Systemic Spread and Propagation of a Plant-Pathogenic Virus in European Honeybees, Apis mellifera

Li, Ji Lian; Cornman, R. Scott; Evans, Jay D.; Pettis, Jeffery S.; Zhao, Yan; Murphy, Charles; Peng, Wen Jun; Wu, Jie; Hamilton, Michele; Boncristiani, Humberto F.; Zhou, Liang; Hammond, John; Chen, Yan Ping

cDNA library construction and virus-specific primer design. Total RNA was extracted by homogenizing the viral solution with TRIzol LS reagent (Invitrogen), a solution of phenol and guanidine isothiocyanate used for isolating total RNA from liquid samples according to the manufacturer's instructions. The resultant RNA pellets were resuspended in DNase-and RNase-free water (Invitrogen) in the presence of ribonuclease inhibitor (Invitrogen). The quantity and purity of RNA were measured with a NanoDrop spectrophotometer (NanoDrop Technologies). The cDNA library was constructed using a CloneMiner cDNA library construction kit (Invitrogen) per the manufacturer's protocol. First-strand cDNA was synthesized from extracted RNA using Superscript II reverse transcriptase with a biotin-conjugated attB2 oligo(dT) primer. After cDNA synthesis, the products were size fractionated by column chromatography to remove excess primers, adapters, and small cDNAs and cloned into an attP-containing donor vector, pDONR 222. The BP (recombination between attB and attP sites) reaction products were transformed into ElectroMAX DH10B T1 phage-resistant cells, and the transformed cells were plated onto LB agar medium supplemented with kanamycin (50 g/ml). The positive clones were purified using the Wizard Plus miniprep DNA purification system (Promega). A total of 50 cDNA clones were randomly selected and sequence analyzed to confirm the presence of the insert.