Figure 11.

Mab 55A10 and 55B20 detect the 3CL protease/DL as well as mature 3CL protease and 3DL in bee pupae transfected with synthetic DWV RNA. Honey bee pupae transfected with synthetic RNA of the DWV strain 1414 (rDWV 1414) and mock-transfected control pupae were harvested three days post-transfection (p. t.). The pupae were homogenized and total bee protein was resolved via SDS-PAGE. (A) Western blot analysis using the anti-VP1 Mab VP1A1. The mock-transfected pupa showed no signal indicating no background infection of the bees. In contrast, a typical VP1 pattern with bands at 47, 42, and 39 kDa appeared in the DWV RNA-transfected pupae, indicating successful infection and viral protein expression. (B) Western blot analysis using Mab 55A10 anti-Pol, which targets the 3DL of DWV. The antibody did not show a reactivity against proteins of the DWV-free bee pupae. In the DWV-infected bee pupae, a strong reactivity against a 90 kDa protein and a weak signal indicative of a 55 kDa protein occurred. These reactivities could be assigned to the 3CL protease/DL precursor and the mature 3DL. A 65 kDa band, which cannot yet be assigned more precisely, and a 130 kDa band, which could represent a high-molecular precursor molecule, were marked with an asterisk. (C) Western blot analysis using Mab 55B20 anti-Prot, which targets the 3CL protein of DWV. Mab 55B20 showed a strong reactivity with proteins in the uninfected bee pupae, with very strong bands at 75 and 130 kDa and a weaker band at 40 kDa. Despite the strong background reactivities, additional DWV-specific protein bands appeared in the infected pupae. The protein bands at 90 kDa and 33 kDa could be assigned to the 3CL/DL precursor and the mature 3CL protease.