Table 3. Evaluation of AHSV-1 DIVA rRT-PCR using horse and vaccine samples from Thailand* .

No.	Sample ID	Ct value by target gene (reference)
		VP7 AHSV rRT-PCR (20)	VP2 AHSV-1 serotyping rRT-PCR (22)	VP5 (this study)
				AHSV-1 DIVA rRT-PCR-1	AHSV-1 DIVA rRT-PCR 2
Testing of probes with initial batch samples reported in (9)
1	110983/63	17.98	17.02	17.57	Undetermined
2	111495/63	22.61	20.72	22.06	Undetermined
3	111406/63	20.88	19.19	20.04	Undetermined
4	111146/63	27.49	22.70	24.69	Undetermined
5	112080/63	17.96	16.37	16.72	Undetermined
6	111789/63	19.86	18.31	18.24	Undetermined
7	111367/63	20.67	18.78	20.29	Undetermined
8	AHSV-1 OBP VACCINE 10−3	24.52	24.17	Undetermined	27.65
9	AHSV-1 OBP VACCINE 10−4	27.56	27.90	Undetermined	30.54
10	AHSV-1 OBP VACCINE 10−5	31.81	32.15	Undetermined	34.78
11	AHSV-1 RSArah1/03	15.21	15.38	Undetermined	17.97
Validation testing of probes with samples from clinically affected horses in this study†
1	111146/63	18.04	17.40	18.25	Undetermined
2	111147/63-5	17.12	16.89	17.32	Undetermined
3	111147/63-19	21.30	20.12	20.14	Undetermined
4	111147/63-20	26.56	27.30	28.57	Undetermined
5	111147/63-21	23.05	22.98	23.81	Undetermined
6	111147/63-22	30.15	29.80	31.47	Undetermined
7	111162/63-A	21.67	22.75	23.16	Undetermined
8	111162/63-B	22.40	22.06	22.23	Undetermined
9	111164/63-1	28.60	28.39	28.45	Undetermined
10	111164/63-4	26.85	27.36	27.72	Undetermined
11	111367/63-B	16.44	17.69	17.51	Undetermined
12	111406/63-A	24.12	23.97	23.82	Undetermined
13	111406/63-B	18.34	18.09	18.26	Undetermined
14	111496/63	20.84	22.19	22.36	Undetermined
15	111790/63	23.62	23.83	23.88	Undetermined
16	112080/63	21.04	21.12	21.23	Undetermined
17	112590/63	26.71	29.12	29.04	Undetermined
18	112594/63	25.59	22.34	22.28	Undetermined
19	112680/63	23.62	22.84	22.50	Undetermined
20	113308/63	25.20	18.98	19.15	Undetermined
21	113480/63	25.52	26.15	25.97	Undetermined
22	113481/63	27.01	20.59	20.45	Undetermined
23	113489/63	24.58	26.19	25.94	Undetermined
24	113561/63-1	20.79	19.12	18.93	Undetermined
25	113561/63-3	33.45	19.66	19.34	Undetermined
26	113869/63	27.49	22.18	21.99	Undetermined
27	113870/63-11	19.68	35.29	34.38	Undetermined
28	113870/63-5	27.43	28.98	28.70	Undetermined
29	113871/63-N	27.32	31.34	32.02	Undetermined
30	113871/63-5140	28.32	21.76	21.31	Undetermined
31	113908/63	26.16	23.53	23.45	Undetermined
Validation testing of probes with samples from vaccinated horses in this study
1	116187/63	27.48	34.19	Undetermined	35.62
2	122045/63	31.60	33.12	Undetermined	35.37
3	135560/63	38.59	36.71	Undetermined	38.79
4	137720/63	34.29	33.36	Undetermined	35.32
5	118696/63	32.31	32.79	Undetermined	34.37
6	120865/63	32.23	34.03	Undetermined	35.16
7	136979/63	32.33	31.40	Undetermined	32.92
8	121673/63-A	33.24	31.16	Undetermined	33.22
9	121673/63-B	33.89	32.81	Undetermined	34.72
10	121673/63-C	34.37	32.39	Undetermined	33.85
11	124916/63-A	36.45	35.11	Undetermined	36.53
12	124916/63-B	36.45	33.69	Undetermined	35.51

*Samples that were initially submitted for testing (9) were used in the first round of designed assay optimization. Subsequently, EDTA blood and tissues samples were taken from clinically affected and vaccinated horses for assay validation. Ct values were used for classification of positive AHSV detections. AHSV, African horse sickness virus; Ct, cycle threshold; DIVA, Differentiating Infected from Vaccinated Animals; ID, identification; rRT-PCR, real-time reverse transcription PCR; VP, viral protein. †AHSV-1 serotyping rRT-PCR was not carried out for the clinically affected and vaccinated horse samples used in the confirmatory rRT-PCR reactions to test the DIVA assays. Cycle threshold values not registered after 40 cycles were reported as undetermined.