Table 2.

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

Expression plasmids with authentic ORFs							RNA	Virus
VP1	VP3	VP4	VP6	VP7	NS1	NS2	Transcripts	Recovery
-	-	-	-	-	-	-	Capped	-
+	+	+	+	+	+	+	Capped	+
+	+	+	+	-	+	+	Capped	-
+	+	+	+	+	-	+	Capped	+
Expression plasmids with optimized ORFs							RNA	Virus
VP1	VP3	VP4	VP6	VP7	NS1	NS2	Transcripts	Recovery
+	+	+	+	+	+	+	Capped	+
-	+	+	+	+	+	+	Capped	-
+	-	+	+	+	+	+	Capped	-
+	+	-	+	+	+	+	Capped	+
+	+	+	-	+	+	+	Capped	+
+	+	+	+	-	+	+	Capped	+
+	+	+	+	+	-	+	Capped	+
+	+	+	+	+	+	-	Capped	-
+	+	-	-	-	-	+	Capped	+
+	+	+	+	+	+	+	Uncapped	+
+	+	-	+	+	+	+	Uncapped	-
+	+	+	-	+	+	+	Uncapped	-
+	+	+	+	-	+	+	Uncapped	-
+	+	+	+	+	-	+	Uncapped	+
+	+	-	-	-	-	+	Uncapped	-
+	+	+	+	+	+	+	minSeg-5	-
+	+	+	+	+	+	+	delSeg-5	-

AHSV expression plasmids in the first transfection mix are indicated. In the upper panel, non-optimized ORFs in expression plasmids were used, except for ORF-VP3. In the lower panels, expression plasmids with optimized AHSV-ORFs were used. Then, monolayers were transfected with 10 full length in vitro synthesized capped or uncapped run-off RNA transcripts, except for minSeg-5 RNA (without) and delSeg-5 RNA (Seg-5 RNA with a small out-of-frame deletion). Virus recovery was microscopically determined by CPE and confirmed by IPMA, and indicated as positive (+) or negative (-)