dsRNA-mediated reduction of virus in honey bees.

<p>(A) Schematic of experiment. Honey bees were inoculated with SINV-GFP or co-injected with (B) nucleic acid treatments including: virus-specific dsRNA (SINV dsRNA); non-specific dsRNA corresponding to Drosophila C virus (DCV) sequence (DCV dsRNA); poly(I:C); NTPs; non-specific dsRNA corresponding to luciferase sequence (LUC dsRNA); or SINV dsDNA visualized on a 1% agarose gel. (C) Fluorescence microscope images of a mock-infected bee (left panel), a SINV-GFP infected bee (middle panel), or a bee infected with SINV-GFP in the presence of non-specific dsRNA DCV. (D) Western blot analysis using α-GFP and α-actin antibodies demonstrated reduced SINV-GFP in bee protein lysates pooled from ten bees treated with dsRNA (lanes 1, 2, and 5) and poly(I:C) (lane 3), but not in bees treated with NTPs (lane 4) or dsDNA (lane 6) compared to bees inoculated with virus alone (virus). Mock-infected (mock) bees injected with buffer had no detectable GFP. (E) Boxplot of GFP fluorescence (510 nm, relative arbitrary units) in individual honey bee lysates (10 bees per condition). A Welch two-sample t-test was performed to compare honey bee lysates from each treatment group to virus-infected bees and determined statistically significant differences between bees co-inoculated with virus-specific dsRNA SINV p=0.023, with non-specific dsRNA DCV p=0.020, and with poly(I:C) p=0.0038, whereas co-injection of NTPs p=0.41 or dsDNA p=0.36 were not significantly different from virus alone (virus). Bees injected with virus only (virus) exhibited greater fluorescence compared to mock-infected controls (m); p=0.00001. (F) Relative abundance of SINV-GFP in individual bees (5 per condition) from different treatment groups was assessed by RT-qPCR. The bees treated with virus only had the highest SINV copy number 3 days p.i. (1.65x10⁶ copies <u>+</u> 4,100 copies per 500 ng RNA, ~1.65x10⁸ per bee), as compared to those in which either virus-specific SINV dsRNA (1.66x10⁵ +1,050) or non-specific DCV dsRNA (2.32x10⁵<u>+</u> 1,070) was co-injected with virus.