Search Results (21)

In this study, honeybee viruses were identified in naturally infected honeybee colonies (Apis mellifera carnica). From nine selected samples of clinically affected and ten samples of healthy honeybee colonies, different strains of honeybee viruses were first detected using quantitative real-time RT-PCR methods. Twenty-two nucleotide sequences of the complete genomes of honeybee viruses were identified using the Illumina Next-Generation Sequencing (NGS) method: acute bee paralysis virus (ABPV) (n = 4), black queen cell virus (BQCV) (n = 3), chronic bee paralysis virus (CBPV) (n = 2), deformed wing virus (DWV) (n = 5), Lake Sinai virus (LSV) (n = 4), sacbrood bee virus (SBV) (n = 1), Apis rhabdovirus-1 (ARV-1) (n = 1), bee macula-like virus (BeeMLV) (n = 1) and Hubei partiti-like virus 34 (HPLV34) (n = 1). The nucleotide sequences of ABPV, BQCV, DWV and SBV are the first complete genomes of these viruses identified in Slovenia and they represent an important contribution to our understanding of the genetic diversity of honeybee viruses. ARV-1, BeeMLV and HPLV34 were detected and sequenced for the first time in Slovenia. Full article

The Megaselia scalaris and Senotainia tricuspis parasitoid flies of the honeybee Apis mellifera were found to infest apiaries of different European and Mediterranean countries but their prevalence and impact on apiary health are little known. Therefore, in this study, quantitative PCR (qPCR)-based methods were developed for their rapid detection directly in hive matrices. The newly developed qPCR assays were targeted at the mitochondrial cytochrome oxidase subunit I (COI) gene for the M. scalaris and the cytochrome B (cytB) gene for the S. tricuspis. The tests were preliminarily applied to 64 samples of adult honeybees and hive debris collected in the Abruzzo and Molise regions, Central Italy, and the Republic of Kosovo showing that both flies occur in the two countries and more frequently in Italy. The positive apiaries in Italy were re-sampled by capturing viable forager bees and isolating emerging flies to carry out the genotyping and analyses aimed at defining if these flies can transmit honeybee pathogens. Genotyping based on the COI and cytB gene sequencing for M. scalaris and S. tricuspis, respectively, identified one S. tricuspis genotype and diverse genotypes of M. scalaris highly similar to those from distant countries. Some fly isolates harbored the DNA or RNA of honeybee microbial pathogens Paenibacillus larvae, deformed wing viruses A and B (DWVA and B), black queen cell virus (BQCV), chronic paralysis virus (CBPV), and Nosema ceranae. The results indicated that these parasites should be efficiently controlled in apiaries by using rapid detection methods to facilitate the large screening studies and early detection. Full article

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. Full article

Verifying the inclusivity of molecular detection methods gives indications about the reliability of viral infection diagnosis because of the tendency of viral pathogens to undergo sequence variation. This study was aimed at selecting inclusive probes based on reverse transcription–quantitative PCR (RT-qPCR) assays for the diagnosis of the most widespread and detrimental viruses infecting honeybees, namely the acute bee paralysis virus (ABPV), the black queen cell virus (BQCV), the chronic paralysis bee virus (CBPV), the deformed wing virus variants A (DWVA) and B (DWVB), and the sacbrood virus (SBV). Therefore, previously described detection methods were re-evaluated in silico for their specificity and inclusivity. Based on this evaluation, selected methods were modified, or new ones were designed and tested in duplex RT-qPCR reactions. The limits of detection (LODs), effect of multiplexing on sensitivity and the viral RNA quantification potential in bees and hive debris were assessed. This study made available diagnostic assays able to detect an increased number of virus variants compared with previously described tests and two viral pathogens in a single PCR reaction. Full article

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. Full article

Viruses contribute significantly to the global decline of honey bee populations. One way to limit the impact of such viruses is the introduction of natural antiviral compounds from fungi as a component of honey bee diets. Therefore, we examined the effect of crude organic extracts from seven strains of the fungal genus Talaromyces in honey bee diets under laboratory conditions. The strains were isolated from bee bread prepared by honey bees infected with chronic bee paralysis virus (CBPV). The antiviral effect of the extracts was also quantified in vitro using mammalian cells as a model system. We found that three extracts (from strains B13, B18 and B30) mitigated CBPV infections and increased the survival rate of bees, whereas other extracts had no effect (B11 and B49) or were independently toxic (B69 and B195). Extract B18 inhibited the replication of feline calicivirus and feline coronavirus (FCoV) in mammalian cells, whereas extracts B18 and B195 reduced the infectivity of FCoV by ~90% and 99%, respectively. Our results show that nonpathogenic fungi (and their products in food stores) offer an underexplored source of compounds that promote disease resistance in honey bees. Full article

Several pathogens are important causes of the observed pollinator decline, some of which could be transmitted between different pollinator species. To determine whether honeybee viruses can be transmitted to butterflies, a total of 120 butterflies were sampled at four locations in Slovenia. At each location, butterflies from three families (Pieridae, Nymphalidae, Hesperiidae/Lycenidae) and Carniolan honeybees (Apis mellifera carnica) were collected. The RNA of six honeybee viruses, i.e., acute bee paralysis virus (ABPV), black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), deformed wing virus A (DWV-A), Sacbrood bee virus (SBV), and Lake Sinai virus 3 (LSV3), was detected by a specific quantitative method (RT-PCR). The presence of ABPV, BQCV, LSV3, and SBV was detected in both butterflies and honeybees. All butterfly and bee samples were negative for CBPV, while DWV-A was detected only in honeybees. The viral load in the positive butterfly samples was much lower than in the positive bee samples, which could indicate that butterflies are passive carriers of bee viruses. The percentage of positive butterfly samples was higher when the butterflies were collected at sampling sites with a higher density of apiaries. Therefore, we believe that infected bees are a necessary condition for the presence of viruses in cohabiting butterflies. This is the first study on the presence of pathogenic bee viruses in butterflies. Full article

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. Full article

To determine the presence and the prevalence of four different honeybee viruses (acute bee paralysis virus—ABPV, black queen cell virus—BQCV, chronic bee paralysis virus—CBPV, deformed wing virus—DWV) in wild bumblebees, pooled randomly selected bumblebee samples were collected from twenty-seven different locations in the territory of Croatia. All samples were prepared and examined using the RT-PCR methods for quantification of mentioned honeybee viruses. Determined prevalence (%) of identified positive viruses were in the following decreasing order: BQCV > DWV > ABPV, CBPV. Additionally, direct sequencing of samples positive for BQCV (n = 24) and DWV (n = 2) was performed, as well as a test of molecular phylogeny comparison with those available in GenBank. Selected positive field viruses’ strains showed 95.7 to 100% (BQCV) and 98.09% (DWV) nucleotide identity with previously detected and deposited honeybee virus strains in the geographic areas in Croatia and neighboring Slovenia. In this article, the first detection of four honeybee viruses with genetic characterization of high diversity strains circulating in wild bumblebees in Croatia is presented. Full article

Veterinarians are educated in prevention, diagnosis and treatment of diseases in various vertebrate species. As they are familiar with multifactorial health problems in single animals as well as in herd health management, their knowledge and skills can be beneficial for the beekeepers and honeybee health. However, in education and in practice, honeybees are not a common species for most veterinarians and the typical veterinary diagnostic methods such as blood sampling or auscultation are not applicable to the superorganism honeybee. Honeybee colonies may be affected by various biotic and abiotic factors. Among the infectious agents, RNA-viruses build the largest group, causing covert and overt infections in honeybee colonies which may lead to colony losses. Veterinarians could and should play a more substantial role in managing honeybee health—not limited to cases of notifiable diseases and official hygiene controls. This review discusses the veterinary diagnostic approach to adult bee examination with a special focus on diagnosis and differential diagnosis of the common virus diseases Acute Bee Paralysis Virus (ABPV)-Kashmir Bee Virus (KBV)-Israeli Acute Paralysis Virus (IAPV)-Complex, Chronic Bee Paralysis Virus (CBPV) and Deformed Wing Virus (DWV), as well as coinfections like Varroa spp. and Nosema spp. Full article

Chronic Bee Paralysis Virus (CBPV), a widespread honey bee RNA virus, causes massive worker bee losses, mostly in strong colonies. Two different syndromes, with paralysis, ataxia and flight incapacity on one hand and black hairless individuals with shortened abdomens on the other, can affect a colony simultaneously. This case report presents two Apis mellifera carnica colonies with symptoms of paralysis and hairless black syndrome in 2019. Via RT-PCR, a highly positive result for CBPV was detected in both samples. Further problems, such as a Nosema infection and Varroa infestation, were present in these colonies. Therapy methods were applied to colony 1 comprising queen replacement, shook swarm method and Varroa control, whereas colony 2 was asphyxiated after queen loss and colony weakening. After therapy, colony 1 was wintered without symptoms. Beekeeping and sanitary measures can save a CBPV-infected colony, while further complications result in total colony loss. Full article

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. Full article

Throughout a honey bee queen’s lifetime, she is tended to by her worker daughters, who feed and groom her. Such interactions provide possible horizontal transmission routes for pathogens from the workers to the queen, and as such a queen’s pathogen profile may be representative of the workers within a colony. To explore this further, we investigated known honey bee pathogen co-occurrence, as well as pathogen transmission from workers to queens. Queens from 42 colonies were removed from their source hives and exchanged into a second, unrelated foster colony. Worker samples were taken from the source colony on the day of queen exchange and the queens were collected 24 days after introduction. All samples were screened for Nosema spp., Trypanosome spp., acute bee paralysis virus (ABPV), black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), Israeli acute paralysis virus (IAPV), Lake Sinai virus (LSV), and deformed wing virus master variants (DWV-A, B, and C) using RT-qPCR. The data show that LSV, Nosema, and DWV-B were the most abundant pathogens in colonies. All workers (n = 42) were LSV-positive, 88% were Nosema-positive, whilst pathogen loads were low (<1 × 10⁶ genome equivalents per pooled worker sample). All queens (n = 39) were negative for both LSV and Nosema. We found no evidence of DWV transmission occurring from worker to queen when comparing queens to foster colonies, despite DWV being present in both queens and workers. Honey bee pathogen presence and diversity in queens cannot be revealed from screening workers, nor were pathogens successfully transmitted to the queen. Full article

The ecological and economic importance of bees for pollination and biodiversity is well established. The health of bees is, however, threatened by a multitude of factors, including viruses. In this study, we screened 557 colonies from 155 beekeepers distributed all over Belgium to monitor the prevalence and distribution of seven widespread viruses in Belgian honey bees (Apis mellifera). Several of these viruses have been linked with an increased risk for colony loss. Although these viruses can severely impact honey bees and can even cause the death of larvae or adults, colonies with a low viral load usually appear asymptomatic (covert infection). The presence of viruses was determined by real-time RT-PCR. The three most prevalent viruses in Belgian honey bees are Deformed wing virus B (DWV-B or VDV-1), Black queen cell virus (BQCV), and Sacbrood virus (SBV). These viruses were found in more than 90% of the honey bee colonies, but often with a high Ct value, which indicates that they are present at low viral loads (less than 3 log10 genome copies per bee). In certain colonies, however, DWV-B, BQCV, or SBV was detected with a low Ct value, representing a high viral load (in some cases, more than 7 log10 genome copies per bee) and with an increased likelihood of development of clinical symptoms. Deformed wing virus A (DWV-A), Acute bee paralysis virus (ABPV), and Chronic bee paralysis virus (CBPV) were found in less than 40% of the colonies. Kashmir bee virus (KBV) was not found in any of the analyzed Belgian honey bees. Most of the honey bee colonies are infected with multiple viruses, albeit with low virus loads. The impact of viruses can however become critical in the presence of other detrimental factors such as parasites (Nosema sp., Varroa sp.) and pesticides. Full article

Honey bee viruses are associated with honey bee colony decline. Israeli acute paralysis virus (IAPV) is considered to have a strong impact on honey bee survival. Phylogenetic analysis of the viral genomes from several regions of the world showed that various IAPV lineages had substantial differences in virulence. Chronic bee paralysis virus (CBPV), another important honey bee virus, can induce two significantly different symptoms. However, the infection characteristics and pathogenesis of IAPV and CBPV have not been completely elucidated. Here, we constructed infectious clones of IAPV and CBPV using a universal vector to provide a basis for studying their replication and pathogenesis. Infectious IAPV and CBPV were rescued from molecular clones of IAPV and CBPV genomes, respectively, that induced typical paralysis symptoms. The replication levels and expression proteins of IAPV and CBPV in progeny virus production were confirmed by qPCR and Western blot. Our results will allow further dissection of the role of each gene in the context of viral infection while helping to study viral pathogenesis and develop antiviral drugs using reverse genetics systems. Full article