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

Sequence identity of TRSV genomic segments and morphology of the virus isolates. Sequence analysis of cDNA libraries from purified virus preparation revealed overlapping and nonoverlapping clones of different lengths. About 75% of the clones (n Ï­ 40) matched the genome sequences of common honeybee viruses, including BQCV, DWV, and Israeli acute paralysis virus (IAPV). Unexpectedly, about 20% of the clones (n Ï­ 10) matched the sequences of TRSV for two genomic segments in the NCBI database. By assembling sequence fragments from different cDNA clones, we obtained a 1,545-bp length of nucleotide sequences encoding the RNA helicase and covering~21% of the coding region of the polyprotein gene of genomic RNA-1. We also obtained a 2,024-bp long sequence encoding the complete capsid protein. A BLAST search of the helicase sequence showed highest identity with a TRSV strain isolated from bud blight disease of soybean (GenBank accession no. U50869), with 88% homology at the nucleotide level and 96% homology at the amino acid level. A BLAST search of the DNA fragment encoding the capsid protein showed strongest similarity to a TRSV strain from bean (GenBank accession no. L09205), with 96% homology at the nucleotide level and 99% homology at the amino acid level. The cDNA sequences were used to design two primer sets, TRSV-F1/R1 and TRSV-F2/R2 (Fig. 1) , for the subsequent studies of TRSV replication and distribution in honeybees and Varroa mites.