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Abstract

Bluetongue virus (BTV) is the causative agent of bluetongue, a disease of ruminant livestock that occurs almost worldwide between latitudes 3 ° S and 5 ° N. There are 24 serotypes of BTV (currently identified by serum neutralization assays). Since 1998, eight strains of six BTV serotypes (1, 2, 4, 8, 9 and 16) have invaded Europe. The most variable BTV protein is major outer-capsid component VP2, encoded by segment 2 (Seg-2) of the double-stranded RNA virus genome. VP2 represents the major target for neutralizing (and protective) antibodies that are generated in response to BTV infection, and is therefore the primary determinant of virus serotype. RT-PCR primers and assays targeting Seg-2 have been developed for rapid identification (within 24 h) of the six European BTV types. These assays are sensitive, specific and show perfect agreement with the results of conventional virus-neutralization methods. Previous studies have identified sequence variations in individual BTV genome segments that allow different isolates to be grouped on the basis of their geographical origins (topotypes). The assays described in this paper can detect any of the BTV isolates of the homologous serotype that were tested from different geographical origins (different Seg-2 topotypes). Primers were also identified that could be used to distinguish members of these different Seg-2 topotypes, as well as field and vaccine strains of most of the European BTV serotypes. The serotype-specific assays (and primers) showed no cross-amplification when they were evaluated with multiple isolates of the most closely related BTV types or with reference strains of the remaining 24 serotypes. Primers developed in this study will be updated periodically to maintain their relevance to current BTV distribution and epidemiology (http://www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/ReoID/rt-pcr-primers.htm).

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2019-11-12
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vol. , part 10, pp. 2811 – 2823

Electrophoretic analysis of cDNA products from Seg-2 of BTV-1 isolates using the newly designed type-specific primer pairs 1A , 1A and 1A , and a vaccine-specific primer pair, 1V.

Electrophoretic analysis of cDNA products from Seg-2 of BTV-2 isolates using the newly designed type-specific primer pairs 2A, 2W , 2W and 2W .

Electrophoretic analysis of cDNA products from Seg-2 of BTV-4 isolates using the primer pairs 4W , 4W , 4F and 4V.

Electrophoretic analysis of cDNA products from Seg-2 of BTV-8 isolates using the primer pairs 8W and 8W .

Electrophoretic analysis of cDNA products from Seg-2 of isolates of BTV-9 using type-specific primer pairs 9A and 9A and primer pairs 9E , 9W and 9W , specific to field or vaccine strains, respectively.

Electrophoretic analysis of cDNA products from Seg-2 of BTV-16 isolates using the newly designed type-specific primer pairs 16A, 16E and 16E .

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