Centre for Ecology and Hydrology, Natural Environmental Research Council, Wallingford, Oxfordshire, United Kingdom.
PLoS One. 2013 Sep 18;8(9):e74508. doi: 10.1371/journal.pone.0074508. eCollection 2013.
We sequenced small (s) RNAs from field collected honeybees (Apis mellifera) and bumblebees (Bombuspascuorum) using the Illumina technology. The sRNA reads were assembled and resulting contigs were used to search for virus homologues in GenBank. Matches with Varroadestructor virus-1 (VDV1) and Deformed wing virus (DWV) genomic sequences were obtained for A. mellifera but not B. pascuorum. Further analyses suggested that the prevalent virus population was composed of VDV-1 and a chimera of 5'-DWV-VDV1-DWV-3'. The recombination junctions in the chimera genomes were confirmed by using RT-PCR, cDNA cloning and Sanger sequencing. We then focused on conserved short fragments (CSF, size > 25 nt) in the virus genomes by using GenBank sequences and the deep sequencing data obtained in this study. The majority of CSF sites confirmed conservation at both between-species (GenBank sequences) and within-population (dataset of this study) levels. However, conserved nucleotide positions in the GenBank sequences might be variable at the within-population level. High mutation rates (Pi>10%) were observed at a number of sites using the deep sequencing data, suggesting that sequence conservation might not always be maintained at the population level. Virus-host interactions and strategies for developing RNAi treatments against VDV1/DWV infections are discussed.
我们使用 Illumina 技术对野外采集的蜜蜂(Apis mellifera)和熊蜂(Bombus pascuorum)的小 RNA 进行测序。将 sRNA 读段组装,将得到的连续序列用于在 GenBank 中搜索病毒同源物。在 A. mellifera 中获得了与 Varroa destructor virus-1 (VDV1) 和 Deformed wing virus (DWV) 基因组序列匹配的结果,但在 B. pascuorum 中没有获得匹配结果。进一步分析表明,流行的病毒群体由 VDV-1 和 5'-DWV-VDV1-DWV-3' 的嵌合体组成。通过使用 RT-PCR、cDNA 克隆和 Sanger 测序,确认了嵌合体基因组中的重组连接点。然后,我们通过使用 GenBank 序列和本研究中获得的深度测序数据,关注病毒基因组中的保守短片段(CSF,大小>25nt)。CSF 位点在种间(GenBank 序列)和种群内(本研究数据集)水平上都得到了确认。然而,在 GenBank 序列中的保守核苷酸位置在种群内水平上可能是可变的。使用深度测序数据观察到许多位点的高突变率(Pi>10%),表明序列保守性并不总是在种群水平上保持。讨论了病毒-宿主相互作用和开发针对 VDV1/DWV 感染的 RNAi 治疗的策略。