Table 3.

Requirements of expression plasmids and run-off RNA transcripts for BTV recovery

Expression plasmids with optimized ORFs						RNA	Virus
VP1	VP3	VP4	VP6	NS1	NS2	Transcripts	Recovery
-	-	-	-	-	-	Capped	-
+	+	+	+	+	+	Capped	+
-	+	+	+	+	+	Capped	-
+	-	+	+	+	+	Capped	+
+	+	-	+	+	+	Capped	+
+	+	+	-	+	+	Capped	+
+	+	+	+	-	+	Capped	+
+	+	+	+	+	-	Capped	-
+	-	-	-	-	+	Capped	+
+	+	+	+	+	+	Uncapped	+
+	-	+	+	+	+	Uncapped	-
+	+	-	+	+	+	Uncapped	+
+	+	+	-	+	+	Uncapped	+
+	+	+	+	-	+	Uncapped	+
+	-	-	-	-	+	Uncapped	-
+	+	-	-	-	+	Uncapped	-

BTV expression plasmids with optimized ORFs in the first transfection mix are indicated. Then, monolayers were transfected with 10 full length in vitro synthesized capped or uncapped run-off RNA transcripts. Virus recovery was microscopically determined by CPE and confirmed by IPMA, and indicated as positive (+) or negative (-)