Fig. 1.

Schematic for the DWV passaging paradigm. (a) Each injection experiment included bees that were in vitro reared but unmanipulated (Control, no inject), PBS injected (Control, PBS inject), and bees injected with deformed wing virus (DWV+). At 4 days post-injection (4 DPI), pupae were collected and crude inocula were created from individual pupae. This paradigm resulted in one ‘passage’. Inocula created from single bees were subsequently injected into two new pupae, and again harvested 4 DPI, and passaged inocula were again prepared from individual pupae. This method was repeated for five passages. All passaged inocula were assessed for DWV titres via qPCR. Passage 1 inocula and a subset of inocula from Passage 5 were also assessed for virulence differences in survival assays. The starting inoculum, Passage 1 inocula, and all Passage 5 inocula were sequenced to identify nucleotide variation that shifted through passaging. (b) From one starting DWV population, multiple lineages were established in Passage 1, and a total of 47 final viral populations (Passage 5), were created through this passaging paradigm.