Identification and differentiation of the twenty six bluetongue virus serotypes by RT-PCR amplification of the serotype-specific genome segment 2.

Narender S Maan,Manjunatha N Belaganahalli,Peter P C Mertens,Sushila Maan,Kyriaki Nomikou,Eileen N Ostlund,Donna J Johnson,Binu T Velayudhan

doi:10.1371/journal.pone.0032601

Narender S Maan, Manjunatha N Belaganahalli + Show 6 more

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https://doi.org/10.1371/journal.pone.0032601

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Abstract

Bluetongue (BT) is an arthropod-borne viral disease, which primarily affects ruminants in tropical and temperate regions of the world. Twenty six bluetongue virus (BTV) serotypes have been recognised worldwide, including nine from Europe and fifteen in the United States. Identification of BTV serotype is important for vaccination programmes and for BTV epidemiology studies. Traditional typing methods (virus isolation and serum or virus neutralisation tests (SNT or VNT)) are slow (taking weeks, depend on availability of reference virus-strains or antisera) and can be inconclusive. Nucleotide sequence analyses and phylogenetic comparisons of genome segment 2 (Seg-2) encoding BTV outer-capsid protein VP2 (the primary determinant of virus serotype) were completed for reference strains of BTV-1 to 26, as well as multiple additional isolates from different geographic and temporal origins. The resulting Seg-2 database has been used to develop rapid (within 24 h) and reliable RT–PCR-based typing assays for each BTV type. Multiple primer-pairs (at least three designed for each serotype) were widely tested, providing an initial identification of serotype by amplification of a cDNA product of the expected size. Serotype was confirmed by sequencing of the cDNA amplicons and phylogenetic comparisons to previously characterised reference strains. The results from RT-PCR and sequencing were in perfect agreement with VNT for reference strains of all 26 BTV serotypes, as well as the field isolates tested. The serotype-specific primers showed no cross-amplification with reference strains of the remaining 25 serotypes, or multiple other isolates of the more closely related heterologous BTV types. The primers and RT–PCR assays developed in this study provide a rapid, sensitive and reliable method for the identification and differentiation of the twenty-six BTV serotypes, and will be updated periodically to maintain their relevance to current BTV distribution and epidemiology (http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/rt-pcr-primers.htm).

Highlights

Bluetongue (BT) is an arthropod-transmitted hemorrhagic disease of wild and domestic ruminants
We previously reported RT-PCR based typing assays for six European bluetongue virus (BTV) serotypes 1, 2, 4, 8, 9 and 16 [16]
We describe a complete set of primer-pairs, together with an initial evaluation of their use in RT-PCR amplification assays to identify the 26 serotypes of BTV

Summary

Introduction

Bluetongue (BT) is an arthropod-transmitted hemorrhagic disease of wild and domestic ruminants. BT can have major effects on animal health, in naıve ruminant populations, restricting international trade in livestock It is listed as a ‘notifiable’ disease by the Office International des Epizooties (OIE) [4,5,6]. The BTV outer-capsid proteins VP2 and VP5, which are encoded by genome segments 2 and 6 (Seg-2 and Seg-6) respectively, are primarily involved in cell-attachment and entry during the early stages of infection. They contain epitopes ( VP2) that bind neutralising antibodies generated during infection of the mammalian host [11,12,13,14]. The specificity of these reactions in serum neutralisation or virus neutralisation tests (SNT or VNT), shows a close correlation with variations in the amino acid (aa) sequence of VP2, and has been used to identify 26 distinct serotypes (including the recent identification of BTV25 from Switzerland and BTV-26 from Kuwait) [15,16,17, 18,19,20]

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