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The Asian yellow-legged hornet Vespa velutina nigrithorax, a major predator of honeybees, is spreading in Europe in part due to a lack of efficient control methods. In this study, as a first step to identify biological control agents, we characterized viral RNA sequences present in asymptomatic or symptomatic hornets. Among 19 detected viruses, the honey bee virus Deformed wing virus-B was predominant in all the samples, particularly in muscles from the symptomatic hornet, suggesting a putative cause of the deformed wing symptom. Interestingly, two new viruses closely related to Acyrthosiphon pisum virus and Himetobi P virus and viruses typically associated with honey bees, Acute bee paralysis virus and Black queen cell virus, were detected in the brain and muscles, and may correspond to the circulation and possible replication forms of these viruses in the hornet. Aphid lethal paralysis virus, Bee Macula-like virus, and Moku virus, which are known to infect honey bees, were also identified in the gut virus metagenome of hornets. Therefore, our study underlined the urgent need to study the host range of these newly discovered viruses in hornets to determine whether they represent a new threat for honey bees or a hope for the biocontrol of V. velutina.

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.

In this study, seven bee viruses of significant importance for bee health in Türkiye were investigated using one-step RT-PCR. For this purpose, larvae from 1183 hives and adult bees from 1196 hives were sampled from 400 apiaries in 40 provinces. The prevalence of viral infections in hives was as follows: acute bee paralysis virus (ABPV), 6.4%; black queen cell virus (BQCV), 77%; chronic bee paralysis virus (CBPV), 3.2%; deformed wing virus (DWV), 63.8%; Israel acute bee paralysis virus (IAPV), 7%; Kashmir bee virus (KBV), 2.7%; sacbrood virus (SBV), 49.7%. Moreover, 50 different combinations of viral infections were identified in the hives. While dual infections (36.1%) were the most common in hives, triple infections with BQCV, DWV, and SBV were found to have the highest prevalence (22.1%). At least one viral infection was detected in all of the apiaries tested. Phylogenetic analysis showed that the isolates from this study generally exhibited the highest similarity to previously reported Turkish isolates. When similarity ratios and the locations and types of amino acid mutations were analyzed, it was observed that the isolates from our study exhibited high similarity to isolates from various countries, including China, the United Kingdom, Syria, and Germany.

Honey bee viruses are serious pathogens that can cause poor colony health and productivity. We analyzed a multi-year longitudinal dataset of abundances of nine honey bee viruses (deformed wing virus A, deformed wing virus B, black queen cell virus, sacbrood virus, Lake Sinai virus, Kashmir bee virus, acute bee paralysis virus, chronic bee paralysis virus, and Israeli acute paralysis virus) in colonies located across Canada to describe broad trends in virus intensity and occurrence among regions and years. We also tested climatic variables (temperature, wind speed, and precipitation) as predictors in an effort to understand possible drivers underlying seasonal patterns in viral prevalence. Temperature was a significant positive predictor of the total number of viruses per sample, which was highest in British Columbia (mean = 5.0). Lake Sinai virus (LSV) was the most prevalent overall (at 89%) and had the highest infection intensity, at an average of 3.9 × 10 8 copies per bee. Acute bee paralysis virus was the least prevalent virus (at 4.7%) and had the lowest infection intensity (1.9 × 10 5 copies per bee). Surprisingly, including Varroa abundance as a covariate did not significantly improve model fit for any virus. All viruses, except Kashmir bee virus, varied by region, and one or more climatic variables were significant predictors for six of the nine viruses. Although climatic effects were often inconsistent among individual viruses, we show that climatic variables can be better predictors of virus intensity and occurrence than Varroa mite abundance, at least when infestation rates are low.

In the last few years, the isolation and amplification of DNA or RNA from the environment (eDNA/eRNA) has proven to be an alternative and non-invasive approach for molecular identification of pathogens and pests in beekeeping. We have recently demonstrated that bee pollen and bee bread represent suitable biological material for the molecular identification of viral RNA. In the present study, we extracted total RNA from different bee products (pollen, n = 25; bee bread, n = 17; and royal jelly, n = 15). All the samples were tested for the presence of six of the most common honey bee-associated viruses—Deformed wing virus (DWV), Acute bee paralysis virus (ABPV), Chronic bee paralysis virus (CBPV), Sacbrood virus (SBV), Kashmir bee virus (KBV), and Black queen cell virus (BQCV)—using a reverse transcription polymerase chain reaction (RT-PCR). We successfully detected six records of DWV (10.5%, 6/57), four of ABPV (7.0%, 4/57), three of Israeli acute paralysis virus (IAPV) (5.3%, 3/57), and two of BQCV (3.5%, 2/57). Using ABPV primers, we also successfully detected the presence of IAPV. The obtained viral sequences were analyzed for phylogenetic relationships with the highly similar sequences (megablast) available in the GenBank database. The Bulgarian DWV isolates revealed a high homology level with strains from Syria and Turkey. Moreover, we successfully detected a DWV strain B for the first time in Bulgaria. In contrast to DWV, the ABPV isolates formed a separate clade in the phylogenetic tree. BQCV was closely grouped with Russian isolates, while Bulgarian IAPV formed its own clade and included a strain from China. In conclusion, the present study demonstrated that eRNA can be successfully used for molecular detection of honey bee-associated viruses in bee products. The method can assist the monitoring of the health status of honey bee colonies at the local, regional, and even national levels.

The invasion of non-native bees to new ecological territories could spread novel pathogens causing emerging infectious diseases (EIDs) in native species. We provide novel information on the prevalence, load, and co-infection network of honey bee viruses, trypanosoma, microsporidia and neogregarinorida pathogens in native Bombus dahlbomii and non-native Bombus terrestris and Bombus ruderatus. Apicystis bombi and Crithidia bombi were highly prevalent (> 78%) in three bumble bee species, with high loads of these pathogens. Nosema bombi was detected only in B. terrestris (37%) and B. ruderatus (15%). Lotmaria passim was detected in low prevalence (< 6%) and low loads in three bumble bee species. Deformed wing virus (genotype A) was detected only in B. terrestris (20%) and B. ruderatus (6%). Black queen cell virus was detected in B. terrestris (34%), B. ruderatus (22%) and B. dahlbomii (23%). Chronic bee paralysis virus, Kashmir bee virus and Acute bee paralysis virus were detected with low prevalence (7%) and titers in the three bumble bee species. The proximity of apiaries and collection sites was not a significant factor in the presence of viruses in bumble bees. The three bumble species were found to be co-infected with Apicystis bombi and C. bombi; a significant positive correlation was found between these two parasites, especially in B. terrestris. Multiple infections with N. bombi, A. bombi, C. bombi and viruses in B. terrestris and B. ruderatus were also detected. This suggests that the invasion and successful establishment of exotic bumble bees in a new area also entails the possible establishment of the pathogens that they carry, which could also be present in native bee species. This finding evidences a potential link between the population decline of B. dahlbomii and the pathogens that were detected with high levels and prevalence.

The health of honey bees is threatened by multiple factors, including viruses and parasites. We screened 557 honey bee (Apis mellifera) colonies from 155 beekeepers distributed all over Belgium to determine the prevalence of seven widespread viruses and two parasites (Varroa sp. and Nosema sp.). Deformed wing virus B (DWV-B), black queen cell virus (BQCV), and sacbrood virus (SBV) were highly prevalent and detected by real-time RT-PCR in more than 95% of the colonies. Acute bee paralysis virus (ABPV), chronic bee paralysis virus (CBPV) and deformed wing virus A (DWV-A) were prevalent to a lower extent (between 18 and 29%). Most viruses were only present at low or moderate viral loads. Nevertheless, about 50% of the colonies harbored at least one virus at high viral load (>107 genome copies/bee). Varroa mites and Nosema sp. were found in 81.5% and 59.7% of the honey bee colonies, respectively, and all Nosema were identified as Nosema ceranae by real time PCR. Interestingly, we found a significant correlation between the number of Varroa mites and DWV-B viral load. To determine the combined effect of these and other factors on honey bee health in Belgium, a follow up of colonies over multiple years is necessary.

The honey bee queen is the central hub of a colony to produce eggs and release pheromones to maintain social cohesion. Among many environmental stresses, viruses are a major concern to compromise the queen’s health and reproductive vigor. Viruses have evolved numerous strategies to infect queens either via vertical transmission from the queens’ parents or horizontally through the worker and drones with which she is in contact during development, while mating, and in the reproductive period in the colony. Over 30 viruses have been discovered from honey bees but only few studies exist on the pathogenicity and direct impact of viruses on the queen’s phenotype. An apparent lack of virus symptoms and practical problems are partly to blame for the lack of studies, and we hope to stimulate new research and methodological approaches. To illustrate the problems, we describe a study on sublethal effects of Israeli Acute Paralysis Virus (IAPV) that led to inconclusive results. We conclude by discussing the most crucial methodological considerations and novel approaches for studying the interactions between honey bee viruses and their interactions with queen health.

Knowledge regarding the honey bee pathogens borne by invasive bee pests remains scarce. This investigation aimed to assess the presence in Aethina tumida (small hive beetle, SHB) adults of honey bee pathogens belonging to the following groups: (i) bacteria (Paenibacillus larvae and Melissococcus plutonius), (ii) trypanosomatids (Lotmaria passim and Crithidia mellificae), and (iii) viruses (black queen cell virus, Kashmir bee virus, deformed wing virus, slow paralysis virus, sacbrood virus, Israeli acute paralysis virus, acute bee paralysis virus, chronic bee paralysis virus). Specimens were collected from free-flying colonies in Gainesville (Florida, USA) in summer 2017. The results of the molecular analysis show the presence of L. passim, C. mellificae, and replicative forms of deformed wing virus (DWV) and Kashmir bee virus (KBV). Replicative forms of KBV have not previously been reported. These results support the hypothesis of pathogen spillover between managed honey bees and the SHB, and these dynamics require further investigation.

The viral loads of acute bee paralysis virus (ABPV), black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), deformed wing virus (DWV), Lake Sinai virus 3 (LSV3), and sacbrood bee virus (SBV) were determined in samples with the use of quantitative TaqMan real-time reverse transcription and polymerase chain reaction (RT-qPCR). A total of 108 samples of healthy adult honeybees from four differently located apiaries and samples of honeybees showing different clinical signs of viral infections from 89 apiaries were collected throughout Slovenia. The aim of this study was to discover correlations between viral loads and clinical signs in adult honeybees and confirm previously set threshold viral load levels between healthy and clinically affected honeybees. Within this study, two new RT-qPCR assays for quantification of LSV3 and SBV were developed. Statistically significant differences in viral loads of positive samples were identified between healthy and clinically affected honeybees for ABPV, CBPV, DWV, and SBV, while for BQCV and LSV3, no statistical differences were observed between both groups. Despite high detected LSV3 prevalence and viral loads around 6.00 log10 viral copies/bee, this lineage probably has a limited impact on the health status of honeybee colonies. The determined viral loads between 3.94 log10 and 13.17 log10 in positive samples for six viruses, collected over 10 consecutive months, including winter, present additional information of high viral load variations in healthy honeybee colonies.