Requirements and comparative analysis of reverse genetics for bluetongue virus (BTV) and African horse sickness virus (AHSV)

Table 1 Overview of rescued AHSV4LP variants demonstrating the improvements of the RG method

Name	Seg of AHSV4LP¹	Segment exchange²	600 ng	300 ng	300 ng	Virus rescue	CPE³ VP5	IPMA⁴
			a	b	c			VP2	NS3
AHSV4LP	1–10	-	+	+	+	+	+	4	+
Serotyped 3	1, 3–5, 7–10	(2, 6) 3	-	+	nd	+	+	3	nd
Serotyped 6	1, 3–5, 7–10	(2/6) 6	-	+	nd	+	+	6	nd
AHSV1LP	1, 3–10	(2) 1	nd	+	nd	+	+	1	nd
AHSV2LP	1, 3–10	(2) 2	-	+	nd	+	+	2	nd
AHSV3LP	1, 3–10	(2) 3	-	+	nd	+	+	3	nd
AHSV4LP	1–10	-	+	+	+	+	+	4	+
AHSV5LP	1, 3–10	(2) 5	-	-	+	+	+	5	nd
AHSV6LP	1, 3–10	(2) 6	-	+	nd	+	+	6	nd
AHSV7LP	1, 3–10	(2) 7	nd	+	nd	+	+	7	nd
AHSV8LP	1, 3–10	(2) 8	nd	+	nd	+	+	8	nd
AHSV9LP	1, 3–10	(2) 9	nd	+	nd	+	+	9	nd
AHSV4LP	1–10	-	+	+	+	+	+	4	+
Without Seg-10	1–9	-	-	-	-	-	-	nd	nd
Seg-10 of AHSV2	1–9	(10) 2	nd	nd	+	+	+	nd	+
Seg-10 of AHSV3	1–9	(10) 3	-	-	+	+	+	nd	+
mutAUG1	1–9	(10) AUG1	+	nd	nd	+	+	nd	+
mutAUG1 + 2	1–9	(10) AUG1 + 2	nd	+	nd	+	Small	nd	+
mutAUG1 + 2 & STOPS	1–9	(10) AUG1 + 2&STOPS	nd	+	nd	+	Small	nd	+
delLD	1–9	(10) delLD	-	+	nd	+	Small	nd	+
delTMR1	1–9	(10) delTMR1	nd	nd	+	+	-	nd	-
delTMR2	1–9	(10) delTMR2	nd	nd	+	+	-	nd	±
AUG total	1–9	(10) AUGtotal	nd	nd	+	+	-	nd	-
AHSV1LP + (mutAUG1 + 2 & STOPS)	1, 3–9	(2) 1, (10) AUG1 + 2&STOPS	nd	-	+	+	Small	1	+
AHSV8LP + (mutAUG1 + 2 & STOPS)	1, 3–9	(2) 8, (10) AUG1 + 2&STOPS	nd	+	+	+	Small	8	+
AHSV8LP + (AUG total)	1, 3–9	(2) 8, (10) AUGtotal	nd	nd	+	+	-	8	-

(1) In total, a mixture of 600 or 300 ng in equal molar amounts of seven expression plasmids were transfected followed by transfection of indicated RNAs of Seg-1 to 10. (2) Exchanged Seg-2[VP2] or Seg-10[NS3/NS3a] are indicated. AHSV1LP to AHSV9LP and all Seg-10 mutants have been described previously, except for Seg-10 ‘AUG total’ containing AUG- > GCC mutations for all 13 AUG codons of the NS3/NS3a ORF. Expression plasmids contained authentic ORFs, except for ORF-VP3 (a & b), or all seven expression plasmids with ORFs optimized for plasmid stability and eukaryotic expression (c). In total, 600 or 300 ng expression plasmids were used. Virus rescue was determined and confirmed by IPMA and indicated as successful (+), unsuccessful (-), or not done (nd). (3) After immunostaining with α-VP5 MAb, plaque morphology was microscopically scored as normal CPE (+), small CPE (small), or immunostained plaques without CPE (-). (4) IPMA results with the respective α-VP2 GP serum is indicated by the serotype number, and with α-NS3 MAbs as positive (+), weak (±), negative (-) or not done (nd)

ISSN: 1743-422X