In this study honeybee viruses in naturally infected honeybee colonies (Apis mellifera carnica) were determined and identified. Furthermore, the development of the viral infection after honeybee brood virus inoculation was studied. Two new sets of primers, TaqMan probes and standards for quantitative method of real-time reverse transcription and polymerase chain reaction (RT-qPCR) for detection and quantification of Lake Sinai virus lineage 3 (LSV3) and sacbrood bee virus (SBV) were developed and implemented. 89 samples of clinically affected honeybees sampled from April 2016 to September 2020 and 108 samples of clinically healthy honeybee colonies sampled over one year period from October 2018 to October 2019, were examined with RT-qPCR methods, for presence of six honeybee viruses: acute bee paralysis virus (ABPV), black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), deformed wing virus (DWV), LSV3 and SBV. ABPV was detected in 29,63 % of clinically healthy and 79,78 % of clinically affected honeybee colonies, BQCV was detected in 98,15 % of clinically healthy and 96,63 % of clinically affected honeybee colonies. CBPV was not detected in any sample of clinically healthy but was detected in 19,10 % of clinically affected honeybee samples. DWV was detected in 39,81 % of healthy and 69,66 % of affected honeybee colonies, LSV3 was detected in 67,59 % of healthy and 48,31 % of affected honeybee colonies. SBV was detected in 35,19 % of healthy and in 22,47 % of affected honeybee colonies. There were statistically significantly higher viral copy numbers detected in clinically affected honeybee colonies infected with ABPV, CBPV, DWV in SBV (p < 0,0001), but with BQCV (p= 0,6597) and LSV3 (p=0,0062) there were no statistically significant differences detected in viral copy numbers between healthy and affected honeybee colonies. From nine samples of clinically affected and ten samples of healthy honeybee colonies in which different strains of honeybee viruses were detected by Sanger sequencing, 22 nucleotide sequences of complete genomes of honeybee viruses were determined with next generation sequencing (NGS) method (ABPV n=4, BQCV n=3, CBPV n=2, DWV n=5, LSV n=3, SBV n=1, Apis Rhabdovirus-1 (ARV-1) n=1, Bee Macula-like virus (BeeMLV) n=1, Hubei partiti-like virus 34 (HPLV34) n=1). Nucleotide sequences of complete genomes of ABPV, BQCV, DWV and SBV are first nucleotide sequences of complete genomes of this viral species determined in Slovenia. ARV-1, BeeMLV and HPLV34 were detected in Slovenia for the first time and they represent important contribution to genetic diversity of honeybee viruses identified in Slovenia and over the world. With the goal to research the replication of honeybee viruses and development of viral infections, we implemented the method of in vitro rearing of honeybee larvae. In the developing phase of honeybee larva from day one to day five, with inoculation of field strains of LSV3 and SBV per os, viral replication and dying of larvae were studied. The replication of LSV3 and SBV in the larvae was not determined and significant differences in dying of larvae depending on different viral doses were not detected. The implemented methods have great potential for further studies of viral, bacterial and other infections on the developing stages of honeybees.
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