First Complete Genome of Lake Sinai Virus Lineage 3 and Genetic Diversity of Lake Sinai Virus Strains From Honey Bees and Bumble Bees

Abstract

The complete genome of Lake Sinai virus 3 (LSV3) was sequenced by the Ion Torrent next-generation sequencing (NGS) technology from an archive sample of honey bees collected in 2010. This strain M92/2010 is the first complete genome sequence of LSV lineage 3. From October 2016 to December 2017, 56 honey bee samples from 32 different locations and 41 bumble bee samples from five different locations were collected. These samples were tested using a specific reverse transcriptase-polymerase chain reaction (RT-PCR) method; 75.92% of honey bee samples and 17.07% of bumble bee samples were LSV-positive with the RT-PCR method. Phylogenetic comparison of 557-base pair-long RNA-dependent RNA polymerase (RdRp) genome region of selected 23 positive samples of honey bees and three positive bumble bee samples identified three different LSV lineages: LSV1, LSV2, and LSV3. The LSV3 lineage was confirmed for the first time in Slovenia in 2010, and the same strain was later detected in several locations within the country. The LSV strains detected in bumble bees are from 98.6 to 99.4% identical to LSV strains detected among honey bees in the same territory.

Determination of Genetically Identical Strains of Four Honeybee Viruses in Bumblebee Positive Samples

“…The result of testing by reaction (RT-PCR) method showed that and 75.92% of honeybee samples and 17.07% of bumblebee samples were LSV positive. The identified strains of LSV from bumblebees share from 98.6% to 99.4% nucleotide identity to the closest honeybee LSV strains collected from the same geographical area in Slovenia [5]. LSVs have also been also detected, with similar incidences and titres in historical European honeybee samples.…”

“…Nucleic acids of five honeybee viruses (ABPV, BQCV, CBPV, DWV and SBV) were detected using a specific method of reverse transcription and polymerase chain reaction (RT-PCR), as previously described [6]. The LSV was detected with a specific RT-PCR method [5] using previously described primers LSV 1765-F and LSV 2368-R [19]. Results were evaluated based on the size of RT-PCR products in the agarose gel as positive in the case of the expected product size: for ABPV 452 nucleotides (nt), for BQCV 770 nt, for CBPV 570 nt, for DWV 504 nt, for SBV 814 nt and for LSV 603 nt.…”

“…In the case of a positive result, the selected RT-PCR products of a single virus were directly sequenced with the Sanger sequencing protocol, using the same primers as used for specific RT-PCR as described previously [5,9,16]. Individual sequences were analysed using the DNASTAR 5.05 (Lasergen, WI, USA) programme and 158 positive samples of four viruses (ABPV: n = 33, BQCV: n = 75, LSV: n = 25 and SBV: n = 25) detected in honeybees and bumblebees together with closely related sequences from GenBank and interpreted according to the results of the nucleotide sequence matching between honeybee and bumblebee samples.…”

“…Regarding the most pathogenic bee viruses, such as ABPV, CBPV, DWV, SBV, and the detection of several novel RNA viruses, studies have been carried out over the previous decade in relation to honeybee losses and the evaluation of their impact on honeybee health [3]. The increased number of investigations of viral infections in honeybee colonies and the use of new technologies, such as the next generation of sequencing (NGS), resulted in the detection of several new RNA and DNA viruses, such as the Lake Sinai virus (LSV) in 2010 [2][3][4][5]. For some of these viruses, it is not yet clear what impact they have on the honeybee colonies, since some viral infections in bees are present as permanent or subclinical infections [6][7][8].…”

The Comparison of Honeybee Viral Loads for Six Honeybee Viruses (ABPV, BQCV, CBPV, DWV, LSV3 and SBV) in Healthy and Clinically Affected Honeybees with TaqMan Quantitative Real-Time RT-PCR Assays

“…LSV replicates in honeybees that often harbour high viral burdens (10 8 –10 9 viral copies/bee) [ 5 ]. Although there are no obvious clinical symptoms attributed to LSV-positive honeybee families, their abundance and association with colonies of poor health are indications that different genetic strains of LSV might have a negative impact on honeybee health [ 28 ].…”