RNA-Seq of Apis mellifera pupa injected with hemolymph extract containing 5 DWV USA Infectious Clones
Full-length infectious cDNA clones of individual DWV genotypes from a typical Varroa-infested honeybee colony were constructed using five sequenced 9.7 kb DWV cDNA constructs. (This source was the same as the one used to prepare the NGS library V99, BioSample SAMN08430537.) These constructs were extended to include the missing 468 nt 5  terminal sequences designed according to North American DWV data. The DWV cDNA construct was preceded by the T7 RNA polymerase promoter to enable in vitro transcription of the DWV cDNA (GenBank Acc. MG831200 to MG831204) and a ribozyme sequence designed to cut the in vitro transcripts to produce a precise 5  terminus. The unique restriction site PmeI located downstream of the 3  terminal poly(A) tail was used to linearize the DWV cDNA constructs to produce templates to the full-length in vitro RNA transcripts. Constructs were then treated with RNAse-free DNAse, column purified and resuspended in PBS prior to injection in honey bee pupa. Pink-eye honey bee pupae, from a Texas brood frame with low Varroa infestation and which had no observable Varroa mite feeding exposure, were intra-abdominally injected into the hemolymph to introduce 5 microgram of purified RNA transcript; separate pupae were used for each of five transcripts (GenBank Acc. MG831200-MG831204). After a 3 day-long incubation (3 dpi) to allow replication of DWV, the infected pupae were homeogenized and filtered to prepare an extract containing clone-derived DWV particles. After a 3 day-long incubation (3 dpi) to allow replication of DWV, the infected pupae was homeogenized and filtered to prepare an extract containing clone-derived DWV particles. The extracts were quantified using qPCR, then diluted and mixed to achieve equal proportions of each clone-derived DWV particle. A pink-eye honey bee pupa, from a Florida brood frame with low Varroa infestation and which had no observable Varroa mite feeding exposure, was intra-abdominally injected into the hemolymph to introduce the filtered clone derived extract mixture containing a total of 10^7 copies of DWV RNA genomes. After a 3 day-long incubation (3 dpi) to allow replication of DWV, total RNA was extracted for Illumina TruSeq library preparation and sequencing.