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

TRSV is a positive-stranded RNA virus replicating through the production of a negative-stranded intermediate; therefore, the presence of negative-stranded RNA constitutes proof of active viral replication. To investigate the replication of TRSV in bees, negative-stranded RT-qPCR was performed using a tagged primer system (28) . Amplification and sequence analysis of a 462-bp negative-strand-specific product in different tissues showed that active replication of TRSV occurs in most tissues (Fig. 4) . A single peak on the melting curve analysis corroborated the specificity of the amplicon. The lack of amplification following RT-qPCR of total RNA without primers in the reverse transcription reaction mixture ruled out any nonspecific effect from self priming due to the secondary structure of viral RNA or false priming by antigenomic viral RNA or cellular RNAs. Among tissues with detectable levels, the relative abundance of negative-stranded TRSV varied significantly (P Ͻ 0.001; one-way analysis of variance [ANOVA]). The brain had the lowest detectable level of negativestranded TRSV and was chosen as the calibrator. The abundance of TRSV in other tissues relative to the brain ranged from 56-fold to 957-fold. The concentration of TRSV in additional body tissues showed the following ranking: muscle Ͼ hypopharyngeal gland Ͼ leg Ͼ fat body Ͼ trachea Ͼ hemolymph Ͼ antenna Ͼ nerve Ͼ wing. The replication of TRSV was not evident in the salivary gland, gut or compound eye (Fig. 5) , although the presence of PCR inhibitors in the latter is a possibility (27) .