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Volume 28, Number 12—December 2022
Research

Development of Differentiating Infected from Vaccinated Animals (DIVA) Real-Time PCR for African Horse Sickness Virus Serotype 1

Yifan Wang, Jasmine Ong, Oi Wing Ng, Tapanut Songkasupa, Eileen Y. KohComments to Author , Jeslyn P.S. Wong, Kanokwan Puangjinda, Charlene Judith Fernandez, Taoqi Huangfu, Lee Ching Ng, Siow Foong Chang, and Him Hoo Yap
Author affiliations: Animal and Veterinary Service, National Parks Board, Singapore (Y. Wang, J. Ong, O.W. Ng, E.Y. Koh, C.J. Fernandez, T. Huangfu, S.F. Chang, H.H. Yap); National Institute of Animal Health, Bangkok, Thailand (T. Songkasupa, K. Puangjinda); Environmental Health Institute, National Environment Agency, Singapore (J.P.S. Wong, L.C. Ng)

Main Article

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.

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Page updated: November 21, 2022
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