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

Strand-specific RT-qPCR. In order to determine the ability of TRSV to replicate in different tissues of honeybees, RNA samples were further analyzed for the presence and abundance of negative-stranded RNA, a replicative intermediate, using strand-specific reverse transcription coupled with quantitative PCR (RT-qPCR). For each tissue sample, the firststrand cDNA was synthesized from total RNA using Superscript III reverse transcriptase (Invitrogen) with Tag-TRSV-F1 (5=-AGCCTGCGCA CGTGGcatgaatgttgttatccaat-3=), where the capitalized sequence corresponding to Tag was published by Yue and Genersch (61) . The synthesized cDNAs were then purified using a MinElute PCR purification kit (Qiagen) followed by a MinElute Reaction Clean kit (Qiagen) to remove short fragments of oligonucleotides and residue of enzymatic reagents to prevent amplification of non-strand-specific products (28) . cDNA derived from negative-stranded RNA was amplified using the Brilliant SYBR green qPCR master mix (Stratagene) with a 0.4 M concentration each of the Tag (3=-AGCCTGCGCACCGTGG-5=) and TRSV-R1 primers in a 25-l volume according to the manufacturer's protocol. To normalize the qPCR result, amplification of a housekeeping gene, the ␤-actin gene, was performed for each sample with a previously reported primer set (62) .