Arbovirus Molecular Research Group, Vector-Borne Viral Diseases Programme, Institute for Animal Health, Woking, Surrey, United Kingdom.
PLoS One. 2012;7(2):e32601. doi: 10.1371/journal.pone.0032601. Epub 2012 Feb 28.
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).
蓝舌病（BT）是一种由节肢动物传播的病毒性疾病，主要影响世界热带和温带地区的反刍动物。全世界已确认有 26 种蓝舌病病毒（BTV）血清型，其中包括 9 种来自欧洲和 15 种来自美国。BTV 血清型的鉴定对于疫苗接种计划和 BTV 流行病学研究非常重要。传统的分型方法（病毒分离和血清或病毒中和试验（SNT 或 VNT））速度较慢（需要数周时间，取决于参考病毒株或抗血清的可用性），并且可能无法得出明确的结论。对 BTV-1 至 26 型的参考株以及来自不同地理和时间来源的多个额外分离株的基因组第 2 节（Seg-2）编码 BTV 外壳蛋白 VP2（病毒血清型的主要决定因素）的核苷酸序列分析和系统发育比较已经完成。由此产生的 Seg-2 数据库已用于为每个 BTV 型快速（24 小时内）和可靠的基于 RT-PCR 的分型检测。广泛测试了多个引物对（每个血清型至少设计了三个），通过扩增预期大小的 cDNA 产物初步鉴定了血清型。通过对 cDNA 扩增子的测序和与以前表征的参考株的系统发育比较来确认血清型。RT-PCR 和测序的结果与所有 26 种 BTV 血清型的 VNT 完全一致，与测试的现场分离株也完全一致。血清型特异性引物与其余 25 种血清型的参考株或其他密切相关的异源 BTV 型的多个分离株没有交叉扩增。本研究中开发的引物和 RT-PCR 检测方法为 26 种 BTV 血清型的鉴定和区分提供了一种快速、敏感和可靠的方法，并将定期更新以保持其与当前 BTV 分布和流行病学的相关性（http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/rt-pcr-primers.htm）。
