Sacbrood Virus Research Articles

Патогенные вирусы и клещ Варроа на пасеках России

In the European part of Russia, a study was conducted on the presence of six pathogenic viruses in bee families: SAC brood virus, black Queen virus, wing deformity virus, Kashmir virus, Israeli acute paralysis virus, and acute paralysis virus. The prevalence of pathogenic viruses was studied in apiaries using the molecular genetic method OT-PCR. The tick load on bee families was determined. The heterogeneous distribution of different types of viruses in apiaries was established. With a low tick load, there was a high viral load on bee families in the southern and Central regions of the European part of Russia, and a low one in the Northern region.

Designing of rt-lamp primers and detection of sac brood virus from indian honey bee Apis cerana indica (F.)

Designing of rt-lamp primers and detection of sac brood virus from indian honey bee <i>Apis cerana indica</i> (F.)

Occurrence and Molecular Phylogeny of Honey Bee Viruses in Vespids.

Since the discovery that honey bee viruses play a role in colony decline, researchers have made major breakthroughs in understanding viral pathology and infection processes in honey bees. Work on virus transmission patterns and virus vectors, such as the mite Varroa destructor, has prompted intense efforts to manage honey bee health. However, little is known about the occurrence of honey bee viruses in bee predators, such as vespids. In this study, we characterized the occurrence of 11 honey bee viruses in five vespid species and one wasp from four provinces in China and two vespid species from four locations in France. The results showed that all the species from China carried certain honey bee viruses, notably Apis mellifera filamentous virus (AmFV), Deformed wing virus (DWV), and Israeli acute paralysis virus (IAPV); furthermore, in some vespid colonies, more than three different viruses were identified. In France, DWV was the most common virus; Sacbrood virus (SBV) and Black queen cell virus (BQCV) were observed in one and two samples, respectively. Phylogenetic analyses of IAPV and BQCV sequences indicated that most of the IAPV sequences belonged to a single group, while the BQCV sequences belonged to several groups. Additionally, our study is the first to detect Lake Sinai virus (LSV) in a hornet from China. Our findings can guide further research into the origin and transmission of honey bee viruses in Vespidae, a taxon of ecological, and potentially epidemiological, relevance.

Development of a Bacillus thuringiensis based dsRNA production platform to control sacbrood virus in Apis cerana.

Sacbrood virus (SBV) is a fatal viruses that infects the Asian honey bee, Apis cerana in Korea. Recently, RNA interference (RNAi) has been suggested as a promising strategy for the suppression of honey bee viruses. For the efficient control of SBV infection using RNAi, simple and cost-effective methods to produce double-stranded RNA (dsRNA) are needed. To develop a dsRNA production platform using Bacillus thuringiensis (Bt), pBTdsSBV-VP1 vector was constructed in which the SBV vp1 gene was located between two oppositely oriented cyt promoters. Both strands of the vp1 gene were bidirectionally transcribed under the control of the sporulation-dependent cyt promoter in Bt cells transformed with pBTdsSBV-VP1, and the resulting dsRNA was easily extracted from the Bt transformant, Bt 4Q7/pBTdsSBV-VP1, by inducing its autolysis. The replication of SBV was dramatically suppressed in A. cerana workers who ingested the dsRNA produced from the Bt 4Q7/pBTdsSBV-VP1. In this study, we successfully silenced SBV in its host, A. cerana, by the application of exogenous dsRNA produced from an entomopathogenic bacteria, Bt. These results suggested that Bt could be a useful dsRNA production platform to control viral pathogens in their host insects. © 2019 Society of Chemical Industry.

Outcomes of honeybee pupae inoculated with deformed wing virus genotypes A and B

Deformed wing virus and Varroa destructor virus-1 have a high percentage of nucleotide identity and might be considered as closely related viruses: DWV genotype A (DWV-A) and DWV genotype B (DWV-B) respectively. They have been implicated in overwinter colony losses in association with Varroa destructor infestations that vectored both DWV variants. In this study, we performed experimental inoculations of honeybee pupae with viral suspensions prepared from honeybee heads naturally infected by either DWV-A or DWV-B. Two outcomes were observed: the inoculated pupae exhibited either higher rates of bees with deformed wings or higher mortality rates than control bees. For both DWV variants, the viral loads quantified in the head of inoculated bees were significantly greater than those in control bees (p < 0.01). These outcomes were not correlated to the virus genotype (DWV-A or DWV-B) detected in the inocula by RT-quantitative PCR targeting the VP3 coding sequence (RT-qPCR). However, the highest mortality rates found in our study were correlated with an increase in sacbrood virus (SBV) load. Despite the fact that only either DWV-A or DWV-B was expected to be inoculated, we observed increased mortality in honeybees that were infected with both DWV and SBV.

High resistance to Sacbrood virus disease in Apis cerana (Hymenoptera: Apidae) colonies selected for superior brood viability and hygienic behavior

Sacbrood virus (SBV) caused significant colony collapse in Korean Apis cerana. Therefore, breeding of resistant bees to counter this viral disease is urgently needed. Considering that hygienic behavior in honey bees confers colony-level resistance against brood diseases, we utilized this trait for selecting A. cerana colonies. In addition, the brood survival rate was evaluated after colonies were SBV-inoculated. Over four selective generations, dead brood removal and brood survivorship in selected colonies were higher than those in the controls, which were not selectively bred for those traits (P < 0.01, 99.3 vs. 89.9% for removal of pin-killed pupae; P < 0.01, 99.0 vs. 63.9% for removal of SBV-killed larvae; and P < 0.01, 70.0 vs. 9.2% for brood survivorship). Following SBV inoculation, selected colonies showed an increase in the number of surviving pupae and adults, whereas control colonies collapsed mostly. Our results confirm the feasibility of selecting SBV-resistant A. cerana.

Characterization of sacbrood virus and its infection in Apis cerana in the Qiandao Lake region in Zhejiang Province, China

Abstract Chinese sacbrood virus (CSBV) is a disastrous virus that fatally threatens honeybees (Apis cerana) in China. Here, we identified the complete genome of CSBV using the rapid amplification of cDNA ends (RACE) method and compared the obtained sequences with those of other SBVs through multiple sequence alignment. The results showed that the full-length of CSBV genome from Zhejiang Province (CSBV-ZJ) is 8824 bp and contains a single, large open reading frame (ORF) that encodes 2842 amino acids flanked by 190 nucleotides (nts) of the 5′-untranslated region (UTR) and 108 nts of the 3′-UTR. The multiple genome comparison showed that SBVs have five conserved domains and that CSBV-ZJ belong to SBV type II. The phylogenetic analysis demonstrated that the CSBV-ZJ strain was similar to the CSBV from Fuzhou (CSBV-FZ) strain. Colonies collected from most of the sampling sites in the area surrounding Qiandao Lake were carriers of CSBV. The haplotypes of the VP1 gene formed a network with radiated and reticular structures. In conclusion, this characterization of the CSBV-ZJ genome will help us understand the SBV prevalence mechanism and aid the protection of honeybees.

Identification of the Novel Host Protein Interacting With the Structural Protein VP1 of Chinese Sacbrood Virus by Yeast Two-Hybrid Screening.

Chinese sacbrood virus (CSBV) is the major cause and lead to the collapse of Apis cerana colonies. VP1, the structural protein of CSBV, shows the highest variation in the amino acid sequences among proteins from different CSBV strains as well as exhibits excellent immunogenicity. However, its function with host protein still remains unclear. To clarify its function with host protein, we screened out host cellular proteins that interact with VP1 using the membrane protein yeast two-hybrid system. In addition, we verified interactions between heat shock protein 70 cognate 5 (Hsp70-c5) and VP1 using glutathione S-transferase (GST) pull-down and co-immunoprecipitation assays. VP1 and Hsp70-c5 were colocalized in the cytoplasm and nucleus. Using western blot and real-time polymerase chain reaction (PCR), Hsp70-c5 expression in CSBV-infected larvae was upregulated compared with that in healthy larvae. We observed that when we silenced Hsp70-c5, VP1 expression was significantly downregulated. These results demonstrate that Hsp70-c5 is involved in at least one stage(s) of the viral life cycle.

Molecular detection and phylogenetic analysis of the honeybee (Apismellifera) sacbrood virus in Turkey

Sacbrood virus (SBV) is widely distributed in honeybees. It is an infectious and contagious disease, which affects both the larvae and adult stages of honeybees (Apis mellifera). This study reports the detection of sacbrood virus in Turkey in 2017. Phylogenetic analysis based on partial sequences encoding the polyprotein gene was carried out in SBV-infected bees from four different apiaries. Phylogenetic analysis revealed noteworthy results. Although European-South American, Asian, and Korean genotypes have a maximum of 5.3% variance with each other, the variation rates between them and the Turkish genotype were 10.4%, 11.4%, and 12.5%, respectively. Sequences of Turkish isolates did not fit into the previously reported genotype groups; furthermore, they created a completely separate branch according to phylogeny. This new branch was therefore named the Turkish genotype.

Occurrence of multiple honeybee viruses in the ectoparasitic mites Varroa spp. in Apis cerana colonies

In this study, we investigated the prevalence of honeybee viruses in Varroa destructor and Varroa underwoodi infesting Apis cerana colonies in China. Deformed wing virus (DWV) was the most prevalent virus in these two mite species, followed by Israeli acute paralysis virus (IAPV), Black queen cell virus (BQCV), Kashmir bee virus (KBV), Chronic bee paralysis virus (CBPV), Apis mellifera filamentous virus (AmFV) and Sacbrood virus (SBV) in V. destructor, while in V. underwoodi, it was followed by CBPV, AmFV, BQCV, IAPV and KBV. In addition, multiple viruses were commonly detectable in both mite species.

Dynamics of Apis cerana Sacbrood Virus (AcSBV) Prevalence in Apis cerana (Hymenoptera: Apidae) in Northern Taiwan and Demonstration of its Infection in Apis mellifera (Hymenoptera: Apidae).

Since 2016, Apis cerana sacbrood virus (AcSBV) has been recorded in Taiwan. It is epizootic in Apis cerana (Hymenoptera: Apidae) and causing serious loss of A. cerana. Herein, we performed a long-term survey of AcSBV prevalence in the populations of A. cerana in Northern Taiwan from January 2017 to July 2018. The surveillance of AcSBV prevalence in A. mellifera (Hymenoptera: Apidae) populations was starting and further confirmed by sequencing since April 2017; thus, these data were also included in this survey. In our survey, the average prevalence rates of AcSBV were 72 and 53% in A. cerana and A. mellifera, respectively, in 2017, which decreased to 45 and 27% in 2018. For the spatial analysis of AcSBV in two honey bee populations, Hsinchu showed the highest prevalence, followed by New Taipei, Yilan, Taipei, and Keelung, suggesting that AcSBV might have come from the southern part of Taiwan. Interestingly, the AcSBV prevalence rates from A. cerana and A. mellifera cocultured apiaries gradually synchronized. The result of phylogenetic analysis and comparison of the annual AcSBV prevalence in A. cerana-only, A. mellifera-only, and A. cerana/A. mellifera cocultured sample sites indicate cross-infection between A. cerana and A. mellifera; however, AcSBV may lose the advantage of virulence in A. mellifera. The evidence suggested that the transmission of AcSBV might occur among these two honey bee species in the field. Therefore, A. mellifera may serve as a guard species to monitor AcSBV in A. cerana, but the cross-infection still needs to be surveyed.

Detection of replicative Kashmir Bee Virus and Black Queen Cell Virus in Asian hornet Vespa velutina (Lepelieter 1836) in Italy

Information concerning the pathogenic role of honey bee viruses in invasive species are still scarce. The aim of this investigation was to assess the presence of several honey bee viruses, such as Black Queen Cell Virus (BQCV), Kashmir Bee Virus (KBV), Slow Paralysis Virus (SPV), Sac Brood Virus (SBV), Israeli Acute Paralysis Virus (IAPV), Acute Bee Paralysis Virus (ABPV), Chronic Bee Paralysis Virus (CBPV), in Vespa velutina specimens collected in Italy during 2017. Results of this investigation indicate that among pathogens, replicative form of KBV and BQCV were detected, assessing the spillover effect of both these viruses from managed honey bees to hornets.

Detection of Sacbrood Virus Infecting Apis cerana (AcSBV) Using Polyclonal Antibody Raised against Recombinant VP3 Capsid Protein Expressed in Escherichia coli

In Korea, sacbrood virus infecting Apis cerana (AcSBV) is a small RNA virus belonging to the family Iflaviridae. AcSBV causes larval death, and even the collapse of entire bee colonies and its viral capsid is composed of four structural proteins (VP1, VP2, VP3 and VP4). In this study, we synthesized VP3 gene based on genomic sequences to insert into a His-tag expression plasmid of pET-28a. The resultant recombinant vector was then transformed into Escherichia coli BL21 (DE3) to express the recombinant VP3 protein (rVP3). The rVP3 protein was strongly expressed in an insoluble fraction, with a molecular size of around 26 kDa. Purification of the rVP3 protein using an affinity Ni-NTA resin, followed by SDS-PAGE separation, resulted in a high purity of the protein and used as an antigen to raise the polyclonal antibodies. Mice were then immunized with the purified rVP3 protein and the antiserum collected at 4SUPth/SUP week and 6SUPth/SUP week was able to detect the rVP3 at a concentration of 500 ng and 250 ng. Furthermore, IgG antibody levels reacted with wild-type larva crude protein extract, AcSBV-infected larva crude protein extracts and rVP3 protein were quantitatively analyzed by ELISA and qualitatively analyzed by western blotting. This is the first report on production of polyclonal antibodies directed against the rVP3 protein of AcSBV and its use for detection and diagnosis of virus from field samples.

Does viral load alter behavior of the bee parasite Varroa destructor?

The invasive mite Varroa destructor has negatively impacted global apiculture, by being a vector for many viruses of the honey bee (Apis mellifera). Until now, most studies have been limited to varroa-honey bee or virus-honey bee interactions. The aim of this study is to bridge the important research gap of varroa-virus interactions by correlating varroa behavior with viral load. Ten-minute video recordings of 200 varroa mites were analyzed, and average speeds of the mites were compared to individual qPCR viral loads for deformed wing virus (DWV) and sacbrood virus (SBV). Statistically significant models reveal that colony, DWV, and SBV all might play a role in mite behavior, suggesting that the varroa-virus interaction needs to be an integral part of future studies on honey bee pathogens.

Validation of quantitative real-time RT-PCR assays for the detection of six honeybee viruses

Acute bee paralysis virus (ABPV), Black queen cell virus (BQCV), Chronic bee paralysis virus (CBPV), Deformed wing virus (DWV), Sacbrood virus (SBV) and Varroa destructor virus 1 (VDV1) are the six main honeybee viruses reported in Europe. We assessed the accuracy (trueness and precision) of reverse transcriptase quantitative TaqMan® PCR methods (RT-qPCR) for quantifying ABPV, BQCV, DWV, VDV1 and SBV loads. Once the systematic bias in quantitative results had been corrected (overestimation in ABPV and BQCV quantification and underestimation in that of SBV and VDV1), measurements were taken to determine the viral load ranges for which quantification uncertainty was below ± 1 log10 equivalent of genome copies per bee (hereafter reported as genome copies/bee). The accuracy range of RT-qPCR was found to be between 6.4 and 10.4 log10 genome copies/bee for ABPV, between 3.0 and 10.0 log10 genome copies/bee for BQCV, between 2.4 and 10.4 log10 genome copies/bee for DWV and between 3.4 and 10.4 log10 genome copies/bee for SBV. Outside these ranges, the results’ uncertainty is higher. VDV1 RT-qPCR accuracy was outside accuracy limits for all viral loads. Using these RT-qPCR methods, we quantified viral loads in naturally-infected honeybees. The viral load distribution and clinical signs reported with the honeybee samples allowed us to define a threshold that could be used to differentiate between covert and overt infections. These methods will be useful in diagnosing the main viral infections impairing honeybee health.

Multiple Virus Infections in Western Honeybee (Apis mellifera L.) Ejaculate Used for Instrumental Insemination.

Instrumental insemination of Apis mellifera L. queens is a widely employed technique used in honeybee breeding that enables the effective control of mating. However, drone semen represents a potential source of honeybee viruses. In this study, 43 semen doses collected from apparently healthy drones, and consequently used in instrumental insemination, were analysed using PCR or RT-PCR to detect the presence of viral genome of 11 honeybee viruses. In 91% of samples, viral infection was detected. The survey revealed genomes of five viruses, namely Deformed wing virus (DWV), Acute bee paralysis virus (ABPV), Black queen cell virus (BQCV), Sacbrood virus (SBV), and A. mellifera filamentous virus (AmFV) in 84%, 19%, 14%, 2%, and 67% of samples, respectively. Single infection (30% of samples) as well as multiple infection (61% of samples) of two, three or four pathogens were also evaluated. To the best of our knowledge, this is the first study describing the presence of the BQCV and SBV genome sequence in drone ejaculate. Phylogenetic analysis of BQCV partial helicase gene sequence revealed the high similarity of nucleotide sequence of described Czech strains, which varied from 91.4% to 99.6%. The findings of our study indicate the possibility of venereal transmission of BQCV and SBV.

Prevalence of bee viruses in Apis cerana cerana populations from different locations in the Fujian Province of China.

Prevalence of honeybee viral diseases has recently been causing major problems in the beekeeping industry, causing economic losses worldwide. Honeybees are susceptible to a variety of diseases and various pathogens. Among these pathogens, prevalence viruses, along with other factors, are seriously threatening the health of bee species. In the present study, samples were collected from 80 Apis cerana cerana (A. c. cerana) colonies from three different locations, Cangshan, Fuan, and Yongtai, in the Fujian Province of China. All samples were screened using the reverse transcription polymerase chain reaction (RT‐PCR) method for detection of seven honeybee viruses, namely, Chinese sacbrood virus (CSBV), deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), acute bee paralysis virus (ABPV), and Kashmir bee virus (KBV). Our results showed that CSBV was the most prevalent as it was detected in (90%), of the samples, DWV was detected in (81.25%), and IAPV was detected in (26.25%). In contrast, insignificant prevalence results were obtained from all apiaries for BQCV, CBPV, APBV, and KBV, which were not detected in any sample. Here, we are providing the first report on the molecular detection of honeybee viruses, especially the prevalence of IAPV, from different regions in the Fujian Province of China with a high prevalence of bee viruses, on A. c. cerana, and there is great concern for the presence of honeybee viruses in the population of the native honeybee (A. c. cerana) in China.

Direct transmission by injection affects competition among RNA viruses in honeybees.

The arrival of the ectoparasitic mite Varroa destructor on the western honeybee Apis mellifera saw a change in the diversity and prevalence of honeybee RNA viruses. One virus in particular, deformed wing virus (DWV) has become closely associated with V. destructor, leading many to conclude that V. destructor has affected viral virulence by changing the mode of transmission. While DWV is normally transmitted via feeding and faeces, V. destructor transmits viruses by direct injection. This change could have resulted in higher viral prevalence causing increased damage to the bees. Here we test the effect of a change in the mode of transmission on the composition and levels of honeybee RNA viruses in the absence of V. destructor. We find a rapid increase in levels of two viruses, sacbrood virus (SBV) and black queen cell virus (BQCV) after direct injection of viral extracts into honeybee pupae. In pupae injected with high levels of DWV extracted from symptomatic adult bees, DWV levels rapidly decline in the presence of SBV and BQCV. Further, we observe high mortality in honeybee pupae when injected with SBV and BQCV, whereas injecting pupae with high levels of DWV results in near 100% survival. Our results suggest a different explanation for the observed association between V. destructor and DWV. Instead of V. destructor causing an increase in DWV virulence, we hypothesize that direct virus inoculation, such as that mediated by a vector, quickly eliminates the most virulent honeybee viruses resulting in an association with less virulent viruses such as DWV.

Viral transmission in honey bees and native bees, supported by a global black queen cell virus phylogeny.

In recent decades, we have realized that honey bee viruses are not, in fact, exclusive to honey bees. The potential impact of Apis-affiliated viruses on native pollinators is prompting concern. Our research addresses the issue of virus crossover between honey bees and native bees foraging in the same localities. We measured the presence of black queen cell virus (BQCV), deformed wing virus (DWV) and sacbrood virus (SBV) in managed Apis mellifera (honey bees) and native Andrena spp. (subgenus Melandrena) bee populations in five commercial orchards. We identified viral presence across sites and bees and related these data to measures of bee community diversity. All viruses were found in both managed and native bees, and BQCV was the most common virus in each. To establish evidence for viral crossover between taxa, we undertook an additional examination of BQCV where 74 samples were sequenced and placed in a global phylogenic framework of hundreds of BQCV strains. We demonstrate pathogen sharing across managed honey bees and distantly related wild bees. This phylogenetic analysis contributes to growing evidence for host switching and places local incidence patterns in a worldwide context, revealing multispecies viral transmission.

Occurrence of honey bee (Apis mellifera L.) pathogens in commercial and traditional hives

This work was aimed at the detection of the differences in the occurrence of seven bee pathogens between bee colonies kept in commercial and traditional ways. The research was conducted on 120 apparently healthy, commercially kept colonies in DB hives and 24 traditionally kept colonies in primitive, so-called trmka hives on the Pester Plateau. Brood samples were taken from all colonies to assess the occurrence of bee brood disease agents (Paenibacillus larvae, Melissococcus plutonius, Ascosphaera apis and sacbrood virus – SBV) and adult bee pathogens (deformed wing virus – DWV, chronic bee paralysis virus – CBPV and acute bee paralysis virus – ABPV). PCR diagnostics was used in all cases, in compliance with the existing methods adopted by OIE. Concerning bee brood disease-causing agents, in commercial hives P. larvae (16.67% samples), A. apis (15.83%) and the SBV (96.67%) were confirmed, whilst in traditional hives, SBV was the only one detected (33.33%). M. plutonius was not found in any sample. As for adult bee diseases, in both commercial and traditional hives all of the three viruses were detected (DWV, ABPV, CBPV), but their occurrence in the former (100.00, 100.00 and 83.33%, respectively) was significantly (p<0.001) higher than in the latter (33.33% occurrence of each). No commercially kept colonies were free from all disease causes, while in the traditionally kept group there were 66.66% of such colonies. It can be concluded that the traditional way of beekeeping provides significantly better conditions for maintenance of bee health and their resistance to pathogens.