Iwanowicz Deborah D, Wu-Smart Judy Y, Olgun Tugce, Smart Autumn H, Otto Clint R V, Lopez Dawn, Evans Jay D, Cornman Robert
Leetown Science Center, U.S. Geological Survey, Kearneysville, WV, United States of America.
Entomology, University of Nebraska-Lincoln, Lincoln, NE, United States of America.
PeerJ. 2020 Jul 17;8:e9424. doi: 10.7717/peerj.9424. eCollection 2020.
Lake Sinai Viruses (LSV) are common RNA viruses of honey bees () 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 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.
Here 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).
The first sample set in our pilot assessment compared cDNA pools from managed hives in California ( = 8) and Maryland ( = 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, = 24 paired samples), collected from managed 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 (four of 63 specimens tested, 6.3%) at a similar rate as (nine of 115 specimens, 7.8%), but only one 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.
西奈湖病毒（LSV）是蜜蜂常见的RNA病毒，其数量常常很高，但与明显的疾病并无关联。LSV在基因上具有异质性且分布广泛，但尽管在调查中经常被检测到，人们并不了解影响其在蜜蜂中分布的生态和地理因素。对于它们在其他物种中的分布更是知之甚少。对LSV流行情况和生态学的更好理解一直受到LSV进化枝内高序列多样性的阻碍。
在此，我们报告了一种新的聚合酶链反应（PCR）检测方法，该方法与目前已知的谱系兼容，引物简并度最低，可产生适合终点PCR和宏基因组测序的预期365 bp扩增子。我们使用Illumina MiSeq平台对三个样本集进行了初步宏基因组评估，每个样本集代表一个可能构成LSV多样性的不同变量（地理、组织和物种）。
我们初步评估中的第一个样本集比较了来自加利福尼亚州（n = 8）和马里兰州（n = 6）管理蜂箱的cDNA文库，这些蜂箱之前已针对LSV2进行过评估，证实引物能在实际样本中共扩增不同的谱系。第二个样本集包括来自北达科他州管理蜂箱的不同组织（胸部与腹部，n = 24对样本）的cDNA文库。两种组织类型中LSV的终点检测结果常常不同；在一对测序样本中LSV宏基因组组成相似，但在另一对样本中则不同。总体而言，LSV1和中间谱系在这些样本中很常见，而与LSV2聚类的变体则很少见。第三个样本集包括从内布拉斯加州林肯市附近不同景观中采集的单个传粉者标本的cDNA。我们在熊蜂（63个测试标本中有4个，6.3%）中检测到LSV的比例与蜜蜂（115个标本中有9个，7.8%）相似，但只有一个熊蜂测序文库产生了足够的数据用于组成分析。测序样本通常包含多个不同的LSV谱系，包括单个标本。虽然这些研究是探索性的，而非对假设进行具有统计学效力的检验，但它们说明了高通量测序在理解LSV在物种内和物种间传播方面的实用性。
