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Development and evaluation of real time RT-PCR assays for detection and typing of Bluetongue virus

Richard, Yolande; Maan, Sushila; Maan, Narender Singh; Belaganahalli, Manjunatha N.; Potgieter, Abraham C.; Kumar, Vinay; Batra, Kanisht; Wright, Isabel M.; Kirkland, Peter D.; Mertens, Peter P.C.

Authors

Yolande Richard

Sushila Maan

Narender Singh Maan

Manjunatha N. Belaganahalli

Abraham C. Potgieter

Vinay Kumar

Kanisht Batra

Isabel M. Wright

Peter D. Kirkland

Peter P.C. Mertens



Abstract

Bluetongue virus is the type species of the genus Orbivirus, family Reoviridae. Bluetongue viruses (BTV) are transmitted between their vertebrate hosts primarily by biting midges (Culicoides spp.) in which they also replicate. Consequently BTV distribution is dependent on the activity, geographic distribution, and seasonal abundance of Culicoides spp. The virus can also be transmitted vertically in vertebrate hosts, and some strains/serotypes can be transmitted horizontally in the absence of insect vectors. The BTV genome is composed of ten linear segments of double-stranded (ds) RNA, numbered in order of decreasing size (Seg-1 to Seg-10). Genome segment 2 (Seg-2) encodes outer-capsid protein VP2, the most variable BTV protein and the primary target for neutralising antibodies. Consequently VP2 (and Seg-2) determine the identity of the twenty seven serotypes and two additional putative BTV serotypes that have been recognised so far. Current BTV vaccines are serotype specific and typing of outbreak strains is required in order to deploy appropriate vaccines. We report development and evaluation of multiple ‘TaqMan’ fluorescence-probe based quantitative real-time type-specific RT-PCR assays targeting Seg-2 of the 27+1 BTV types. The assays were evaluated using orbivirus isolates from the ‘Orbivirus Reference Collection’ (ORC) held at The Pirbright Institute. The assays are BTV-type specific and can be used for rapid, sensitive and reliable detection / identification (typing) of BTV RNA from samples of infected blood, tissues, homogenised Culicoides, or tissue culture supernatants. None of the assays amplified cDNAs from closely related but heterologous orbiviruses, or from uninfected host animals or cell cultures.

Citation

Richard, Y., Maan, S., Maan, N. S., Belaganahalli, M. N., Potgieter, A. C., Kumar, V., …Mertens, P. P. (2016). Development and evaluation of real time RT-PCR assays for detection and typing of Bluetongue virus. PLoS ONE, 11(9), https://doi.org/10.1371/journal.pone.0163014

Journal Article Type Article
Acceptance Date Sep 1, 2016
Publication Date Sep 23, 2016
Deposit Date Nov 23, 2016
Publicly Available Date Nov 23, 2016
Journal PLoS ONE
Electronic ISSN 1932-6203
Publisher Public Library of Science
Peer Reviewed Peer Reviewed
Volume 11
Issue 9
Article Number e0163014
DOI https://doi.org/10.1371/journal.pone.0163014
Public URL http://eprints.nottingham.ac.uk/id/eprint/38905
Publisher URL http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0163014
Copyright Statement Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0

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Development and Evaluation of Real Time RT-PCR Assays for Detection and Typing of Bluetongue Virus.pdf (771 Kb)
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Copyright Statement
Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0





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