Sindbis virus expressing enhanced GFP (SINV-GFP) was produced by transfecting in vitro transcribed RNA from the pTE3’2J with GFP inserted into the XbaI site into BHK cells [15 (link),77 (link)]. SINV-GFP was titered on BHK cell monolayers via plaque assay. SINV-GFP (3,750 plaque forming units (PFUs)) was diluted in buffer (2 μl of 10 mM Tris, pH 7.5) and injected into the thorax using a Harbo syringe (from Honey Bee Insemination Service; www.honeybee.breeding.com/HarboAssembly.html) equipped with disposable borosilicate needles made from capillary tubes (0.8-1.10 x 100 m) using a micropipette puller (Sutter Instruments Model P-87). Honey bees were immobilized via incubation in a cold room (4°C) for 20 minutes and with insect pins and forceps during injection; after injection bees recovered at room temperature within 5 minutes. Honey bees were infected with 3,750 PFUs of SINV per bee. This virus dose is modest compared to Drosophila studies which typically utilize 250-2500 PFUs per fly; a newly emerged female worker honey bee (~150 mg) weighs ~ 200x more than an adult female fruit fly (0.8 mg)[15 (link)]. This dose allowed for a natural progression of infection over the course of the experiment and at 3-days post-injection (p.i.) virus abundance could be easily assessed by microscopy and Western-blot analysis. Virus was either injected alone or in conjunction with dsRNA (1 μg).
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