|
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 (-)
