Occurrence of the honey bee viruses in Hungary, investigations of the molecular structure of certain viruses
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Viruses of the honey bee (Apis mellifera Linneaus) have been known for a long time. However, recently the attention of researchers and beekeepers has turned towards the relationship between these viruses and the parasitic mite Varroa destructor (former name V. jacobsoni). Although clinical symptoms indicated the presence of some of the bee specific viruses in Hungary, none has previously been isolated or identified in our country. In July, 1997 unusual adult bee and brood mortality was observed in some colonies of an apiary in Budapest known to be infested with Varroa destructor. Large amounts of virion particles were detected in honey bee pupae experimentally inoculated with bacterium-free extracts 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. The virus was purified by gradient ultracentrifugation and was identified as acute bee paralysis virus (ABPV) by agar-gel immunodiffusion (AGID) tests. Since ABPV is considered to be a common infectious agent of the honey bee, and it is present in high proportions of bee colonies worldwide, a two years survey was undertaken to determine its occurrence in field samples of adult bees and the parasitic mite Varroa destructor in Hungary. Considering the difficulties in the isolation of ABPV, we used polymerase chain reaction following reverse transcription (RT-PCR) to detect the viral nucleic acid in bee samples. We demonstrated the presence of ABPV RNA in 14 of 114 seemingly healthy colonies collected from eight apiaries. The investigation revealed that two third of the apiaries were infected with ABPV at a 12.2 % infection rate. In seven other apiaries out of eight investigated (87.5 %) the presence of the virus was also detected from colonies following a sudden collapse; these colonies were simultaneously infected with Nosema apis or infested with Varroa destructor. Virus specific nucleic acid was also identified in the mites collected from two apiaries falling into the latter category. The amplicon of RT-PCR was sequenced and the nucleic acid sequence was aligned to the complete ABPV sequence deposited in the GenBank database revealing a 93 % identity. Regarding the wide distribution of ABPV in Central Europe with various clinical manifestations, phylogenetic analysis was performed on isolates to reveal the variability of the ABPV genome, and the molecular relationship between virus strains of different geographic origin. A 3071 nt fragment of the ABPV genome including the entire structural protein gene region, has been amplified from one Austrian, three German, three Polish and four Hungarian bee samples employing six different RT-PCR assays. The amplicons were sequenced, and the nucleotide sequences were compiled and aligned. The sequences showed identity rates of 94% to 95% compared to the reference strain. The phylogenetic analysis revealed three distinct genotypes: the ABPV samples from Austria and Germany were grouped together in one branch, while the Polish and the Hungarian strains formed two other distinct clusters. Another comparative and phylogenetic analysis was carried out on a shorter (401 nt) fragment of the ABPV structural protein gene; in this analysis, all ABPV sequences available to date have been included (eleven sequences of probable UK origin deposited in the GenBank database, partial sequences of the samples mentioned above, and additional ten sequences amplified from nine Hungarian and one Polish ABPV specimens). The nucleotide sequences of these virus strains showed identity rates between 89% and 96%, respectively. In the phylogenetic tree constructed with these sequences, the ABPV strains were separated into at least two major branches. One is composed of the British sequences deposited in GenBank, while the other branch comprised the isolates from continental Europe; however, every branch could be sub-divided into several distinct clusters. The RT-PCR assays represent the methodical basis for phylogenetic analysis and classification of new ABPV isolates. To reveal the genetic variability of the non-structural protein genes, the helicase and protease regions of one Hungarian and one Polish ABPV isolates were analyzed. A 4338 nt long sequence was determined, which covers 45.7% of the genome. The sequences were aligned to the reference complete ABPV genome. Sequence analysis revealed 93% identity to the reference strains, while the two Central European strains have shown 97% identity to each other. By the comparison of the deduced amino acid sequences 96% identity to the reference strain and 99% identity within the Central European strains were observed. The investigations supported that the helicase and protease genes are conserved genomic regions of ABPV, with similary low level of sequence divergence as it was observed in the structural protein gene regions of the investigated strains. Within the survey on the occurrence of ABPV in Hungary a virus designated as Hu- B1/97 was isolated from an acute disease outbreak in an apiary causing high mortality among adult bees. In the identification procedure of the virus with ABPV specific immune-serum in AGID test, interestingly, a double precipitation line occured, indicating the presence of two antigenically related but not identical viruses. Discriminating primer pairs designed in the structural protein-coding region of ABPV were used in RT-PCR investigations. Sequencing of the amplicons proved that the virus suspension contains indeed a mixture of two genetically distinct viruses. Homology search demonstrated a new variant of Kashmir bee virus (KBV) as one of the components (nucleic acid identity 83.6 %) while the other virus was closely related to the prototype ABPV strain (93.6 % nucleic acid identity). The sensitivity and the easy application of RT-PCR proved to be extremely useful in the diagnostics of the viral infections of bees. Therefore a diagnostic RT-PCR method was developed and tested for the detection of four important bee viruses in field samples. Specific primer pairs were selected for the amplification of SBV, BQCV, ABPV and KBV genomic fragment in one amplification panel. The amplified products are well-distinguishable by their sizes. The described method is useful for the quick and reliable detection of bee viruses from field samples.