Figure 2. Insertion of a single copy of a target for tick-specific mir-1 into the ORF of LGTV selectively attenuates virus replication in tick-derived ISE6 cells, but not in Vero cells.

(A) Schematic representation of LGTV miRNA targeted in the dE/NS1R. A mir-1 or scrambled mir-1 (mir-1*) target sequence (pixelated box) was fused with the codon-optimized sequence of an E/NS1 stem-anchor region (2171–2488 nt, gray box) and inserted in-frame at nt position 2489 of the LGTV genome (wt). TM1 and TM2 are transmembrane helixes in the C-terminal stem-anchor region of E protein. (B,C) Growth kinetics of E(1) and E(1*) viruses in Vero (B) and ISE6 (C) cells infected at an MOI of 0.01. Titers were determined in Vero cells and presented as an average of two biological replicates (±standard deviation). Differences in growth kinetics were compared using 2-way ANOVA.