revealed a potential link between Nosema and Melissococcus plutonius infections in queens, demonstrating that seasonal opportunism is not confined to just workers. Overall, our findings highlight the intricate interplay between pathogens, metabolic state, and immune response in honey bee queens. Combined with worker and colony-level metrics from the same colonies, our findings illustrate the social aspect of queen health and resilience over the winter dearth. [ABSTRACT FROM AUTHOR]

Jalisco is one of the foremost honey-producing states in Mexico. However, there is no information on viral diseases that affect honey bees (Apis mellifera) in the different beekeeping regions of the state. The objective of this study was to determine the prevalence and intensity of four viral diseases of Apis mellifera during the spring, in six regions of Jalisco. Bee samples from 79 colonies were analyzed, of which, 66 % and 38 % were positive for black queen cell virus (BQCV) and deformed wing virus (DWV), respectively. Two viral diseases were not detected, those caused by the Israeli acute paralysis virus (IAPV) and the chronic bee paralysis virus (CBPV). The infection levels of BQCV were relatively low but elevated for DWV, with infection intensities 8,000 higher than those of BQCV. The prevalence of DWV was significantly higher in the regions of the Highlands, Center, and South, while for BQCV there were no differences between regions. For infection intensity, there were no differences between regions for DWV, but there were for BQCV. The regions with the highest infection levels were the South and Center. Surveys during other seasons of the year are recommended to identify possible seasonal viral effects on the bees and to design control strategies. [ABSTRACT FROM AUTHOR]

Colonies of the western honey bee, Apis mellifera L., are comprised of tens of thousands of genetically related individuals that can easily share and spread pathogens. Honey bee colonies exhibit variations in disease susceptibility that can translate into highly variable pathogen replication among individuals within a colony, between colonies, and between stocks. Here, we investigate the degree of variation in viral titers of common honey bee viruses (DWV-A, DWV-B, IAPV, LSV2, BQCV) within colonies and between colonies of five honey bee stocks in the United States. Our results showed high intra-colony variation in DWV-A, DWV-B, IAPV, and LSV2, but not in BQCV titers. However, the level of variation was not consistent across stocks. Here, we empirically demonstrate that there is significant intra-colony variation in viral titers and that some stocks are more prone to this variability. Our results highlight the need for further studies comparing virus susceptibility among stocks. [ABSTRACT FROM AUTHOR]

Deformed wing virus (DWV) is known as one of the main viruses that affect honeybees’ health all around the world. The virus has two widespread genotypes, DWV-A and DWV-B (VDV-1), transmitted mainly by V. destructor mites. In this study, we collected honeycombs with covered broods from 73 apiaries in eight Lithuanian regions and initially investigated the prevalence of V. destructor mites. Mites were collected from May to the end of July in 2021 from 124 hives. The prevalence of V. destructor infestations in beehives reached 30% and 63% in investigated apiaries. The presence of DWV-A and DWV-B pathogens in mites and broods was examined by RT-qPCR targeting the CRPV-capsid region. The molecular characterization of the virus in mite samples was based on sequence analysis of the RNA-dependent RNA polymerase (RdRp) region. In addition, leader polypeptide (LP), structural protein (Vp3), Helicase, and RdRp genes were used for phylogenetic characterization of dual infection. The prevalences of DWV-B in mites and broods were 56.5% and 31.5%, respectively, while DWV-A was detected in 12.9% of mite samples and 24.7% of brood samples. Some of the examined mite samples harboured dual virus infections. Our findings showed that bee colonies from the same apiary were not always infected by the same viruses. Some bee colonies were virus-free, while others were highly infected. Phylogenetic analysis of 21 sequences demonstrated the presence of highly variable DWV-B and DWV-A genotypes in Lithuania and possible recombinant variants of the virus. This study represents the first molecular characterization of mite-borne pathogens hosted by honeybees (Apis mellifera) in Lithuania.

Simple Summary: The aim of the study was to evaluate the viral load in honey bee colonies after adopting two brood interruption techniques that are used to control varroa mite. We evaluated the efficacy of two integrated pest management (IPM) strategies, "Queen Caging" (QC) and "Trapping Comb" (TC) procedures, in conjunction with an oxalic acid treatment, to control varroa infestations and consequently lower the viral loads of Deformed Wing Virus (DWV) and Acute Bee Paralysis Virus (ABPV). Two distinct apiaries in Slovenia and Italy, each with a different climate, served as the research sites. In the experiment, the adult bee viral load, mite fall, colony strength, and acaricide efficiency were assessed. The study indicated that the TC approach might be more successful in lowering viral loads. Our results also showed that the acaricidal efficacy of the applied IPM protocols is high. Our study is the first attempt to assess viral infections in honey bees after IPM adoption. The results show the potential advantages of using targeted varroa treatments in combination with brood interruption strategies to manage honey bee viruses vectored by varroa mite. Honey bee viruses in combination with varroa mite are very damaging for honey bee colonies worldwide. There are no effective methods to control the viral load in honey bee colonies except regular and effective control of mites. Integrated Pest Management strategies are required to effectively control mites with veterinary medicines based on organic compounds. We evaluated the effect of two brood interruption techniques, queen caging (QC) and trapping comb (TC), followed by an oxalic acid treatment, on the mite fall, colony strength, and viral load of Deformed Wing Virus (DWV) and Acute Bee Paralysis Virus (ABPV). In this paper, we report the data obtained in two experimental sites, in Slovenia and Italy, in terms of the varroacide efficacy, colony strength, and viral load. The number of adult bees after the adoption of the two techniques showed similar decreasing trends in both locations. The viral load of Acute Bee Paralysis Virus did not show any significant reduction after 25 days, reported as the number of Real-Time PCR cycles needed to detect the virus. The viral load of DWV also did not show a significant reduction after 25 days. The acaricidal efficacy of the applied protocols was high in both experimental groups and in both apiaries. Both the queen caging and trapping comb techniques, followed by an oxalic acid treatment, can be considered effective varroa treatment strategies, but further studies should be carried out to evaluate the long-term effects on viral loads to plan the Integrated Pest Management strategy with the right timing before wintering. [ABSTRACT FROM AUTHOR]

Simple Summary: Recently, there has been a serious decline in honey bee colonies due to various factors, including habitat loss, diseases, pathogen infections, chemical exposures, and climatic changes. Among these factors, malnutrition-induced stress plays a significant role in colony decline. In our study, we present a novel perspective on maintaining and developing honey bee colonies. Our research demonstrates that a specific pollen substitute diet significantly improves colony performance during early spring, as evidenced by an increase in population, a greater capped brood area, altered colony weight, honey bee weight (dried head + thorax), and vitellogenin value. This finding could contribute to enhanced colony maintenance and development during the early spring season, with the potential to prevent colony decline. The nutritional quality of a colony significantly affects its health and strength, particularly because it is required for population growth in the early spring. We investigated the impact of various artificial pollen substitute diets on colony performance in the Republic of Korea during early spring, a critical period for colony health and growth. The colonies were provided with different diets, including the commercial product Megabee (positive control), our developed diet Test A, and four upgraded versions (Diet 1, Diet 2, Diet 3, and Diet 4) of Test A. The negative control group received no supplementary feed. Over 63 days, we observed 24 experimental colonies and assessed various parameters at the colony and individual levels. The results revealed that Diet 2 had the highest consumption and had the most positive impact on population growth, the capped brood area, colony weight, honey bees' weight, and vitellogenin levels. These findings suggested that Diet 2 is most attractive to honey bees and thus holds great promise for improving colony maintenance and development during the crucial early spring period. [ABSTRACT FROM AUTHOR]

Viruses are one of many serious threats to honey bee (Apis mellifera L.) health. There are many transmission routes for honey bee viruses, and there is potential for wax comb to act as a reservoir for transmission of viruses. Some work has been done on treating viruses on wax, focusing on irradiation as a potential treatment. However, irradiation is not universally available or economically viable for beekeepers in many regions. With increased colony deaths over winter beekeepers potentially risk further loss from reusing contaminated equipment from dead colonies. Here we explored the use of storage time and temperature on the reduction of waxborne virus levels from winter loss colony wax over 30 days and at –20, 5, and 20 °C. Furthermore, because irradiation has previously worked against waxborne viruses, we performed a dosage experiment with electron-beam irradiation. Winter loss wax was again used, and exposed to 10, 25, 35, and 45 kGy irradiation, including a nonirradiated transport control. Storage time decreased abundance of black queen cell virus and deformed wing virus at times equal or greater than 30 days but temperatures had no significant effect on virus levels. All irradiation doses decreased virus abundance and prevalence, yet only 35 and 45 kGy did so at a greater rate than the effect of transport alone. Graphical Abstract [ABSTRACT FROM AUTHOR]

The deformed wing virus (DWV) belongs to the genus Iflavirus and the family Iflaviridae within the order Picornavirales. It is an important pathogen of the Western honey bee, Apis mellifera, causing major losses among honey bee colonies in association with the ectoparasitic mite Varroa destructor. Although DWV is one of the best-studied insect viruses, the mechanisms of viral replication and polyprotein processing have been poorly studied in the past. We investigated the processing of the protease-polymerase region at the C-terminus of the polyprotein in more detail using recombinant expression, novel serological reagents, and virus clone mutagenesis. Edman degradation of purified maturated polypeptides uncovered the C- and N-termini of the mature 3C-like (3CL) protease and RNA-dependent RNA polymerase (3DL, RdRp), respectively. Autocatalytic processing of the recombinant DWV 3CL protease occurred at P1 Q2118 and P1′ G2119 (KPQ/GST) as well as P1 Q2393 and P1′ S2394 (HAQ/SPS) cleavage sites. New monoclonal antibodies (Mab) detected the mature 3CL protease with an apparent molecular mass of 32 kDa, mature 3DL with an apparent molecular mass of 55 kDa as well as a dominant 3CDL precursor of 90 kDa in DWV infected honey bee pupae. The observed pattern corresponds well to data obtained via recombinant expression and N-terminal sequencing. Finally, we were able to show that 3CL protease activity and availability of the specific protease cleavage sites are essential for viral replication, protein synthesis, and establishment of infection using our molecular clone of DWV-A. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Honey bee viruses have been shown to negatively affect the vigour and longevity of European honey bees (Apis mellifera L). In the present work, beehive materials were tested for their potential to serve as non-invasive samples for honey bee virus detection.

Honey bees (Apis mellifera) play a crucial role in agriculture through their pollination activities. However, they have faced significant health challenges over the past decades that can limit colony performance and even lead to collapse. A primary culprit is the parasitic mite Varroa destructor, known for transmitting harmful bee viruses. Among these viruses is deformed wing virus (DWV), which impacts bee pupae during their development, resulting in either pupal demise or in the emergence of crippled adult bees. In this study, we focused on DWV master variant B. DWV-B prevalence has risen sharply in recent decades and appears to be outcompeting variant A of DWV. We generated a molecular clone of a typical DWV-B strain to compare it with our established DWV-A clone, examining RNA replication, protein expression, and virulence. Initially, we analyzed the genome using RACE-PCR and RT-PCR techniques. Subsequently, we conducted full-genome RT-PCR and inserted the complete viral cDNA into a bacterial plasmid backbone. Phylogenetic comparisons with available full-length sequences were performed, followed by functional analyses using a live bee pupae model. Upon the transfection of in vitro-transcribed RNA, bee pupae exhibited symptoms of DWV infection, with detectable viral protein expression and stable RNA replication observed in subsequent virus passages. The DWV-B clone displayed a lower virulence compared to the DWV-A clone after the transfection of synthetic RNA, as evidenced by a reduced pupal mortality rate of only 20% compared to 80% in the case of DWV-A and a lack of malformations in 50% of the emerging bees. Comparable results were observed in experiments with low infection doses of the passaged virus clones. In these tests, 90% of bees infected with DWV-B showed no clinical symptoms, while 100% of pupae infected with DWV-A died. However, at high infection doses, both DWV-A and DWV-B caused mortality rates exceeding 90%. Taken together, we have generated an authentic virus clone of DWV-B and characterized it in animal experiments.

Invasive vectors can induce dramatic changes in disease epidemiology. While viral emergence following geographical range expansion of a vector is well known, the influence a vector can have at the level of the host's pathobiome is less well understood. Taking advantage of the formerly heterogeneous spatial distribution of the ectoparasitic mite Varroa destructor that acts as potent virus vector among honeybees Apis mellifera, we investigated the impact of its recent global spread on the viral community of honeybees in a retrospective study of historical samples. We hypothesized that the vector has had an effect on the epidemiology of several bee viruses, potentially altering their transmissibility and/or virulence, and consequently their prevalence, abundance, or both. To test this, we quantified the prevalence and loads of 14 viruses from honeybee samples collected in mite-free and mite-infested populations in four independent geographical regions. The presence of the mite dramatically increased the prevalence and load of deformed wing virus, a cause of unsustainably high colony losses. In addition, several other viruses became more prevalent or were found at higher load in mite-infested areas, including viruses not known to be actively varroa-transmitted, but which may increase opportunistically in varroa-parasitized bees.

The Varroa destructor mite is a devastating parasite of honey bees; however the negative effects of varroa parasitism are exacerbated by its role as an efficient vector of the honey bee pathogen, Deformed wing virus (DWV). While no direct treatment for DWV infection is available for beekeepers to use on their hives, RNA interference (RNAi) has been widely explored as a possible biopesticide approach for a range of pests and pathogens. This study tested the effectiveness of three DWV-specific dsRNA sequences to lower DWV loads and symptoms in honey bees reared from larvae in laboratory mini-hives containing bees and varroa. The effects of DWV-dsRNA treatment on bees parasitised and non-parasitised by varroa mites during development were investigated. Additionally, the impact of DWV-dsRNA on viral loads and gene expression in brood-parasitising mites was assessed using RNA-sequencing. Bees parasitised during development had significantly higher DWV levels compared to non-parasitised bees. However, DWV-dsRNA did not significantly reduce DWV loads or symptoms in mini-hive reared bees, possibly due to sequence divergence between the DWV variants present in bees and varroa and the specific DWV-dsRNA sequences used. Varroa mites from DWV-dsRNA treated mini-hives did not show evidence of an elevated RNAi response or significant difference in DWV levels. Overall, our findings show that RNAi is not always successful, and multiple factors including pathogen diversity and transmission route may impact its efficiency. [ABSTRACT FROM AUTHOR]

European foulbrood (EFB) is a severe disease of honey bee (Apis mellifera) larvae caused by the bacterium Linnaeus [Hymenoptera: Apidae]) Melissococcus plutonius (ex White) Bailey and Collins (Lactobacillales: Enterococcaceae). Many beekeepers in North America report severe EFB following blueberry pollination, but it is not clear what factors during pollination are related to clinical disease. Additionally, the impact that other factors such as viral load and hygienic behavior have on EFB has not been studied. In Spring of 2020 we enrolled 60 commercial honey bee colonies in a prospective cohort study. Colonies were inspected 3 times over the season with hive metrics and samples taken for viral testing. Each colony was tested for hygienic behavior twice and the score was averaged. Viral loads were determined by qPCR for deformed wing virus (DWV) A and B. We found no statistical difference in the EFB prevalence or severity between the 2 yards at any timepoint; 50% (n  = 16) of the colonies in the holding yard and 63% (n  = 17) in blueberry developed moderate to severe EFB over the study period. When colonies from both yards were pooled, we found no relationship between viral load or hygienic behavior and development of EFB. These results suggest that other factors may be responsible for driving EFB virulence and hygienic behavior is not likely helpful in managing this disease. [ABSTRACT FROM AUTHOR]

Viruses are responsible for several diseases in animals and have also been linked to the drastic decrease in the number of bee hives worldwide. Considering the necessity to diagnose honey bee viruses, we developed and standardized two multiplex RT-PCRs. We evaluated the presence of six viruses described in Africanized Apis mellifera bees: Deformed wing virus (DWV), Sacbrood bee virus (SBV), Acute bee paralysis virus (ABPV), Israeli acute paralysis virus (IAPV), Black queen cell virus (BQCV), and Chronic bee paralysis virus (CBPV). Therefore, 164 samples of honey bees were collected in 27 apiaries of 12 municipalities in the states of Rio Grande do Sul and Santa Catarina, in southern Brazil. Among the 12 municipalities evaluated, five presented positive samples for BQCV, ABPV and DWV. Cases of co-infection by ABPV and BQCV and BQCV and DWV were observed in both states. In all apiaries with positive samples for viruses, Varroa destructor mite was reported or found. These results demonstrate the high occurrence of BQCV (36%) and the presence of ABPV (3%) and DWV (1%) in apiaries in Rio Grande do Sul, as well as in an apiary in Santa Catarina (75% BQCV and 12% DWV). The results confirm the circulation of these viruses in adult Africanized honey bees in southern Brazil, a factor that may be involved with the decline in the number of honey bees in this region of the country. [ABSTRACT FROM AUTHOR]

Understanding the ecological and evolutionary processes that drive host–pathogen interactions is critical for combating epidemics and conserving species. The Varroa destructor mite and deformed wing virus (DWV) are two synergistic threats to Western honeybee (Apis mellifera) populations across the globe. Distinct honeybee populations have been found to self-sustain despite Varroa infestations, including colonies within the Arnot Forest outside Ithaca, NY, USA. We hypothesized that in these bee populations, DWV has been selected to produce an avirulent infection phenotype, allowing for the persistence of both host and disease-causing agents. To investigate this, we assessed the titre of viruses in bees from the Arnot Forest and managed apiaries, and assessed genomic variation and virulence differences between DWV isolates. Across groups, we found viral abundance was similar, but DWV genotypes were distinct. We also found that infections with isolates from the Arnot Forest resulted in higher survival and lower rates of symptomatic deformed wings, compared to analogous isolates from managed colonies, providing preliminary evidence to support the hypothesis of adaptive decreased viral virulence. Overall, this multi-level investigation of virus genotype and phenotype indicates that host ecological context can be a significant driver of viral evolution and host–pathogen interactions in honeybees. [ABSTRACT FROM AUTHOR]

Deformed wing virus (DWV) is one of the important pathogens of the honey bee (Apis mellifera), which consists of three master variants: types A, B, and C. Among them, DWV types A (DWV-A) and B (DWV-B) are the most prevalent variants in honey bee colonies and have been linked to colony decline. DWV-A and DWV-B have different virulence, but it is difficult to distinguish them via traditional methods. In this study, we established a visual detection assay for DWV-A and DWV-B using recombinase polymerase amplification (RPA) and a lateral flow dipstick (LFD) coupled with the clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein (Cas) 12a fluorescence system (RPA–CRISPR–Cas12a–LFD). The limit of detection of this system was ~6.5 × 100 and 6.2 × 101 copies/μL for DWV-A and DWV-B, respectively. The assays were specific and non-cross-reactive against other bee viruses, and the results could be visualized within 1 h. The assays were validated by extracting cDNA from 36 clinical samples of bees that were suspected to be infected with DWV. The findings were consistent with those of traditional reverse transcription–quantitative polymerase chain reaction, and the RPA–CRISPR–Cas12a assay showed the specific, sensitive, simple, and appropriate detection of DWV-A and DWV-B. This method can facilitate the visual and qualitative detection of DWV-A and DWV-B as well as the monitoring of different subtypes, thereby providing potentially better control and preventing current and future DWV outbreaks. [ABSTRACT FROM AUTHOR]

European honeybees (Apis mellifera L.) were first imported to Australia in the 1820s. The Queensland Beekeepers Association Inc was founded by 13 beekeepers in 1886 to promote beekeeping education and bee biosecurity. Between 2016 and 2023, the number of registered beekeepers in Queensland nearly tripled from approximately 3,000 to well over 9,000. This trend has been mainly attributed to technological changes making beekeeping more accessible to recreational beekeepers. The dramatic increase in new beekeepers is seen by commercial beekeepers as a potential source of increased biosecurity risk. But considering commercial beekeepers still manage over 75% of the 115,000 hives in Queensland, and their apiaries are far more mobile, which type of beekeeper presents the greater risk of spreading undetected pests and diseases? Although Australia remains free of many bee pests and diseases, the industry is constantly under threat. Recent incursions of Varroa destructor (Anderson and Trueman) in New South Wales, Varroa jacobsoni (Oudemans) and Apis cerana (Fabricius) in northern Queensland, braula fly (Braula coeca Nitzsch) in Victoria and New South Wales, and the regular detection of exotic bee swarms at Australian ports, all highlight the need for vigilance. Despite successes in eradicating or containing the spread of previous exotic pest incursions, many challenges remain, including sustaining local research, and developing and maintain high standards of bee biosecurity practice through programs like the National Bee Biosecurity Program (NBBP). Future biosecurity challenges are discussed within the context of the current and future needs of the industry. [ABSTRACT FROM AUTHOR]

Simple Summary: Mexico is an important honey producer, but not much information exists about the health of honey bees in the country. This study analyzed the sanitary status of adult honey bees in five different beekeeping regions of Mexico. Samples from hives were analyzed to identify pests, parasites, and viruses. The mite Varroa destructor was found in 83.5% of the samples, with the Pacific Coast having the highest frequency (>95%) and levels (4.5% ± 0.6). Another mite, Acarapis woodi, was found in only one sample from the Pacific Coast, whereas the fungi Nosema spp. were present in 48.5% of the samples, with the highest frequency in the Yucatan Peninsula (64.6%). For viruses, deformed wing virus (DWV) was more frequently found in the Pacific Coast region (44.7%), and Israeli acute paralysis virus (IAPV) was detected in only 3.2% of the samples. Sacbrood bee virus (SBV) was frequently found in the High Plateau region (36.4%), and the small hive beetle (SHB) was common in the Yucatan Peninsula (39.2%). This information could be useful to design disease control strategies for honey bee colonies in different regions of Mexico. Mexico is a major honey producer, but not much information exists about the health status of honey bees (Apis mellifera L.) in the country. This study was conducted to determine the sanitary status of adult honey bees in Mexico's five beekeeping regions. Samples from 369 apiaries were diagnosed to identify pathogens such as Varroa destructor, which was quantified, Acarapis woodi, Nosema spp., and five viruses. Colonies were also inspected for the presence of the small hive beetle (SHB), Aethina tumida. Varroa destructor was found in 83.5% of the apiaries, with the Pacific Coast region having the highest prevalence (>95%) and rates (4.5% ± 0.6). Acarapis woodi was detected in only one apiary from the Pacific Coast, whereas Nosema spp. were prevalent in 48.5% of the apiaries, with the highest and lowest frequencies in the Yucatan Peninsula and North regions (64.6% and 10.2%, respectively). For viruses, deformed wing virus (DWV) was detected in 26.1% of the apiaries, with the highest frequency in the Pacific Coast region (44.7%). Israeli acute paralysis virus (IAPV) was diagnosed in 3.2% of the samples and sacbrood bee virus (SBV) in 23.3% of them, with the highest frequency in the High Plateau region (36.4%). Chronic bee paralysis and Kashmir bee viruses were not detected. SHB prevalence was 25.2% nationwide, with the highest frequency in the Yucatan Peninsula (39.2%). This study shows that the most common parasites of adult honey bees in Mexico are V. destructor and Nosema spp., and that the most prevalent virus is DWV, whereas SHB is highly prevalent in the Yucatan Peninsula. This information could be useful to design disease control strategies for honey bee colonies in different regions of Mexico. [ABSTRACT FROM AUTHOR]

Pollen is an essential component of bee diets, and rearing bumble bees (Bombus spp.) for commercial use necessitates feeding pollen in mass quantities. This pollen is collected from honey bee (Apis mellifera L.) colonies because neither an artificial diet nor an economical, large-scale pollen collection process from flowers is available. The provenance of honey bee-collected pollen is often unknown, and in some cases has crossed international borders. Both deformed wing virus (DWV) and the fungal pathogen Ascosphaera apis (Claussen) Olive & Spiltoir (cause of chalkbrood disease); occur in honey bee-collected pollen, and infections have been observed in bumble bees. We used these pathogens as general surrogates for viruses and spore-forming fungal diseases to test the efficacy of 3 sterilization methods, and assessed whether treatment altered pollen quality for the bumble bee. Using honey bee-collected pollen spiked with known doses of DWV and A. apis , we compared gamma irradiation (GI), ozone fumigation (OZ), and ethylene oxide fumigation (EO) against an untreated positive control and a negative control. Following sterilization treatments, we tested A. apis spore viability, detected viral presence with PCR, and tested palatability to the bumble bee Bombus impatiens Cresson. We also measured bacterial growth from pollens treated with EO and GI. GI and EO outperformed OZ treatment in pathogen suppression. EO had the highest sterilizing properties under commercial conditions and retained palatability and supported bee development better than other treatments. These results suggest that EO sterilization reduces pathogen risks while retaining pollen quality as a food source for rearing bumble bees. [ABSTRACT FROM AUTHOR]

Spillover of honey bee viruses have posed a significant threat to pollination services, triggering substantial effort in determining the host range of the viruses as an attempt to understand the transmission dynamics. Previous studies have reported infection of honey bee viruses in ants, raising the concern of ants serving as a reservoir host. Most of these studies, however, are restricted to a single, local ant population. We assessed the status (geographical distribution/prevalence/viral replication) and phylogenetic relationships of honey bee viruses in ants across the Asia-Pacific region, using deformed wing virus (DWV) and two widespread invasive ants, Paratrechina longicornis and Anoplolepis gracilipes, as the study system. DWV was detected in both ant species, with differential geographical distribution patterns and prevenance levels between them. These metrics, however, are consistent across the geographical range of the same ant species. Active replication was only evident in P. longicornis. We also showed that ant-associated DWV is genetically similar to that isolated from Asian populations of honey bees, suggesting that local acquisition of DWV by the invasive ants may have been common at least in some of our sampled regions. Transmission efficiency of DWV to local arthropods mediated by ant, however, may vary across ant species.

Abstract Background Varroa destructor is the major ectoparasite of the western honey bee (Apis mellifera). Through both its parasitic life-cycle and its role as a vector of viral pathogens, it can cause major damage to honey bee colonies. The deformed wing virus (DWV) is the most common virus transmitted by this ectoparasite, and the mite is correlated to increased viral prevalence and viral loads in infested colonies. DWV variants A and B (DWV-A and DWV-B, respectively) are the two major DWV variants, and they differ both in their virulence and transmission dynamics. Methods We studied the transmission of DWV between bees, parasitic mites and their offspring by quantifying DWV loads in bees and mites collected in in vitro and in situ environments. In vitro, we artificially transmitted DWV-A to mites and quantified both DWV-A and DWV-B in mites and bees. In situ, we measured the natural presence of DWV-B in bees, mites and mites’ offspring. Results Bee and mite viral loads were correlated, and mites carrying both variants were associated with higher mortality of the infected host. Mite infestation increased the DWV-B loads and decreased the DWV-A loads in our laboratory conditions. In situ, viral quantification in the mite offspring showed that, after an initially non-infected egg stage, the DWV-B loads were more closely correlated with the foundress (mother) mites than with the bee hosts. Conclusions The association between mites and DWV-B was highlighted in this study. The parasitic history of a mite directly impacts its DWV infection potential during the rest of its life-cycle (in terms of variant and viral loads). Regarding the mite’s progeny, we hypothesize that the route of contamination is likely through the feeding site rather than by vertical transmission, although further studies are needed to confirm this hypothesis. Graphical Abstract

Large-scale honey bee colony losses reported around the world have been associated with intoxication with pesticides, as with the presence of pests and pathogens. Among pesticides, neonicotinoid insecticides are the biggest threat. Due to their extensive use, they can be found in all agricultural environments, including soil, water, and air, are persistent in the environment, and are highly toxic for honey bees. In addition, infection by different pests and pathogens can act synergistically, weakening bees. In this study, we investigated the effects of chronic exposure to sublethal doses of imidacloprid alone or combined with the microsporidia Nosema ceranae on the immune response, deformed wing virus infection (DWV), gut microbiota, and survival of Africanized honey bees. We found that imidacloprid affected the expression of some genes associated with immunity generating an altered physiological state, although it did not favor DWV or N. ceranae infection. The pesticide alone did not affect honey bee gut microbiota, as previously suggested, but when administered to N. ceranae infected bees, it generated significant changes. Finally, both stress factors caused high mortality rates. Those results illustrate the negative impact of imidacloprid alone or combined with N. ceranae on Africanized honey bees and are useful to understand colony losses in Latin America. [ABSTRACT FROM AUTHOR]

Deformed wing virus (DWV) is a pathogenic virus of honey bees transmitted by the ectoparasitic mite Varroa destructor. Annual overwintering colony losses, accounting for ~25% of all colonies, are associated with high levels of Varroa‐DWV infestation. Effective miticide treatments are available to control Varroa. However, the absence of coordinated treatment means environmental transmission of mites continues unchecked. We aimed to determine whether rational, coordinated treatment is beneficial, and characterized the DWV population as an indicator of colony health.This study uses coordinated treatment of Varroa in a geographically isolated environment (Isle of Arran, Scotland) over 3 years. The study area contained 50–84 colonies managed by ~20 amateur beekeepers. Sampling and virus analysis to assess strain diversity and viral loads were conducted before and after treatments, and changes in population diversity were quantified by sequence analysis.Over the 3 years analysis of the virus population revealed that the dominant DWV variant shifted from Type A to Type B in all apiaries, regardless of mite levels or proximity to other colonies. During this period the number of managed colonies increased by 47% (57–84 colonies), but despite this, we estimate total mite numbers decreased by 58%.Synthesis and applications. In this study, the beekeepers in Arran significantly improved the number of colonies they managed, without importing any bees onto the island, indicating that an improved focus on management techniques, through the combination of a coordinated miticide programme and an improved understanding of bee diseases, could yield positive results for bee health and sustainability. [ABSTRACT FROM AUTHOR]

Simple Summary: Globally, honey bees are exposed to many challenges, such as the Varroa destructor mite and various viruses, which lead to massive losses. It is generally believed that African honey bees are more resilient and better able to cope with these stressors. This study examined some molecular markers that may be associated with this resilience. Higher resistance to the varroa mite could be related to better olfactory sensing. Higher gene expression levels of the odorant binding protein OBP14 in the antennae of Ethiopian honey bees suggest that reproducing mites might be better detected and cleaned. Resistance or tolerance to viruses could be attributed to a better functioning antiviral RNAi system. Several genes involved in this pathway are upregulated and are positively correlated with the viral load in honey bees. Both mechanisms may contribute to the resilience of African bees to varroa infestation and viral infection. Varroa destructor mites and the viruses it vectors are two major factors leading to high losses of honey bees (Apis mellifera) colonies worldwide. However, honey bees in some African countries show resilience to varroa infestation and/or virus infections, although little is known about the mechanisms underlying this resilience. In this study, we investigated the expression profiles of some key molecular markers involved in olfactory sensing and RNA interference, as these processes may contribute to the bees' resilience to varroa infestation and virus infection, respectively. We found significantly higher gene expression of the odorant binding protein, OBP14, in the antennae of Ethiopian bees compared to Belgian bees. This result suggests the potential of OBP14 as a molecular marker of resilience to mite infestation. Scanning electron microscopy showed no significant differences in the antennal sensilla occurrence and distribution, suggesting that resilience arises from molecular processes rather than morphological adaptations. In addition, seven RNAi genes were upregulated in the Ethiopian honey bees and three of them—Dicer-Drosha, Argonaute 2, and TRBP2—were positively correlated with the viral load. We can conclude that the antiviral immune response was triggered when bees were experiencing severe viral infection and that this might contribute to the bees' resilience to viruses. [ABSTRACT FROM AUTHOR]

Honey bee viruses in combination with varroa mite are very damaging for honey bee colonies worldwide. There are no effective methods to control the viral load in honey bee colonies except regular and effective control of mites. Integrated Pest Management strategies are required to effectively control mites with veterinary medicines based on organic compounds. We evaluated the effect of two brood interruption techniques, queen caging (QC) and trapping comb (TC), followed by an oxalic acid treatment, on the mite fall, colony strength, and viral load of Deformed Wing Virus (DWV) and Acute Bee Paralysis Virus (ABPV). In this paper, we report the data obtained in two experimental sites, in Slovenia and Italy, in terms of the varroacide efficacy, colony strength, and viral load. The number of adult bees after the adoption of the two techniques showed similar decreasing trends in both locations. The viral load of Acute Bee Paralysis Virus did not show any significant reduction after 25 days, reported as the number of Real-Time PCR cycles needed to detect the virus. The viral load of DWV also did not show a significant reduction after 25 days. The acaricidal efficacy of the applied protocols was high in both experimental groups and in both apiaries. Both the queen caging and trapping comb techniques, followed by an oxalic acid treatment, can be considered effective varroa treatment strategies, but further studies should be carried out to evaluate the long-term effects on viral loads to plan the Integrated Pest Management strategy with the right timing before wintering.

Insect wing deformities can be caused by various factors, including the Deformed wing virus (DWV). Symptomatic and asymptomatic specimens of the introduced solitary wild bees Megachile sculpturalis and Megachile disjunctiformis collected at eleven Central European locations were screened for DWV infections. Even though virus spillover is common, and DWV was detected in other bee species collected in the same habitat as Megachile spp., neither DWV-A nor DWV-B were found in any of the samples (N = 54) including two symptomatic males with deformed wings. This indicates that other stressors were responsible for the observed clinical symptoms, thereby highlighting the necessity of differential diagnostics. [ABSTRACT FROM AUTHOR]

The small hive beetle (SHB), Aethina tumida Murray, is a parasite of honey bee colonies and causes the fermentation of honey as well as colony collapse. Outbreaks have been confirmed in Africa, America, Europe as well as Asia, where an outbreak was reported in the Philippines and South Korea in 2014 and 2017, respectively. In South Korea, in September 2016, this honey bee parasite was first identified in apiaries in Miryang, Gyeongnam Province. However, the invasion pathway of SHB has not been identified, and honey bee pathogens harbored by SHB have not been well characterized. Therefore, phylogenetic analysis of SHB with the use of COI gene and detection of fourteen common honey bee pathogens were conducted in this study. The confirmation of the fourteen honey bee pathogens in SHB showed that this beetle carries black queen cell virus and deformed wing virus. Therefore, SHB could have a role in the spread of these viruses. The way of entry of the SHB to South Korea remains undetermined, but the phylogenetic analysis of the COI gene revealed that it was most similar to species found in the USA. There is an urgent need for national-level monitoring and quarantine measures for preventing the spread of SHB infestation in South Korea.

Introduction: Deformed wing virus (DWV) is one of the causative agents of colony collapse disorder. The structural protein of DWV plays a vital role in the process of viral invasion and host infection; however, there is limited research on DWV. Methods and Results: In this study, we screened the host protein snapin, which can interact with the VP2 protein of DWV, using the yeast two-hybrid system. Through computer simulation and GST pull-down and CO-IP assays, an interaction between snapin and VP2 was confirmed. Furthermore, immunofluorescence and co-localization experiments revealed that VP2 and snapin primarily co-localized in the cytoplasm. Consequently, RNAi was used to interfere with the expression of snapin in worker bees to examine the replication of DWV after the interference. After silencing of snapin, the replication of DWV in worker bees was significantly downregulated. Hence, we speculated that snapin was associated with DWV infection and involved in at least one stage of the viral life cycle. Finally, we used an online server to predict the interaction domains between VP2 and snapin, and the results indicate that the interaction domain of VP2 was approximately located at 56-90, 136-145, 184-190, and 239-242 aa and the snapin interaction domain was approximately located at 31-54 and 115-136 aa. Conclusion: This research confirmed that DWV VP2 protein could interacts with the snapin of host protein, which provides a theoretical basis for further investigation of its pathogenesis and development of targeted therapeutic drugs. [ABSTRACT FROM AUTHOR]

The dilution effect hypothesis posits that increasing biodiversity reduces infectious disease transmission. Here, we propose that habitat quality might modulate this negative biodiversity–disease relationship. Habitat may influence pathogen prevalence directly by affecting host traits like nutrition and immune response (we coined the term "habitat–disease relationship" to describe this phenomenon) or indirectly by changing host biodiversity (biodiversity–disease relationship). We used a path model to test the relative strength of links between habitat, biodiversity, and pathogen prevalence in a pollinator–virus system. High‐quality habitat metrics were directly associated with viral prevalence, providing evidence for a habitat–disease relationship. However, the strength and direction of specific habitat effects on viral prevalence varied based on the characteristics of the habitat, host, and pathogen. In general, more natural area and richness of land‐cover types were directly associated with increased viral prevalence, whereas greater floral density was associated with reduced viral prevalence. More natural habitat was also indirectly associated with reduced prevalence of two key viruses (black queen cell virus and deformed wing virus) via increased pollinator species richness, providing evidence for a habitat‐mediated dilution effect on viral prevalence. Biodiversity–disease relationships varied across viruses, with the prevalence of sacbrood virus not being associated with any habitat quality or pollinator community metrics. Across all viruses and hosts, habitat–disease and biodiversity–disease paths had effects of similar magnitude on viral prevalence. Therefore, habitat quality is a key driver of variation in pathogen prevalence among communities via both direct habitat–disease and indirect biodiversity–disease pathways, though the specific patterns varied among different viruses and host species. Critically, habitat–disease relationships could either contribute to or obscure dilution effects in natural systems depending on the relative strength and direction of the habitat–disease and biodiversity–disease pathways in that host–pathogen system. Therefore, habitat may be an important driver in the complex interactions between hosts and pathogens. [ABSTRACT FROM AUTHOR]

Simple Summary: Diseases, particularly those caused by viruses, are a major cause of bee colony losses. However, little is known about the prevalence of the pathogens, particularly virus prevalence, of the honey bee in Egypt, one of the most important countries for beekeeping and agricultural production in Africa. To address this shortfall, we determined the prevalence of ten widespread bee viruses in honey bee colonies in Egypt between two major season, winter and summer, in relation to colony infestation by varroa mites. Honey bees are essential for crop and wild plant pollination. However, many countries have reported high annual colony losses caused by multiple possible stressors. Diseases, particularly those caused by viruses, are a major cause of colony losses. However, little is known about the prevalence of honey bee pathogens, particularly virus prevalence, in Egyptian honey bees. To address this shortfall, we determined the prevalence of widespread bee viruses in honey bee colonies in Egypt—whether it is affected by geography, the season, or infestation with Varroa destructor (varroa) mites. Honey bee worker samples were collected from 18 geographical regions across Egypt during two seasons: winter and summer of 2021. Three apiaries were chosen in each region, and a pooled sample of 150 worker bees was collected from five colonies in each apiary then screened by qPCR for 10 viral targets: acute bee paralysis virus (ABPV), black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), deformed wing virus (DWV) genotypes A (DWV-A), B (DWV-B) and D (Egyptian bee virus), Israeli acute paralysis virus (IAPV), Kashmir bee virus (KBV), sacbrood virus (SBV), and slow bee paralysis virus (SBPV). Our results revealed that DWV-A was the most prevalent virus, followed by BQCV and ABPV; the DWV genotype now spreading across the world, DWV-B, was not detected. There was no difference in varroa infestation rates as well as virus prevalence between winter and summer. However, colonies infected with BQCV had a significantly higher varroa count (adjusted p < 0.05) in the winter season, indicating that there is a seasonal association between the intensity of infestation by varroa and the presence of this virus. We provide data on the current virus prevalence in Egypt, which could assist in the protection of Egypt's beekeeping industry. Moreover, our study aids in the systematic assessment of the global honey bee virome by filling a knowledge gap about the prevalence of honey bee viruses in Egypt. [ABSTRACT FROM AUTHOR]

Deformed wing virus (DWV), notorious for its virulence in the western honey bee (Apis mellifera) when vectored by the ectoparasitic mite Varroa destructor, is also widespread among wild bumble bee species, presumably through spillover from honey bees. Experimental studies on the virulence of DWV in Bombus spp. have provided equivocal results and have until now been confined to bumble bees under laboratory conditions.Here, we inoculated commercially reared Bombus terrestris workers with DWV‐A through feeding or injection and introduced them into experimental colonies placed in the field, thus exposing them to the environment and associated stressors. We monitored the survival of inoculated worker bumble bees and quantified their viral load at 10 days post inoculation.Bombus terrestris workers injected with DWV‐A supported high viral loads and exhibited significantly reduced median survival compared to controls. Bumble bees inoculated by feeding had low or zero detectable viral loads while their mortality did not differ from the control group.Our results demonstrate that, although DWV‐A is pathogenic for commercial B. terrestris, the risks for individual fitness from spillover of DWV‐A during foraging on shared flowers appear limited.The findings of this experiment also highlight the necessity to address the potential context‐dependence of virulence when evaluating the impact of a pathogen in an alternative host. [ABSTRACT FROM AUTHOR]

The deformed wing virus (DWV) belongs to the genus Iflavirus and the family Iflaviridae within the order Picornavirales. It is an important pathogen of the Western honey bee, Apis mellifera, causing major losses among honey bee colonies in association with the ectoparasitic mite Varroa destructor. Although DWV is one of the best-studied insect viruses, the mechanisms of viral replication and polyprotein processing have been poorly studied in the past. We investigated the processing of the protease-polymerase region at the C-terminus of the polyprotein in more detail using recombinant expression, novel serological reagents, and virus clone mutagenesis. Edman degradation of purified maturated polypeptides uncovered the C- and N-termini of the mature 3C-like (3CL) protease and RNA-dependent RNA polymerase (3DL, RdRp), respectively. Autocatalytic processing of the recombinant DWV 3CL protease occurred at P1 Q2118 and P1′ G2119 (KPQ/GST) as well as P1 Q2393 and P1′ S2394 (HAQ/SPS) cleavage sites. New monoclonal antibodies (Mab) detected the mature 3CL protease with an apparent molecular mass of 32 kDa, mature 3DL with an apparent molecular mass of 55 kDa as well as a dominant 3CDL precursor of 90 kDa in DWV infected honey bee pupae. The observed pattern corresponds well to data obtained via recombinant expression and N-terminal sequencing. Finally, we were able to show that 3CL protease activity and availability of the specific protease cleavage sites are essential for viral replication, protein synthesis, and establishment of infection using our molecular clone of DWV-A.

The Varroa destructor mite is a devastating parasite of honey bees; however the negative effects of varroa parasitism are exacerbated by its role as an efficient vector of the honey bee pathogen, Deformed wing virus (DWV). While no direct treatment for DWV infection is available for beekeepers to use on their hives, RNA interference (RNAi) has been widely explored as a possible biopesticide approach for a range of pests and pathogens. This study tested the effectiveness of three DWV-specific dsRNA sequences to lower DWV loads and symptoms in honey bees reared from larvae in laboratory mini-hives containing bees and varroa. The effects of DWV-dsRNA treatment on bees parasitised and non-parasitised by varroa mites during development were investigated. Additionally, the impact of DWV-dsRNA on viral loads and gene expression in brood-parasitising mites was assessed using RNA-sequencing. Bees parasitised during development had significantly higher DWV levels compared to non-parasitised bees. However, DWV-dsRNA did not significantly reduce DWV loads or symptoms in mini-hive reared bees, possibly due to sequence divergence between the DWV variants present in bees and varroa and the specific DWV-dsRNA sequences used. Varroa mites from DWV-dsRNA treated mini-hives did not show evidence of an elevated RNAi response or significant difference in DWV levels. Overall, our findings show that RNAi is not always successful, and multiple factors including pathogen diversity and transmission route may impact its efficiency.

Deformed wing virus (DWV) is one of the important pathogens of the honey bee (Apis mellifera), which consists of three master variants: types A, B, and C. Among them, DWV types A (DWV-A) and B (DWV-B) are the most prevalent variants in honey bee colonies and have been linked to colony decline. DWV-A and DWV-B have different virulence, but it is difficult to distinguish them via traditional methods. In this study, we established a visual detection assay for DWV-A and DWV-B using recombinase polymerase amplification (RPA) and a lateral flow dipstick (LFD) coupled with the clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein (Cas) 12a fluorescence system (RPA–CRISPR–Cas12a–LFD). The limit of detection of this system was ~6.5 × 100 and 6.2 × 101 copies/μL for DWV-A and DWV-B, respectively. The assays were specific and non-cross-reactive against other bee viruses, and the results could be visualized within 1 h. The assays were validated by extracting cDNA from 36 clinical samples of bees that were suspected to be infected with DWV. The findings were consistent with those of traditional reverse transcription–quantitative polymerase chain reaction, and the RPA–CRISPR–Cas12a assay showed the specific, sensitive, simple, and appropriate detection of DWV-A and DWV-B. This method can facilitate the visual and qualitative detection of DWV-A and DWV-B as well as the monitoring of different subtypes, thereby providing potentially better control and preventing current and future DWV outbreaks.

Abstract In 1977, a sample of diseased adult honeybees (Apis mellifera) from Egypt was found to contain large amounts of a previously unknown virus, Egypt bee virus, which was subsequently shown to be serologically related to deformed wing virus (DWV). By sequencing the original isolate, we demonstrate that Egypt bee virus is in fact a fourth unique, major variant of DWV (DWV-D): more closely related to DWV-C than to either DWV-A or DWV-B. DWV-A and DWV-B are the most common DWV variants worldwide due to their close relationship and transmission by Varroa destructor. However, we could not find any trace of DWV-D in several hundred RNA sequencing libraries from a worldwide selection of honeybee, varroa and bumblebee samples. This means that DWV-D has either become extinct, been replaced by other DWV variants better adapted to varroa-mediated transmission, or persists only in a narrow geographic or host range, isolated from common bee and beekeeping trade routes.

Background: Varroa destructor is the major ectoparasite of the western honey bee (Apis mellifera). Through both its parasitic life-cycle and its role as a vector of viral pathogens, it can cause major damage to honey bee colonies. The deformed wing virus (DWV) is the most common virus transmitted by this ectoparasite, and the mite is correlated to increased viral prevalence and viral loads in infested colonies. DWV variants A and B (DWV-A and DWV-B, respectively) are the two major DWV variants, and they differ both in their virulence and transmission dynamics. Methods: We studied the transmission of DWV between bees, parasitic mites and their offspring by quantifying DWV loads in bees and mites collected in in vitro and in situ environments. In vitro, we artificially transmitted DWV-A to mites and quantified both DWV-A and DWV-B in mites and bees. In situ, we measured the natural presence of DWV-B in bees, mites and mites' offspring. Results: Bee and mite viral loads were correlated, and mites carrying both variants were associated with higher mortality of the infected host. Mite infestation increased the DWV-B loads and decreased the DWV-A loads in our laboratory conditions. In situ, viral quantification in the mite offspring showed that, after an initially non-infected egg stage, the DWV-B loads were more closely correlated with the foundress (mother) mites than with the bee hosts. Conclusions: The association between mites and DWV-B was highlighted in this study. The parasitic history of a mite directly impacts its DWV infection potential during the rest of its life-cycle (in terms of variant and viral loads). Regarding the mite's progeny, we hypothesize that the route of contamination is likely through the feeding site rather than by vertical transmission, although further studies are needed to confirm this hypothesis. [ABSTRACT FROM AUTHOR]

Deformed wing virus (DWV), which is an important honey bee virus transmitted by Varroa destructor (V. destructor) , causes colony losses in honey bee colonies. This study aimed to investigate the prevalence and genetic diversity of DWV in honey bees in Türkiye and to determine the role of V. destructor in the transmission of the genetic variants of DWV. Honey bee samples were collected from 62 apiaries, by simple random sampling, during March 2022 and April 2023. The presence of V. destructor in collected bee samples was examined using a stereo microscope. Real-time RT-PCR was used for the detection of DWV-A and DWV-B ( Varroa destructor virus-1 (VDV-1)) viruses. Genetic characterisation of the positive samples was conducted by sequencing polyprotein genomic region. Considering the V. destructor infestation rate of 3% as relevant, out of the 62 apiaries examined, 17 (27.4%) were positive. However, DWV-A and VDV-1 specific RNA was not detected in V. destructor samples. VDV-1 specific RNA was detected in 6.5% (4/62) of the apiaries, whereas DWV-A was not detected in the sampled apiaries. Phylogenetic analysis showed that isolates detected in this study were located in a separate cluster from previously characterised DWV-A and VDV-1 isolates. According to RDP4 and GARD analyses, DWV-VDV-1 recombination breakpoints were detected in field isolates. To the best our knowledge, this is the first report of the presence of VDV-1-DWV recombinants in Türkiye. Further studies are needed to determine the impact of VDV-1-DWV recombinants and their virological and antigenic properties.

Here, we report the complete genome sequence of deformed wing virus (DWV) isolated from honeybees ( Apis mellifera L.) in Iraq, named the Tikrit isolate. The phylogeny revealed that the Tikrit isolate has a close relationship to the Iraqi isolate DWV-Iraq-2023 and related to isolates from France, the United Kingdom, and Israel.

The small hive beetle (SHB), Aethina tumida Murray, is an invasive pest of the honey bee and causes significant damage through the consumption of colony resources and brood. Two assumptions related to honey bee virus transmission have been made about SHB: first, that SHB vectors honey bee viruses and second, that these viruses replicate in SHB based on the detection of both positive and negative strand viral genomic RNA within SHB. To clarify the role of SHB in virus transmission, we sought to address whether selected honey bee viruses replicate in SHB. Sequences derived from five honey bee viruses were identified in the transcriptomes of field-caught SHB from the U.S., but not in those of lab-reared SHB, suggesting that these viruses do not replicate in SHB. To elucidate whether the representative viruses, Israeli acute paralysis virus (IAPV; Dicistroviridae) and Deformed wing virus (DWV; Iflaviridae) replicate in SHB, we tested for replication in vitro in an SHB-derived cell line (BCIRL-AtumEN-1129-D6). Following treatment of the cell line with viral particles or viral RNA, the number of virus genomes was monitored by reverse transcription quantitative PCR (RT-qPCR). In contrast to the positive control, IAPV and DWV RNA levels steadily decreased over a period of 8 days. Collectively, these results from bioinformatic observations and in vitro experiments indicate that IAPV and DWV do not replicate in SHB. These results are consistent with the host specificity of most insect viruses within a single insect order and indicate that while SHB may serve as a mechanical vector of honey bee viruses within and between hives, this insect does not serve as a biological vector for these honey bee viruses., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Food quantity and macronutrients contribute to honey bee health and colony survival by mediating immune responses. We determined if this held true for bees injected with chronic bee paralysis virus (CBPV) and deformed wing virus (DWV), two common honey bee ssRNA viruses. Pollen-substitute diet and syrup consumption rates and macronutrient preferences of two Varroa-resistant stocks (Pol-Line and Russian bees) were compared to Varroa-susceptible Italian bees. Bee stocks varied in consumption, where Italian bees consumed more than Pol-Line and Russian bees. However, the protein: lipid (P:L) ratios of diet consumed by the Italian and Russian bees was greater than that of the Pol-Line bees. Treatment had different effects on consumption based on the virus injected. CBPV was positively correlated with syrup consumption, while DWV was not correlated with consumption. P:L ratios of consumed diet were significantly impacted by the interaction of bee stock and treatment, with the trends differing between CBPV and DWV. Variation in macronutrient preferences based on viral species may indicate differences in energetic costs associated with immune responses to infections impacting different systems. Further, virus species interacted with bee genotype, indicating different mechanisms of viral resistance or tolerance among honey bee genotypes.

Varroa destructor is an ectoparasitic mite associated with significant losses of honeybee colonies globally. The mite vectors a range of pathogenic viruses, the most important of which is the Deformed wing virus (DWV). In the absence of Varroa, DWV exists as a low-level, highly diverse virus population. However, when transmitted by Varroa, certain variants become highly elevated, and may become near-clonal and cause symptomatic infections. Mite transmission between colonies can occur when parasitised workers drift from or rob adjacent hives. These activities can result in elevated mite levels, but the resulting change in the DWV population, the primary determinant of winter colony losses, has not been determined. In reciprocal studies, we investigated the influence of the removal of mites, or their acquisition, on the DWV population. When mites were removed from heavily infested colonies, there was a striking and rapid reduction in virus load. Conversely, siting Varroa-naïve colonies in a mite-infested apiary resulted in the acquisition of mites and concomitant changes in the virus population. We observed both near-clonal and highly divergent virus populations regardless of titre, suggesting changes were stochastic and colony-specific. Our findings have implications for the outcome of strategies in areas with total or patchy implementation of Varroa control plans. [ABSTRACT FROM AUTHOR]

Viruses are one of the most potential risk factors that negatively affect the different life stages of honey bees. This study was conducted to determine the presence of infections caused by the deformed wing virus (DWV), black queen cell virus (BQCV), and acute bee paralysis virus (ABPV) in honey bees in the beekeeping regions of Burdur, along with obtaining information about their prevalence in this particular region. In our study, the adult bees were taken from 31 different beehives and comb samples that had different honey bee breeds and were sampled randomly from 15 beekeeping areas within the region. The collected samples were analyzed using the reverse transcriptase-polymerase chain reaction (RT-PCR), and the prevalence of DWV, BQCV, and ABPV infections were determined to be 74.19% (23/31), 25.81%, (8/31) and 74.19% (23/31), respectively. In this study, the distribution of positive samples and the rates of multiple infections were determined in the colonies. Of the positive honey bee samples, 12 (%38.71) were detected only for one virus, 9 (%29.03) were positive for two viruses (DWV-ABPV) and 8 (%25.81) were positive for all three viruses. In the present study, the presence of the three bee viruses that caused significant damage to the colonies by multiple infections in the Burdur region was determined with RT-PCR. To our knowledge, this is the first report of three mentioned bee infections in honey bees in the Burdur region. The revealed epidemiological conditions lead to the conclusion that serious measures are needed to control these infections in this region. [ABSTRACT FROM AUTHOR]