Skip directly to site content Skip directly to page options Skip directly to A-Z link Skip directly to A-Z link Skip directly to A-Z link
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.

Main Article

References
  1. World Organisation for Animal Health. Infection with African horse sickness virus. In: Terrestrial animal health code. 2021 [cited 2021 Sep 30]. https://www.woah.org/en/what-we-do/standards/codes-and-manuals/terrestrial-code-online-access/?id=169&L=1&htmfile=chapitre_ahs.htm
  2. Meiswinkel  R, Paweska  JT. Evidence for a new field Culicoides vector of African horse sickness in South Africa. Prev Vet Med. 2003;60:24353. DOIPubMedGoogle Scholar
  3. Mellor  PS, Boorman  J. The transmission and geographical spread of African horse sickness and bluetongue viruses. Ann Trop Med Parasitol. 1995;89:115. DOIPubMedGoogle Scholar
  4. Gohre  DS, Khot  JB, Paranjpe  VL, Manjrekar  SL. Observations on the outbreak of South African horse sickness in India during 1960–1961. Bombay Vet Coll Meg. 1965:5–15.
  5. Hazrati  A. Identification and typing of horse-sickness virus strains isolated in the recent epizootic of the disease in Morocco, Tunisia, and Algeria. Arch Razi Inst. 1967;19:13143.
  6. Castillo-Olivares  J. African horse sickness in Thailand: Challenges of controlling an outbreak by vaccination. Equine Vet J. 2021;53:914. DOIPubMedGoogle Scholar
  7. King  S, Rajko-Nenow  P, Ashby  M, Frost  L, Carpenter  S, Batten  C. Outbreak of African horse sickness in Thailand, 2020. Transbound Emerg Dis. 2020;67:17647. DOIPubMedGoogle Scholar
  8. Rodriguez  M, Hooghuis  H, Castaño  M. African horse sickness in Spain. Vet Microbiol. 1992;33:12942. DOIPubMedGoogle Scholar
  9. Toh  X, Wang  Y, Rajapakse  MP, Lee  B, Songkasupa  T, Suwankitwat  N, et al. Use of nanopore sequencing to characterize African horse sickness virus (AHSV) from the African horse sickness outbreak in Thailand in 2020. Transbound Emerg Dis. 2021.PubMedGoogle Scholar
  10. World Organisation for Animal Health. WOAH member’s official African horse sickness status map (September 2020). 2020 [cited 2021 Sep 30]. https://www.woah.org/en/disease/african-horse-sickness
  11. Roy  P, Mertens  PP, Casal  I. African horse sickness virus structure. Comp Immunol Microbiol Infect Dis. 1994;17:24373. DOIPubMedGoogle Scholar
  12. Zientara  S, Weyer  CT, Lecollinet  S. African horse sickness. Rev Sci Tech. 2015;34:31527. DOIPubMedGoogle Scholar
  13. Erasmus  BA. New approach to polyvalent immunization against African horsesickness. In: Proceedings of the 4th International Conference on Equine Infectious Diseases (1976: Lyons, France) Princeton, NJ: Veterinary Publications; 1978.
  14. Mellor  PS, Hamblin  C. African horse sickness. Vet Res. 2004;35:44566. DOIPubMedGoogle Scholar
  15. World Organisation for Animal Health. African horse sickness (infection with African horse sickness virus). In: OIE Terrestrial Manual. 2019 [cited 2021 Sep 30]. https://www.woah.org/fileadmin/Home/eng/Health_standards/tahm/3.06.01_AHS.pdf
  16. Dennis  SJ, Meyers  AE, Hitzeroth  II, Rybicki  EP. African horse sickness: a review of current understanding and vaccine development. Viruses. 2019;11:11. DOIPubMedGoogle Scholar
  17. Crafford  JE, Lourens  CW, Smit  TK, Gardner  IA, MacLachlan  NJ, Guthrie  AJ. Serological response of foals to polyvalent and monovalent live-attenuated African horse sickness virus vaccines. Vaccine. 2014;32:36116. DOIPubMedGoogle Scholar
  18. Weyer  CT, Grewar  JD, Burger  P, Rossouw  E, Lourens  C, Joone  C, et al. African horse sickness caused by genome reassortment and reversion to virulence of live, attenuated vaccine viruses, South Africa, 2004–2014. Emerg Infect Dis. 2016;22:208796. DOIPubMedGoogle Scholar
  19. Bunpapong  N, Charoenkul  K, Nasamran  C, Chamsai  E, Udom  K, Boonyapisitsopa  S, et al. African horse sickness virus serotype 1 on horse farm, Thailand, 2020. Emerg Infect Dis. 2021;27:220811. DOIPubMedGoogle Scholar
  20. Guthrie  AJ, Maclachlan  NJ, Joone  C, Lourens  CW, Weyer  CT, Quan  M, et al. Diagnostic accuracy of a duplex real-time reverse transcription quantitative PCR assay for detection of African horse sickness virus. J Virol Methods. 2013;189:305. DOIPubMedGoogle Scholar
  21. Quan  M, Lourens  CW, MacLachlan  NJ, Gardner  IA, Guthrie  AJ. Development and optimisation of a duplex real-time reverse transcription quantitative PCR assay targeting the VP7 and NS2 genes of African horse sickness virus. J Virol Methods. 2010;167:4552. DOIPubMedGoogle Scholar
  22. Weyer  CT, Joone  C, Lourens  CW, Monyai  MS, Koekemoer  O, Grewar  JD, et al. Development of three triplex real-time reverse transcription PCR assays for the qualitative molecular typing of the nine serotypes of African horse sickness virus. J Virol Methods. 2015;223:6974. DOIPubMedGoogle Scholar
  23. Greninger  AL, Chen  EC, Sittler  T, Scheinerman  A, Roubinian  N, Yu  G, et al. A metagenomic analysis of pandemic influenza A (2009 H1N1) infection in patients from North America. PLoS One. 2010;5:e13381. DOIPubMedGoogle Scholar
  24. Greninger  AL, Naccache  SN, Federman  S, Yu  G, Mbala  P, Bres  V, et al. Rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis. Genome Med. 2015;7:99. DOIPubMedGoogle Scholar
  25. Guthrie  AJ, Coetzee  P, Martin  DP, Lourens  CW, Venter  EH, Weyer  CT, et al. Complete genome sequences of the three African horse sickness virus strains from a commercial trivalent live attenuated vaccine. Genome Announc. 2015;3:3. DOIPubMedGoogle Scholar
  26. Nutz  S, Döll  K, Karlovsky  P. Determination of the LOQ in real-time PCR by receiver operating characteristic curve analysis: application to qPCR assays for Fusarium verticillioides and F. proliferatum. Anal Bioanal Chem. 2011;401:71726. DOIPubMedGoogle Scholar
  27. Robin  X, Turck  N, Hainard  A, Tiberti  N, Lisacek  F, Sanchez  J-C, et al. pROC: an open-source package for R and S+ to analyze and compare ROC curves. BMC Bioinformatics. 2011;12:77. DOIPubMedGoogle Scholar
  28. Potgieter  AC, Page  NA, Liebenberg  J, Wright  IM, Landt  O, van Dijk  AA. Improved strategies for sequence-independent amplification and sequencing of viral double-stranded RNA genomes. J Gen Virol. 2009;90:142332. DOIPubMedGoogle Scholar
  29. von Teichman  BF, Smit  TK. Evaluation of the pathogenicity of African Horsesickness (AHS) isolates in vaccinated animals. Vaccine. 2008;26:501421. DOIPubMedGoogle Scholar
  30. Molini  U, Marucchella  G, Maseke  A, Ronchi  GF, Di Ventura  M, Salini  R, et al. Immunization of horses with a polyvalent live-attenuated African horse sickness vaccine: serological response and disease occurrence under field conditions. Trials Vaccinol. 2015;4:248. DOIGoogle Scholar
  31. Manole  V, Laurinmäki  P, Van Wyngaardt  W, Potgieter  CA, Wright  IM, Venter  GJ, et al. Structural insight into African horsesickness virus infection. J Virol. 2012;86:785866. DOIPubMedGoogle Scholar
  32. House  JA. Recommendations for African horse sickness vaccines for use in nonendemic areas. Rev Élev Méd Vét Pays Trop. 1993;46:7781. DOIPubMedGoogle Scholar
  33. Aksular  M, Calvo-Pinilla  E, Marín-López  A, Ortego  J, Chambers  AC, King  LA, et al. A single dose of African horse sickness virus (AHSV) VP2 based vaccines provides complete clinical protection in a mouse model. Vaccine. 2018;36:700310. DOIPubMedGoogle Scholar
  34. von Teichman  BF, Dungu  B, Smit  TK. In vivo cross-protection to African horse sickness Serotypes 5 and 9 after vaccination with Serotypes 8 and 6. Vaccine. 2010;28:650517. DOIPubMedGoogle Scholar

Main Article

Page created: October 28, 2022
Page updated: November 21, 2022
Page reviewed: November 21, 2022
The conclusions, findings, and opinions expressed by authors contributing to this journal do not necessarily reflect the official position of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors' affiliated institutions. Use of trade names is for identification only and does not imply endorsement by any of the groups named above.
file_external