Abstract
BackgroundLake Sinai Viruses (LSV) are common RNA viruses of honey bees (Apis mellifera) that frequently reach high abundance but are not linked to overt disease. LSVs are genetically heterogeneous and collectively widespread, but despite frequent detection in surveys, the ecological and geographic factors structuring their distribution in A. mellifera are not understood. Even less is known about their distribution in other species. Better understanding of LSV prevalence and ecology have been hampered by high sequence diversity within the LSV clade.MethodsHere we report a new polymerase chain reaction (PCR) assay that is compatible with currently known lineages with minimal primer degeneracy, producing an expected 365 bp amplicon suitable for end-point PCR and metagenetic sequencing. Using the Illumina MiSeq platform, we performed pilot metagenetic assessments of three sample sets, each representing a distinct variable that might structure LSV diversity (geography, tissue, and species).ResultsThe first sample set in our pilot assessment compared cDNA pools from managed A. mellifera hives in California (n = 8) and Maryland (n = 6) that had previously been evaluated for LSV2, confirming that the primers co-amplify divergent lineages in real-world samples. The second sample set included cDNA pools derived from different tissues (thorax vs. abdomen, n = 24 paired samples), collected from managed A. mellifera hives in North Dakota. End-point detection of LSV frequently differed between the two tissue types; LSV metagenetic composition was similar in one pair of sequenced samples but divergent in a second pair. Overall, LSV1 and intermediate lineages were common in these samples whereas variants clustering with LSV2 were rare. The third sample set included cDNA from individual pollinator specimens collected from diverse landscapes in the vicinity of Lincoln, Nebraska. We detected LSV in the bee Halictus ligatus (four of 63 specimens tested, 6.3%) at a similar rate as A. mellifera (nine of 115 specimens, 7.8%), but only one H. ligatus sequencing library yielded sufficient data for compositional analysis. Sequenced samples often contained multiple divergent LSV lineages, including individual specimens. While these studies were exploratory rather than statistically powerful tests of hypotheses, they illustrate the utility of high-throughput sequencing for understanding LSV transmission within and among species.
Highlights
Lake Sinai Viruses (LSV) are single-strand RNA viruses first detected in metagenomic investigations of honey bees (Apis mellifera) (Runckel et al, 2011; Cornman et al, 2012)
Four CA samples and three MD samples testing positive for LSV2 with the approach of Traynor et al (2016) (which used LSV2-specific primers designed by Runckel et al (2011) and fluorescence-based detection of their amplification) and four CA and three MD samples testing negative by that protocol were re-evaluated with the new primers described
We continue to use LSV1 and LSV2 as these labels were established simultaneously in the literature by their original discoverers (Runckel et al, 2011), are consistently identified in phylogenetic analysis as homogeneous clusters well diverged from each other, are in wide usage and recognized as distinct taxa in NCBI databases, and the presence of one or both is nearly ubiquitous in surveys
Summary
Lake Sinai Viruses (LSV) are single-strand RNA viruses first detected in metagenomic investigations of honey bees (Apis mellifera) (Runckel et al, 2011; Cornman et al, 2012). They produce no described disease syndromes in honey bees despite occasionally high prevalence and abundance in managed colonies (Runckel et al, 2011; Cavigli et al, 2016; Traynor et al, 2016; Glenny et al, 2017). While these studies were exploratory rather than statistically powerful tests of hypotheses, they
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