Search

Phylogenetic analysis of 22 Black queen cell virus (BQCV) genotypes collected from honeybee colonies in Poland, Austria and Hungary was performed on a partial helicase enzyme coding region (ORF1) and on a partial structural polypeptide coding region (ORF2). While the phylogeny based on the ORF2 region showed--with the exception of one strain from Poland--clustering of the genotypes corresponding to their geographic origin, the ORF1-based tree exhibited a completely different distribution of the Polish strains: three of them clustered within a branch clearly separated from all other central European BQCVs, while four other Polish strains remained well within the central European BQCV genotypes. In order to investigate this discrepancy in more detail, the nearly complete genome sequences of the three differing Polish strains were determined, together with one Hungarian sample. The sequences were aligned to each other and to the reference strain from South-Africa. Comparison of the different genome regions revealed that the 5'-UTR and the intergenic regions of the BQCV genome are highly conserved with longer homologous sections. ORF1 (non-structural protein coding region) was found more variable compared to ORF2 (structural protein coding region). The 5'-proximal third of ORF1 was particularly variable and contained several deletions/insertions. The sudden changes in the similarity levels of BQCV strains in different genomic regions are indicative of preceding recombination events.

Chronic bee paralysis virus (CBPV) is responsible for chronic paralysis, an infectious and contagious disease of adult honey bees (Apis mellifera L.). The full-length nucleotide sequences of the two major RNAs of CBPV have previously been characterized. The Orf3 of RNA1 has shown significant similarities to the RNA-dependent RNA polymerase (RdRp) of positive single-stranded RNA viruses, whereas the Orf3 of RNA2 encodes a putative structural protein (pSP). In the present study, honey bees originating from 9 different countries (Austria, Poland, Hungary, Spain, Belgium, Denmark, Switzerland, Uruguay and France) were analysed for the presence of CBPV genome. The complete genomic nucleotide sequence of the RdRp (1947bp) and of the pSP (543bp) from 24 honey bee positive samples was determined and the phylogenetic relationship among isolates was investigated. Four distinct genotypes of CBPV were observed.

Microsporidiosis (nosema disease) of the European honeybee ( Apis mellifera L.) is present in bee colonies worldwide. Until recently, Nosema apis had been regarded as the causative agent of the disease, which may have many negative effects on the colony and cause heavy economic losses in apicultures. Another microsporidium species, Nosema ceranae , was reported to infest the Asian honeybee ( Apis ceranae ), but both honeybee species are susceptible to both microsporidia. In the European honeybee N. ceranae was first detected in Spain in the year 2006. As it is difficult to distinguish N. ceranae and N. apis morphologically, a rapid and accurate assay has been developed to differentiate N. apis and N. ceranae based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the partial large subunit ribosomal RNA. The assay was tested on 38 Nosema -infested bee samples, which were collected from geographically distant Hungarian bee colonies representing all regions of the country. Only one sample contained N. apis , and in the other 37 samples N. ceranae was detected, which indicates the dominance of N. ceranae in Hungarian apiaries. This is the first report on the presence of N. ceranae in Hungary.

A survey on the occurrence of six honeybee-pathogenic viruses was carried out using one-step RT-PCR assays. Samples were collected between 1999 and 2004 in 52 Hungarian apiaries located in different regions of the country. The results of the assays on samples of adult honeybees and Varroa destructor mites were compared to similar surveys from France and Austria. The study demonstrates geographical differences in the prevalence of honeybee viruses between Hungary and the older EU member states. The results could serve as a basis for monitoring further changes in the distribution of honeybee viruses in Europe.

Honeybees originating from 10 different countries (Austria, Poland, Germany, Hungary, Slovenia, Nepal, Sri Lanka, the United Arab Emirates, Canada, and New Zealand) located on four continents were analyzed for the presence of deformed wing virus (DWV) nucleic acid by reverse transcription-PCR. Two target regions within the DWV genome were selected for PCR amplification and subsequent sequencing, i.e., a region within the putative VP2 and VP4 structural-protein genes and a region within the RNA helicase enzyme gene. DWV nucleic acid was amplified from 34 honeybee samples representing all the above-mentioned countries with the notable exception of New Zealand. The amplification products were sequenced, and phylogenetic analyses of both genomic regions were performed independently. The phylogenetic analyses included all sequences determined in this study as well as previously published DWV sequences and the sequences of two closely related viruses, Kakugo virus (KGV) and Varroa destructor virus 1 (VDV-1). In the sequenced regions, the DWV genome turned out to be highly conserved, independent of the geographic origins of the honeybee samples: the partial sequences exhibited 98 to 99% nucleotide sequence identity. Substitutions were most frequently observed at the same positions in the various DWV sequences. Due to the high level of sequence conservation, no significant clustering of the samples in the phylogenetic trees could be identified. On the other hand, the phylogenetic analyses support a genetic segregation of KGV and VDV-1 from DWV.

A single-step multiple-target (multiplex) reverse transcription-PCR (RT-PCR) was developed for the simultaneous detection and differentiation of three economically important viruses of the honeybee Apis mellifera L.: Acute bee paralysis virus (ABPV), Black queen cell virus (BQCV) and Sacbrood virus (SBV). Three compatible sets of primers, specific for each virus, were designed in conserved regions of the viral genomes for use in a one-step (single tube) RT-PCR assay. The individual RT-PCR assays and the combined multiplex assay were optimized for highest sensitivity and specificity. The multiplex RT-PCR assay was tested on field samples collected from Austrian honeybee colonies. All three viruses were detected, and their identity was confirmed by sequencing of the PCR products. The described multiplex RT-PCR proved to be an accurate tool for rapid simultaneous detection of ABPV, BQCV and SBV directly in honeybee specimens.

The occurrence, prevalence, and distribution patterns of acute bee paralysis virus (ABPV), black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), deformed wing virus (DWV), Kashmir bee virus (KBV), and sacbrood virus (SBV) were investigated in 90 Austrian honeybee colonies suffering from symptoms of depopulation, sudden collapse, paralysis, or dark coloring by employing reverse transcription-PCR. Infestation with parasites was also recorded. The samples originated from all parts of Austria. The most prevalent virus was DWV, present in 91% of samples, followed by ABPV, SBV, and BQCV (68%, 49%, and 30%, respectively). CBPV was detected in 10% of colonies, while KBV was not present in any sample. In most samples, more than one virus was identified. The distribution pattern of ABPV, BQCV, CBPV, and SBV varied considerably in the different geographic regions investigated, while DWV was widespread in all Austrian federal states. In bees that showed dark coloring and disorientation, CBPV was always detected. Simultaneous infections of DWV and ABPV were most frequently observed in colonies suffering from weakness, depopulation, and sudden collapse. Bees obtained from apparently healthy colonies within the same apiaries showed a similar distribution pattern of viruses; however, the relative virus load was 10 to 126 times lower than in bees from diseased colonies. A limited number of bee samples from surrounding central European countries (Germany, Poland, Hungary, and Slovenia) were also tested for the presence of the above viruses. Variances were found in the distribution of BQCV and SBV.

PCR assays were developed for the direct detection of Paenibacillus larvae in honey samples and compared with isolation and biochemical characterization procedures. Different primer pairs, designed from the 16S rRNA and the metalloproteinase precursor gene regions, and different DNA extraction methods were tested and compared. The sensitivity of the reactions was evaluated by serial dilutions of DNA extracts obtained from P. larvae cultures. The specificity of the primers was assessed by analyzing related Paenibacillus and Bacillus strains isolated from honey. The PCR assays also amplified these related bacteria, but at lower sensitivity. In the next step, the PCR assays were applied to contaminated honey and other bee products originating from 15 countries. Lysozyme treatment followed by proteinase K digestion was determined to be the best DNA extraction method for P. larvae spores. The most sensitive primer pair detected P. larvae in 18 of 23 contaminated honey samples, as well as in pollen, wax, and brood. Honey specimens containing saprophyte bacilli and paenibacilli, but not P. larvae, were PCR negative. Although the isolation and biochemical identification method (BioLog) showed higher sensitivity and specificity, PCR proved to be a valuable technique for large-scale screening of honey samples for American foulbrood, especially considering its rapidity and moderate costs.

Reverse transcription-PCR assays have been established for a quick, sensitive, and specific diagnosis of acute bee paralysis virus (ABPV), a common virus of the honeybee (Apis mellifera), directly from clinical samples. A 3,071-nucleotide fragment of the ABPV genome, which includes the entire capsid polyprotein gene, was amplified from Austrian, German, Polish, and Hungarian ABPV samples and sequenced, and the sequences were compared. The alignment of a smaller fragment with ABPV sequences from the United States and the United Kingdom revealed nucleotide identity rates between 89 and 96%, respectively. Phylogenetic trees which display the molecular relationship between the viruses of different geographic origin were constructed.

Viruses of the honey bee have been known for a long time; however, recently the attention of scientists and apiculturalists has turned towards the relationship between these viruses and the parasitic mite Varroa jacobsoni. Although clinical symptoms indicated the presence of some of the viruses of bees in Hungary, none have previously been isolated or identified. During July unusual adult bee and brood mortality was observed in some colonies of an apiary in Budapest known to be infested with Varroa jacobsoni. Large amounts of acute paralysis virus (APV) were detected serologically in healthy honey bee pupae killed by the injection of a bacteria-free extract of diseased adult bees. Crystalline arrays of 30 nm particles were seen in ultrathin sections of the tissues of injected pupae and naturally infected adult bees. In spite of the application of acaricide treatments the bee population in several colonies had collapsed by the end of summer and the apiary suffered severe wintering losses.