Quantitative RT-PCR assays for identification and typing of the Equine encephalosis virus
Equine encephalosis (EE) is an acute, arthropod-borne, noncontagious, febrile disease of equids. The clinical signs of EE are similar to milder forms of African horse sickness (AHS) and the two diseases can be easily confused. The Equine encephalosis virus (EEV) is a distinct virus species within the genus Orbivirus, family Reoviridae, with ten linear segments of dsRNA genome. Seven distinct serotypes of EEV have been recognised on the basis of sequence analyses of Seg-2. The need for differential diagnosis of similar forms of EE and AHS warranted the development of molecular diagnostic methods for specific detection and identification of EEV. We report the development of quantitative real-time RT-PCR assay for detection of any member of the EEV species targeting the highly conserved EEV Seg-9. Similar serotype-specific qRT-PCR assays were designed for each of the seven EEV serotypes targeting genome Seg-2, encoding the serotype determining VP2 protein. These assays were evaluated using different EEV serotypes and other closely related orbiviruses. They were shown to be EEV virus species–specific, or EEV type–specific capable of detecting 1 to 13 copies of viral RNA in clinical samples. The assays failed to detect RNA from closely related orbiviruses, including AHSV and Peruvian horse sickness virus (PHSV) isolates.
KeywordsEquine encephalosis Equine encephalosis virus EEV TaqMan assays Real-time RT-PCR Serotype-specific assays Serogroup-specific assays Orbivirus Reference Collection Culicoides
This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) (grant no. BB/L004690/1) and Department for Environment, Food and Rural Affairs (Defra) (SE2617) to PM. Department of Biotechnology, Government of India (DBT, GOI) (grant no. BT/IN/Indo-UK/FADH/46/SM/2013.
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Conflict of interest
The authors declare that they have no conflict of interest.
- 2.Attoui H, Maan SS, Anthony SJ, Mertens PPC. 2009. Bluetongue virus, other orbiviruses and other reoviruses: their relationships and taxonomy, p 23-552. In Mellor PS, Baylis M, Mertens PPC (ed), Bluetongue monograph, 1 ed. Elsevier/Academic Press, LondonGoogle Scholar
- 5.Erasmus BJ, Adelaar TF, Smit JD, Lecatsas G, Toms T (1970) The isolation and characterization of equine encephalosis virus. Bulletin de I'office International des Epizooties 74:781–789Google Scholar
- 6.Howell PG, Groenewald D, Visage CW, Bosman AM, Coetzer JA, Guthrie AJ (2002) The classification of seven serotypes of equine encephalosis virus and the prevalence of homologous antibody in horses in South Africa. Onderstepoort J Vet Res 69:79–93Google Scholar
- 8.Westcott DG, Mildenberg Z, Bellaiche M, McGowan SL, Grierson SS, Choudhury B, Steinbach F. 2013. Correction: evidence for the circulation of equine encephalosis virus in Israel since 2001. PLoS One 8Google Scholar
- 15.Attoui H, Mendez-Lopez MR, Rao S, Hurtado-Alendes A, Lizaraso-Caparo F, Jaafar FM, Samuel AR, Belhouchet M, Pritchard LI, Melville L, Weir RP, Hyatt AD, Davis SS, Lunt R, Calisher CH, Tesh RB, Fujita R, Mertens PP (2009) Peruvian horse sickness virus and Yunnan orbivirus, isolated from vertebrates and mosquitoes in Peru and Australia. Virology 394:298–310CrossRefGoogle Scholar
- 18.Mertens PPC, Maan S, Samuel A, Attoui H. 2005. Orbiviruses, Reoviridae, p 466-483. In Fauquet CM, M.A. Mayo, J. Maniloff, U. Desselberger and L.A. Ball (ed), Virus taxonomy eighth report of the international committee on taxonomy of viruses. Elsevier/Academic Press, LondonGoogle Scholar
- 23.Maan S, Maan NS, van Rijn PA, van Gennip RG, Sanders A, Wright IM, Batten C, Hoffmann B, Eschbaumer M, Oura CA, Potgieter AC, Nomikou K, Mertens PP (2010) Full genome characterisation of bluetongue virus serotype 6 from the Netherlands 2008 and comparison to other field and vaccine strains. PLoS One 5:e10323CrossRefGoogle Scholar
- 32.Maan S, Maan NS, Batra K, Kumar A, Gupta A, Rao PP, Hemadri D, Reddy YN, Guimera M, Belaganahalli MN, Mertens PP (2016) Reverse transcription loop-mediated isothermal amplification assays for rapid identification of eastern and western strains of bluetongue virus in India. J Virol Methods 234:65–74CrossRefGoogle Scholar
- 38.Belaganahalli MN (2012) Full genome sequencing and phylogenetic analyses of different Orbivirus species. PhD. University London, LondonGoogle Scholar
- 40.Wechsler SJ, McHolland LE (1988) Susceptibilities of 14 cell lines to bluetongue virus infection. J Clin Microbiol 26:2324–2327Google Scholar
- 46.Leblanc N, Rasmussen TB, Fernandez J, Sailleau C, Rasmussen LD, Uttenthal A, Zientara S, Belak S, Hakhverdyan M (2010) Development of a real-time RT-PCR assay based on primer-probe energy transfer for the detection of all serotypes of bluetongue virus. J Virol Methods 167:165–171CrossRefGoogle Scholar